Poster Flash 1 - 1: CISTUS SALVIFOLIUS TOXICITY IN BEEF HERDS IN SICILY (ITALY): CLINICAL AND PATHOLOGICAL INVESTIGATIONS

S.A. Mignacca*, E. Biasibetti@, M. Mucciarelli#, S. Muscia§, B. Amato, V. Di Marco Lo Presti and M. T. Capucchio@

 *Veterinary practitioner, Enna, Italy; @Department of Veterinary Sciences, University of Turin, Italy; #Department of Life Science and Systems Biology, University of Turin, Italy; §Veterinary practitioner, Palermo, Italy and  Istituto Zooprofilattico Sperimentale della Sicilia “A.Mirri”, Barcellona P.G. (Messina), Italy

Introduction: Cistus spp. toxicosis is sporadically reported in ruminants. The chronic plant consumption is mostly associated with weight loss, urinary tract disease and death. The present work describes the clinical, laboratory and pathological findings regarding a presumed Cystus salvifolius toxicosis in three beef cattle herds located in two different areas of Sicily (Italy).

Materials and methods: Epidemiological data were collected on three farms (70-48-16 animals each) followed by botanical investigations. Affected animals were submitted to clinical evaluation and hematological analyses. Necropsy and histological investigations on most important organs were made on selected animals.

Results: Outbreaks were observed after grazing in wintry pasture where Cystus salvifolius was present. Most of the affected animals were 6 to 36 months old. Clinically disorexia, weight loss, pollakiuria, abortion, neurological symptoms and death were reported. Mean morbidity and mortality were 29% and 21% respectively. Moderate anemia, increase of urea, CPK, AST, LDH and phosphorus and decrease in total serum protein, magnesium, chloride and calcium were detected. At necropsy a severe distention of the urinary bladder and enteritis were present. Urine was cloudy with crystals. Histologically the main findings observed were chronic cystitis, interstitial nephritis, eosinophilic enteritis, non suppurative necrotizing hepatitis and brain degeneration.

Conclusions: On the basis of epidemiology, clinical signs and pathological findings, a diagnosis of presumed Cistus salvifolius toxicosis was made. This is the first report of Cystus salvifolius toxicosis in cattle in Italy. Further studies are needed to correlate the evolution of the disease with the plant intake generating this toxicity.


Poster Flash 1 - 2: Cancelled


Poster Flash 1 - 3: Cancelled


Poster Flash 1 - 4: ASSOCIATION OF EQUINE GAMMAHERPESVIRUS-5 WITH LYMPHOHISTIOCYTIC INTERFACE DERMATITIS ON THE MUZZLE OF TWO HORSES IN THE UNITED STATES

B. Cossic*, A. L. Glaser*, G. E. Duhamel* and J. Peters-Kennedy*

*Department of Biomedical Sciences, and New York Animal Health Diagnostic Center, College of Veterinary Medicine, Cornell University, Ithaca, NY, USA

Introduction: Skin biopsy specimens taken from a 15-year-old Haflinger mix gelding and a 9-year-old Thoroughbred mix gelding with non-painful, non-pruritic, scaly, annular to irregular lesions on the muzzle were submitted for histological evaluation.

Results: Histopathologically, there was a moderate lymphohistiocytic interface dermatitis with mild multifocal hydropic degeneration of the keratinocytes, pigmentary incontinence and moderate to marked compact orthokeratotic to parakeratotic hyperkeratosis. Throughout the epidermis, but predominantly in the stratum basale, there were small numbers of apoptotic keratinocytes, some with satellitosis.  These changes were suggestive of discoid lupus erythematosus-like disease. Additionally, small numbers of keratinocytes within the upper stratum spinosum and stratum granulosum had glassy basophilic intranuclear inclusion bodies that marginated the chromatin, suggestive of herpesvirus infection. PCR assays of DNA extracted from FFPE confirmed the presence of equine gammaherpesvirus-5 (EHV-5). EHV-5 replication within clusters of stratum corneum keratinocytes was further demonstrated by using a virus-specific in situ hybridization method. EHV-5 has a worldwide distribution, and a prevalence ranging between 3 and 100 percent depending on the sampling method and age group. The clinical and pathological features of our cases were similar to those reported by Herder et al., (2012) in a 9-year-old Holsteiner stallion from south Germany with EHV-5-associated erythema multiforme lesions.

Conclusions: These cases provide further evidence of EHV-5 association with dermatitis in horses in the United States, and consideration as a differential diagnosis for facial interface dermatitis in horses. The identification of EHV-5 will assist with the development of improved protocols for the diagnosis of skin diseases in horses.


Poster Flash 1 - 5: AN OUTBREAK OF ENZOOTIC ATAXIA IN CALVES

N. Cuesta-García*, M. Morales, E. Gayo-Roces*, MJ. García-Iglesias*, C. Pérez-Martínez* and JF. García-Marín*

*Animal Health Department, University of León, Spain, Universidad de León. Farm Animal clinician, Zamora, Spain.

Case presentation: In a dairy cow farm, 7-9 month-old calves were fed with commercial feed, barley flour, corn and grass hay ad libitum. After replacing the feed with old barley, 70% of the animals started with progressive ataxia ending with recumbency, with normal response to stimuli and without fever. Calves were administered Vitamin B and Membutone with no improvement. Hypocupremia (56-115 µg/dl mg/L, reference range 125-150).

Pathology results: Post-mortem examination was performed on two calves with acute clinical signs, and on one calf with subacute clinical signs. Histopathology revealed demyelination, axonopathy and neuronal degeneration mainly located on the red nucleus, medulla oblongata and ventral horns of the spinal cord, with milder changes in the cerebellum and peripheral nerves.

Diagnosis: A diagnosis of copper deficiency was established and the remaining calves were given a supplementary oral treatment based on copper sulphate monohydrate and Vitamin B. Gradually, all the affected animals returned to normal.

Conclusions: The clinical signs, the neural lesions and the response to treatment were consistent with a diagnosis of Enzootic Ataxia in calves similar to that described in lambs. The mineral levels analysed in water, feed and land were not altered, therefore, an indirect form of deficiency was suspected. The farm vet confirmed the old barley may have been treated with sulphates, which could have formed copper sulphurs that are insoluble in the ruminant digestive tract. Copper deficiency should be included in the differentials of calves with neurological signs, however a histopathological study is essential to support the diagnosis.


Poster Flash 1 - 6: BACILLUS ANTHRACIS AS A CAUSE OF BOVINE ABORTION – A CHALLENGING CASE REGARDING DIAGNOSIS AND BIOSAFETY

M. Dettwiler*, K. Mehinagic*, S. Gobeli Brawand, S. Rottenberg* and H. Posthaus*

*Institute for Animal Pathology and Institute of Veterinary Bacteriology, Department of Infectious Diseases and Pathobiology, Vetsuisse Faculty, University of Bern, Switzerland

Introduction: Bacillus (B.) anthracis causes anthrax, a potentially fatal zoonosis. Diseased ruminants usually die suddenly with significant bacteremia. Necropsy of anthrax cases is highly risky due to the release of resistant endospores causing environmental contamination. We describe the necropsy of an unusual anthrax case and its challenges for pathologists.

Materials and Methods: On a farm with a previously reported B. anthracis isolation from a deceased bovine, several cows developed unspecific feverish illness. Blood cultures repeatedly remained negative for B. anthracis. To determine the etiology, the veterinary authorities demanded a necropsy of one cow. Our necropsy room was divided into three risk zones. Personnel were equipped with disposable suits and HEPA-filtered respirators. Necropsy was started and continued only after negative microscopic testing of Giemsa-stained smears from blood, liver, spleen and kidney, respectively. Immediately after necropsy, carcass, equipment and necropsy room were decontaminated.

Results: The cow showed vaginal discharge and a severe ventral edema, while blood smears were negative for B. anthracis. Spleen, liver and kidneys were inconspicuous and smears were negative for B. anthracis. Sublumbal lymph nodes were severely enlarged and necrotic. The uterus showed hemorrhagic placentomes and contained an autolytic fetus. Blood, spleen, liver and kidney were sterile in bacterial cultures, but turned out to contain antibiotics. Nevertheless, B. anthracis was detected in high numbers in smears and culture of placenta and lymph node. Swabs taken subsequently from necropsy room and equipment were all negative for B. anthracis.

Conclusions: Non-septicemic anthrax cases may exhibit unexpected lesions mimicking less hazardous disease.


Poster Flash 1 - 7: FORENSIC RELEVANCE OF AGONAL WOUNDS IN SLAUGHTER PIGS

K. Barington and H. E. Jensen

Department of Veterinary and Animal Sciences, University of Copenhagen, Denmark

Introduction: Determining the vitality of wounds is central in forensic pathology. In porcine wounds established seconds postmortem (PM) a vital reaction is absent. In the present study, we examined if a reaction of vitality is present in pigs traumatized seconds prior to death, and if cooling of the tissue interfered with the interpretation.

Materials and Methods: A stab wound was inflicted in the sulcus jugularis of 26 slaughter pigs at 30-60 seconds prior to cardiac arrest. All wounds were subjected to gross and histological examination. Wounds were sampled 1 h PM (n=11, before cooling) and 24 h PM (n=15, after cooling). All samples were assessed for hemorrhage, fibrin, extravasated leukocytes, hyperleukocytosis and pavement of leukocytes.

Results: Grossly and histologically, blod clots (hemorrhage and fibrin) were present in all wounds. Leukocytes were present at the edges of all wounds sampled 1 h PM and in 13 of the 15 wounds (86.7%) sampled 24 h PM. The leukocytes were at all locations observed in relation to hemorrhage and consisted of mature non-degenerated neutrophils and mononuclear cells. In none of the wounds, hyperleukocytosis or pavement of leukocytes was present.

Conclusions: A vital reaction was not present in wounds inflicted seconds before death. Moreover, cooling of the wounds did not interfere with reactions of relevance for forensic interpretation.


Poster Flash 1 - 8: INHIBITION OF IMMUNE RESPONSE IN SHEEP INFECTED WITH BLV

M. Wasiak, A. Kycko and M. Reichert

Department of Pathology, National Veterinary Research Institute, Pulawy, Poland

Introduction: Bovine leukemia virus (BLV) naturally infects cattle but can also be experimentally transmitted to sheep. Infection accompanied by a long latency period in which the animal is in an aleukemic state and after few years acute leukemia develops in sheep or persistent leukocytosis in cows. How isit possible that BLV is cheating on immune cells and what is the way to escape from them?

Materials and Methods: Flow cytometry analysis of T cell activation in sheep infected with BLV by assessment of CD25 expression on T helper cells and T cytotoxic cells. Enzyme linked immunosorbent assay for determination of inhibitory cytokines: TGF-β1 and IL-10.

Results: Percentages of CD4+CD25+ cells and CD8+CD25+ cells were not statistically different in control (43,6% and 7,28%; n=9), latent (43,4% and 5,77%; n=9) and leukemic (34,% and 3,07%; n=6) group. Concentration of TGF-β1 in plasma was highest in the latent group (129,5 ng/ml) in comparison to the control (76,54 ng/ml) and the leukemic (47,03 ng/ml) group. Concentration of IL-10 in plasma was lowest in the leukemic group (6,8 pg/ml) in comparison to the latent group (7,5 pg/ml) and the control group (8,12 pg/ml).

Conclusions: BLV induces TGF-β1 secretion in early stage after infection and latency period. TGF-β1 inhibits the expression levels of IL-2 receptor (CD25) on T cells down-regulating their activity. High level of TGF-β1 in plasma of infected sheep could explain a non-effective immune response resulting in inability to totally eliminate virus, but this is not sufficient to explain cancer transformation.


Poster Flash 1 - 9: MYCOPLASMAL AND VIRAL CO-INFECTIONS IN BOVINE RESPIRATORY DISEASE COMPLEX IN SWISS CATTLE

K. Mehinagic*, P.Pilo and N. Stokar-Regenscheit*

*Institute of Animal Pathology and Institute for Veterinary Bacteriology, Vetsuisse Faculty, University of Bern, Switzerland

Introduction: Bovine respiratory disease complex (BRDC) is a common and economically significant disease of cattle worldwide. Respiratory disease is the most commonly observed disease in dairy calves in Switzerland. The aim of the project was to detect Mycoplasma (M.) bovis and primary viral agents in BRDC cases in Swiss cattle at the chronic and lethal stage of the disease, since these are usually underdiagnosed in routine diagnostics.

Materials and Methods: The study was performed retrospectively including 104 BRDC necropsy cases. HE sections were classified according to the type of pneumonia and chronicity. Furthermore, immunohistochemistry for Bovine Respiratory Syncytial Virus (BRSV), Parainfluenza-3 (PI-3), M. bovis and PCR for M. bovis were performed on all cases and correlated to the morphological type of pneumonia.

Results: Histomorphologically, out of 104 cases, 76% were classified as bronchopneumonia, 15% as broncho-interstitial pneumonia and 9% as interstitial pneumonia. In 86% (n=89) of the cases at least one of the investigated agents was detected. Out of these, 49% (n=44) showed a co-infection with two or three agents, whereas in 51% (n=45) only one agent was detected. PI-3 was the predominant agent present, with 44% (n=39) as single infection, and in 44% (n=39) in co-infection with M. bovis.

Conclusions: The results show that viral agents are persisting in chronic and lethal BRDC, suggesting an ongoing impairment of defense mechanisms in the lung, increasing the lethality of BRDC. These results support new preventive and treatment perspectives like the development of immunotherapy or vaccines for severe and chronic BRDC cases.


Poster Flash 1 - 10: PATHOPIG - STRENGTHENING POST MORTEM DIAGNOSTICS AND EARLY DETECTION OF PIG DISEASES

M. Schediwy*, S. Balmer, G. Rosato, T. Sydler, D. Hadorn, H. Posthaus*, P. J. Bless, M. Harisberger # and C. Gurtner *

*Institute of Animal Pathology, Department of Infectious Diseases and Pathobiology, Vetsuisse Faculty, University of Bern, Switzerland and Federal Food Safety and Veterinary Office (FSVO), Bern, Switzerland and Institute of Veterinary Pathology, Vetsuisse Faculty, University of Zurich, Switzerland and #Division Pig Health Service, SUISAG, Sempach, Switzerland

Introduction: Post mortem diagnostics is important for the early detection of emerging animal diseases and zoonoses, and contributes to nationwide surveillance programs. However, over the last years there has been a decline of post mortem examinations of pigs in Switzerland. Possible reasons are of financial or logistical nature or lack of awareness of the importance of necropsies as a diagnostic tool. To counteract this decline the Federal Food Safety and Veterinary Office together with Swiss pathology laboratories and the Swiss Pig Health Service established a project in 2014 called PathoPig to support financially post mortem diagnostics of diseased pigs.

Materials and Methods: Farmers with herd health problems contacted the veterinarian who submitted up to three animals representing the health problem to a pathology laboratory. At least one of four submitting criteria had to be fulfilled in order to get the financial support.

Results:  From 2014 to 2016, a total of 1897 pigs were sent for a post mortem examination, resulting in an increase in the number of pig necropsies in Switzerland. In the majority of cases, a conclusive pathological diagnosis was obtained. The most commonly affected organ system was the gastrointestinal tract. The results of the follow-up investigations showed that in the majority of cases the herd health improved.   

Conclusions: The project led to an increase of porcine necropsies and improvement of pig herd health. The improvement of collaboration between the different stakeholders strengthens post mortem diagnostics, disease awareness and early detection of pig diseases in Switzerland.


Poster Flash 2 - 1: AUDITORY AND NERVOUS SYSTEM PATHOLOGY IN A MOUSE MODEL OF POMPE DISEASE

F.D. Franzoso*, C. Ciron*, J. Hordeaux*, C, Bagarie* , J. Deniaud* and MA. Colle*

*PAnTher, INRA, École nationale vétérinaire, agro-alimentaire et de l’alimentation Nantes-Atlantique (Oniris), Université Bretagne Loire, (UBL), Nantes, F-44307, France

Introduction: Hearing loss was recently recognized as one of the symptoms of Pompe disease (glycogenosis of type II), a lysosomal storage disorder. It is still unknown whether central nervous system involvement interferes with auditory function. We investigated this hypothesis in a knock-out mouse model of Pompe disease.

Materials and Methods: Auditory brainstem response (ABR) was recorded at 9 and 12 months. Sections of the brain and ear of the 6neo/6neo mice and wild type B6/129 mice (age of 9, 12 and 15 months) were fixed, embedded in paraffin and stained with Hematoxylin-Eosin, PAS-Luxol Fast Blue and Masson’s Trichome.

Results:  The ABR demonstrated an alteration in nerve conduction velocity within the auditory brainstem at 9 and 12 months of age. The pathology of the specific nervous structures of the auditory tract, such as spiral ganglion, superior olivary nucleus, cochlear nucleus, inferior colliculi, auditory cortex showed severe cytoplasmic neuronal vacuolisation with marked accumulation of PAS positive glycogen inclusions in our mouse model. The tympanic membrane, Reissner membrane and organ of Corti presented increased numbers of granular PAS positive deposits at age of 15 months when compared to the 9 months and wild-type mice.

Conclusion: We observed severe pathologic changes of the affected structures of nervous system and mild changes in the auditory system that could explain the moderate to severe auditory physiologic dysfunction reported in the 6neo/6neo mice. This mouse model can be used to compare the hearing deficits and pathology observed in Pompe disease affected human patients.


Poster Flash 2 - 2: PDGFRB FUNCTION IN ANAPLASTIC LARGE CELL LYMPHOMA

S. Roos1, I. Garces de los Fayos Alonso1,2,3, M. Schlederer2, M. Kothmayer1,2, G. Wodarz1,2, O. Pusch4, S. Lagger1 and L. Kenner1,2,3

1 Unit of Laboratory Animal Pathology, University of Veterinary Medicine, Vienna, Austria

2 Institute of Experimental Pathology, Medical University of Vienna, Vienna, Austria

3 Ludwig Boltzmann Institute for Cancer Research, Vienna, Austria

4 Center for Anatomy and Cell Biology, Medical University of Vienna, Vienna, Austria

Introduction: Anaplastic large-cell lymphoma (ALCL) is a malignant non-Hodgkin lymphoma (NHL) most commonly diagnosed in children and young adults. A majority of the tumours carry the translocation t(2;5)(p23;q35), resulting in the fusion of the Nucleophosmin (NPM) gene to the Anaplastic lymphoma kinase (ALK) gene. The oncogenic fusion protein (NPM-ALK) consequently contributes to the pathogenesis and progression of over 50% of ALCL cases. Recent studies from our lab identified AP-1 transcription factors JUNB and cJUN as downstream effectors of NPM-ALK, which directly up-regulate platelet derived growth factor receptor beta (PDGFRB) expression in lymphoma cells. Therapeutic inhibition of PDGFRB with the kinase inhibitor Imatinib resulted in rapid, complete, and sustained remission in a late-stage therapy-resistant ALCL patient. Despite these findings the underlying molecular mechanisms of PDGFRB signaling in ALCL still remain unclear.

Materials and Methods: To investigate the mechanisms of PDGFRB signalling in ALCL, we have crossed a murine ALCL model, which expresses NPM-ALK under the CD4 promoter, to PDGFRB floxed mice and a CD4 promoter driven Cre recombinase, to yield specific deletion of PDGFRB in T cells (CD4-NPM-ALK-CD4ΔPDGFRB).

Results: Intriguingly, CD4-NPM-ALK-CD4ΔPDGFRB mice show significantly prolonged survival rates, reduced tumour growth and size (n=28). IHC stainings revealed a reduced proliferation rate, increased apoptosis and reduced vascularisation (n=9).

Conclusions: Altogether our data further support the significance of PDGFRB in ALCL as its absence leads to decreased tumour development in NPM-ALK mice. Further experiments are needed to identify the involved pathways.


Poster Flash 2 - 3: SOLID LIPID NANOPARTICLES AS NEW SYSTEM FOR THE DELIVERY OF TEMOZOLOMIDE IN A MOUSE MODEL OF MELANOMA

E. Biasibetti*, L. Battaglia, R.Veglio*, B. Ferrara, E. Muntoni, L. Cangemi, R. Evangelista*, M.T. Capucchio* and C. Dianzani

* Department of Veterinary Sciences, University of Turin, Italy, Department of Drug Science and Technology, University of Torino, Torino, Italy,

Introduction: This preliminary work aims to study a new system for the delivery of temozolomide (TMZ), based on solid lipid nanoparticles (SLN) to melanomas, by employment of in vitro and in vivo models.

Materials and Methods: SLN biocompatibility on human lymphocytes, and SLN-TMZ cytoxicity on human and mouse melanoma cells were assayed (WST-1 and clonogenic assay), as well as anti-angiogenic activity (tubuli-forming assay on HUVEC). Twenty female C57BL6/J mice (according with the University Ethical Committee and European guidelines) were subcutaneously injected with B16-F10 cells (1x105 in 100 μl/mouse). Ten days after the induction, mice were treated three times a week by injection of: TMZ (G1), SLN-TMZ(G2), or PBS(G3). The treatment was carried out for two weeks (6 i.v./mouse; 0.5 µmoles TMZ/g). Animals were sacrificed two days after the last injection and submitted to a complete necropsy followed by histological and immunohistochemical investigations (Ki67-Dako-; Cleaved Caspase 3(Asp175)-Cell Signaling-; and CD31-ABCAM-).

Results: SLN were biocompatible on lymphocytes, and SLN-TMZ exerted an increased antitumoral activity on melanoma cell lines compared to free TMZ. Tumor growth obtained in G2 treated mice was significantly lower than either G3 or G1 and only one animal showed pulmonary metastasis (G3). All the tumors were characterized by diffuse and severe necrotic areas. Ki67 and CD31 expression showed a negative trend in G2, while Caspase 3 staining did not demonstrate a significant induction of tumor apoptosis.

Conclusions: Despite encouraging results, further investigations should be performed concerning the mechanism of cytotoxicity and the biodistribution of SLN.


Poster Flash 2 - 4: COMPARISON OF SKIN PENETRATION, PATHOLOGICAL AND CLINICAL EFFECTS OF NOVEL DRUG TRANSPORTERS -THERMORESPONSIVE NANOGELS AND CORE MULTISHELL NANOCARRIERS- IN A MURINE MODEL OF ATOPIC DERMATITIS

H. Pischon*, M.  Radbruch*, F.Du, M. Guilbudagian, M. Calderon, M. Unbehauen, R .Haag, A.D. Gruber* and L. Mundhenk*

*Institute of Veterinary Pathology and † Institute of Chemistry and Biochemistry, Freie Universität Berlin, Germany

Introduction: Core multishell nanocarriers (CMS) and thermoresponsive dPG_pNIPAM based Nanogels (NG) are promising novel concepts for topical delivery of drugs to treat inflammatory skin diseases. These systems are designed to enhance dermal drug uptake, however, the carriers themselves might also penetrate into inflamed skin, possibly eliciting biological effects themselves. Here, we evaluated their penetration, histopathological and clinical effects after topical application in a murine dermatitis model.

Materials and Methods: Atopic dermatitis was chemically induced in SKH-1 mice. Fluorescently labeled, unloaded CMS, NG or solvents were topically applied to the inflamed skin. Clinical parameters including transepidermal water loss, skin hydration, erythema, ear thickness and a clinical score were assessed. Fluorescence microscopy was performed to localize the carriers. Epidermal thickness and degree of inflammation were characterized histologically.

Results: Both CMS and NG were localized in the stratum corneum only, with no penetration into the viable skin layers or beyond. No adverse clinical or histological effects of the carriers were observed in any of the parameters evaluated.

Conclusions: Unloaded CMS and NG seem to not aggravate atopic dermatitis in mice unlike certain nanoparticles. They thus may serve as the basis for novel promising and biocompatible concepts for drug delivery in atopic dermatitis.


Poster Flash 2 - 5: SARCOMERIC GENES EXPRESSION AND MICRORNAS PROFILES IN THE HEARTS OF CATS WITH HYPERTROPHIC CARDIOMYOPATHY

E. Lepri, G.Guelfi, V.Stefanetti, L.Imperial, C.Brachelente, M.Sforna, F.Porciello and G.Vitellozzi

Department of Veterinary Medicine, University of Perugia, Italy

Introduction:  Primary Hypertrhophic Cardiomiopathy (HCM) affects many breeds of cats as well as human beings. In human primary HCM more than 800 different causative mutations in sarcomeric genes are known, most commonly involving 6 genes, namely TNNT2, MYH7, MYBPC3,TPM1,TNNI3, and ACTC. MRNA from these genes was investigated in the myocardium of 10 cats with feline primary HCM, 4 cats with secondary HCM and 4 control cats.

Materials and Methods: RNA was extracted from formalin-fixed and paraffin-embedded tissues. RNA was retro-transcribed into cDNA and submitted to real time PCR. Furthermore, extracted RNA was examined with Next Generation Sequencing (NGS).

Results:  All the six investigated genes were up-regulated in primary-HCM compared with control (P <0.01). Primary-HCM had a statistically significant increase (P <0.01) for TNNT2, MYH7, MYBPC3 and TPM1 compared to secondary-HCM and non-statistically significant difference in TNNI3 expression, while ACTC1 was found to be more expressed in secondary-HCM (P <0.01). MiRNAs NGS showed statistically significant difference (P<0.01) in primary-HCM vs control only for miR-370-3p (FC 24.88, FDR 0.01). In secondary-HCM vs control, differences were observed in the expression of miR-375-3p (FC 575.77, P<0.01, FDR 0.01), miR-338-3p (FC -3.57, P<0.01, FDR 0.05), miR-484 (FC 3.42, P<0.01, FDR 0.06) and miR-370-3p (FC 5.70, P<0.01, FDR 0.04). A four-fold increase in expression of miR-370-3p in primary-HCM compared to secondary-HCM (P <0.01) was found.

Conclusions: These data suggest a more complex pathogenesis of feline HCM than single base mutations in MYBPC3 gene as detected in Ragdoll and Maine Coon breeds.


Poster Flash 2 - 6: VASCULOGENIC MIMICRY- ASSOCIATED ULTRAESTRUCTURAL FINDINGS IN HUMAN AND CANINE INFLAMMATORY BREAST CANCER CELL LINES

L. Barreno*, S. Cáceres†, A. Alonso-Díez*, A.V. Montaña ‡, M.L. Garcia‡, JC Illera† and L. Peña*

*Veterinary Clinical Hospital, Pathology Service, †Dept. of animal Physiology, and ‡ ICTS National Center of Electron Microscopy. Complutense University of Madrid, Spain

Introduction: Canine inflammatory mammary cancer (IMC) and human inflammatory breast cancer (IBC) are the most aggressive mammary neoplasia in both species. Exacerbated angiogenesis and vasculogenic mimicry are prominent characteristics. There is very little information regarding ultrastructural characteristics of mammary cell lines. The aim was to study by transmission (TEM) and scanning electron microscopy (SEM) the recently established IMC cell line IPC-366 and IBC cell line SUM149 in adherent and non-adherent conditions.

Materials and Methods: Several pellets from both cell lines in adherent and non-adherent conditions (mammospheres, cancer stem cells) were obtained and processed for TEM and SEM.

Results: TEM revealed similar ultrastructural features in both adherent cell lines: round to oval cells with surface microvilli and high numbers of peroxisomes in close apposition to lipid droplets. Some cells showed several or unique cytoplasmic empty spaces (endothelial-like cells formation). Few SUM-149 cells appeared joined by tight junctions. TEM on mammospheres revealed groups of joined cells (tight junction) with a central lumen (compatible with acini or capillary arrangement). Endothelial–like cells were more frequent than in adherent conditions, and contained Weibel-Palade cytoplasmic bodies. SEM on mammospheres confirmed this features and two cellular shapes: rounded and flattened cells.     

Conclusions:  IPC-366 and SUM-149 share ultrastructural characteristics supporting canine IMC as a model for the human disease. This study demonstrates the morphological transformation of neoplastic epithelial cells in culture into endothelial-like cells, confirming the vasculogenic mimicry phenomenon.


Poster Flash 2 - 7: HISTOCHEMICAL ASSESSMENT OF COPPER STORAGE IN THE LIVER IN VETERINARY HISTOPATHOLOGY: IS IT A RELIABLE DIAGNOSTIC TOOL?

G.C.M. Grinwis* and H. Fieten

* Department of Pathobiology and Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, The Netherlands

Introduction: Hereditary copper-associated chronic hepatitis is recognized in an increased frequency in several dog breeds, including the Labrador retriever. Since the therapeutic strategy of copper-associated hepatitis is partly based on the assessment of the amount of copper in the liver, accurate measurement of the amount of copper is of major importance. To evaluate the accuracy of histological assessment of hepatic copper levels, semi-quantitative grading of the amount of copper is compared to quantitative assessment of the hepatic copper levels in liver biopsies of dogs.

Materials and Methods: Biopsies of healthy Labrador retrievers (n=16) were stained with rubeanic acid and rhodanine for semi-quantitative copper measurement. The amount of copper in these biopsies was graded 0 to 3 according to a previously published grading scheme. In simultaneously harvested liver biopsies of these dogs, quantitative copper levels were measured using instrumental neutron activation analysis.

Results: The semi-quantitative grading of the copper level in the liver biopsies was similar in both histochemical stains in all 4 grades. Quantitative copper levels ranged from 113 to 895 mg/kg dry liver weight (< 400 mg/kg is regarded as normal).

Conclusions: A significant variation between the semi-quantitative analysis of the amount of copper in the liver and the quantitative copper level was noted in all 4 copper score groups. This indicates that the translation of the semi-quantitative copper score into a therapeutic approach by the clinician needs to be performed cautiously.


Poster Flash 2 - 8: PATHOLOGICAL FINDINGS IN THE PITUITARY GLAND OF 201 DOGS AND CATS

L. Polledo*, G. Grinwis, P. Graham*, M. Dunning* and K. Baiker*

* University of Nottingham, United Kingdom and Utrecht University, Netherlands.

Introduction: Pituitary lesions are uncommonly described in the veterinary literature besides classic neoplasia. Approximately 10% of pituitary glands from asymptomatic humans contain abnormalities; there is no data on their incidence in small animals. Pituitary glands were collected from 136 dogs and 65 cats for histopathological evaluation to begin to evaluate the incidence of pituitary abnormalities in small animals.

Materials and Methods: Samples were collected during routine necropsies and stained with HE, PAS, reticulin and immunohistochemistry to characterize any pituitary abnormalities.

Results: Pituitary abnormalities were identified in 36/136 dogs and 10/65 cats. Proliferative lesions (hyperplasia/neoplasia) were seen in 12 dogs and 1 cat: The cat had nodular hyperplasia, 11 dogs demonstrated ACTH-positive corticotroph adenomas and 1 a primary pituitary carcinoma. Corticotroph adenomas were uniformly PAS positive with a loss of the reticulin network. Most adenomas were >2 mm (7/11) but only one dog had been diagnosed with hyperadrenocorticism ante-mortem. The pituitary carcinoma caused neurological signs due to mass effect. Other pituitary abnormalities included: secondary metastatic neoplasia in 2 dogs (nasal and salivary adenocarcinoma respectively), cysts in 18 dogs and 8 cats, hypophysitis in 4 dogs and 1 cat. Of these, only 2 dogs had clinical signs consistent with pituitary disease identified antemortem, these were suffering with hypophysitis and had signs consistent with hypopituitarism.

Conclusions: Pituitary changes seem to appear commonly in dogs and cats and despite often being incidental need consideration on digital imaging.


Poster Flash 2 - 9: AN HISTOLOGICAL AND IMMUNOHISTOCHEMICAL ASSESSMENT OF CANINE FREE MOVABLE THYROID CARCINOMAS

C. Gola*, R. De Maria, E. Morello, S. Iussich and F. Guscetti*

*Institute of Veterinary Pathology, University of Zurich, Zurich Switzerland, Department of Veterinary Sciences, University of Torino, Grugliasco Italy

Introduction: Canine free movable thyroid carcinomas are, according to a few reports, mostly curable through surgical excision. This retrospective study aimed at providing a basic histological and immunohistochemical assessment of a collection of cases.

Materials and Methods: Thirty-four tumors were classified using thyreoglobulin and calcitonin immunostains. Further histological parameters evaluated included mitotic count (MC, mitoses per 10 high-power fields) and assessment of vascular invasion. Indices for proliferation (PI) and apoptosis (AI) were derived from Ki-67 and cleaved-Lamin A immunostains. P53 immunostain was carried out using a canine-specific antibody.

Results: Twenty-two (74.7%) tumors were of follicular cell origin (FTC) and were well-differentiated, 12 (35.3%) were of medullary origin (MTC). The MC showed a mean of 9.76 and a median of 7.5 (range 1-36). Invasion into peritumoral blood vessels was detected in 28/34 cases. The Ki-67 PI showed a mean of 5.02% and a median of 4.37% (range 0-15.5%), the cLam-A-based AI showed a mean of 0.22% and a median of 0.16% (range 0-1.25%). The  PI/AI ratio showed a mean of 57.39 and a median of 22.51 (range 0-831.9). P53 immunohistochemistry was negative. The Ki-67 PI was highly correlated with the mitotic count (p<0.0001; Spearman’s test). Similarly to the AI and the PI/AI ratio, the PI did not correlate with any of the remaining histologic parameters. A higher mitotic count was correlated with MTC compared with FTC (p= 0.022; linear regression).

Conclusions: This data contributes significantly to the sparse literature on this tumor type, providing the basis for further assessments.


Poster Flash 2 - 10: THE SECRECT LIFE OF EMBOLI IN CANINE MAMMARY CARCINOMA.

B. Brunetti*, B. Carlà*, I. Porcellato, G. Sarli*, C. Benazzi*, L. Mechelli and L.V. Muscatello*

* Department of Veterinary Medical Sciences, University of Bologna, Italy and Department of Veterinary Medicine, University of Perugia, Italy

Introduction: The presence of neoplastic emboli is an independent prognostic factor for metastasis and recurrence. The aim of this study is to characterize the morphology, morphometry, kinetics and prognosis of neoplastic emboli in canine mammary carcinomas (CMCs).

Materials and Methods: Thirty CMCs with massive embolization were selected. Morphological diagnosis and grading of primary tumor (PT) were made; pattern, perimeter, area, elongation ratio (E.R.) of emboli and E.R. of vessels were measured with ImageJ. Serial sections were immunostained with antibodies against Ki67 and Caspase-3. Ki67 and apoptotic indices were counted both in PT and emboli. A twenty-four-month follow-up was evaluated.

Results: Ten tubulopapillary, 6 solid, 6 comedocarcinoma, 2 inflammatory, 2 lipid-rich, 1 micropapillary, 1 papillary intraductal, 1 complex and 1 anaplastic carcinomas were collected. In all cases emboli pattern was morphologically similar to PT. PT graded 2 (6/30) or 3 (22/30).  The comparison of the mean proliferation index was not significant (p=0.72) in PT (33.35±14.38%) versus emboli (34.66 ±14.78%), while apoptotic index was significantly higher (p=0.005) in emboli (2.91±2.62%) compared to PT (1.7±1.71%). Survival was worst in a subset of tumors and emboli with high Ki67 and low apoptotic indices. Emboli perimeter and area were not associated with prognosis. Emboli and vessel E.R. were highly and significantly associated (R=0.93; P<0.0001).

Conclusions: The neoplastic emboli had a similar morphology to the PT, high Ki67 and low apoptotic indices. They showed high plasticity adapting to the vessels shape, as confirmed by the relationship between the two elongation parameters.


Poster Flash 3 - 1: NEW INSIGHTS INTO THE PATHOGENESIS OF IDIOPATHIC CARDIAC FIBROSIS IN EUROPEAN CAPTIVE CHIMPS

K. Baiker1, V. Strong1,2, S. Moittie1,2, S. Redrobe2, C. Macdonald2, M. Liptovszky2, M. Cobb1 and K. White1

1School of Veterinary Medicine and Science, University of Nottingham, UK and 2 Twycross Zoo, East Midlands Zoological Society, UK

Introduction: Idiopathic cardiac fibrosis or also known as ‘fibrosing cardiomyopathy’ is a life threatening progressive cardiac disease in captive great apes all over Europe and USA with a high mortality rate and currently unknown etiology.

Materials and Methods: A protocol for a standardized cardiac post-mortem examination was developed. 20 captive chimps from European zoos have undergone detailed examination. Specials stains, immunohistochemistry, PCR for cardiotropic viruses and Vitamin E, Se and Vitamin D levels were investigated.

Results: Histopathological evaluation showed mild to marked interstitial (reactive) fibrosis and replacement fibrosis often without any overall changes of the cardiac outline in 17 of 20 captive chimps and no evidence of fibrosis in adult chimp hearts from an African sanctuary. Immunohistochemistry identified a T-cell dominant infiltration within the scar tissue and high numbers of macrophages. For the first time, myofibroblasts, which are not part of normal cardiac tissue, are shown in affected hearts. A PCR for cardiotropic viruses in a subset showed no convincing evidence of a potential anthroponotic or otherwise viral aetiopathogenesis. Vitamin D levels in blood samples taken in winter and summer showed variations within seasons and low or deficient levels in cases of cardiac failure.

Conclusions: This presentation will discuss pro-inflammatory macrophages stimulating tissue-resident fibroblasts differentiating into new, pro-fibrotic myofibroblasts producing the unwanted, increased extracellular matrix and the vital role of Vitamin D in this process. It will also highlight a potentially easy therapeutic intervention to protect our captive great ape population affected by idiopathic cardiac fibrosis.


Poster Flash 3 - 2: IN VIVO TROPISM OF MODIFIED VACCINIA VIRUS ANKARA IN INFECTED CHICKEN EMBRYOS

M. C. Langenmayer, A.-T. Lülf, S. Adam-Neumair, G. Sutter and A. Volz

Institute for Infectious Diseases and Zoonoses, LMU Munich, Munich, Germany

Introduction: The Modified Vaccinia virus Ankara (MVA) is a replication deficient, highly attenuated vaccinia virus serving as a promising viral vector platform for the development of vaccines against infectious diseases and cancer. The objective of our research is to deepen the understanding of the MVA infection in different host systems to further improve our knowledge on the biodistribution of MVA-based vaccines and the in vivo cell tropism of the virus.

Materials and Methods: In this study we used wildtype MVA and a recombinant MVA expressing fluorescent reporter proteins GFP and mCherry under transcriptional control of specific viral early and late promoters. The chorioallantoic membrane (CAM) of embryonated chicken eggs was inoculated with 1000 plaque-forming units of wildtype and recombinant MVA. PBS was used as inoculation control. The infection was analyzed by fluorescence microscopy, accompanied by histology, immunohistochemistry, and virus titration of several parenchymatous organs.

Results: MVA infection resulted in small to medium-sized proliferative pocks at the inoculation site. Wildtype and recombinant MVA displayed generalized spread beyond the inoculation site at the CAM. Target tissues in this avian in vivo model were CAM, liver, and hematopoietic tissues, which displayed viral gene expression suggesting productive MVA replication.

Conclusions: Our analyses revealed new insights of the course of MVA infection in the embryonated chicken egg and provides a good model to further elucidate the MVA-avian host interactions.


Poster Flash 3 - 3: HISTOPATHOLOGY OF CENTRAL NERVOUS SYSTEM LESIONS AND RELATED ETIOLOGY IN CETACEANS STRANDED ALONG THE SPANISH COASTS

M. de Miguel Chaves*, J.L. Crespo, C. Rubio-Guerri, D. García, M. Melero*, J.M. Sánchez-Vizcaíno*, and M.A. Jiménez Martínez*

* Complutense University of Madrid, Spain and, L´Oceanogràfic of Valencia, Spain.

Introduction: Central nervous system (CNS) diseases have been reported in free-ranging cetaceans, often associated with Dolphin Morbillivirus (DMV), herpesvirus, Toxoplasma gondii, and Brucella sp. The purpose of this study was to analyze CNS tissue from cetaceans stranded in Spain between 2012 and 2016, and relate the morphology of the lesions with known etiologies.

Materials and Methods: CNS lesions from 11 striped dolphins (Stenella coeruleoalba), 1 Risso's dolphin (Grampus griseus), from the Spanish Mediterranean coasts, and 2 common dolphins (Delphis delphi) from the northwestern coasts, were retrospectively selected from the histological archives and compared with available serology, culture, PCR and immunohistochemistry (IHC) for Brucella sp., Dolphin Morbillivirus and herpersvirus.

Results: Seven animals had multifocal lymphoplasmacytic meningoencephalitis (striped dolphins=7; common dolphin=1), positive for DMV (PCR and IHC) in 4 striped dolphins. Complementary tests for the studied agents were negative in the remaining four animals. Severe multifocal histiocytic and lymphoplasmacytic meningoencephalitis associated with Brucella sp. (rose Bengal and culture positive) was found in 5 animals (striped dolphins =4; common dolphin=1). The Risso´s dolphin had severe focally extensive cerebral necrosis consistent with trauma.

Conclusions: Chronic meningoencephalitis was the most common lesion in the CNS in our study, regardless of the species. Brucella sp. followed by DMV, were the most frequent etiologies. Herpesvirus was not detected within CNS lesions. Lesions consistent with Toxoplasma gondii were not seen contrary to other reports in the Mediterranean. Brucella sp. induced a more severe, mixed meningoencephalitis, than DMV. CNS disease played a significant role in death when present.


Poster Flash 3 - 4: BAT BITES ARE ONLY SKIN DEEP, BUT DOG BITES GO CLEAN TO THE BONE: IMPLICATIONS OF COMPARATIVE PATHOGENESIS IN RESERVOIR HOSTS FOR HUMAN RABIES

L. Begeman*, C. GeurtsvanKessel*, S. Finke, C.M. Freuling, M. Koopmans*, T. Müller, T.J.H. Ruigrok and T. Kuiken*

*Department of Viroscience and Department of Neuroscience, Erasmus Medical Centre, Rotterdam, The Netherlands, and Institute of Molecular Virology and Cell Biology, Friedrich Loeffler Institute, Federal Research Institute for Animal Health, Greifswald-Insel Riems, Germany

Introduction: Rabies, caused by lyssavirus infection, is an invariably fatal disease of all mammals. Usually, lyssaviruses are transmitted to people by bites of infected carnivores and bats. Bat-acquired rabies is becoming more common, and its diagnosis may be missed because the clinical presentation differs from that of carnivore-acquired rabies. Our aim was to compare rabies pathogenesis between carnivores and bats to identify underlying mechanism for the differences in clinical presentation of human rabies.

Materials and Methods:  We performed a literature review for references on lyssavirus infection in bats using the major search engines, with relevant search terms. Literature on lyssavirus infections in carnivores and people was identified through searches in handbooks and literature reviews.

Results: Our results showed three differences in rabies pathogenesis between bats and carnivores. First, bat lyssavirus infections start in the skin, while carnivore lyssavirus infections likely start in the skin and muscle. Second, bat lyssavirus excretion may occur via tongue as well as salivary gland, while carnivore lyssavirus excretion via the tongue has not been examined. Third, some bats lack lymphocytic encephalitis at the end stage of rabies, in contrast to carnivores, where lymphocytic encephalitis is reported in all end-stage cases.

Conclusions: The preferential entry into sensory nerves of the skin in bat-acquired rabies versus motor nerves of the muscle in carnivore-acquired rabies, influences the pattern of spread into the central nervous system. This difference in pattern of virus spread provides a mechanistic explanation for most of the differences in clinical presentation of human rabies.


Poster Flash 3 - 5: CETACEAN MORBILLIVIRUS INFECTION AND CENTRAL NERVOUS SYSTEM ASPERGILLOSIS IN ABSENCE OF HISTOPATHOLOGIC MORBILLIVIRAL LESIONS AND IMMUNOHISTOCHEMICAL CEMV-STAINING IN THE MEDITERRANEAN STRIPED DOLPHIN (STENELLA COERULEOALBA)

A. Canturri*, M. Cuvertoret*, L. Pérez*, L. Ganges, A. Jeffers, MJ. McMenamy, L. Cosby and M. Domingo*†

*Servei de Diagnòstic Patologia Veterinària, Departament de Sanitat I Anatomia Animal, Universitat Autònoma de Barcelona (UAB), IRTA-CReSA, Campus UAB, Spain, and Agri-food and Biosciences Institute, Veterinary Sciences Division, Dept. of Virology, Stoney Road, Stormont, Belfast, BT4 3SD, UK

Introduction: Cetacean morbillivirus (CeMV) causes mass mortalities and sporadic disease in cetaceans, with bronchointerstitial pneumonia, lymphoid depletion, mononuclear encephalitis, syncytial cells and viral inclusions as the main microscopic lesions in stranded dolphins. CeMV-Inmunohistochemistry (IHC) and/or RT-PCR aid in confirming the diagnosis of CeMV-disease. Secondary protozoal or mycotic lesions have been commonly observed in brain and lung tissues.

Materials and Methods: Four striped dolphins stranded between 12th and 27th of February 2017 were necropsied. CeMV-IHC was performed with a MoAb recognizing the N-protein of CeMV, and a RT-PCR targeting the Phosphoprotein of CeMV was routinely performed according to published procedures. Results were re-confirmed in another laboratory. 

Results: Microscopic lesions of morbilliviral disease were not observed, and CeMV-IHC was negative in target organs. Two dolphins had large focal granulomatous-necrotizing brain lesions, visible at brain slicing. Culture of one case yielded Aspergillus fumigatus. No culture was attempted from second case. Despite absence of morbilliviral lesions and antigen, the 4 striped dolphins were CeMV RT-PCR positive in lung and diaphragmatic lymph nodes, and, in one case, also in the brain.

Conclusions: CeMV infection detected by RT-PCR, without characteristic microscopic lesions, and with negative CeMV-Inmunostaining may occur in stranded striped dolphins. Two of four cases showed CNS Aspergillosis, a disease associated with overt CeMV disease and immunosuppression. These cases raise the need to perform CeMV-RT-PCR in target organs to be able to detect morbilliviral infection, as routine histopathologic and inmunohistochemical investigation would not detect participation of this virus in the process leading to stranding and/or death.


Poster Flash 3 - 6: INCIDENCE OF SYSTEMIC AMYLOIDOSIS AND VIABILITY IN A CAPTIVE POPULATION OF CUVIER´S GAZELLES (GAZELLA CUVIERI)

L. Salgado Muro*, E. Valderrábano Cano, A. Ferreira Prado, J.L. Rois, and M.A. Jiménez Martínez *

* Dept. of Animal Medicine and Surgery, Complutense University of Madrid and †Marcelle Natureza S.L., Lugo. 

Introduction: Renal amyloidosis has been reported previously in Cuvier´s gazelles (Gazella cuvieri) with implications for survival. The amyloidoses are a group of diseases characterized by the deposition of intracellular or extracellular insoluble protein fibrils. The purpose of this study was to retrospectively determine the presence of amyloid in a captive population of Cuvier´s gazelles, characterize associated lesions, determine the type of amyloid and analyse risk factors for this species.

Material and methods: Eight adult (>9 years) Cuvier´s gazelles from a zoo in northern Spain died and were necropsied. Samples were processed at the Zoo and Wildlife Pathology Service of the Complutense Veterinary Hospital for histopathology, stained with Congo red, and processed for electron microscopy.

Results: Five gazelles (62.5%) contained amyloid in kidneys, livers or spleens. The most affected organ was the kidney. Amyloid deposition was more severe in the renal medullary interstitium followed by the glomeruli, and was associated with severe interstitial nephritis. The liver contained amyloid along sinusoids and surrounding central veins, with associated hepatocellular atrophy. Splenic amyloidosis was multifocal and predominant in the red pulp. Renal disease was the cause of death in two of these gazelles. The five animals with amyloidosis had underlying chronic inflammatory processes. 

Conclusions: Our results show a high incidence of systemic, not just renal, amyloidosis in Cuvier´s gazelles, with implications for survival. Given the presence of concurrent inflammation, amyloidosis secondary to chronic inflammatory disease was presumed. However, a genetic/hereditary etiology was not ruled out given the low genetic variability of these gazelles.


Poster Flash 3 - 7: LESION PROFILE IN RAPTORS MONITORED DURING THE 2016-2017 H5N8 OUTBREAK IN NORTHERN GERMANY

R. Ulrich*, J. Schinköthe*, O. Krone and T. Harder

*Department of Experimental Animal Facilities and Biorisk Management, Friedrich-Loeffler-Institute, Germany and Leibnitz Institute for Zoo and Wildlife Research, Germany and Institute of Diagnostic Virology, Friedrich-Loeffler-Institute, Germany

Introduction: During winter 2016-2017, Germany was affected by an outbreak of highly pathogenic avian influenza (HPAI; H5N8 influenza A virus), which caused mass mortality in poultry and various species of wild birds. This study was performed in order to unravel the potential thread of H5N8-induced HPAI on raptors.

Materials and Methods: 34 raptors (15 white-tailed sea eagles, 14 common buzzards, 2 northern goshawks, 1 rough-legged buzzard, 1 red kite, and 1 peregrine falcon) collected in Northern Germany between 9th November 2016 and 30th March 2017 underwent necropsy under biosafety level 3 conditions followed by histopathological, immunohistological and virological investigations.

Results: Morphologic lesions consistent with HPAI, influenza A virus nucleoprotein-antigen and/or H5- and N8-specific RNAs were detected in more than 82 % of the raptors, and led to a diagnosis of HPAI as the major reason of disease and/or death. Among the raptors with confirmed HPAIV infection, characteristic gross lesions were scarce and included hemorrhages, typhlitis, and myocarditis. In contrast, histopathological findings were common and included encephalitis, myocarditis, splenitis and typhlitis. Real time RT-PCR revealed H5- and H8-specific RNAs in brain and/or lungs.

Conclusions: Gross lesions are scarce in H5N8-induced HPAI in raptors and correct diagnosis relies on histopathological, immunohistological and virological confirmation. The spectrum of wild bird species affected by the 2016-2017 H5N8-virus differs from a previous HPAIV H5N1 epizootic in 2005-2006. In particular, multiple white-tailed sea eagles succumbed to H5N8-induced HPAI. HPAIV H5N8 infection therefore represents an emerging threat for this endangered species.


Poster Flash 3 - 8: NOVEL SPECIES OF CHLAMYDIA ISOLATED FROM SNAKES ARE TEMPERATURE-SENSITIVE AND EXHIBIT DECREASED SUSCEPTIBILITY TO AZITHROMYCIN

E. Staub*, H. Marti*, R. Biondi, A. Levi, M. Donati, H.M.B. Seth-Smith* and N. Borel*

*Institute of Veterinary Pathology, Vetsuisse Faculty, University of Zurich, Switzerland and DIMES, Microbiology, Policlinico S. Orsola, University of Bologna, Italy

Introduction: Chlamydiae have recently been recognized as emerging pathogens in snakes, though little is known about their growth characteristics in vitro. This study aims to characterize chlamydiae isolated from cloacal and choanal swab samples of different snake species.

Materials and Methods: Isolation was attempted on 18 swabs collected from 197 captive snakes originating from different Swiss snake collections. In contrast to previously reported isolation protocols, cultures were grown both at 37 and 28 °C. We then investigated the morphology, infectivity and growth characteristics of successfully isolated strains (n = 4) at 28 °C and 37 °C using immunofluorescence and transmission electron microscopy. These isolates were further analyzed by whole-genome sequencing and their in vitro antibiotic susceptibility was determined in comparison to the human C. pneumoniae strain K6.

Results: Isolates from two individual snakes from one farm (isolates A and B) grew better at 37 °C, whereas the cloacal and choanal isolates (C, D) of a snake from another farm grew better at 28°C. All isolates were sensitive to tetracycline and moxifloxacin, though the minimal inhibitory concentration (MIC) of isolate B was slightly higher (0.5-1 μg/ml) compared to the others (0.06-0.125 μg/ml). Interestingly, only C. pneumoniae K6 was sensitive to azithromycin, while all snake isolates were intermediate to resistant (2-4 µg/ml). Genomic analysis revealed that the snake isolates represent novel chlamydial species clustering within the Chlamydiaceae family.

Conclusions: We report the isolation of snake chlamydiae displaying various novel in vitro characteristics including temperature sensitivity and decreased susceptibility to azithromycin.


Poster 1: INCREASED EXPRESSION OF BASIC TRANSCRIPTION FACTOR (BTF3) IN CANINE LYMPHOMA REVEALED BY TWO –DIMENSIONAL ELECTROPHORESIS.

A. Kycko*, M. Reichert*, W. Łopuszyński and A. Śmiech

*Department of Pathology, National Veterinary Research Institute in Puławy, Poland and Department of Pathological Anatomy, University of Life Sciences in Lublin, Poland

Introduction:  Proteomic methods allow for identification of potential novel markers of animal diseases. The aim of the study was identification of protein(s) differentially expressed in canine lymphoma by comparative proteomic analysis of fresh frozen neoplastic and non-neoplastic tissues.

Materials and Methods: Protein lysates obtained from fresh frozen tissues of canine B-cell lymphomas (n=4) and lymph nodes of dogs non-affected with lymphoma (n=6) were analysed by two-dimensional electrophoresis (2DE) using gel strips with the pH range 3-10. Spots showing over-4-fold  differential expression between the two groups were submitted to protein identification by liquid chromatography-tandem mass spectrometry. Furthermore, archival paraffin-embedded tissues of canine lymphomas (n=30) and non-neoplastic canine lymph nodes (n=7) were analysed by immunohistochemistry for an expression of a protein characterised by the highest difference in expression between the two groups of 2D gels; basic transcription factor 3 (BTF3).

Results: Two spots showed over-4-fold increased expression in each lymphoma 2DE gel compared to non-neoplastic ones. In spots of the highest overexpression, BTF3 was identified. IHC analysis of BTF3 in canine lymphomas revealed diffuse, mild to moderate cytoplasmic and marginal staining in cells of neoplastic appearance. In normal lymph nodes cytoplasmic staining was observed in macrophages and centroblasts.      

Conclusions: The proteomic study allowed for identification of BTF3 overexpression in canine lymphoma. As indicated by IHC results, BTF3 overexpression in 2DE gels was related to presence of high amount of proliferating neoplastic cells expressing the protein. Upregulation of BTF3, a protein involved in regulation of tumour-associated genes, was previously reported in several human cancers.


Poster 2: LACRIMAL GLAND TUMORS IN DOGS: A HISTOLOGICAL AND IMMUNOHISTOCHEMICAL STUDY

L. Nordio*, N. Perelli*, D. Multari^ and C. Giudice*

*Department of Veterinary Medicine, Università degli Studi di Milano, Milan, Italy ^Centro Veterinario Oculistico, Fontane di Villorba (TV), Italy.

Introduction: Lacrimal gland tumors (LGT) are well known neoplasm affecting both the nictitating membrane and the main lacrimal glands in dogs, however extensive histological and immunohistochemical studies are currently lacking. In the last years an increasing number of LGT with a complex (epithelial and myoepithelial) phenotype have been observed in our routine histopathology service. The aim of the present study was to review histological and immunohistochemical features of lacrimal gland tumors in the canine species.

Material and Methods: Nictitating membrane (NM) and main lacrimal gland (LG) biopsies from the departmental archive (2010 – 2016) were reviewed. HE sections were re-evaluated on light microscopy: since a specific classification for LGT is currently lacking, the classification proposed by Goldschmidt (2011) for mammary gland tumors was applied. Immunohistochemistry for pancytokeratin (CK AE1/AE3), CK 14; alpha-smooth-muscle-actin, calponin and p63 was performed.

Results: One-hundred-sixty-one NM and LG specimens were reviewed: 106 neoplastic lesions were diagnosed, 28 were epithelial gland tumors (25 NM; 3 LG). 24/28 were carcinomas: 7 complex; 5 solid; 2 simple tubular, 2 tubulo-papillary, 2 comedocarcinoma, 1 ductal, 4 carcinoma and malignant myoepithelioma, 1 malignant myoepithelioma. Four were benign tumors: 2 complex adenomas, 2 multilobular adenomas. CKAE1/AE3 stained tubular cells while CK14, Alpha-SMA, Calponin and p63 stained myoepithelial cells, when present.

Discussion: In the present cohort of cases NM and LG complex carcinomas and adenomas were highly represented among lacrimal gland tumors. NM and LG carcinomas outnumbered adenomas and were characterized by marked intratumoral variability. A specific classification for lacrimal tumors is needed.


Poster 3: A CASE OF CANINE MEDULLOBLASTOMA, FROM ATAXIA TO IMMUNOREACTIVITY TO DOUBLECORTIN AND OLIG2

M.N. Lucas*, M. Debreuque, D. Pi, J.L. Thibaud, G. Mogicato**, M. Delverdier*, M. Pumarola-Batlle and C. Lacroux*

*Pathology, **Anatomy and Medicine Departments, University of Toulouse, ENVT, Toulouse, France, MICEN VET, Créteil, France, and Department of Animal Medicine and Surgery, Veterinary Faculty, Universitat Autònoma de Barcelona, Barcelona, Spain

Introduction: Medulloblastomas are extremely rare malignant neoplasms arising within the cerebellum of young dogs, and assumed to originate from embryonal cells of the cerebellar external granular layer, with often variable immunoreactivity for one or more neuronal markers.

Materials and Methods: Harrow, a 4-year-old male Dogue de Bordeaux was referred for exploration of a 2-month history of progressive ataxia and tetraparesia, with right head-tilt and nystagmus. High field Magnetic Resonance Imaging located an ill-defined, hyperintense lesion within the cerebellum extending to the right myelencephalon. Worsening of the dog’s condition led to the decision of euthanasia. A necropsy was followed by brain routine histopathology and immunohistochemistry.

Results: On gross examination, there was a soft, pale pinkish, ill-defined mass located within the caudal aspect of the vermis, extending into the right lateral cerebellar hemisphere.

Histopathologic analysis revealed a basophilic, densely cellular neoplasm, replacing several ventro-caudal cerebellar folia, diffusely infiltrating adjacent folia, cerebellar white matter and nuclei, extending along pia mater and into the brain stem, notably vestibular nuclei, through right cerebellar peduncles and fourth ventricle. Tumor cells were small, arranged in uniform sheets supported by a scanty stroma, with minimal cytoplasm, round-to-oval, hyperchromatic nuclei with marked anisokaryosis and high mitotic activity. Immunohistochemistry highlighted a massive Doublecortin and Olig2 expression within neoplastic cells, and occasional expression of NeuN, Neurofilaments and Synaptophysin, consistent with a neuroblastic origin.

Conclusions: This case reported the diagnosis of a medulloblastoma from the clinical and neuroimaging presentation to the  Doublecortin and Olig2 immunoreactivity of original tumor cells.


Poster 4: CANINE DILATATIVE CARDIOMYOPATHY (DCM): A MORPHOMETRIC APPROACH

S. Gasparini*, S. Fonfara, U. Hetzel* and A. Kipar

*Institute of Veterinary Pathology, Vetsuisse Faculty, University of Zurich, Switzerland, and Department of Clinical Studies, Ontario Veterinary College, University of Guelph, Canada

Introduction: Myocardial inflammation, fibrosis and neovascularisation have been shown to be key features of dilated cardiomyopathy (DCM), with cardiomyocytes suspected to play a role in the pathogenesis. While these changes are particularly obvious as focal lesions, the present study aimed to quantify the diffuse changes in the myocardium with DCM.

Materials and Methods: Using the Visiopharm Integrator System (VIS, version 4.5.1.324, Visiopharm, Hørsholm, Denmark) on scanned slides (NanoZoomer-XR C12000; Hamamatsu, Japan) we quantified the overall cell number (DAPI fluorescence), the number of macrophages (Iba-1-positive) and small vessels (α-SMA-positive), and the proportion of cardiomyocytes, interstitial collagen fibres and fibre-free interstitial space (van Gieson stained sections) in the myocardium of 15 dogs with DCM and 6 control dogs.

Results: DCM hearts exhibited a significantly lower overall cell count, and the proportion of space occupied by cardiomyocytes was significantly lower than in control hearts. Instead, there was a significantly higher amount of interstitial collagen and of macrophages in the interstitial space. The fibre-free interstitial space was significantly increased, suggesting interstitial oedema in the DCM hearts, while the number of interstitial vessels was not altered.

Discussion: These results suggest that diffuse interstitial fibrosis and macrophage infiltration as well as a potential increased vascular permeability, in combination with a - likely gradual - loss of cardiomyocytes are key pathomechanisms of canine DCM.  Whether these processes result from an initial cardiomyocyte injury with subsequent macrophage recruitment, or are driven by macrophages that persist after an initial inflammatory stimulus, requires further investigation.


Poster 5: ALPHA-FETOPROTEIN EXPRESSION IN NORMAL UTERUS AND IN FELINE ENDOMETRIAL ADENOCARCINOMA

E. Rodrigues*‡, D. Lopes*‡, M. Teixeira*‡, A. Teixeira da Silva‡P, E. Freitas*‡, R. Payan-Carreira†# and M.A. Pires*†‡

*Veterinary Sciences Department, Animal and Veterinary research Centre (CECAV), Laboratory of Histology and Anatomical Pathology (LHAP), PEscola Superior de Saúde, Instituto Politécnico de Bragança #Zootecny Department. 5000-801 Vila Real, Portugal.

Introduction: Alpha-fetoprotein (AFP) is a glycoprotein synthesized during embryonic and fetal development that has been associated with human endometrial adenocarcinoma. AFP studies in animals are scarce and never in uterine neoplastic lesions. The queen’s uterus is an uncommon location for neoplasms, but the Feline Endometrial Adenocarcinoma (FEA) seems to be more common than reported. The study of molecules foreseeing to understand the prospective malignancy and prognosis of this is important to support clinical decision.

Materials and Methods: Tissue samples from control animals [in the proliferative (PS, n=6) and in secretory (SS, n=6) stages] and 16 cases of FEA were used evaluated for AFP expression by immunohistochemistry with anti-AFP antibody. The intensity was recorded in the surface epithelium and surface and deep glands - the normal tissues - and in the neoplastic epithelium of FEA.

Results: The immunolabelling for AFP was weaker in the surface epithelium than in the glandular epithelium, being stronger in PS compared to SS. Still, the intensity of labeling was weaker in the neoplastic epithelium than either stage of the cycle.

Conclusions: This work describes for the first time the AFP presence in the feline uterus and in FEA. FEA is reported as similar to the papillary serous carcinoma in women. In the later, AFP expression was reported in some types of endometrial carcinomas, often related to particular histological types. Nevertheless, due to the limited information on FEA, more studies are necessary to correlate AFP expression with clinical aspects and prognostic of these lesions.


Poster 6: BLOOD LYMPHOCYTE SUBSET POPULATIONS IN HEALTHY BOXERS AND

IN BOXERS AFFECTED BY CANINE LEISMANIOSIS (L. INFANTUM)

B. Passeri, C. Mangia, E. DeAngelis, D. Aliani, A.M. Cantoni, G. Melis  and L. Kramer

Department of Veterinary Science, University of Parma, Parma- Italy

Introduction: Leishmania infantum infects dogs in several Mediterranean countries, including Italy. It is known, however, that many infected dogs will not develop clinical manifestations of canine leishmaniosis (CL). Several studies have been aimed at identifying possible breed predisposition to disease and the Boxer breed, among others, has been reported to be more predisposed. The aim of the present study was to analyze peripheral blood lymphocyte subset populations in healthy boxers and in boxers affected by leishmaniosis.

Materials and Methods: Nine healthy, L. infantum-negative boxers and 6 boxers with CL were studied. Infection status was determined by antibody titre and PCR. Peripheral blood mononuclear cells (PBMC) were isolated by density gradient and analyzed in flow cytometry for the expression of:  CD3, CD4,CD8, CD21, CD11a/CD18, CD25 and Foxp3. Double staining was carried out for CD4/CD8 and CD4/FoxP3, while triple staining was carried out for CD4/CD25/FoxP3.  Results were analyzed with the Kolmogorov-Smirnov test, with significant differences between healthy and sick dogs for each subset at p ≤ 0.05.

Results: No significant differences were observed between sick dogs and healthy dogs for CD3+, CD4+CD8- or CD4+/Foxp3+ cells; levels of CD4-/CD8+ and CD4+/CD8+ lymphocytes tended to be higher in dogs with CL, even though this was not significant.  The levels of CD4+/CD25+/FoxP3+ were extremely low in both groups.

Conclusions: Further studies with a larger population of animals are necessary to confirm or exclude a role for lymphocyte subset variation in the development of CL in predisposed breeds.


Poster 7: CANCER ASSOCIATED FIBROBLASTS IN CANINE SQUAMOUS CELL CARCINOMA: CORRELATION WITH HISTOPATHOLOGICAL GRADES

L.H. Pulz* T.M. Vargas , O. Monteiro and R.F. Strefezzi

*Department of Pathology, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, Brazil,  Department of Veterinary Medicine, Centro Universitário Fundação de Ensino Octávio Bastos - São João da Boa Vista, Brazil and Department of Veterinary Medicine, Faculdade de Zootecnia e Engenharia de Alimentos, Universidade de São Paulo, Brazil

Introduction:  Representing 54% of all cutaneous neoplasms in dogs, squamous cell carcinomas (SCCs) present variable features, ranging from well-differentiated to highly malignant and invasive tumours. Previous studies have indicated that alterations in the underlying connective tissue are associated with tumour development and progression. Increased density of carcinoma associated fibroblasts (CAFs) in the stroma of human and feline SCCs was found to correlate with lymph node metastasis and higher mortality. The aim of this study was to characterize the CAFs density in canine SCCs and compare with histopathological grades.

Materials and Methods: Samples from 18 dogs were graded based on cellular differentiation, degree of keratinization, nuclear polymorphism, number of mitoses and pattern of invasion. Canine tumours were grouped into low-grade (histopathological grades 1 and 2) and high-grade SCCs (grades 3 and 4). Stromal myofibroblasts were identified through immunostaining using monoclonal antibody against alpha-smooth muscle actin (α-SMA). The percentage of positive α-SMA stromal cells was assessed semiquantitatively and graded as 0, when α-SMA labelling < 50% of the stromal cells, and 1 for α-SMA labelling ≥ 50%.

Results: CAFs within the stroma were identified as spindle-shaped cells with cytoplasmic immunoreactivity for α -SMA. They were most frequently located adjacent to the neoplastic cells. The percentage of stromal myofibroblasts was significantly higher in poorly differentiated canine SCCs when compared with well-differentiated tumours (P=0.0090).

Conclusions: The present study demonstrated that high-grade SCCs present more CAFs than low-grade SCCs, reinforcing the hypothesis that myofibroblasts may contribute to tumour progression, recurrence and metastasis.


Poster 8: CANINE SMOOTH MUSCLE TUMORS OF SOFT TISSUE: A CASE SERIES OF 23 CASES

G. Avallone*, V. Pellegrino*, P. Roccabianca, M. Tecilla, C. Benazzi* and G. Sarli

*DIMEVET, University of Bologna, Italy and DIMEVET, University of Milano, Italy

Introduction: Canine smooth muscle tumors (STMs) occur mostly in the gastrointestinal and female genital tracts while soft tissue SMTs are rarely reported. In humans soft tissue leiomyomas are prevalent in cutaneous and subcutaneous tissues, while leiomyosarcomas develop more frequently in retroperitoneal, pelvic or intra-abdominal tissues in women. This study describes the clinicopathological features of canine soft tissue SMT.

Materials and Methods: Cases were retrospectively collected, clinical data retrieved, and diagnoses confirmed by re-evaluation of H E slides and by immunohistochemistry. Mitotic count, atypia, necrosis, and histological grade were assessed. Estrogen (ER) and progesterone (PR) receptor expression was immunohistochemically evaluated.

Results: Twenty-three cases were collected: 8 leiomyomas and 15 leiomyosarcomas (7 grade I, 6 grade II, 2 grade III). Thirteen dogs were female (5 spayed), and 10 male (1 neutered). Eleven SMTs were subcutaneous (5 in the limbs, 4 perineal, 2 perianal); 5 were pelvic, 5 retroperitoneal, 1 intra-abdominal, 1 in the oral cavity. Seven of 10 retroperitoneal/pelvic SMTs were found in female (70%) and 3 in male dogs. Two perineal leiomyosarcomas and 1 pelvic leiomyoma expressed ER. One retroperitoneal leiomyoma expressed PR. All ER and/or PR positive SMTs occurred in intact females.

Conclusions: Canine soft tissue SMTs were more frequently subcutaneous (limbs and perianal/perineal region), and retroperitoneal (including pelvic). Retroperitoneal SMTs were more common in female dogs paralleling human SMTs. ER and PR expression in some cases suggests the existence of a distinct subset of canine soft tissue SMTs that may bear different behavior to be assessed in future studies.


Poster 9: CANINE VULVAR FIBROMATOSIS: A RARE LESION

B. Brunetti*, M. Levi*, O. Martini°, D. Zambelli*,  E.L. Laddaga and L.V. Muscatello*

*Department of Veterinary Medical Sciences, University of Bologna, Italy, °Clinical practioner, † Ospedale veterinario I Portoni Rossi

Introduction: Vulvar fibromatosis in human medicine is a rare locally aggressive fibroproliferative neoplasm, with no reported potential for metastasis and typically characterized by an unpredictable clinical course. This study describes a case of diffuse fibromatosis leading toenlargement of the vulva.

Materials and Methods: A 13-year-old entire female Labrador Retriever presented for examination to a veterinary practioner, with a history of of a slow (1 month) progressive vulvar thickening. Clinical evaluation and 2 punch biopsies of 8 mm each were performed. The samples were processed for staining with HE, Masson’s and Weigert-Von Gieson trichrome (WVG) stain and for immunohistochemistry for vimentin, desmin, smooth muscle actin, S-100 and oestrogen. The dog was controlled after 1 year.

Results: The clinician observed a uniform increase in vulvar thickness in absence of nodular masses. Histologically, the lesion was composed of an abundant amount of collagen fibers associated with few fibroblasts organized around all the structures of the submucosa, i.e. smooth muscle, adipose tissue and follicular unit. The fibroblasts presented no signs of atypia and no mitotic activity. Masson’s stain showed a diffuse collagen deposition. The WVG stain revealed an increase of reticular fibers in the submucosa. The fibroblasts stained with vimentin and mildly for oestrogen; all the other antibodies were negative. After 1 year without any type of therapy the lesion has remained unvaried.

Conclusions:  These findings are consistent with vulvar fibromatosis, a tumoral lesion that, to the best of our knowledge, has never been described in dogs.


Poster 10: CARDIAC CHEMODECTOMA AND HEMANGIOSARCOMA: A MORPHOLOGIC AND ECOGRAPHIC COMPARISON

A.S. Mihai (Ionita), R.I. Rizac , F. Leca, C.M. Constantinescu, T. Soare and M. Militaru

University of Agronomical Sciences and Veterinary Medicine of Bucharest, Romania

Introduction: The most frequent dog heart tumors are hemangiosarcomas and chemodectomas. The antemortem diagnosis is difficult, therefore this study’s purpose is to correlate the gross and the histopathologic differences with echographic findings.

Materials and Methods: The paper is studying two dogs ultrasonographycally diagnosed with heart base tumors. Postmortem, gross and histopathologic examinations were performed.

Results: For the first case the gross examination showed a friable red-cherry multinodular structure, unencapsulated and spongious in section. The histopathologic examination revealed vascular spaces with highly cellular stroma, lined by elongated, anaplastic endothelial cells with anisocytosis, ovoid-round hyperchromatic nuclei, with frequent mitoses. This was diagnosed as a hemangiosarcoma. The second case was a multinodular tumor, grey to dark-red and firm with uneven surface. The histopathologic examination revealed cells structured in lobules separated by fine septa of connective tissue, aligned around and along the capillaries. They had a polygonal to columnar aspect with central, round nuclei with no mitoses and cellular  atypia. This was diagnosed as a chemodectoma. The hemangiosarcoma location was within the right atrium infiltrating the aortic and pulmonary artery origin and was characterized by a heterogeneous aspect, with multiple hypoechogenic cavities. The chemodectoma was located between the aortic artery and the pulmonary trunk, extending toward the atria and encircling the pulmonary artery, revealing homogeneous and hyperechogenic aspect.

Conclusions: Ultrasonography helps monitoring the location, evolution and size of the cardiac tumors, representing important criteria for a clinical approach and a presumptive diagnostic, but histopathology establishes the final diagnoses.


Poster 11: CLASSIFICATION OF CANINE PANCREATIC ISLET CELL TUMORS USING VARIOUS BIOMARKERS

C. de Vries*, B. Konukiewitz, W. Weichert, G. Klöppel, H. Aupperle* and K. Steiger

*Department of Pathology, LABOKLIN GmbH Co.KG, Bad Kissingen, Germany
 
Institute of Pathology, Technical University of Munich, Munich, Germany

Introduction: Canine islet cell tumors (ICTs) are classified based on histomorphology (adenoma/carcinoma). However, this is often not predictive for their biological behavior. In human pancreatic neuroendocrine neoplasms a prognostic grading based on the Ki67-index distinguishes between low grade (PanNET; G1: < 3% or G2: 3-20%) and high grade tumors (PanNEC; G3: > 20%). Somatostatin receptor 2A (SSTR2A) is a clinico-pathological biomarker used in human PanNET. The aim of this study is to correlate Ki67-index and hormonal profile of canine ICTs with their metastatic behavior, and to analyze SSTR2A expression.

Materials and Methods: Sixteen canine ICTs (11 males, 5 females, 3-12 years; 4/16 metastatic) collected between July 2011 and August 2016 were analyzed immunohistochemically (synaptophysin, insulin, glucagon, gastrin, VIP, Ki67, SSTR2A, ATRX and p53). 

Results: Computer-assisted analysis revealed a Ki67-index below 2% (0.20 to 1.36%) in all ICTs (corresponding to PanNET G1). Insulin was expressed in 13/16 tumors. Of the three insulin-negative tumors, two were negative for all hormones, one expressed VIP. Most insulin-negative tumors (2/3) were metastatic. ATRX and p53 expression was normal in all ICTs. SSTR2A was at least moderately expressed in 11 tumors (score 2+, 8/11; score 3+, 3/11).

Conclusions: The Ki67-index graded all canine ICTs (including those with metastases) as G1-PanNETs (according to the human WHO-classification) and showed no correlation with the ICTs' biological behavior. Negativity for hormone expression might be superior to Ki67 to predict the metastatic potential. Similar to humans, SSTR2A seems to be a potential receptor target for diagnosis and treatment of canine ICTs.


Poster 12: CLINICOPATHOLOGICAL AND GENETIC FINDINGS IN CANINE HYPOPHOSPHATASIA. A CASE REPORT

P. Syrjä*, A. Lappalainen#, R. Viitmaa, K. Kyöstilä and H. Lohi,   

*Veterinary Pathology, Dept. of Veterinary Biosciences, University of Helsinki, Finland, #Dept. of Equine and Small Animal Medicine, University of Helsinki, Finland Erivet clinic, Estonia and Department of Molecular Genetics, Folkhälsan Institute of Genetics, Research Programs Unit, Molecular Neurology, University of Helsinki, Helsinki, Finland    

Introduction: A 12-weeks-old Karelian Bear dog puppy was scheduled for euthanasia due to poor growth, hind leg weakness and poor musculature. Since 3 of 8 puppies in the litter were similarly affected, an inherited disease was considered and a clinicopathological, as well as a genetic investigation, were performed.

Materials and Methods: The puppy underwent clinical, neurological and radiographic examination. A post-mortem investigation was conducted. Exome sequencing was performed to identify possible disease-causing gene variants.

Results: Clinical signs included general weakness, plantigrade, palmograde stand. Radiographs showed “hour-glass” shaped diaphyses, with irregular bone formation within the epiphyseal area. Post-mortem examination revealed severe failure of enchondral and intramembranous ossification. Epiphyseal cartilage retention, poorly mineralized metaphyseal primary and secondary spongiosa, with retention of secondary spongiosa in the marrow cavity, was noted. The diaphyseal compact cortical bone was hypoplastic. Diffuse severe thyroidal C-cell hypertrophy, indicating chronic hypercalcemia or hyperphosphatemia, was present. Mild eosinophilic hepatitis, bronchopneumonia, urinary cystitis and extraocular myositis were diagnosed by histopathology. The puppy was homozygous for a missense mutation in the alkaline phosphatase 1 gene, coding tissue-non-specific alkaline phosphatase.

Conclusions: Defective mineralization, hypercalcemia, hyperphosphatemia and low serum ALP activity are cardinal signs in hypophosphatasia, a disease caused by various mutations of the ALPL1 gene in man. The lesions in this case indicate failure of ossification with decreased skeletal mineralization. The findings support the diagnosis of hypophosphatasia and functional relevance of the ALPL-mutation in this case.


Poster 13: PRODUCTION OF PROTEINASE AND PHOSPHOLIPASE BY MALASSEZIA PACHYDERMATIS ORIGINATING IN DOGS WITH AND WITHOUT EXTERNAL OTITIS

E.C. Chiurco*, G.H. Nardi, M.A. Lallo* and S.D. Coutinho*

*Environmental and Experimental Pathology, Paulista University, São Paulo, Brazil and   Center for Zoonoses Control, São Paulo, Brazil

Introduction: Malassezia spp. are commensal yeasts isolated from animal and human microbiome, but they can also cause opportunistic infections. It is suggested that the production of exoenzymes is related to the pathogenicity of these fungi. The aim of this work was to compare the frequency of Malassezia pachydermatis in dogs with and without otitis, as well as, the production of phospholipase and proteinase.

Materials and Methods: In this research 111 dogs were studied, 53 with and 58 without external otitis. Clinical samples were collected from right and left ear canals. Samples were seeded on Petri dishes, studied macro-and-micromorphologically and identified by phenotypic characteristics.  The production of phospholipase and proteinase was carried out using egg yolk and bovine serum albumin as substrates, respectively.

Results: Malassezia pachydermatis was detected in the two groups with no differences (75% (40/53) of the diseased animals; 76% (44/58) of the healthy animals). No statistical differences were observed related to enzymes production between the two groups, with or without otitis. Phospholipase was produced by 72% (47/65) and 86% (60/70) of the strains originated in dogs with and without otitis, respectively. Proteinase was produced by 94% (66/70) and 91% (59/65) of the strains originating in healthy and diseased dogs, respectively.

Conclusions: The yeast was isolated in the same proportion in diseased and healthy animals. These results suggest that the pathogenicity of M. pachydermatis must be related to other virulence factors and not just the production of proteinase and phospholipase.


Poster 14: MORPHOLOGICAL AND INMUNOHISTOCHEMICAL DIAGNOSIS OF AN ENDOMETRIAL STROMAL SARCOMA IN A CAT

E. Ruiz Riera*, J. Lite Diaz†, I. Borges* and G. Albinyana Grapí*

*Citopat Veterinaria Laboratories, Barcelona; Abat Marcet Veterinary Hospital, Terrassa (Spain)

Introduction: A 12-year-old queen showed vaginal discharge; after clinical examination a pedunculated uterine mass was found.

Materials and Methods: After ovariohysterectomy, uterus was submitted for histopathological evaluation. The following immunohistochemical markers were used on sections of the uterus: AE1/AE3 cytokeratin, vimentin, desmin, alpha-smooth muscle actin, estrogen receptor, caldesmon and CD10.

Results: A sarcomatous growth with an epithelial component was histologically observed, and the diagnosis of endometrial stromal sarcoma was given. CD10 immunostaining confirmed the endometrial stromal origin, and finally, endometrial stromal nodule was the histological classification of the tumour. Spindle-shaped neoplastic cells and endothelium strongly reacted to vimentin and desmin, while glandular epithelium was strongly marked by cytokeratin. Some endothelium cells were slightly marked by caldesmon. CD10 was multifocally positive, with extensive strongly marked areas alternated with areas of more attenuated positivity. The rest was negative. The last actualization in women uterine sarcomas classifies these tumours according to the latest WHO classification: endometrial stromal nodules, low-grade endometrial stromal sarcomas and undifferentiated endometrial sarcomas. The present case could be classified in an ''endometrial stromal nodule''. They are considered to have excellent prognosis and patients are cured by hysterectomy.

Conclusions: Although there are scarce endometrial stromal sarcomas in veterinary bibliography, this case shows the possibility of using immunochemistry procedures (vimentin, desmin, cytokeratin, CD10) in order to establish a diagnosis and prognosis of the patient.


Poster 15: COORDINATED EXPRESSION OF KERATINS 7 AND 20 IN PRIMARY AND NODAL FELINE MAMMARY CARCINOMAS

J.P. Boccia*, L.M.G. Silveira*, C.S. Alves*, M.A. Lallo* and J.G. Xavier*,

*Environmental and Experimental Pathology, Paulista University, Brazil and Methodist University of São Paulo, São Bernardo do Campo, Brazil

Introduction: Mammary tumors are common in cats, and usually have an aggressive behavior, with reported elevated rates of metastases. Regional lymph nodes are frequently affected, with eventual diagnosis from metastatic tumor without evidence of the primary lesion, being important in the search for histogenic origin. Particularly the expression of keratins (K) is suitable for the identification of carcinomas of unknown origin. The aim of this study was to characterize the K 7 and K 20 expression in feline primary and metastatic mammary carcinomas. 

Materials and Methods: 20 feline mammary carcinomas and 4 nodal metastases were submitted to immunostaining with anti-human mouse monoclonal antibodies for K 7 (OV-TL 12/30) and K 20 (Ks20-8), which are amplified by the system LSAB (DAKO).

Results: The K7 + / K20 -  profile was present in 60% of the cases of primary tumors, in which was observed double negativity in 30% of samples and double positivity in 10% of them, reinforcing the adequacy of feline mammary carcinoma as a model in comparative mammary carcinogenesis. In the nodal lesions, immunophenotypic difference was observed in relation to the primary tumor in 50% of the samples, indicating the possibility of divergence between the immunohistochemical profile of primary and secondary lesions.

Conclusions: The findings corroborate a previous study, and contribute to the efficacy of the primary site survey in metastatic tumors of unknown origin, also warning of the complexity of the process, with the possibility of immunophenotypic variations between primary and secondary lesions.


Poster 16: CONCURRENT LEUKODYSTROPHY AND CEREBELLAR CORTICAL DEGENERATION IN A JUVENILE LABRADOR RETRIEVER

B. Cossic*, J. R. Cross and A. D. Miler*

*Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY, USA and VCA Advanced Veterinary Care Center, IN, Fishers, USA

Introduction: A 7-month-old male intact Labrador retriever presented with a history of epilepsy that progressed with cerebellar signs consistent with a neuroanatomic diagnosis of diffuse.  Magnetic resonance imaging revealed symmetric hyperintensities in the forebrain and circumferentially around the midbrain and pons.

Results: Histologic evaluation of the central nervous system (CNS) revealed widespread bilaterally symmetrical white matter vacuolation with loss of myelin and retention of unaffected axons throughout the entire brain including the mixed white and gray matters sections of brainstem.  Loss of myelin was confirmed via Luxol fast blue and Bielschowsky silver stain confirmed the lack of axonal pathology.  In the white matter, blood vessels were cuffed by concentric rings of astrocyte processes that were strongly positive for glial fibrillary acidic protein.  In addition, Purkinje cells in the cerebellum were commonly degenerate with hypereosinophilic cytoplasm, shrunken borders, and pyknotic nuclei. Immunohistochemistry for microtubule-associated protein 2 and synaptophysin confirmed the Purkinje cell loss. Secondary degeneration was noted in the cerebellar nuclei.

Conclusions: This case is unusual due to the simultaneous occurrence of a leukodystrophy and Purkinje cell degeneration which has not been reported in the veterinary literature and is equally rare in human neuropathology. 


Poster 17: HISTOLOGICAL, IMMUNOHISTOCHEMICAL AND MOLECULAR CHARACTERIZATION OF A FOLLICULAR LYMPHOMA IN A DOG

S. Muller †, S. Le Calvez*, P. Martin‡ and J. McKay*

*IDEXX Laboratories Ltd., Wetherby, UK, † IDEXX Vet Med Labor, Ludwigsburg, Germany and Vets Now Referrals, Glasgow, UK

Introduction: A follicular lymphoma was diagnosed in a 5-year-old, neutered female Labrador dog with unilateral right submandibular lymph node enlargement.

Materials and Methods: Histological sample was fixed in 4% neutral buffered formalin for 24 hours, processed, embedded in paraffin, sectioned at 4 μm and stained with hematoxylin and eosin. Immunohistochemistry was performed for the expression of CD3 and PAX5. Clonality status was determined by analysis of B- and T-cell antigen receptor gene rearrangement.

Results: The surgically excised lymph node sample showed loss of normal node architecture. Medullary cords and sinus were replaced by a monomorphic population of large lymphoid cells. Anisocytosis and anisokaryosis were moderate. The follicles were composed of intermediate-size centrocytes with indented, heterochromatic nuclei and large-size centroblasts that had large nucleoli. The number of centroblasts was an average of 6-10 /400x high power field consistent with follicular lymphoma grade II (World Health Organization). Immunohistochemistry showed follicles composed of strongly and uniformly PAX5-positive B-cells. PCR test for the antigen receptor clonal rearrangement identified a monoclonal B-cell antigen receptor.

Conclusions: Follicular B-cell lymphoma needs to be differentiated from the other indolent lymphomas of dogs (mantle cell lymphoma and T-zone lymphoma). Dogs tend to present with lack clinical illness. Although the true incidence of canine indolent lymphomas is unknown, the tumors may have been under recognized. Recognition of their architectural features, routine application of immunophenotyping, and molecular clonality assessment should alleviate this. Animals with follicular lymphoma of grade I and II should be considered to have indolent lymphoma and treated appropriately.


Poster 18: A REVIEW OF THE CAUSE OF DEATH OF SUSPECTED ANIMAL ABUSE CASES IN KOREA, 2013-2016

JY. Jung, K. Lee, H. Lee, SS. Yoon, BJ. So and E. Choi

Animal Disease Diagnostic Research Division, Animal and Plant Quarantine Agency, Gimcheon 39660, Republic of Korea

Introduction: Cases of suspected animal abuse submitted to the diagnostic laboratories are increasing worldwide. Animal abuse is often legally defined as gross neglect, physical trauma, sexual abuse, hoarding, cockfighting, or dog fighting. In recent years, companion animal abuse and neglect has gained more and more attention in Korea.

Materials and Methods: A retrospective study was conducted about suspected animal abuse necropsy cases submitted between January 2013 and December 2016 at the animal disease diagnostic research division, Animal and Plant Quarantine Agency. All submission information, necropsy photographs, necropsy reports, and final reports were reviewed. 

Results: A total of 56 cases including 25 dogs and 31 cats were analyzed. The cause of death of animal abuse was determined in 52.0% (13/25) of dogs. The most common cause of death in dogs was trauma (38.5%, 5/13), followed by poisoning (30.8%, 4/13), malnutrition (23.1%, 3/13), and asphyxia (7.7%, 1/13). However, only 6 cases (19.3%) were confirmed in cats, and all of them died by trauma.      

Conclusions: In this report, the most common cause of death in dogs and cats was trauma. We couldn’t determine the specific type of trauma. In Korea, the number of stray cats and sudden death are increasing recently. For this reason, suspected animal abuse cases were submitted frequently, although only 20% in cats was confirmed as animal abuse. Skilled veterinarians are necessary to confirm cause of death and prevent acts of animal neglect and abuse.      


Poster 19: EXPRESSION OF MATRIX METALLOPROTEINASES IN FELINE ENDOMETRIAL ADENOCARCINOMA

E. Freitas*‡, F. Seixas*†‡, A. Radar, R. Payan-Carreira†# and M.A. Pires*†‡

*Veterinary Sciences Department, Animal and Veterinary research Centre (CECAV), Laboratory of Histology and Anatomical Pathology (LHAP), #Zootecny Department. 5000-801

Introduction: Even though the queen’s uterus is an uncommon location for neoplasms, Feline Endometrial Adenocarcinoma (FEA) seems to be more common than previously noted. Tissue matrix metalloproteinases (MMPs) are involved in the extracellular matrix degradation, promoting tumor progression and metastasis, as well epithelial-mesenchymal transition.

Materials and Methods: We evaluate MMP-1,-2 and -9 expression in FEA (n=21) compared to normal uterus, in either proliferative (n=6; PS) and secretory (n=8; SS) stages. The indirect immunohistochemistry was used, with anti-MMP-1, anti-MMP-2 and anti-MMP-9 antibodies. The intensity was evaluated as null (zero), weak (1), moderate (2) or intense (3).

Results: FEA showed a decreased expression in MMP-1 and -9 (p < 0,05) compared with the PS stage, which was also significantly higher in both FEA and PS in comparison with the SS. However, the intensity of labelling for MMP-2 did not differ between FEA and the normal uterus, neither between PS and SS.

Conclusions: This study showed that, in the feline cyclic uterus, MMP-1 and -9 presented a greater expression in the PS and this might be related to the cellular remodeling preceding the larger glandular expression in SS. Conversely, the expression of MMP-1 and -9 in FEA was significantly lower compared with the PS, in contrast to what is described in the literature for women. As the MMPs activity could be modulated by its inhibitors (TIMPs), additional studies were needs to clarify the MMPs/TIMPs relevance in FEA.


Poster 20: GALECTIN-3 EXPRESSION CORRELATES WITH BCL2 EXPRESSION AND PROLIFERATIVE ACTIVITY IN CANINE CUTANEOUS MAST CELL TUMOURS

T.H.M. Vargas*, C.N. Barra*, L. H. Pulz†* and R. F. Strefezzi*

*Laboratório de Oncologia Comparada e Translacional, Departamento de Medicina Veterinária, Faculdade de Zootecnia e Engenharia de Alimentos and Departamento de Patologia, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, Brazil

Introduction: Mast cell tumours (MCTs) are important neoplasms in dogs and present variable biological behaviour. Evasion of apoptosis is an important hallmark of the cancer and is associated with tumour progression and patient prognosis. BCL2 (B-cell lymphoma 2) is an anti-apoptotic protein related with progression in tumours such as lymphoma and mast cell tumour. Galectin-3 (Gal-3) is known to influence several biological functions such as adhesion, migration, invasion, angiogenesis, cell proliferation and apoptosis. In human cancer cells, Gal-3 is capable of bind to BCL2 and to mimic its ability of stabilizing the mitochondrial membrane. The objective of this study was to compare the expression of Gal-3 with BCL2 and Ki67 expressions in MCTs, in order to verify their correlation.         

Materials and Methods: Twenty-two samples of canine cutaneous MCTs were treated surgically, with no adjuvant therapy, and a minimum follow-up period of 180 days. The expressions of Gal-3, Ki67 and BCL2 were investigated using immunohistochemical methods. Immunostaining for Gal-3 versus BCL2 and Gal-3 versus Ki67 were compared using Spearman’s and Pearson’s correlation tests, respectively.

Results: Gal-3 and BCL2 expressions were positively correlated (p = 0.0071). Similar results were obtained when Gal-3 and Ki67 were compared (p = 0.0185). These findings corroborate human literature data and suggest that Gal-3 may influence apoptosis and cell proliferation in MCTs.

Conclusions: Gal-3 expression is positively correlated with anti-apoptotic protein BCL2 and cell proliferation in canine mast cell tumours.


Poster 21: GROSS EVALUATION, SAMPLING PROCEDURES AND HISTOLOGICAL CHARACTERIZATION OF FELINE MIDDLE EAR DISEASES

A.M. Cantoni*, V. Bertani*, P. Orlandini†, B. Passeri* and A. Corradi*

*Pathological unit, Department of Veterinary Science, University of Parma, Italy and Practitioner, Brescia, Italy

Introduction: Study of ear diseases is hampered by the difficult specimen preparation, resulting in the belief that these are uncommon in cats. The purpose of this study was to characterize gross and microscopic lesions of inflammatory and neoplastic origin, particularly of the middle ear.

Materials and Methods: Ears from 26 cats were samplingduring necropsies, using a previously described technique (Sula et al. 2014) and were examined grossly and processed for routine histologic examination of the external, middle, and internal ear.

Results: This sampling technique allows to have on a single slide the external, middle and internal ear andthis facilitates  a single, complete and exhaustive evaluation of these structures. Gross and microscopic evaluation of feline ears highlighted numerous pathological conditions: 3 chronic external and medial otitis, 1 nasopharyngeal polyp, 1 ceruminous cystomatosis, 1 squamous cell carcinoma and 1 ceruminous adenocarcinoma with metastasis. Furthermore, we identified 1 case of mucoperiosteal exostoses.

Conclusions: The sampling technique for the ear is simple to be performed and useful to evaluate all the anatomical structures. Focusing on middle ear alterations, these were frequently not reported in clinical history, but became evident during gross and, particularly, histopathological evaluations. We concluded that histologic evidence of middle ear diseases in cats is far greater than clinical literature reported; this suggests that ears, when possible, should be histologically evaluated. Furthermore, we reported for the first time mucoperiosteal exostoses in a domestic cat; this condition was described only in a single report in African lions.


Poster 22:SENESCENCE mARKERS IN CANINE tESTES: P21 BUT NOT H2AX IS INCREASED WITh AGE

S. E. Merz*, A. Breithaupt*, R. Klopfleisch* and A. D. Gruber*

* Department of Veterinary Pathology, Freie Universität Berlin, Germany

Introduction: Many studies in man and mice have shown that senescent cells accumulate with age. Additionally, a connection with tumor cell proliferation is assumed. However, most studies have been conducted in cell culture.

Materials and Methods: Fibroblasts and Leydig cells in normal formalin-fixed, paraffin-embedded (FFPE) testes from 15 dogs < 2 years versus 15 dogs between 7-15 years were examined for age-dependency of the expression of senescence markers H2AX and p21 and their correlation with morphological changes. Both markers were also tested in the microenvironment of 15 canine Leydig cell tumors, 15 seminomas and 15 Sertoli cell tumors.

Results:  : An age-dependent increase in p21 expression from 0.8 ± 0.8 to 4.1 ± 3.4 / 200 fibroblasts (p = 0.0002) and from 1.5 ± 1.4 to 7.6 ± 5.0 / 200 Leydig cells (p = 0.0005) was noted. Although H2AX expressing cells showed no increase with age, they were increased in the microenvironment of seminomas (p = 0.0049) and Sertoli cell tumors (p = 0.0006) in comparison to young individuals. Leydig cells were increased from 42.3 ± 5.0 / 10 HPF in young to 124.5 ± 18.4 / 10 HPF (p < 0.0001) in aged testes. Only a slight decrease in spermatogenesis was seen but no significant changes in fibrosis, tubular diameter or epithelial thickness.

Conclusions: Although both markers are approved senescence markers in vitro, only p21 but not H2AX expressing cells increase with age in canine testes. P21 may thus hold potential to serve as senescence marker in ex vivo FFPE material.


Poster 23: HISTOPATOLOGICAL AND IMMUNOHISTOCHEMICAL STUDIES OF APOCRINE GLAND TUMOURS IN DOGS

J. S. de Vasconcelos*, R. A. S. Siqueira, R. B. de Lucena, A. F. M. Dantas* and A. Alves

*Universidade Federal de Campina Grande, Campus de Patos, Paraíba, Brasil, Universidade Federal da Paraíba, Campus II, Areia, Paraíba, Brasil and CECAV, Universidade de Trás-os-Montes e Alto Douro, Vila Real, Portugal.

Introduction: The apocrine gland tumors are uncommon growths in the dog arising from the glandular or ductular components of sweat glands. They can be benign or maligant, and accounted for 4% of canine skin tumors. The purpose of this study was to determine coordinate expression of p63, citokeratin 14 (CK14) and vimentin using commercially available antibodies in canine apocrine tumours.

Materials and Methods: Seven tissue samples were obtained from the city of João Pessoa, Paraíba, Brazil and from the histopathology archive of the University of Trás-os-Montes e Alto Douro, Vila Real. Tumours were classified according to the WHO diagnostic criteria for epithelial tumours of domestic animals. Sections were immunostained for CK14 (1:800; LL002, Thermo Scientific), vimentin (1:100; NCL-L-VIM-V9, Novocastra) and p63 (1:150; 4A4; Neomarkers).

Results: According to the clasification, 3 apocrine adenomas, 1 apocrine carcinoma, 1 mixed apocrine carcinoma and 2 apocrine ductal carcinomas were diagnosed. CK14, p63 and vimentine positivity was observed in basal/myoepithelial cells of all tumors (7/7). Secretory epithelial cells were negative for p63 (7/7), positive to CK14 in adenomas (2/3) and in carcinomas only one shows positivity (1/4). Secretory epithelial cells were negative to vimentine in adenomas (3/3) and positive in carcinomas (2/4).

Conclusions: In conclusion coordinate expression of p63, CK14 and vimentin identified by IHC may be helpful in differentiating between some canine apocrine tumors, in particular loss of CK14 and acquisition of vimentin expression in adenocarcinomas may be useful to differentiate a benign versus malignant, even additional investigations are needed to confirm this hypothesis.


Poster 24: IATROGENIC SPREAD OF TUMOR CELLS DUE TO SUBCUTANEOUS SURGICAL SEEDING OF A FELINE MENINGIOMA

J. Hoppe, M. Deutschland*, A. Guddat and A.D. Gruber

Institute of Veterinary Pathology, Freie Universität Berlin, Germany, *Tierneurologie Berlin, Germany and Praxis Mahlsdorf, Germany

Introduction: Surgery-associated tumor metastasis is rare in veterinary medicine and mainly reported for tumors in the pleural or abdominal cavity. Here, we report on two subcutaneous metastases of a feline meningioma at the site of surgery one year after removal of the primary tumor.

Materials and Methods: A ten-year-old neutered male domestic shorthair cat presented with progressive neurological symptoms. Magnetic resonance imaging (MRI) of the brain revealed a mass compressing the frontal and temporal lobes. The mass was removed via craniotomy, histologically examined, and no remnants were detected by a control MRI. One year later, the neurological symptoms recurred with a new intracranial mass as well as two subcutaneous masses at the site of surgery. All three masses were removed and examined histologically and immunohistochemically.

Results: The primary mass was diagnosed as benign feline meningioma. The recurring intracranial lesion as well as both subcutaneous masses also showed typical histological patterns of feline meningioma. Immunohistochemistry for S100, vimentin and cytokeratin was also consistent with feline meningioma.  There was no evidence of discontinuities of the cranial bone at the times of the first and second surgeries.

Conclusions: The findings indicate surgery-associated subcutaneous spread of meningioma cells, similar to previous observations on so called needle path metastases described for transabdominal diagnostic punctures of tumors of the prostate gland and urinary bladder in the dog. In light of the frequency of surgical removals of feline intracranial meningiomas, such iatrogenic subcutaneous spread of meningioma cells still seems to be a rare exception.


Poster 25: IMMUNOHISTOCHEMICAL EXPRESSION OF NETRIN-1 IN CANINE LYMPHOMAS

A. Tortereau*, N. Milhau*, E. Rhumy*, M. Castets, T. Marchal* and P. Mehlen

*VetAgro-Sup, Université de Lyon, Marcy l’Etoile, France and Dependence Receptors, Cancer and Development Laboratory - Equipe labellisée 'La Ligue', LabEx DEVweCAN, Centre de Cancérologie de Lyon, INSERM U1052-CNRS UMR5286 Université de Lyon Centre Léon Bérard, Lyon, France

Introduction: Lymphomas are one of the most frequent cancers in both humans and dogs, treated with conventional polychemotherapies, with variable success. Netrin-1, the ligand of dependence receptors DCC and UNC5A-D, has been shown to be overexpressed in some lymphoma subtypes in humans and could thus participate to tumorigenesis by blocking apoptosis induced by its unbound dependence receptors. As canine malignancies represent a robust spontaneous model for human lymphomas, and in light of the dependence receptor model, roles of netrin-1 in canine lymphoma development and, its potential use as a therapeutic target should be investigated.

Materials and Methods: We assessed by immunohistochemistry the expression of netrin-1 in 58 and 65 formalin-fixed paraffin-embedded samples of canine normal lymphoid tissues and lymphomas, respectively. Localization of the immunostaining was recorded and a semi-quantitative analysis of netrin-1 expression was performed. Subtypes, aggressiveness, transformation and extension of lymphomas were considered.

Results: Normal lymphoid B and T cells expressed a nucleolar form of netrin-1. Nucleolar expression was present in 77% and 73% of B and T cell lymphomas respectively. It was decreased or lost in high grade lymphomas. No cytoplasmic expression of netrin-1 was observed in low grade lymphomas whereas it was present in respectively 10% and 13% of B-cell and T-cell high grade lymphomas.

Conclusions: Translocation from nucleolar to cytoplasmic expression of netrin-1 in high grade lymphomas is a promising preliminary result in understanding netrin-1's role in canine lymphomas. Targeting netrin-1 could prove interesting as a therapeutic tool in such lymphoma subtypes.


Poster 26: LTA4H AND FXR1 GENE AND PROTEIN EXPRESSION IN CANINE ORAL MELANOMA

L. Nordio*, F. Genova*, V. Serra*, C. Bazzocchi*, M. L. Longeri*, D. Stefanello*, M. Rondena^ and C. Giudice*

*Department of Veterinary Medicine, Università degli Studi di Milano, Milan, Italy

^Laboratorio Veterinario San Marco, Padova, Italy

Introduction: Canine oral melanoma is a common neoplasm that is usually considered malignant, although the correlation between histology and prognosis is still controversial. Therefore research of new biological markers is ongoing. In this study, LTA4H (an enzyme of the arachidonic acid cascade) and FXR1 (a RNA binding protein) are investigated. Both of them have been previously investigated for their possible role in ocular melanoma metastatization processes, and are studied here in canine oral melanoma. 

Materials and Methods: 29 samples of formalin-fixed/paraffin embedded canine oral melanomas were analyzed. Routine HE sections were evaluated on light microscopy and received a histological description, including mitotic activity index (MI). Immunohistochemical staining for LTA4H, FXR1 and Ki-67 was performed.LTA4H and FXR1 were semi-quantitatively scored and Ki67 was quantitatively scored. The expression of LTA4H and FXR1 genes was also quantified by RT-PCR.

Results: Most of the cases were epithelioid, poorly pigmented melanomas. MI ranged 0.1-9.2 (median 1.1). Ki-67 index ranged 7.9-44.4% (median 19%). Immunolabelling was positive in 29/29 LTA4H and 28/29 FXR1, with variable intensity and percentage of positivity. RT-PCR relative expression values ranged 0,49-9,11 for LTA4H and 0,15-10,33 for FXR1 (in 4 cases was under the detection limit)

Conclusions: LTA4H and FXR1 were detected in all tested melanomas. Marked differences among cases at RT-PCR emerged, although target genes expression levels were not consistent with histological parameters or Ki-67 index. Conversely, immunohistochemical positivity was elevated in most cases with negligible expression differences. Further work is ongoing to clarify the prognostic significance of different levels of expression.


Poster 27: ORAL CAVITY TUMOURS IN DOGS: A RETROSPECTIVE STUDY OF 136 CASES

M. Mikiewicz, K. Paździor-Czapula, I. Otrocka-Domagała, M. Gesek and P. Myrdek

Department of Pathological Anatomy, Faculty of Veterinary Medicine, University of Warmia and Mazury in Olsztyn, Poland

Introduction:  Oral cavity tumours are commonly observed in dogs. They can develop spontaneously or secondary to chronic inflammation. The aim of the study was to evaluate the incidence of the inflammatory, hyperplastic and neoplastic lesions of the canine oral cavity.

Materials and Methods: Canine oral cavity tumours (n=136), diagnosed routinely in 2016, were classified as inflammatory, hyperplastic and neoplastic (benign and malignant).  

Results: The majority (72.8%) of the evaluated tumours represented benign neoplastic, hyperplastic or inflammatory lesions, with the most common being gingival hyperplasia (24.3%), and peripheral odontogenic fibroma (24.3%). The other benign or hyperplastic lesions were represented by viral filiform papilloma (3.7%), calcinosis circumscripta (1.5%), and single cases of squamous papilloma, plasma cell tumour, granular cell tumour, acanthomatous ameloblastoma, and giant cell granuloma. Inflammatory lesions constituted 14.7% of the evaluated cases. Half of these cases represented lympho-plasmacytic stomatitis, and the remaining half – chronic stomatitis of purulent, pyogranulomatous, mixed, ulcerative or eosinophilic types. In four of these cases, the inflammatory infiltrates were accompanied by epithelial dysplasia. Malignant tumours constituted 27.2% of the evaluated cases, with the most frequent being melanoma (11%, mostly high-grade), followed by fibrosarcoma (4.4%), squamous cell carcinoma (3.7%), undifferentiated round cell tumour (2.2%), mast cell tumour (2.2%), poorly differentiated sarcoma (2.2%), and single cases of osteosarcoma, and epitheliotropic lymphoma.

Conclusions:   The majority of canine oral cavity tumours are benign, hyperplastic or inflammatory. However, approximately 30% of canine oral cavity tumours are malignant, and therefore surgical excision followed by histopathological examination is recommended in each case.  


Poster 28: FELINE MESENCHYMAL NASAL HAMARTOMA IN TWO SIBLINGS

J.M. Monné Rodríguez*,  C. Cambi, A. Pratesi  and R. Verin*

* Department of Veterinary Pathology and Public Health, Institute of Veterinary Science, University of Liverpool, United Kingdom University of Liverpool Small Animal Practice,  University of Liverpool, United Kingdom,  Small Animal Teaching Hospital, University of Liverpool, United Kingdom.

Introduction: Mesenchymal nasal hamartoma (MNH) is a rare benign entity affecting young cats that has similar histological features to nasal hamartoma (NH) in children. In both species the lesions are benign, albeit they can be local invasive hence leading to misdiagnosis. The underlying cause for these lesions is undetermined, however, congenital origin is suspected.

Materials and Methods: Two 1 year old cats from the same litter, with respiratory signs and monolateral epistaxis were concomitantly diagnosed by means of X-ray and rhinoscopy with a nasal osteolytic mass partially protruding from the nostrils. Complete surgical removal was undertaken and. tissues were fixed and routinely stained for histological examination (H E stain).

Results: Both cats showed a mesenchymal proliferation expanding the submucosa to form an exophytic nodule lined by respiratory epithelium and composed of 15 mm in diameter plump spindloid cells arranged in streams and supported by a moderate loose to dense fibrous stroma. Large blood filled cavities were multifocally observed together with clusters of osteoid lined by well-differentiated osteoblasts and occasional osteoclasts. Mitotic index was low (less than 1 mitosis per 10 HPF). Numerous inflammatory cells including neutrophils, macrophages, lymphocytes and plasma cells were observed as well as numerous areas of haemorrhage with associated haemosiderophages.

Conclusions: The histological features were compatible with feline MNH. The concomitant presence of the nasal masses in two siblings supports the congenital origin of the lesion. MNH should be included in the differential diagnosis of osteolytic lesions in the nasal cavity of cats.


Poster 29: MULLERIAN DUCT SYNDROME IN A UNILATERAL CHRYPTORCHID DOG WITH A SERTOLI CELL TUMOR AND CYSTIC ENDOMETRIAL HYPERPLASIA

E. Wydooghe*, L. Van Brantegem, M. Switonski, I. Szczerbal, J. Nowacka-Woszuk and A. Van Soom*

*Department of Reproduction, Obstetrics and Herd Health and  Department of Pathology,  Bacteriology and Poultry Diseases, Faculty of Veterinary Medicine, Ghent University, Belgium and Department of Genetics and Animal Breeding, Poznan University of Live Sciences, Poland

Introduction: A 9-year-old Miniature Schnauzer with unilateral cryptorchidism and male external genitalia presented as lame. Clinical examination showed alopecia on the back and an abdominal mass was palpated. Ultrasonography confirmed the mass and X-rays showed no pulmonary metastases. During laparotomy the mass was identified as gonadal tissue connected to the right uterine horn. The left uterine horn was connected to the left intrascrotal gonad. Both uterine horns gathered in the uterine body which ended in the cervix. Connection between female reproductive organs and urethra could not be demonstrated. Gonads and uterus were surgically removed, blood was collected.

Materials and Methods: FFPE tissue samples were stained with HE. Chromosomal analysis and PCR was performed on the blood.

Results: Microscopic examination of the gonads identified atrophic testes with no ovarian tissue. The abdominal testicle had a Sertoli cell tumor. Epididymis and plexus pampiniformis were present, together with an underdeveloped oviduct. Both uterine horns showed cystic endometrial hyperplasia. The dog’s karyotype was 78, XY (normal male) and the SRY-gene (Sex determining region) was present. Further, the MISR2-gene was sequenced and a known substitution in exon 3, causing PMDS in Miniature Schnauzer, in a homozygous status was found.

Conclusions: Surgery and microscopic examination confirmed the presence of testicular tissue, ductus deferens and a uterus in this 78 XY (SRY+) phenotypical male animal, with a nonsense mutation in MISR2-gene, resulting in the diagnosis of persistent Mullerian duct syndrome, a XY disorder of sex development. Furthermore a Sertoli cell tumor and cystic endometrium hyperplasia were present.


Poster 30: MYCOBACTERIUM AVIUM SUBSUP. HOMINISSUIS MENINGOENCEPHALITIS IN A CAT

H. Madaramea, M. Saitob, K. Ogiharac, H. Ochiaid, M. Obae, T. Omatsue, Y. Tsuyukif and T. Mizutanie

aVeterinary Teaching Hospital, bDepartment of Veterinary Medicine, cSchool of Life and Environmental Science, dResearch Institute of Biosciences, Azabu University, Sagamihara, Japan. eResearch and Education Center for Prevention of Global Infectious Diseases of Animals, Tokyo University of Agriculture and Technology, Fuchu, Japan. fSanritsu Zelkova Veterinary Laboratory, Kawasaki, Japan

Introduction: Infection of the CNS by Mycobacterium. avium complex (MAC) is exceedingly rare in cats.  MAC has been further subdivided, although, subspecies or genomic differences of MAC in terms of neuropathology have not yet been a focus of research.  We reported here on Mycobacterium avium subsp. hominissuis (MAH) meningoencephalitis in a cat.

Materials and Methods: A 33-month-old, neutered female poorly nourished Abyssinian cat with indoor access died following sudden onset of widespread neurologic signs about half a year after birth. Complete necropsy, histopathological examination, bacterial culture, PCR, sequencing and genomic analysis of hsp65 gene were carried out.

Results: External examination of the brain showed greenish-yellow discolored arachnoid predominantly covering the basilar regions of the brain.  The coronal slice of the fixed brain revealed bilaterally symmetrical, non-encapsulated, firm and cream-yellow colored mass lesions mimicking cerebral tuberculomas, particularly in the areas of the basal nuclei and piliform lobes. Histopathology of the brain was characterized as a pyogranulomatous meningoencephalitis with extensive cerebral infarctions.  Histiocytes contained piles of acid-fast bacilli distributed in a random fashion.  Acid-fast bacilli infection was also observed in systemic organs, including lungs, skin, intestine, bone marrow and lymph nodes.  There was generalized lymphadenopathy that was most evident in mesenteric lymph nodes, which also had the most severe granulomatous lymphadenitis.  MAH group was isolated and identified from the brain.

Conclusions: MAH infection should be considered in cats with neurologic signs in zoonotic aspects.  This is a first case of MAH infection observed in the brain in either humans or animals.


Poster 31: P-GLYCOPROTEIN IS OVEREXPRESSED IN HUMAN AND CANINE INFLAMMATORY BREAST CANCER CELL LINES BUT NOT IN THE XENOTRASPLANTED MICE TUMORS

A.  Alonso-Díez*, M. Levi, S. Caceres**, L. Barreno*, G. Sarli, C. Benazzi, J.C. Illera** and L. Peña*

*Dept. Animal Medicine, Surgery and Pathology, Complutense University of Madrid, Spain ** Dept. of Animal Physiology, Complutense University of Madrid, Spain and Department of Veterinary Medical Sciences, University of Bologna, Italy.

Introduction:   Multidrug resistance (MDR) to anti-cancer drugs is a major issue for the treatment of breast cancer. Overexpression of the transmembrane protein P-Glycoprotein (PGP) is involved in MDR mechanism. PGP acts as an ATP-dependent drug transporter and pumps a wide spectrum of drugs out of the cell. Inflammatory breast cancer is a special type of breast cancer with the worst prognosis and a high mortality due to the lack of effective treatments. Therefore, many investigations are conducted to find effective therapies. The aim of the present study was to determine the immunolabelling of PGP in human and canine inflammatory mammary cancer cell lines and in the corresponding xenograft tumors in mice.

Materials and Methods: Immunohistochemistry against PGP was performed in human and canine inflammatory mammary cancer cell lines (SUM-149 and IPC-366, respectively) growing in adherent and non-adherent (mammospheres) conditions and in the corresponding mice xenografts.   

Results:  Adherent cells of both cell lines showed the characteristic membranous expression of PGP, with less than 5% having cytoplasmic immunolabelling. A cytoplasmic and membranous clumped immunolabelling was found in the mammospheres of the cell lines. In contrast, tumors of the corresponding xenotrasplanted mice did not express PGP in most of the cells: <5% of cells expressed a slight clumped membranous staining.

Conclusions:  The lack of PGP immunolabelling found in the mice xenografts suggests a loss of MDR related to PGP expression in the murine model. Although further studies are necessary, caution should be considered in experimental therapeutic studies carried out in the xenograft mice.  


Poster 32: PRELIMINARY EVALUATION OF IMMUNOHISTOCHEMICAL EXPRESSION OF INHIBIN-α AND GATA- 4 IN NORMAL AND NEOPLASTIC CANINE GONADS

V. Bertani*, R. di Lecce*, C. Rossi, A. Corradi* and A.M. Cantoni*

*Pathological unit, Department of Veterinary Science, University of Parma, Italy and Practitioner, Brescia, Italy

Introduction: Inhibin-α is a protein produced by gonadal stromal cells responsible for the physiological regulation of the productive axis in both genders, and is connected to mechanisms of cell differentiation and tumor suppression. GATA-4 is a transcription factor which is differentially expressed depending on gonadal developmental stages and pathological conditions.

Materials and Methods: Immunohistochemistry for Inhibin-α and GATA-4 was performed on normal gonads of both genders at different developmental stages and also on neoplasms. Immunostaining was evaluated with a semiquantitative score.

Results: Inhibin-α was expressed in Sertoli cells of normal testis of prepubertal dogs.  Meanwhile in mature dogs, Leydig cells were also positive. In both sex-cord stromal tumors (Leydig and Sertoli cell tumors) neoplastic cells were positive. In normal ovary, follicular epithelial cells of primordial follicles and granulosa cells of remnant follicle stages were positive. Granulosa cell tumors expressed Inhibin-α in neoplastic cells, meanwhile papillary cystadenomas were negative. GATA-4 was expressed in normal testis of prepubertal and pubertal dogs in Sertoli and Leydig cells. In both sex-cord stromal tumors, neoplastic cells were positive. In normal ovary, prepubertal dogs had positive stromal and epithelial cells. In pubertal and mature females, granulosa cells were positive with viable expression in stromal cells. Granulosa cell tumors expressed GATA-4 in neoplastic cells and papillary cystadenoma was negative.

Conclusions: Inhibin-α can be used as marker of normal and neoplastic sex-cord stromal cells in testis and ovarian granulosa cells. GATA-4 has a similar expression in testis and a constant expression in normal and neoplastic ovarian granulosa cells.


Poster 33: SUBCUTANEOUS PANNICULITIS-LIKE T-CELL LYMPHOMA IN A DOG

V. Baldassarre1, M. Campigli2, V. Giacobbi1, C. Masserdotti3, M. Rondena1 and P. Roccabianca4  

1Laboratorio di Analisi Veterinarie San Marco, Padova, Italy, 2Clinica Veterinaria San Marco, Padova, Italy, 3 IDEXX Laboratories, Brescia, Italy, 4 Department of Veterinary Medicine, University of Milan, Italy

Introduction:  Subcutaneous panniculitis-like T-cell lymphoma (SP-TCL) is an uncommon extranodal gamma delta T cell lymphoma included in the human WHO classification. SP-TCL develops in adult patients with no well-defined treatment and variably aggressive course. SP-TCL is characterized by skin nodules or plaques composed of neoplastic lymphocytes infiltrating the subcutaneous fat mimicking lymphocytic panniculitis. SP-TCL has been included in the revised canine REAL classification among subcutaneous lymphomas but reports are anecdotal. The aim of this report is to describe a case of subcutaneous panniculitis-like T-cell lymphoma in a dog.

Materials and Methods:  A 6 years old female Golden retriever was referred for an erythematous plaque in the dorsal neck. Fine needle aspiration cytology and immunocytochemistry were performed. After wide surgical excision, histopathology, immunohistochemistry (anti- CD3, -CD79a and -CD20 antibodies), and PARR were performed.

Results:  Cytology revealed medium to large sized atypical lymphocytes in association with well differentiated adipocytes, consistent with large cell lymphoma. Histology showed a multifocal to coalescing infiltration of large to medium sized pleomorphic lymphoid cells prevalently in the subcutis, rimming adipocytes and extending to the deep dermis. CD3+ positivity and TCR clonal rearrangement confirmed the neoplastic T cell origin of the lymphoid cells.

Conclusions:  Cytomorphology, and growth pattern of this lymphoma paralleled descriptions of primary SP-TCL. The dog developed multiple waxing and waning subcutaneous nodules and lymphomatous multicavitary effusion two years after diagnosis. Cytopathology and flow cytometry revealed a CD3+, CD5+, CD4- CD8-, CD79- T-cell lymphoma consistent with a SP-TCL. The dog did not respond to chemotherapy. 


Poster 34: SYSTEMIC MYCOBACTERIUM MICROTI INFECTION IN TWO CATS

A. Rieger and M. Majzoub-Altweck

Institute of Veterinary Pathology at the Centre for Clinical Veterinary Medicine, Ludwig-Maximilians-University, Munich, Germany

Introduction: Infections with Mycobacterium microti, a member of the Mycobacterium tuberculosis complex, infrequently occur worldwide in various species. Cats seem to be particularly susceptible and nowadays, M. microti constitutes the most common etiological agent for feline tuberculosis. Affected animals mostly present with skin lesions such as cutaneous nodules and non-healing ulcers, while systemic spreading is only seen sporadically.

Materials and Methods: M. microti was identified by PCR in two domestic short hair cats that presented with cutaneous nodules and enlarged lymph nodes. The animals were euthanized and submitted for post-mortem examination. Collected tissue samples were routinely processed for pathohistologic evaluation (H E, Giemsa, modified Ziehl-Neelsen, PAS reaction) and electron microscopic analysis.

Results: At necropsy, both cats presented with multiple caseous granulomas in the lung and mediastinal as well as several other lymph nodes. In one case the liver and the omentum were involved, as well. Histologically affected sites displayed multifocal to coalescing (pyo)granulomatous inflammation with central necrosis. Numerous intralesional acid fast bacilli were detected.

Conclusions: Feline tuberculosis caused by M. microti is an important differential diagnosis in cats presenting with cutaneous lesions and/or systemic signs. Due to their zoonotic potential, mycobacterial infections should always be ruled out in such cases.


Poster 35: Cancelled


Poster 36: TONSILLAR LYMPHANGIOMATOUS POLYPS IN DOGS: A CLINICOPATHOLOGICAL DESCRIPTION OF 7 CASES

S. Maes* and D. Caliari

*IDEXX Laboratories, Belgium and IDEXX Laboratories, Italy

Introduction: Benign tonsillar proliferations are rarely described in dogs, and mostly include inflammatory polyps. 1 lymphangiomatous polyp has been previously reported in an 8 years old female dog. No other benign vascular proliferations of the tonsil are known.

Materials and Methods: 7 cases were recorded in our case load in a 27-month period. Submitting veterinarians were contacted for follow up information. Hematoxylin and eosin-stained slides were reviewed.

Results: Both sexes were almost equally affected, and most dogs were old (7-11 years). There was no specific breed overrepresented. In all cases, no distinct clinical signs were related to the tonsillar mass, which was detected during routine examinations or intubation for a non-related operative treatment. All lesions were unilateral, polypoid with a smooth surface, and ranging 20 - 49 mm in length, and 5 - 21 mm in diameter. Microscopically, lesions consisted of a submucosal polypoid proliferation of thin-walled vascular channels filled with few lymphoid cells, surrounded by edematous connective tissue. Interspersed in between, there were varying amounts of lymphoplasmacytic and follicular lymphoid aggregates. Marginal excision or complete tonsillectomy was conducted. In all patients andthere was no recurrence noted in the follow up period ranging from 1 to 28 months.


Poster 37: TUMOR-INFILTRATING LYMPHOCYTES AND CD4+, CD8+ AND FOXP3+ EXPRESSION IN CANINE CARCINOMA IN MIXED TUMOR

B.E. Lopes-Neto*, M.F. Gärtner and D.C.S. Nunes-Pinheiro*

* State University of Ceara, Brazil, and  Institute of Biomedical Sciences Abel Salazar, University of Porto, Portugal

Introduction: Abundant T-lymphocyte infiltration is frequently found in canine malignant mammary tumours. The aim of the study was to assess the tumor-infiltrating lymphocytes (TIL) and the expressions for subpopulations of T-lymphocytes in canine carcinoma in mixed tumour (CMT).

Materials and Methods: Ten samples of CMT were selected from female dogs treated at the Veterinary Hospital of the State University of Ceara (UECE), Brazil. The TIL were characterized as the peri and/or intratumoral area, distribution, and intensity. Immunohistochemistry was performed in CMT samples using anti-CD4+, anti-CD8+ and anti-FoxP3+ monoclonal antibodies. The statistic was performed using an unpaired t-test, Kaplan-Meier assessment and Cox regression analysis with P value

Results: The TIL component were distributed both in the peri and intratumoral area, multifocally dispersed with discrete intensity. The correlation of TIL with tumour grade showed a slight positive relationship (ρ = 0.28). The survival curve had a negative correlation with TIL, being more evident in samples with intense infiltrations. The immunodetection of CD4+ and CD8+ in TIL was more concentrated in areas surrounding the malignant tumor region. FoxP3+ immunodetection showed lower concentration in TIL population.

Conclusions: The results of this study indicated the relationship of tumor infiltrating T-cells with CMT and TIL might be associated with tumor malignancy criteria, as well as a lower survival of the animals. However, more studies need be done to understand the role of the TIL in canine mammary tumours.


Poster 38: ASSOCIATION BETWEEN THE NUMBER OF LEISHMANIA SPP WITH T-LYMPHOCYTE INFILTRATE IN SKIN GRANULOMATOUS LESIONS IN DOGS

R.M van Ginkel *, A. Gama *, N. Machado *, E. Teixeira and M.A. Pires*

*Laboratory of Histology and Anatomical Pathology (LHAP), Veterinary Sciences Department, Animal and Veterinary research Centre (CECAV). 5000-801 Vila Real, Portugal.

Introduction: Leishmania spp is a disease endemic to the Mediterranean region. It is commonly found in canines, who serve as the reservoir host. This study strived to find an association between the amount of Leishmania amastigotes and the number of T-lymphocytes present in canine skin lesions, from the Northern/central region of Portugal, Southwestern Europe.

Material and methods: A streptavidin–biotin immunohistochemical method was performed in ten canine skin lesions characterized by a diffuse/nodular granulomatous dermatitis compatible with Leishmaniosis, using a canine hyperimmune serum and anti-CD3 as primary antibodies. Leishmania amastigotes were counted in five fields per tissue and categorized in four classes: 0 (I), 1-10 (II), 11-30 (III), >30 (IV) leishmania/field. T-lymphocytes were also counted in five fields per lesions, to determine the number of cells/40x field. The cases were categorized in low and high counts, according to median value. 

Results: Tissues were from six males and four females, from different breeds, with ages between sixteen months to ten years. T-lymphocytes ranged from 77 to 387 cells per field (40X), median 196, with cases categorized in low (n=5) and high (n=5) counts. The Leishmania counts were classified in class I (n=2); II (n=3) and III (n=5). Although higher T-lymphocyte numbers were more common in tissues with increased amastigotes, no significant differences were observed (p = 0,34).

Conclusion: In our study, no association was found between the number of T-lymphocytes and the number of amastigotes. Additional studies with a larger series are welcome, in order to confirm this finding.


Poster 39: ENCAPSULATING PERITONEAL SCLEROSIS: CASE REPORT IN MAINE COON CAT

L. Sonck*, L. Van Brantegem*and K. Chiers *

*Department of veterinary pathology, bacteriology and poultry diseases, University of Ghent, Belgium

Introduction:  Case report about a 10 month old Maine Coon cat presented with acute vomition and a copious amount of ascites. Blood examination, ultrasound, x-ray and explorative laparatomy could not make a diagnosis. Necropsy and histology revealed a case of encasing peritoneal sclerosis.

Materials and Methods: A necropsy was performed, histologic samples were taken from all abdominal organs and lungs. Slides were stained using standard HE, Giemsa and Masson’s trichrome. Immunohistochemistry was performed for CD3, CD20, MAC387, MHCII, c-kit, vimentin, alpha-smooth muscle actin, desmin and cytokeratin.

Results: On necropsy, all abdominal organs were covered in a severely thickened layer of peritoneum, encasing and distorting their shape. Histologically, the peritoneum consisted of dense collagenous tissue with active fibroblasts, multifocal aggregates of lymphocytes and plasma cells, neovascularisation and dilated lymphatics and a diffuse influx of mast cells. Immunohistochemistry revealed aggregates of CD3 and CD20 positive cells, induction of MHCII in the underlying tissues and a strong proliferation of both alpha-smooth muscle actin and vimentin positive cells (myofibroblasts).

Conclusions: A diagnosis of encasing peritoneal sclerosis was made, previously called sclerosing encapsulating peritonitis. This is a rare disorder in which a chronic peritonitis leads to a strong fibrotic reaction. It is a well-known complication in human medicine as a reaction to peritoneal dialysis in end stage kidney patients. In veterinary medicine, half of cases are idiopathic; the rest is linked with singular causes such as peritonitis, steatitis, ingestion of fiber glass…


Poster 40: IMMUNOHISTOCHEMICAL ANALYSIS OF T-LYMPHOCYTES (CD3+) IN FELINE MAMMARY LESIONS

F. Seixas*, D. Antunes* and M. A. Pires*

*Laboratory of Histology and Anatomical Pathology (LHAP), Veterinary Sciences Department and †Animal and Veterinary research Centre (CECAV), University of Trás-os-Montes e Alto

Introduction: Lymphocytes were originally thought to form the basis of a 'cancer immunosurveillance' and during the past decade, insights have been focused on mechanisms underlying the dynamic interplay between immune cells and tumour cells. In humans and dogs, several studies suggest that certain types of inflammatory cells may intervene on tumourigenesis and tumour progression. Herein we aim to study CD3+ cell population in a series of feline mammary lesions.

Materials and Methods: Paraffin-embedded tissue sections from 71 mammary lesions were analysed by immunohistochemistry to demonstrate CD3+ T-lymphocytes. Cell counting was done blindly using an x40 objective (high power field, HPF). Positive cells were evaluated in the tumour, in the periphery of the tumour and in the non-tumoural mammary gland adjacent to tumours, when present in ten fields of each. Four non-neoplastic (“normal”) glands from queens devoid of mammary tumours were also studied.

Results: Statistical significant differences were achieved between CD3+ counts on non-neoplastic lesions, benign and malignant tumours, with carcinomas exhibiting the highest CD3+ cell counts inside and in the periphery of the tumour. Although differences were observed with histological grade, no statistic significance was achieved. Regarding the non-neoplastic mammary gland, the gland adjacent to malignant tumours showed the lowest counts.

Conclusions: This study suggests a positive relationship between T-lymphocytes and increased feline mammary lesions aggressiveness that may be associated with cancer immunoediting.


Poster 41: WALKING AGAINST CANCER: THE IMPACT OF LONG-TERM EXERCISE TRAINING ON MAMMARY TUMORS

A.I. Faustino-Rocha1, P.A. Oliveira2, A. Gama2, H. Vala 2,3, F. Esteves3, R. Ferreira4 and M. Ginja2

1Faculty of Veterinary Medicine, Lusophone University of Humanities and Technologies, Lisbon, 2CITAB, Department of Veterinary Sciences, University of Trás-os-Montes and Alto Douro, Vila Real, 3CI DETS, ESAV, IPV. Portugal and 4Department of Chemistry, University of Aveiro, Aveiro, Portugal

Introduction:  Breast cancer is one of the most frequent cancers among women worldwide. The present work evaluated the effects of long-term exercise training on the development of chemically-induced mammary cancer.

Materials and Methods: Procedures followed the European legislation and were approved by the Portuguese Ethics Committee (DGAV, approval nº008961). Thirty 7-week-old female Sprague-Dawley rats were intraperitoneally injected with the carcinogen N-methyl-N-nitrosourea (MNU) (50mg/Kg). After this, they were randomly divided into two experimental groups: sedentary (n=15) and exercised (n=15). Exercised animals were trained on a treadmill for 35 weeks. At the end of the study, the mammary tumors were collected, fixated in formalin and histologically evaluated. The expression of estrogen receptor (ER)α was assessed by immunohistochemistry.

Results: An incidence of 100% was observed. A total of 51 mammary tumors was developed: 28 in sedentary group and 23 in exercised one (p>0.05). At histopathological analysis, it was observed that each mammary tumor exhibited more than one histological pattern. A total of 121 mammary lesions was counted: 71 in sedentary group and 50 in exercised group (p=0.056). The number of malignant lesions was higher in sedentary group when compared with the exercised one (39 versus 21) (p=0.020). All mammary lesions exhibited nuclear immunostaining for ERα; the immunoexpression was higher in exercised group when compared with the sedentary one (p<0.05).

Conclusions: Long-term exercise training reduced the number and malignancy of mammary tumors. Additionally, it increased the ERα immunoexpression that is an indicator of tumor differentiation and better response to hormone therapy.


Poster 42: NEW THERAPEUTIC APPROACHES ON CHRONIC KIDNEY DISEASE

A. Nogueira1, H. Vala2,4, C. Nóbrega2, CA Pires3, B. Colaço3,4, PA Oliveira3,4and  MJ. Pires3,4

1IPB, Bragança. Portugal, 2CI DETS, ESAV, IPV. Portugal, 3UTAD. Portugal, 4CITAB, UTAD. Portugal

Introduction: The main research goals in the CKD patients are the research of new therapeutic approaches capable of slowing down the progression to end-stage renal disease. The aim of this work was to evaluate the effects of chronic administration of chaetomellic acid A (CAA), which selectively blocks H-Ras farnesylation, on kidney chronic lesions in 5/6 nephrectomized Wistar rats, an animal model of chronic renal disease.

Material and Methods: Sixty male Wistar rats were sham-operated (SO) or submitted to 5/6 nephrectomy (RMR). One week after surgery, surviving animals were distributed into four groups: SO -SO rats receiving no treatment (n=13); SO+CAA- SO rats receiving CAA treatment (n=13); RMR- RMR rats receiving no treatment (n=14); RMR+CAA- RMR rats receiving CAA treatment (n=13). CAA was intraperitoneally administered three times a week for six months. Renal fibrosis was evaluated by ultrasonography and histopathological analysis. All experimental procedures followed the European (European Directive 2010/63/EU) and National (Decree-Law 113/2013) legislation on the protection of the animals used for scientific purposes.

Results: The kidneys of the RMR animals treated with CAA showed a significantly decrease in the medullary echogenicity (p<0.05) compared with the RMR rats that received no treatment. Glomerulosclerosis and arteriolosclerosis scores were significantly lower (p<0.001) in the RMR+CAA group when compared with the RMR group.

Conclusion: These data suggest that pharmacological inhibition of H-Ras proteins activation may be a future strategy in the prevention of end-stage renal disease.


Poster 43: CHRONIC MILD STRESS INCREASES DENTIN HIPERSENSITIVITY AND INDUCES ASTROGLIOSIS IN THE PREFRONTAL CORTEX AND HYPOTHALAMUS IN RATS

F.M. Barbosa*, D. Cabral**, F. Kabadayan*, E.F. Bondan**, M.F.M. Martins**, T.B. Kirsten**, L.V. Bonamin**, N. Queiroz-Hazarbassanov***, M.M. Bernardi*,** and C.H.C. Saraceni*

*Graduate Program in Dentistry, University Paulista, São Paulo, Brazil, **Department of Environmental and Experimental Pathology, University Paulista, São Paulo, Brazil, and ***Department of Pathology, School of Veterinary Medicine, University of São Paulo, Brazil.

Introduction: Although depression and pain are common comorbidities, their interaction is not fully understood. This study evaluated the nociceptive response induced by dentin hypersensitivity after dental erosion in rats that were submitted to unpredictable chronic mild stress (UCMS)-induced depressive-like behavior.

Materials and Methods: Adult male rats were subjected to UCMS (depression [D] group) or not (no depression [ND] group) for 30 days and received either acidic solution to induce dental erosion (E) or water (W), thus forming the WND, END, WD, and ED groups. After the end of treatment, depressive-like parameters (i.e., sucrose preference and immobility in the forced swim test) and dentin hypersensitivity were evaluated. Plasma tumor necrosis factor α (TNF-α) and corticosterone levels were measured, and astrocytic glial fibrillary acidic protein (GFAP) expression was evaluated in the prefrontal cortex, hippocampus, amygdala, and hypothalamus.

Results: Administration of the acidic solution potentiated dentin hypersensitivity and increased corticosterone levels in the ED group compared with the WD group. TNF-α levels only increased in the ED group. The ED group exhibited an increase in astrocytic GFAP expression in the hypothalamus and prefrontal cortex, but decreased in the hippocampus.

Conclusions: These results suggest that UCMS exacerbated the nociceptive response associated with dentin hypersensitivity, concomitant with an increase in plasma corticosterone levels. Hypothalamic and prefrontal cortex astrogliosis in the ED group may be attributable to the increase in corticosterone associated to UCMS procedure. The reduction of astrocytic GFAP expression in the hippocampus in the ED group supports the association between depression and dentin hypersensitivity.


Poster 44: DIGITAL IMAGE ANALYSIS IN MURINE MODELS OF ACUTE PNEUMONIA WITH PATHOGEN-SPECIFIC HISTOPATHOLOGIES AND LESION QUANTIFICATION

K. Dietert*, K. Reppe, B. Gutbier, X. Jiang, N. Baal, M. Witzenrath, H. Hackstein, L.E. Sander and A.D. Gruber*

*Institute of Veterinary Pathology, Freie Universität Berlin, Germany; Department of Infectious Diseases and Pulmonary Medicine, Charité-Universitätsmedizin Berlin, Germany; Institute for Clinical Immunology and Transfusion Medicine, Universities of Giessen and Marburg Lung Center (UGMLC), Germany

Introduction: The character of inflammation, lesion distribution, severity and time course of experimentally induced pneumonia in mice vary widely between different pathogens. Here, we provide an overview on the most relevant differences in histopathologies and introduce an approach to digital image analyses which allows movement from a descriptive pathology approach into pathogen-specific statistically testable data acquisition.

Material and Methods: Lesions and tissue immune cell responses of acute experimental murine pneumonia induced by Streptococcus pneumoniae, Staphylococcus aureus, Klebsiella pneumoniae or influenza A virus as well as PBS-challenged and naïve lungs were compared in terms of relevant histopathologic parameters amenable to two latest software-based image analysis tools.

Results: Histopathology revealed several distinct, model-specific lesion patterns with a clear necessity of pathogen-specific adaptations of parameters for quantitative data analysis. For example, only Klebsiella and staphylococci tended to cause abscesses whereas solely Klebsiella and pneumococci induced pleuritis and steatitis with bacteria visible by hematoxylin and eosin staining. Moreover, results from digital image analyses differed among the models used but allowed for a model-specific reliable numeric data quantification. 

Conclusion: We have identified a spectrum of pathogen-specific lung lesions in murine models of pneumonia which should be considered when selecting a model for comparative infection research. Pathogen-related lesion patterns underscore the necessity of model-specific algorithms for digital image data analyses. We introduce a combination of two latest image analysis tools that allow reliably reproducibly and quantification of agent-specific lung lesions and immunological patterns in tissues that may set a standard for future mouse models of pneumonia.


Poster 45: DISTRIBUTION OF T AND B LYMPHOCYTES AND MACROPHAGES IN COLON FROM RATS WITH TNBS-INDUCED COLITIS PREVIOUSLY FED WITH FUNCTIONAL FOOD

I. Gjurovski1, T. P. Ivanovska2, L. P. Tozi2, K. Mladenovska2, S. K. Kunovska3 and T. Ristoski1

1Department of Pathology, Faculty of veterinary Medicine, Skopje, Macedonia; 2Faculty of Pharmacy, Skopje, Macedonia and 3Institute of Pathology, Faculty of Medicine, Skopje, Macedonia

Introduction: This work aims to analyze the distribution of lymphocytes and macrophages in the colon of rats with trinitrobenzenesulphonic acid (TNBS) induced colitis which were previously orally administered functional foods containing microencapsulated probiotic L. casei 01 and prebiotic oligofructose-enriched inulin.

Materials and Methods: We used female Wistar rats divided in three groups (n=6, 180-250 g, 10-14 weeks old) one of which was a control group receiving only drinking water and plain ayran. The second group received plain ayran and the third  received ayran containing microencapsulated synbiotic. TNBS (30 mg/kg) colitis was induced in the second and third group. Colon specimens were routinely processed. Distribution of lymphocytes and macrophages by CD3, CD79 and MAC387 monoclonal antibodies was analyzed.

Results: The immunohistochemical staining showed a large concentration of CD3 and CD79 positive cells in the mucosa and submucosa of the colon of TNBS group that received only plain ayran. The staining with the same antibodies in the other two groups showed reduced distribution. The MAC387 antibody was also in greater concentration in the mucosa and submucosa of the colon in the TNBS group that received only plain ayran compared with the other two groups.

Conclusions: This study shows the potential anti-inflammatory effects of the ayran-containing microencapsulated synbiotic, characterised by the reduction of inflammatory cells.


Poster 46: ECTOPIC PREGNANCY CONCOMITANT WITH A UTERINE LEIOMYOMA IN A LABORATORY MOUSE

S. Kitz*, D.C. Schäfer and G. Pellegrini*

*Laboratory for Animal Model Pathology (LAMP), Institute of Veterinary Pathology, Vetsuisse Faculty, University of Zurich, Switzerland and Laboratory Animal Services Center (LASC), University of Zurich, Switzerland

Introduction:  Ectopic pregnancy, i.e. a pregnancy occurring outwith the uterine cavity, is a leading cause of maternal death within the first trimester in humans. In laboratory animals, ectopic pregnancies are rare with the highest incidence reported in primates, followed by guinea pigs, rabbits, hamsters, rats and mice. The underlying causes and mechanisms leading to extrauterine implantation of the conceptus are still ill defined.

Materials and Methods:  A 19-week-old, female Sall1CreER x Sall1 fl mouse (C57BL/6 background) was electively euthanized because of severe abdominal distension. A full necropsy with histology and immunohistology was performed.

Results:  The animal was in poor body condition and presented with a large, irregular firm mass in the cranial abdomen, and a smaller spherical nodule in the right uterine horn, the uterus appeared otherwise normal. Histologically the mass was a severe pyogranulomatous reaction centred on multiple necrotic foetal structures (cartilage, bones, skeletal muscles, skin), surrounded by a thick fibrous capsule. The mass found in the uterus was diagnosed as a leiomyoma.

Conclusions:  Based on the pathological findings, we consider this a case of primary abdominal pregnancy, as in the latter, the zygote enters the peritoneal cavity and directly adheres to tissues other than the uterus, while secondary forms occur after a pathological rupture of the oviduct or uterus. In humans, several factors, including tubal and uterine primary conditions, are known to increase the risk of ectopic pregnancy. It might be speculated that the uterine leiomyoma had predisposed to the abdominal implantation of the conceptus in this mouse.


Poster 47: EFFECT OF SODIUM VALPROATE ON THE ALCL3 TOXICITY IN THE BRAIN OF RATS

F. Moayer* and H. Ashrafzade+

*  Department of Pathobiology, School of Veterinary Medicine, Islamic Azad University, Karaj Branch, Karaj, Iran. + Department of Pathobiology, School of Veterinary Medicine, Islamic Azad University, Science and Research Branch, Tehran, Iran

Introduction: Valproate (VPA) is an anti-convulsant and mood-stabilizing drug that is approved to treat seizures, manic episodes associated with bipolar disorder, and to prevent migraine headaches. According to recent hypothesis, there is a close relationship between Aluminum and Alzheimer's disease. In this study, the effects of VPA on experimental induction of amyloidosis by Alcl3 in rat brain were investigated.

Materials and Methods: 24 wistar rats received 3.7×10-4 g/kg/bw Alcl3 by microinjection into CA1 sector of the hippocampus and were then randomly divided into 2 groups. Rats in treatment group were treated with VPA at the dose of 500 mg/kg/day on alternate day thrice/week for a period of two weeks and the control group were not treated with any drug. At the end of period, after euthanasia, the brain samples were stained with H E and Congo-Red. Immunohistochemical analysis was made by using NCL-β-Amyloid Antibody.

Results: Histopathologial and immunohistochemical evaluation showed focal amyloidosis in brains of the control group, particularly in hippocampus and around the vessels. Neuronal degeneration, edema, and gliosis with neurophagia were detected in brain matrix of rats in this group. The focal amyloidosis and other pathological changes were decreased significantly in treatment group.

Conclusions: Valproate may work by suppressing repetitive neuronal firing through inhibition of voltage-sensitive sodium channels and is thought to also be a histone deacetylase inhibitor. The present results clearly demonstrate VPA can reduce Alcl3 toxicity.


Poster 48: INFLUENCE OF ROUTE OF INFECTION ON MURINE EXPERIMENTAL ENCEPHALITOZOONOSIS

C. R. S. Moyses*, A. M. Alvares-Saraiva*, E. C. Perez*, D. D. Spadacci-Morena, J. G. Xavier*, P. R. D. Rocha*and M. A. Lallo*

*Environmental and Experimental Pathology, Paulista University, Brazil and Department of Pathophysiology, Butantã Institute, Brazil

 

Introduction: We analyzed the pattern of histopathological lesions caused by the experimental infection of mice by Encephalitozoon cuniculi by two routes of inoculation, oral or intraperitoneal.

Materials and Methods: Balb/c mice immunosuppressed or not with cyclophosphamide were inoculated with E. cuniculi either orally (VO) or intraperitoneally (IP). After 14 and 21 days post-infection (DPI) animals were euthanized and tissue samples were collected from liver, intestines and lungs, fixed in 10% buffered formalin solution and routinely processed for Hematoxylin-Eosin.

Results: Mice inoculated by the oral route had fewer lesions than those inoculated by the intraperitoneal route. Multifocal periportal hepatitis with a predominance of lymphoplasmocytic infiltrates and eventual influx of eosinophils was observed in both routes of infection. Apparently independent of infection route, IP or VO, the inflammatory infiltrate is present in the same place of the liver. Focal interstitial pneumonia and pleural thickening were observed in all infected animals, regardless of the route of inoculation. The kidneys of Balb/c mice infected by the IP route showed discrete interstitial lymphoplasmocytic infiltrates. Only in the mice inoculated VO  was nephrosis observed. Discrete lymphoplasmocytic enteritis was observed only in the VO of infection. Immunosuppression decreased the number and size of inflammatory areas in both groups, however more degenerative lesions were observed.

Conclusions: We conclude that the oral route of E. cuniculi infection induced fewer lesions than IP route, probably due to the complexity and efficiency of the intestine-associated immune system.


Poster 49: REDUCTION IN VEGFA EXPRESSION AND TUMOR PROGRESSION IN MICE SUBMITTED TO COMBINED TREATMENT WITH ELECTROCHEMOTHERAPY AND RESVERATROL

C.H.M.Brunner*, V.Borelli*, C.V.Cardoso*, E.S. de Carvalho Junior*, M.J. dos Santos Junior*, S.R. Kleeb, M.A. Lallo*and J.G. Xavier*,

*Environmental and Experimental Pathology, Paulista University, Brazil and Methodist University of São Paulo, São Bernardo do Campo, Brazil

Introduction:  Electrochemotherapy is an oncologic treatment that induces a transient reduction of blood flow, stabilizing the transcriptional factor HIF1a, a potent inductor of VEGF expression, resulting in local angiogenesis.  Resveratrol, a grape-derived compound, increases the expression of sirtuin 1, which represses HIF1a. The aim of this study was to evaluate the effect of electrochemotherapy and resveratrol on the development of Ehrlich tumor in mice, and the local expression of VEGF.

Materials and Methods: 38 mice BALB-C, female adult, received 0.1 mL subcutaneous inoculum containing 5.0x106 cells of Ehrlich´s tumor. 30 days later, the tumors were measured, and the animals were randomly divided into control group, without manipulation, an electrochemotherapy group, which received intratumoral application of electrochemotherapy,  a resveratrol group, which received a single intraperitoneal inoculum of resveratrol, and a combined group, that received both treatments. The mice were euthanized 7 days later, the tumors were measured and then, submitted to histological and immunohistochemical processing to evaluate the expression of VEGFA.

Results: All treated animals showed reduction of the tumor growth (ANOVA, Tukey-Kramer, with p < 0.01), but   with the combined therapies showed the smallest tumor expansion. Histologically,  extensive necrosis in the tumor central areas was noted with elevated cellularity in perivascular sites. There was a reduction of VEGFA immunoexpression in the animals subjected to treatment with resveratrol alone as well those receiving combined therapy.

Conclusions: The treatment with electrochemotherapy and resveratrol was associated with a reduction in tumor progression, suggesting their potential utility in adjuvant therapy of neoplasia.


Poster 50: COMPARATIVE MUSCLE PATHOLOGY AND REGENERATIVE PATTERN IN A MOUSE MODEL OF POMPE DISEASE (GLYCOGENOSIS TYPE II)

F.D. Franzoso1*, E. Gougeon1*, L. Lagalice1, L. Dubreil1, J. Deniaud1, K. Rouger and M-A. Colle

1 PAnTher, INRA, École nationale vétérinaire, agro-alimentaire et de l’alimentation Nantes-Atlantique (Oniris), Université Bretagne Loire (UBL), Nantes, F-44307, France

* and § These authors contributed equally to this work

Introduction: Pompe disease is a lysosomal storage disorder caused by an inherited deficiency of acid α-glucosidase (GAA). Severe glycogen accumulation is detected within the nervous system, skeletal and cardiac muscles. The resulting muscle pathology and the myogenic regenerative process is not fully understood. We investigated the histopathology of the skeletal muscle in the mouse model 6neo/6neo of Pompe disease with a focus on the satellite cell behavior.

Materials and Methods: Tibialis anterior and Triceps brachialis muscle sections from 6neo/6neo and B6/129 mice (1.5, 4, 6 and 9 months) were stained with HE, Masson’s Trichome and PAS. Immunohistochemistry was performed using primary antibodies directed against Laminin, Pax7, MyoD, Ki67, developmental Myosin Heavy Chains (MHCd). Image acquisition and analysis were performed using ZEISS LSM780 confocal microscope and Fiji software.

Results: Skeletal muscle of 6neo/6neo mice presented age-progressive glycogen PAS positive deposition within the myofibers. Muscle lesions were age-dependent and characterized by increasing presence from 6 months of vacuolization, irregular shaped, split fibers, centronucleation, and necrosis, as well as mild inflammatory infiltration and fibrosis. Numbers of Pax7+-, Pax7+/Ki67+-, MyoD+-satellite cells and MHCd+ fibers were not different from those obtained in control mice.

Conclusions: Skeletal muscle pathologic features in the 6neo/6neo mice model increased in severity with age, showing similar lesions as in the infantile form of human disease. Muscle damage was not associated with activation of satellite cells, revealing a severe defect of muscle regeneration, as reported in human patients. Possible underlying mechanisms could be used as targets for novel therapeutic strategies.


Poster 51: HMGA2 COOPERATES WITH EITHER P27 DEFICIENCY OR CDK4R24C MUTATION IN PITUITARY TUMORIGENESIS

M. Fedele*, O. Paciello, D. De Biase, M. Monaco**, M. Vitiello*, D. Rea, A. Luciano, C. Arraand A. Fusco*

*Institute of Experimental Endocrinology and Oncology - CNR c/o Department of Molecular Medicine and Medical Biotechnologies, University of Naples “Federico II”, Naples, Italy. Department of Veterinary Medicine and animal production, University of Naples “Federico II”, Naples, Italy. **Department of  Experimental Oncology, Functional Genomic Unit, National Cancer Institute “Fondazione Giovanni Pascale”, IRCCS, Naples, Italy. Animal facility, National Cancer Institute “Fondazione Giovanni Pascale”, IRCCS, Naples, Italy.

Introduction: Hmga1 or Hmga2 genes are chromatin regulating factors that act as cellular oncogenes when overexpressed and play a pivotal role in the development of pituitary adenomas (PA) in humans. Impairment of cell cycle control represents a feature of human pituitary adenomas, therefore the aim of our work was to experimentally evaluate the possible synergism between Hmga2 overexpression and the alterations of other cell cycle regulators such as p27 deficiency or CDK4 mutations.

Materials and Methods: Hmga2/T transgenic mice were crossed with either p27kip1-null mice or Cdk4R24C mice in order to obtain double mutant mice. Genotypes were analyzed by PCR with genomic DNA isolated from mouse tail. Mice were euthanized at any sign of disease and pituitary tissues were recovered for histological and molecular analysis. Kaplan-Meyer survival curves were used to analyze the percentage of tumor-free mice. Differences were analyzed by Log-Rank (Mantell-Cox) test. P values < 0.05 were considered statistically significant.

Results: Increased incidence and decreased latency in the development of pituitary lesions appeared in double mutant Hmga2/T;Cdk4R24C mice and increased features of malignancy such as anisokaryosis and anisocytosis were shown by pituitary tumors of both Hmga2/T;p27-ko and Hmga2/T;Cdk4R24C double mutant mice as compared with single mutant. Moreover, double mutant mice developed pituitary carcinomas.

Conclusions: Taken together, the results reported here indicate a cooperation between Hmga2 overexpression and either p27 or CDK4 impairment in promoting pituitary tumor development and progression. Furthermore, mice developing pituitary carcinomas may represent the first mouse model for this rare human pituitary neoplasm.


Poster 52: EXPRESSION OF MUC-1 AND MUC-2 IN ILEUM OF GF MICE AFTER ADMINISTRATION OF L.REUTERI AND CHALLENGE WITH PCV-2

V. Karaffová*, J. Pistl*, M. Levkutová*, T. Csank*, V. Revajová*, R.. Nemcová*,  M. Levkut, jr.* and M. Levkut *†

*University of Veterinary Medicine and Pharmacy, Komenského 73, Košice,

Neuroimmunological Institute, SAS, Bratislava

Introduction: Porcine circoviruses (PCV) are present worldwide, infecting domestic pigs and wild boars alike. PCV2 has been linked to several pathological conditions in pigs, named together as porcine circovirus diseases (PCVDs). Lactic acid bacteria as well as  Lactobacillus reuteri may exert their antiviral effects by several mechanisms including direct interaction with viruses, production of antiviral inhibitory substances or by stimulation of immune system. The goal of this paper was to study the effect of L. reuteri on mRNA expression of MUC-1 and MUC-2, and evaluate the amount of infected virus in faeces after challenge of germ free mice with PCV2.

Materials and Methods: Four week-old GF mice (n= 30) were divided into three groups: PCV-2 (0.3 ml/ mouse per os on day 7), LPCV-2 (109 CFU/0.1 ml; from 1 to 7 day of experiment + PCV-2) and controls. Samples of ileum were homogenized and total of RNA was isolated. Primers for MUC-1and MUC-2 were used. The PCV-2 was quantified by RT-PCR. Amplification and detection of specific products was performed using CFX 96 RT system (Bio-Rad, USA) with predefined program.

Results: Relative mRNA expression of MUCs was upregulated in LPCV group compared to control and PCV (P<0.01) groups during both sampling (5, 14 day post infection). However, the mean amount of PCV-2 in faeces was significantly lower from 3 dpi in PCV-2 group compared to the LPCV-2 group.

Conclusions: Mucoprotective effect of L. reuteri in ileum of infected mice and decrease amount of PCV-2 in faeces was observed.


Poster 53: IS THERE A RELATIONSHIP BETWEEN MORPHOMETRY OF INTESTINE AND EXPRESSION OF IGA IN GF MICE AFTER APPLICATION OF PROBIOTIC BACTERIA AND INFECTION WITH PCV2?

M. Šefcová*, M. Levkut, Jr.*, V. Karaffová*, J. Pistl*, T. Csank*, R. Nemcová*, M. Levkutová*, R. Herich*, and M. Levkut†*

*University of Veterinary Medicine and Pharmacy, Komenského 73, Košice, Slovakia and †Neuroimmunological Institute, SAS, Bratislava

Introduction: Porcine circovirus type 2 (PCV2) has been linked to several pathological conditions in pigs. PCV2 infection has a significant impact on the pig immune system. Microbial stimulation plays an important role in the development of the immune system, its total absence results in morphological and immunological changes. The aim of the study is to compare the effect of the PCV2 and Lactobacillus reuteri on the expression of IgA and morphological parameters of the intestine of gnotobiotic mice.

Materials and Methods: Four week-old GF mice (n= 30) were divided into three groups: PCV-2 (0.3 ml/ mouse per os on day 7), LPCV-2 (109 CFU/0.1 ml; from 1 to 7 day of experiment + PCV-2) and controls. Samples of ileum were homogenized and total RNA was isolated. Primers for IgA were used. The PCV-2 was quantified by RT-PCR. Amplification and detection of specific products was performed using CFX 96 RT system (Bio-Rad, USA) with predefined program. Morphologically, height and area of villi and depth of crypt was measured in the jejunum.

Results: Relative mRNA expression of IgA was upregulated in LPCV group compared to control and PCV groups (P<0.001) on 5 dpi and (P<0.01) on 14 dpi. Morphometry showed increased villi in PCV2 group (P<0.01) compared to controls on 14 dpi. Depth of crypt was increased in LPCV group (P<0.001) compared to control and PCV group (5 and 14 dpi).

Conclusions: Application of Lactobacillus reuteri stimulated onset of IgA production and simultaneously supported the growth of epithelial cells represented by increased crypt depth.


Poster 54: GENOMIC AND MOLECULAR BASIS FOR SUITABILITY ASSESSMENT OF ANIMAL MODELS: DIFFERENCES IN CLCA GENES AND PROTEIN EXPRESSION BETWEEN HUMANS, MICE AND CATS

 

L. Mundhenk*#, N.A. Erickson*#, N. Klymiuk and A.D. Gruber*

*Institute of Veterinary Pathology, Freie Universität Berlin, Germany, and Institute of Molecular Animal Breeding and Biotechnology, Ludwig-Maximilians-Universität München, Germany,

 #contributed equally to this work

 

Introduction: Several chloride channel regulators, calcium-activated (CLCA) molecules play a role in chronic inflammatory airway disease with mucus overproduction, including asthma. However, contradictory data on their function in mucus cell metaplasia and dyskrinia between mice and humans raise the question of species-specific differences of CLCA members in the respiratory tract. In particular, mouse models have proven unsuitable so far.

Materials and Methods: The CLCA genome cluster and its proteins were characterized in silico in humans, mice, and cats. The expression patterns of feline CLCA members were established on the mRNA-level via RT-qPCR and on the protein level using immunohistochemistry. The data were compared with orthologs in man and mice.

Results: Identical to humans but in contrast to mice, the feline CLCA-genome cluster consists of four separate genes. The protein structures appear conserved between the different species as seem the expression patterns of most CLCA-proteins in the airways except for CLCA3 which is unique in each of the species tested.

Conclusions: On the genomic level, the cat seems as a much better model for the human CLCA family than the mouse. However, due to striking differences in the expression patterns of the CLCA3 genes among all species tested, both mice and cats appear unsuitable to fully mimic the human CLCA3 and another model species needs to be identified.


Poster 55: GLIOTOXIC INJURY IN THE RAT BRAINSTEM IS AMELIORATED BY CURCUMIN

E.F. Bondan*,**, M.F.M. Martins** and C.V. Cardoso*

*Department of Environmental and Experimental Pathology, University Paulista (UNIP), São Paulo, Brazil, and **Department of Veterinary Medicine, University Cruzeiro do Sul, São Paulo, Brazil

Introduction: Curcumin (Cur) exhibits antioxidant, anti-inflammatory, anti-cancer and anti-fibrotic effects in many pathological conditions. Ethidium bromide (EB) injection into the brain induces focal oligodendrocyte and astrocyte loss, leading to primary demyelination, neuroinflammation and peripheral astrogliosis, with increased immunoreactivity to glial fibrillary acidic protein (GFAP). This study aimed to evaluate the effects of Cur administration on remyelination and astrocytic response following gliotoxic injury.

Materials and Methods: Wistar rats were injected with 10 microlitres of 0.1% EB into the cisterna pontis and treated or not with Cur (100 mg/kg/day, intraperitoneal route) during the experimental period. Brainstem sections were collected at 15, 21 and 31 days after EB injection for transmission electron microscopy and GFAP immunohistochemical investigation. Astrocytic reactivity and remyelination status were assessed by morphometry.

Results: By 15 days after EB injection the center of the lesion was filled with myelin debris among foamy macrophages and demyelinated axons. No astrocytic processes were found in this site and an initial association between naked axons and remyelinating cells was seen at peripheral locations. Invasive Schwann cells were associated with demyelinated axons or already forming thin myelin lamellae around single axons in astrocytic-free areas. Oligodendrocytes began to form thin myelin sheaths in areas presenting astrocytic prolongations. By 21-31 days, it was noted that Cur-treated rats presented increased oligodendroglial remyelination. GFAP-stained area around lesion was significantly smaller in Cur-treated rats in all periods when compared to untreated animals.

Conclusions: Results show that Cur has beneficial effects on remyelination and reduces glial scar development following gliotoxic injury.


Poster 56: HYPERCALORIC DIET INDUCES ASTROGLIOSIS IN SEVERAL AREAS OF THE RAT BRAIN

E.F. Bondan*,**, J. Molina*, P.D. Dossa*, L.B. Viebig*, L.V. Bonamin*, M.F.M. Martins*,**, A. Joaquim*, T.B. Kirsten*, C.V. Cardoso* and M.M. Bernardi*

*Department of Environmental and Experimental Pathology, University Paulista (UNIP), São Paulo, Brazil, and **Department of Veterinary Medicine, University Cruzeiro do Sul, São Paulo, Brazil

Introduction: The consumption of high fat diets is associated with chronic low-grade inflammation in peripheral tissues, which contributes to the development of comorbidities such as insulin resistance and cardiovascular disease. It also promotes hypothalamic inflammation and morphological changes in astrocytes, including the increased expression of the astrocytic biomarker glial fibrillary acidic protein (GFAP), which is seen as a sign of neuroinflammation. Because of their physical proximity to blood vessels and their function in transporting nutrients, astrocytes are directly affected by nutrient excess, leading to hypothalamic astrogliosis. It remains obscure if this process occur only in the hypothalamus or affect other central nervous system (CNS) areas. In this context, this study aimed to observe the impact of a hypercaloric diet on GFAP expression in several brain areas.

Materials and Methods: Male Wistar rats were fed with normocaloric diet (ND group) or with highly palatable hypercaloric diet (Ensure ®) (HD group) from postnatal day 23 to 65. At postnatal day 90, rats from both groups were euthanized and brains were collected and submitted to GFAP immunohistochemical study. The following areas were chosen for investigation: parietal and frontal cortex, corpus callosum, nucleus accumbens, arcuate and periventricular nuclei of hypothalamus, molecular and granular layers of cerebellum and pons. Astrocytic reactivity was assessed by morphometry.

Results: GFAP immunoreactivity was increased in all analyzed regions from the HD group compared to the ND group (p<0.001).

Conclusions: The consumption of hypercaloric diet induced extensive astrogliosis in all observed areas of the brain, providing an indication of neuroinflammation.


Poster 57: IDIOPATHIC CORVILLOSUM (PERICARDITIS FIBRINOSA) IN LABORATORY GUINEA PIG

A. Drzewiecka*, J. Faron*, M. Węgrzyńska*, P. Schulz and J.Pajdak

*Department of Toxicology, Institute of Industrial Organic Chemistry Branch, Pszczyna, Poland and Department of Microbiology and Clinical Immunology, Faculty of Veterinary Medicine, University of Warmia and Mazury in Olsztyn, Poland, and Department of Epizootiology, Faculty of Veterinary Medicine, University of Warmia and Mazury in Olsztyn, Poland

Introduction: Corvillosum (the hairy heart or bread butter pericarditis) occurs when, due to fibrinous pericarditis, epicardium is covered with shaggy fibrin deposits, and this disease is very rare in laboratory animals. This is a case of fibrinous pericarditis in laboratory guinea pig with unknown etiology (idiopathic).

Materials and Methods: The SPF (Specific Pathogen Free) Guinea pigs Dunkin-Hartley, complete  with health reports were obtained  for skin sensitization tests. After quarantine period animals were introduced to the experiment and during administration of the test item one guinea pig died without clinical signs. The gross examination was conducted and samples of heart and pericardium were taken, fixed with 10% formalin solution, embed with paraffin and stained with HE, iron hematoxylin-picric acid-acid fuchsin (van Gieson), and iron hematoxylin-periodic acid-Schiff reagent-alcian blue (AB-PAS).

Results: Macroscopic examination revealed an enlarged pericardium sac filled with yellowish, gelatinous content with admixture of  fibrin tufts. During histopathological examination a eosinophilic homogeneous mass of fibrin was noted with ingrowth of granulation tissue  covering the epicardium. There was congestion and infiltration of inflammatory cells into the epicardium and ingrowths of adipose tissue into myocardium (adipositascordis) . No lesions in fibers of heart muscle were observed. Additionally fibrosis of pericardial sac with infiltration of inflammatory cells and an eosinophilic homogeneous mass of fibrin attached was also noted. The animal died due to cardiac tamponade caused by fibrinous pericarditis exacerbated by too tight a squeeze of the animal during sensitization test.

Conclusions: Corvillosum causing sudden death can significantly affect results of experiments.


Poster 58: IMMUNE CELL PROFILE OF BRAIN INFLAMMATION CAUSED BY ENCEPHALITOZOON CUNICULI IN MICE

P.R.D. Rocha*, M. A. Lallo*, A. M. Alvares-Saraiva*, Nicolle G. T. Q. Hazarbassanov¥, E. C. P. Hurtado*, C. O. S. Massocco¥ and G.F.Machado

*Graduate Program in Environmental and Experimental Pathology, Paulista University,Sao Paulo State University, Brazil. ¥ Department of Pathology, University of Sao Paulo, Brazil

Introduction: Encephalitozooonosis is a zoonosis caused by Encephalitozoon cuniculi, an intracellular pathogen, causing mostly encephalitis, nephritis and pneumonia in immunocompetent and immunocompromised individuals. Although brain lesions have already been well described in rabbits, the pathogenesis of brain disease in encephalitozoonosis is totally unclear. The aim of this study was to evaluate the immune response in the brain of mice experimentally infected with Encephalitozoon cuniculi.

Materials and Methods: C57Bl/6 male mice, 8-10 weeks old, were intravenously infected with E. cuniculi spores and intraperitoneally immunossupressed with 75mg/kg of cyclophosphamide. Non-infected mice were controls. After 30 days post-infection, the brain was collected, processed and evaluated by flow cytometry to characterize phagocytes (microglia and invading macrophages) and lymphocytes (TCD4+, TCD8+, B+). Up to 1 million events were counted from each animal.

Results: Both microglia and invading macrophages increased significantly (at least 8 times) in infected animals compared to control. The total number of microglia was 33 times higher than invading macrophages. Although there was a low absolute percentage of total lymphocytes, TCD4+ increased more than 2000 times and TCD8+ increased more than 100 times in infected animals compared to control, whereas B lymphocytes showed no difference between infected animals compared to control.

Conclusions: These results suggest that phagocytes, especially microglia, play a significant role in the brain immune response against encephalitozoonosis, regardless of an immunossupressive condition. Further investigation is needed to better characterize the inflammatory pathway of neuroinflammation due to E. cuniculi.


Poster 59: PATHOMORPHOLOGICAL PATTERN OF THE MOUSE LIVER AFTER INTRATRACHEALLY INSTILLATION OF SANDING DUST FROM PAINT CONTAINING DIFFERENT NANOSIZED TITANIUM DIOXIDE PARTICLES

J. Szarek§, A. Mortensen, A. T. Saber, N. R. Jacobsen, M. Levin, I. K. Koponen, K. A. Jensen, U. Vogel* and H. Wallin†‡

  • Department of Pathophysiology, Forensic Veterinary Medicine and Administration, University of Warmia and Mazury in Olsztyn, Poland, and The National Research Centre for the Working Environment, Copenhagen, Denmark, and *Department of Micro and Nanotechnology, Technical University of Denmark, Copenhagen, Denmark, and †‡Institute of Public Health, University of Copenhagen, Copenhagen K, Denmark

Introduction: Inclusion of nanomaterials (NM) in paints is a common nanotechnology application. This study focuses on the liver pathomorphology in mice of dusts from sanding of paints containing NM. We compared the influence of nanotitanium dioxide and dusts generated by sanding boards coated with paints with different amounts of two different types of uncoated nanotitanium dioxide (10.5 nm and 38 nm).

Materials and Methods: The experiment was conducted in the C57BL/6 mice (Taconic, Ry, Denmark.), female, 8 weeks old. Animals were intratracheally instilled with single dose of 18, 54, 162 µg of nanotitanium dioxide or 54, 162, 486 µg of sanding dusts. Particle (3.24 mg/ml) and dust (9.72 mg/ml) suspensions were dispersed in 2% serum in nano-pure water by sonication. Each control and treatment group consisted of 22-24 and 6 mice, respectively. Microscopical pattern of liver (HE staining) in mice was evaluated one, 3 and 28 days post-instillation.

Results: Several histological lesions of similar type were observed in the liver from the exposed mice when compared to the controls. The most often recorded inflammatory lesions were polymorphonuclear cell foci. Necrotic changes were represented by microfoci of necrosis, eosinophilic necrosis of single hepatocytes and hepatocytes with pycnotic nuclei. Hyperplasia of bile ducts epithelium and sinusoidal dilatation were observed sporadically in mice exposed to carbon black as compared to the livers from the mice exposed to either TiO2 NM or to sanding dusts with TiO2NM.

Conclusions: For all exposures mild histological lesions were observed in the mice liver compared to vehicle controls.


Poster 60: PREPARATION OF THE CHICKEN CORNEAS FROM ICE TEST FOR MICROSCOPIC EVALUATION – FIXATION

A. Drzewiecka*, J. Faron*, D. Krakowian*, P. Schulz and J. Pajdak

*Department of Toxicology, Institute of Industrial Organic Chemistry Pszczyna Branch, Poland and Department of Microbiology and Clinical Immunology, Faculty of Veterinary Medicine, University of Warmia and Mazury in Olsztyn, Poland, and Department of Epizootiology, Faculty of Veterinary Medicine, University of Warmia and Mazury in Olsztyn, Poland

Introduction: The isolated chicken eye (ICE) test is an alternative method accepted by the OECD for identification of severe irritants. Histopathological examination of the corneas is not required, however OECD guidelines (no. 438) encourages this implementation as a helpful tool in assessing the effects of chemicals that are difficult to classify using current methods.

Materials and Methods: Following fixatives were tested: 10% formalin solution, 4% formalin solution, Davidson’s fluid, modified Davidson’s fluid, 4% paraformaldehyde solution, 6% glutaraldehyde solution and mixture of 2% formalin with 2.5% glutaraldehyde (1:1). All eyeballs (total 10 eyeballs/fixative) were incubated at 32 ± 1.5oC for 240 minutes (in accordance with OECD no.438) and then fixed for 24 hours (5 eyeballs/fixative) and 168 hours (5 eyeballs/fixative). From each eyeball one sample of cornea with the surrounding sclera for both time points (24 and 168 hours of fixation) were collected, embedded in paraffin and stained with HE. However from eyeballs fixed with Davidson’s and modified Davidson’s fluids samples were taken after 24 and 48 hours of fixation. 

Results: Histopathological evaluation of corneas revealed no significant differences due to prolonged fixation time (up to 168 hours), regardless of fixatives.

Conclusions: Only fixation in 10% formalin solution and 4% paraformaldehyde solution didn’t show any negative impact on the quality of obtained histopathological slides. For other fixatives, a significant effect on the cornea was observed, i.e. change of their shape, varying degrees of opacity, difficulty in cutting of paraffin block, and a number of histopathological lesions in the cornea.


Poster 61: THE ROLE OF THE C-TYPE LECTIN RECEPTOR DCIR IN THEILER’S MURINE ENCEPHALOMYELITIS VIRUS INFECTION

M. Stoff*, M. Ciurkiewicz*, ‡, S. Mayer, T. Störk*, B. Lepenies and A. Beineke*, ‡

*Department of Pathology and Research Center for Emerging Infections and Zoonoses, Immunology Unit, University of Veterinary Medicine Hannover, Germany, Center for Systems Neuroscience, Hannover, Germany

Introduction: Theiler’s murine encephalomyelitis virus (TMEV) infection is an infectious mouse model for the investigation of neurological disorders including multiple sclerosis and epilepsy. C-type lectin receptors (CLR) are involved in the regulation of immune homeostasis and serve as pattern recognition receptors for different antigens by recognition of carbohydrate structures in a Ca2+-dependent manner.

Materials and Methods: To investigate the functional role of the CLR dendritic cell immunoreceptor (DCIR) during the acute phase of TMEV-induced encephalitis, five-week old wildtype (WT) C57BL/6 and DCIR-/- mice were intracerebrally infected with 1 x 105 plaque forming units of TMEV (Daniels strain) into the right hemisphere and necropsied 7 and 14 days post infection (dpi). Flow cytometry of spleen tissue as well as histological and immunohistochemical analyses (IHC) of the hippocampus were performed.

Results: Histological examination of the brain revealed a significant reduction of hippocampal damage in DCIR-/- mice compared to WT mice at 14 dpi as determined by NeuN-specific IHC. In addition, significantly lower numbers of CD107b+ macrophages/microglia and infiltrating CD3+ T-lymphocytes and a decrease of TMEV-infected cells within the hippocampus of DCIR-/- mice compared to WT controls were detected. Flow cytometry revealed a significant increase of CD4+CD44+ and CD8+CD62L+ T cells in spleen tissue of DCIR-/- mice, indicative of peripheral T cell activation.

Conclusions: Results show an inhibitory effect of DCIR upon antiviral immunity in the TMEV model. Moreover, genetic ablation of DCIR displays protective properties on neuronal integrity of the hippocampus in infected mice.


Poster 62: VACCINATION WITH DIFFERENT TOLL-LIKE RECEPTOR AGONISTS BY DISSOLVING MICRONEEDLE PATCHES TO PIGS: ASSESSMENT OF LOCAL IMMUNE RESPONSE BY MACROSCOPIC AND HISTOLOGIC EVALUATION

S. Vreman*, J. McCaffrey, A. Rebel*, N. Stockhofe-Zurwieden* and A. Moore

*Wageningen Bioveterinary Research, Wageningen University Research, The Hague, The Netherlands,  †Xeolas, Pharmaceuticals, Dublin;Ireland;and  ‡School of Pharmacy University College Cork, Cork, Ireland.

Introduction: Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) is an important pig pathogen. Currently, there is no adequate vaccination program for PRRSV. We aim to develop an efficacious vaccine by using dissolving microneedle (DMN)-patches in the skin, which has a high number of antigen presenting cells (APCs). This may result in equivalent or stronger immune responses compared to traditional systemic routes. Toll like receptor (TLR) agonists (adjuvants) can stimulate APCs, and could work in combination with DMN-patches as a promising adjuvant. In this study we examined how three different TLR-agonists could adjuvant the inactivated PRRSV.

Materials and Methods: Pigs (n=6) were administered with six DMN patches with iPRRSV in combination with different TLR-agonists and placebo patch. At 6 hours and 24 hours after vaccination the skin was graded from 0-3 and punch biopsies were taken for histologic evaluation (graded 0-3) at 24 hours.

Results: At 6 hours after vaccination, one TLR agonist displayed a grade 2 to 3 reaction in all pigs, which was visible with the same intensity at 24 hours. The other two TLR-agonists induced only a mild and occasionally moderate skin reaction (grade 0 to 2 ) comparable to the control patch. The histologic grading of the 24 hour skin biopsies was consistent with the macroscopic findings.

Conclusions: The three TLR-agonists induced a different response in the skin. Further investigation is ongoing (IHC for evaluation of immune cells) to elucidate how this skin reaction impacts on vaccine efficacy or whether it induces local side effect.


Poster 63: VISUALIZATION AND QUANTIFICATION OF HEPATOCELLULAR NECROSIS USING CRESYL VIOLET STAIN

M. Henrich* and K. Köhler*

* Institute for Veterinary Pathology Justus-Liebig University Giessen, Giessen, Germany

Introduction: In some cases, assessment of the presence and extent of hepatocellular necrosis can be difficult in hematoxylin and eosin stained slides. One hallmark of cell injury is the failure of membranes to keep up the osmotic homeostasis with subsequent influx of water. This results, in ribosome detachment and dissociation. Hepatocytes are highly productive cells and therefore contain large numbers of ribosomes. As cresyl violet stains RNA in intact ribosomes, a dissociation of these structures results in gradual loss of staining intensity. Therefore, cresyl violet staining of hepatocytes can help to identify and quantify hepatocellular damage.

Materials and Methods: Liver samples of different animals with suspected hepatocellular necrosis were stained with cresyl violet. Staining intensity was assessed and hepatocellular damage was quantified in relation to intact cells.

Results: Damaged hepatocytes showed a visible decrease in staining intensity of cresyl violet. The extent and distribution of damaged cells could be easily assessed in cresyl violet stained liver sections.

Conclusions: Cresyl violet is a helpful staining in cases in which hepatocellular damage is not easily identified in hematoxylin and eosin stained slides. However, differentiation of postmortem degeneration from damage in vivo is not possible with this staining method.


Poster 64: RESTORATION OF INTERNALIN B INCREASES THE VIRULENCE OF THE EPIDEMIC F2365 LISTERIA MONOCYTOGENES STRAIN

I.M. Rodríguez-Gómez*, J.J. Quereda, P. Titos-Jiménez*, L. Carrasco*, M.A. Nahori,  J. Meza-Torres, P. Cossart, J. Pizarro-Cerdáand J. Gómez-Laguna*

*I.M. Rodríguez-Gómez, P. Titos-Jiménez, L. Carrasco and J. Gómez-Laguna, University of Córdoba, Spain and J.J. Quereda, M.A. Nahori,  J. Meza-Torres, P. Cossart and J. Pizarro-Cerdá, Institute Pasteur, France

Introduction: Mediating host cell specific internalization, Internalin B (InlB) is one of the virulence factors of Listeria monocytogenes. The strain F2365 naturally lacks InlB avoiding the internalization of L. monocytogenes through this route and reducing, therefore, the degree of cell invasion. The aim of this study was to characterize the degree of tissue damage in the spleen and liver of mice infected with either the F2365 strain or a mutant strain whose mutation has been repaired and is able to produce the so-called InlB protein (F2365InlB+).

Materials and Methods: To carry out the study sixteen 8-week-old female BALB/c mice were injected intravenously with 104 CFU of the above indicated strains. Mice were euthanized at 48 and 72 hours after infection (hpi). Spleen and liver were collected from each animal to perform the histopathological analysis.

Results: Histologically, the main lesion observed in both organs was necrosis accompanied by inflammatory infiltrate mainly composed of degenerated neutrophils as well as macrophages and lymphocytes in a lesser extent. These necrotic foci were found in the spleen and liver of infected animals regardless of the bacterial strain used. However, the number of necrotic foci was higher in those animals infected with the strain F2365InlB+. Moreover, when compared between organs, spleens were much more affected than livers. The number of megakaryocytes was substantially reduced in those mice infected with the strain F2365InlB+ at 72hpi.

Conclusions: In conclusion, our data suggest a successful rescue of InlB in F2365InlB+ infected animals, which increased the virulence of this bacterial strain.


Poster 65: EVALUATION OF ANTIGENIC PRESERVATION IN HISTOLOGIC SLIDES STORED AT DIFFERENT TEMPERATURES

V. Nascimento*, A.T. Silva‡, F. Seixas†, M.L. Pinto†, A. Gama†, L. Lourenço†, A.F. Vieira*and M.A. Pires†

*Escola Superior de Saúde, Politécnico do Porto, Escola Superior de Saúde, Instituto Politécnico de Bragança, Laboratory of Histology and Anatomical Pathology (LHAP), Animal and Veterinary Research Centre (CECAV), 5000-801 Vila Real, Portugal.

Introduction: Immunohistochemistry (IHC) technique is currently applied in paraffin embedded tissues to identify antigens in multiple lesions, neoplasias or in normal tissues. However, there are several factors that affect the antigen detection, together with the loss or decrease of antigenicity over time, especially in histologic slides stored for long periods of time before the IHC technique is performed. The aim of this work was to evaluate the loss of antigenicity in histologic slides maintained in the refrigerator or freezer over different periods of time, to verify the storage method that allows more effective antigenicity preservation.

Material and methods: Five cases of canine uterus and ovaries (at different phases of the oestric cycle) and one case of canine normal thymus, preserved in the refrigerator (between 4 to 6ºC) and in the freezer (temperatures between -18 to -20ºC) for different storage periods (from one week to one year) were studied using indirect immunohistochemistry. Three Antibodies (vimentin, desmin and broad-spectrum keratin) were performed to ascertain the extension and intensity of immunorreactivity.

Results: After comparison of the immunolabelling, there were no significant changes between the cases preserved in the freezer and those stored in the refrigerator, when the time period was less than one week. However, for slides stored for longer periods, there was a better antigen preservation in the freeze slides.

Conclusions: Preserving antigenicity is a major factor in obtaining reliable results; storing the slides previously cut in the cold is a strategy that should be improved for this purpose.


Poster 66: HEPATIC HISTOPATHOLOGICAL LESIONS IN ACUTE CONTROLED HAEMORRHAGE FOLLOWED BY VOLUME REPLACEMENT WITH A CRYSTALLOID OR COLLOID SOLUTION

Vala H1,2, Pina R1, Cruz R1, Venancio C2, Esteves F1; Silva A3; Mesquita J1; Ortiz AL4 and Ferreira D5

1CI DETS, ESAV, IPV. Portugal, 2CITAB, UTAD. Portugal, 3REQUIMTE, Faculty of Pharmacy, University of Porto, Portugal, 4Department of Veterinary Medicine, University of Cambridge, 5ICAAM, UE

Introduction: The aim of this work was to evaluate the effect of different intravenous solutions used for volume replacement following acute controlled haemorrhage, one crystalloid (RL) and one colloid (HES130/0.4) in the integrity of the hepatic tissue in a pig animal model.

Material and Methods: Hepatic samples were collected from animals submitted to passive arterial blood bleeding and reperfusion with a crystalloid (RL) (G1) and with a synthetic colloid (HES 130/0.4) (G2). Samples were also collected from animals that were not subjected to acute bleeding nor volume replacement (G3, control group). All procedures were carried out under personal and project licenses approved by the Ethical Committee of the national regulatory office. Samples were collected and fixed in 10% neutral-buffered formalin, for a maximum of 24hours, embedded in paraffin wax and 3µm sections were stained for routine histopathology with haematoxylin and eosin.

Results: The histopathological assessment revealed no statistically significant differences between the three groups. However, some lesions were more often expressed in some groups. More severe hepatocellular hydropic degeneration and hepatocellular steatosis was seen in G1, which is the only group in which haemorrhage was observed and within which oedema was not present. Hyperaemia was only observed in G2 and G3. Necrosis was not present in any of the groups.

Conclusion: Hepatic histopathological lesions following controlled bleeding and intravenous volume replacement with RL or HES130/0.4 were subtle. Our results suggest that HES130/04 may be associated with better hepatic perfusion when used for intravenous volume replacement when compared to RL.


Poster 67: OPINIONS OF VETERINARY EXPERTS ON ANIMALS ISSUED IN 1995-2015

I. Babińska*, J. Szarek*, D. Kusiak*, A. Lis*, A. Łyko*, M. Szweda*, M. Maciejewska*, K. Popławski* and M. Z. Felsmann

*Department of Pathophysiology, Forensic Veterinary Medicine and Administration, University of Warmia and Mazury in Olsztyn, Poland, and Institute of Veterinary Medicine, University of Life Sciences, Poznań, Poland

Introduction: In its complexity and diversity, animal breeding and rearing gives rise to numerous conflicting situations which can generate economic losses. The objective of the paper is to analyse expert opinions and to determine the most common reasons for appointing veterinarians as an expert.

Materials and Methods: This study was carried out on 172 expert opinions issued by our department between 1995-2015. The material was divided into two groups: the years 1995-2005 (73 opinions) and 2006-2015 (99 opinions).

Results and Conclusions: It was found that among 172 opinions 70 referred to dogs, 25 were on cattle, 18 to horses, 9 to cats and 8 to pigs. The remaining 42 opinions were related to different animal species (e.g. wild boars, eagles, rabbits, chinchillas, roe deer, cormorants, turtles and foxes). Criminal judicial bodies were the most common ordering party, followed by civil judicial bodies, natural persons, public administration authorities and insurance companies. The most common reason for appointing an expert was cruelty against animals (46.3%) and a necropsy was carried out in 37.2% cases. In case of companion animals the most common causes of deaths were gunshot wounds (18.0%), poisonings (12.0%) and drowning/suffocation (7.0%). Opinions on livestock concerned assessment of prophylaxis and treatment choices, evaluation of husbandry conditions and animal welfare. The cases of wild-living animals mainly referred to poaching, illegal ownership of hunting trophies or identification of carcasses or meat from game animals. About 6.0% of all opinions encompassed varied aspects, e.g. animal origin products and compensation for traffic accidents involving animals.


Poster 68: TRENDS IN EUROPEAN VETERINARY PATHOLOGY IN THE LAST YEARS IN THE LIGHT OF THE CONGRESSES OF THE EUROPEAN SOCIETY OF VETERINARY PATHOLOGY AND EUROPEAN COLLEGE OF VETERINARY PATHOLOGISTS

J. Szarek§, A. Dzikowski§, I. Babińska§, M. Z. Felsmann, K. Popławski§, D. Gulda, K. Wąsowicz§ and B. Szynaka*

  • Department of Pathophysiology, Forensic Veterinary Medicine and Administration, University of Warmia and Mazury in Olsztyn, Poland, and Institute of Veterinary, University of Life Sciences, Poznań, Poland, and Department of Sheep, Goat and Fur Bearing Animal Breeding, University of Science and Technology, Bydgoszcz, Poland, and *Department of Medical Pathomorphology, Medical University of Bialystok, Poland

Introduction: The annual congresses of the European Society of Veterinary Pathology (ESVP) and European College of Veterinary Pathologists (ECVP) are a supranational platform for contacts and knowledge, information and skill sharing between scientists from all over Europe and beyond. The objective of this paper is to depict the current research directions in veterinary pathology in Europe.

Materials and Methods: The analysis was carried out based on the abstracts and agendas of the 7 annual ESVP and ECVP congresses organized in 2010 – 2016. In total, 1,444 presentations were evaluated - 41 plenary lectures, 319 short oral presentations and 1,081 posters, and in 2016 also 3 science slams. The calculations were generated with the Statistica 9 pl StatSoft package.

Results: It was found that infectious and parasitic diseases (467 presentations, 32.34%) and oncology (450 presentations, 31.16%) were the most commonly discussed topics. Organ pathology was also frequently addressed (327 presentations, 22.65%), with the subsequent places taken by research on different topics (140 presentations, 9.70%) and toxicopathology (67 presentations, 4.64%). Among the most commonly presented issues, there were a substantial number of presentations on neurology (129 - 8.93%) and mammary gland diseases (101 - 6.99%).

Conclusions: The analysis showed that infectious and parasitic disease topics will be referred to less frequently in upcoming years, representing a downward tendency. A statistically significant positive trend has been stated for organ pathology topics to predict that this subject matter will be addressed more often at the ESVP and the ECVP congresses in the forthcoming years.


Poster 69: EQUINE EOSINOPHILIC GRANULOMA VS. CUTANEOUS MAST CELL TUMOUR: ONE, TWO OR MANY ENTITIES?

A.M. Elbahi*,, A. Kipar†, and L. Ressel*,

*Department of Pathology, Infection and Public Health, Institute of Veterinary Science, Institute of Global Health, Faculty of Health and Life Sciences, University of Liverpool, Liverpool, United Kingdom, and Institute of Veterinary Pathology, Vetsuisse Faculty, University of Zurich, Zurich, Switzerland

Introduction: Equine cutaneous mast cell tumours (MCT) and eosinophilic granuloma (EG) contain areas morphologically consistent with eosinophilic granuloma (EG) and mast cell aggregates (MCA) in different proportions. It is therefore debated whether these are different entities. The aim of the study was to thoroughly investigate MCT and EG to unravel similarities and differences and collect data that can explain their pathogenesis.

Materials and Methods: A total of 95 cutaneous lesions (EG, MCT) were re-examined to assess morphological criteria, including lesion size and tissue location, hair follicle association, proportion and distribution of infiltrating eosinophils, lymphocytes (T/B cells) and macrophages, and cell proliferation.  

Results: Based on the presence and proportion of EG and MCT components, lesions fell into five categories: C-I (EG+ MCA-); C-II (EG>MCA); C-III (EG=MCA); C-IV (EG<MCA); C-V (EG- MCA+). C-I lesions were more superficially located than the others, and were the only lesions associated with hair follicles. T and, to a much lesser extent, B lymphocytes were always present, but decreased from C-I to C-V, while macrophages were generally found in similar proportions. Viable, MCA-infiltrating eosinophils were most abundant in C-V.

Conclusions: Our results confirm EG as a chronic inflammatory process based on the interaction between eosinophils, macrophages and T cells, they may involve hair follicles. Eosinophils and macrophages seem to be equally relevant in MCTs, though whether they then have a similar functional state remains to be examined. Similarly, further studies are needed to elucidate whether both cell types are responsible for the possible EG/MCT "transition stages".


Poster 70: ISOLATION AND DETERMINATION OF EXOSOMES INFECTIVITY IN CATTLE INFECTED WITH BOVINE LEUKEMIA VIRUS (BLV)

M. Szczotka*, E. Iwan* and A. Szczotka-Bochniarz**

Department of Biochemistry* and Department of  Swine Diseases**, National Veterinary Research Institute, Pulawy, Poland

Introduction: The aim of the study was to isolate and determine infectivity of exosomes in samples from BLV infected cattle.

Materials and Methods: Exosomes were isolated from the blood sera and supernatant of dendritic cells (DCs) from BLV infected cell cultures and control cows. Immunological status of animals was determined by ELISA and qPCR. Exosomes were isolated by differential centrifugation. Filtered supernatant was ultracentrifuged at 120,000 x g for 3 h, at 4 0C. The pellet of EVs was resuspended in PBS and stored at -80°C for further analysis. Purified protein from each sample was used for Western Blot. Proteins separated on SDS-PAGE were transferred to an Immobilon-P PVDF membrane. BLV proteins were also detected using monoclonal antibodies specific to a D-D9 epitope on BLV gp51 and a BLV p24. Lisosomal markers: CD63, CD9 and flotillin-1 were determined. The visualization was performed with chemiluminescence method.  

Results: The presence of viral proteins gp51 and protein p24 was detected in exosomes isolated from the sera and supernatants of DCs culture infected with BLV, while these proteins were absent in exosomes isolated from healthy cattle samples. Cellular markers: CD63, CD9 and flotillin-1 were present in both BLV infected and negative samples.

Conclusions: BLV markers: gp51 and p24 were present in exosomes isolated from the sera and supernatants of the BLV infected dendritic cell culture. These findings suggest that BLV proteins are released into exosomes in sera and supernatant, and could be transferred into recipient cells as an alternative route not requiring virus infection.


Poster 71: ACUTE AND SUBACUTE NATURAL AFLATOXICOSIS IN NEONATAL CALVES IN A DAIRY HERD

J. Ashrafihelan*, H. Hamaliand  M. Khordadmehr*

*Departments of Pathobiology and Clinical Sciences, Faculty of Veterinary Medicine, University of Tabriz, Tabriz, Iran

Introduction: Aflatoxins have been an important cause of animal disease for 30 years. The toxins occur naturally in animal feeds either in the field or during storage when moisture content and temperatures are sufficiently high for mold growth.

Materials and Methods: The clinical signs and morphopathological features of aflatoxicosis in 2 to 3-day-old dairy calves which were exposed naturally in February 2017 are described. According to the history and clinical examinations, clinical signs included anorexia, ataxia, reluctance to move, convulsion and neonatal mortality in 2 to 3-day-old calves in this herd. Also, black mold as Aspergillus niger was observed in storage animal feeds especially in roughage and silage.

Results: At necropsy, there was sanguinous ascites and multifocal hemorrhages under serosal and mucosal surfaces of gastrointestinal tract, mesenteric lymph nodes, kidneys, and meninges. Intramural edema and hemorrhage were seen in the wall of the gall bladder. Histopathological examinations revealed hemorrhagic enteritis, abomasitis and nephritis. There were multifocal large extramedullary haematopoiesis sites especially with accumulation of megakaryocytes in the liver and spleen. Moreover, the liver showed mild fatty changes in hepatocytes, centriacinar fibrosis with bile duct proliferation. In the brain, there were regions of perivascular and perineuronal edema, and perivascular hemorrhages.

Conclusions: According to these results, this herd suffered from hepatoencephalopathy due to simultaneous acute and subacute aflatoxicosis in which aflatoxins orginally produced in animal feeds and transferred during pregnancy and also postnatally via milk, and it was the cause of neonatal mortality in the herd.


Poster 72: ALTERED EXPRESSION OF FOUR PROTEINS REVEALED BY COMPARATIVE ANALYSIS OF PROTEIN PROFILES OF OVINE PULMONARY ADENOCARCINOMA AND NON-NEOPLASTIC OVINE LUNGS

A. Kycko and M. Reichert

*Department of Pathology, National Veterinary Research Institute in Puławy, Poland

Introduction:  Ovine pulmonary adenocarcinoma (OPA) is a neoplastic disease caused by jaagsiekte sheep retrovirus. The aim of the present experiment was an identification of proteins associated with OPA development by comparison of  protein profiles of OPA and non-neoplastic lung tissues of sheep using two dimensional electrophoresis (2DE) utilising gels with pH gradient 5-8.

Materials and Methods: Protein lysates obtained from frozen lung tissues of OPA-affected sheep (n=5) and non-neoplastic lungs (n=4) were submitted for analysis with two-dimensional electrophoresis (2DE) using pH range 5-8 in first dimension of separation. After comparative analysis of 2DE gels, selected gel spots presenting over-3-fold overexpression in OPA  were submitted for protein identification using liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Results: As a result of 2DE gels analysis, 4 spots showed at least 3-fold higher expression in each OPA sample compared to non-neoplastic ones. The proteins identified in these spots with LC-MS/MS were pyruvate kinase 2, valosin containing protein (VCP), enolase 1 and cathepsin H.    

Conclusions: The proteomic methods allowed for identification of four proteins, overexpression of which has been previously reported mainly in human cancers which indicates that these proteins might be associated with OPA development. Previously reported proteomic study of OPA revealed. that using pH gradient 3-10 in 2DE,  insufficient separation of  some proteins in the gels. Application of gels with pH 5-8 in 2DE in the present study allowed for more detailed analysis of the protein profiles.


Poster 73: AN IN SITU APPROACH TO UNRAVEL THE PATHOGENESIS OF CUTANEOUS MAST CELL TUMOUR AND EOSINOPHILIC GRANULOMA IN HORSES

F. Romero-Palomo, A.M. Elbahi, K. Köhler#, L. Ressel and A. Kipar†,§

Institute of Veterinary Pathology, Vetsuisse Faculty, University of Zurich, Zurich, Switzerland, Department of Pathology, Infection and Public Health, Institute of Veterinary Science, and  §Institute of Global Health, University of Liverpool, United Kingdom, and #Institute of Veterinary Pathology, Faculty of Veterinary Medicine, University of Giessen, Germany

Introduction: Cutaneous mast cell tumors (MCT) in horses are consistently infiltrated by abundant eosinophils, often in conjunction with other leukocyte populations. This can result in the presence of eosinophilic granuloma (EG)-like lesions within MCTs, suggesting a related pathogenesis. The aim of this study was to characterize the differential expression of selected cytokines and chemokines as potential mediators in the pathogenesis of cutaneous MCT and EG.

Materials and Methods: We assessed a population of 15 equine skin lesions, 8 MCTs and 7 pure EGs, by RNA-ISH (RNAscope® technology) and immunohistology for the expression of cytokines and chemokines with roles in MC, eosinophil, macrophage and lymphocyte cross talk, i.e. RANTES/CCL5, eotaxin/CCL11, Mip1α /CCL3, IL-4, IL-5, IL-13, SCF/CKIT-L and TGF-β.

Results: Both types of lesions exhibited similar general expression patterns. While TGF-β was expressed across most cell types, RANTES, eotaxin, Mip1α, IL-4, IL-5, and IL-13 expression was seen in lymphocytes, and, selectively, in other cells: MCs (IL-5), palisading histiocytes (RANTES, TGF-β), macrophages (eotaxin, Mip1α), and fibroblastoid cells (eotaxin). The latter was only present in association with infiltrating neutrophils in ulcerated MCTs. SCF was only detected in spindle cells of vessel walls. Eosinophils did not express any of the markers. Overall, RANTES and eotaxin signaling was more intense in EG.

Conclusions: The results suggest that neither condition has a specific cytokine/chemokine expression pattern. Instead, MCT growth and/or recruitment of MC and eosinophils in MCT and EG are likely the result of a cellular cross-talk involving all relevant mediators, individually orchestrated in each specific case.


Poster 74: CORRELATION BETWEEN PREDISPOSING FACTORS AND LUNG INFLAMMATORY LESIONS IN PIGS NATURALLY INFECTED WITH MYCOPLASMA HYOPNEUMONIAE

F. Parisi, L. Giuliotti, M.N. Benvenuti, G. Rocchigiani, C. Salvadori and A. Poli

Dipartimento di Scienze Veterinarie, Università di Pisa, Italy

Introduction: Mycoplasma hyopneumoniae and several predisposing factors are involved in the pathogenesis of enzootic pneumonia. The aim of this study was to investigate the correlation between farm management factors and pulmonary alterations in slaughtered swines.

Materials and Methods: One thousand one hundred and three lungs of fattened pigs from 18 different vaccinated and unvaccinated batches from Italian herds were macroscopically examined. For each slaughtered batch, data related to production system, management practices and vaccination protocols were collected. One hundred lung tissue samples, were fixed in formalin and zinc salts for histopathological and immunohistochemistry studies (using antibodies against CD3, CD79 and Iba1).

Results: Our study revealed that open cycle, growing-finishing weight lower than 30 Kg, presence of slatted systems and mixed natural and forced ventilation systems are predisposing factors. The histopathological investigation revealed that vaccinated subjects have less severe lung lesions and more evident BALT hyperplasia. In vaccinated animals not affected by pneumonia, a higher count of B- and T-lymphocytes and a lower number of macrophages were observed in the BALT compared to unvaccinated animals. In vaccinated animals with pneumonia, a higher number of T-lymphocytes and macrophages and a lower of B-lymphocytes were evident in BALT and in inter-alveolar septa than in unvaccinated ones.

Conclusions: Our study underlined the main risk factors involved in the pathogenesis of enzootic pneumonia. A good tissue immune response was evident in vaccinated pigs without lung lesions but a lower Th2 response and a higher non-protective cell-mediated response was observed in vaccinated animals with pneumonia.


Poster 75: SALIVAPRINT: SHEEP SALIVA ELECTROPHORETIC PROTEIN PROFILE IN A BIOINFORMATICS APPROACH

E. Esteves1, M. Fernandes1, I. Cruz1, F. Esteves2,3, N. Rosa1, MJ. Correia1, H. Vala2,3 and M. Barros1

1 Institute of Health Sciences, Center for Interdisciplinary Research in Health, Universidade Católica Portuguesa, Viseu, Portugal, 2 CI DETS, ESAV, IPV. Portugal, 3 CITAB, UTAD. Portugal

Introduction: Sheep saliva can be used for individual monitoring and decision aiding in therapeutic intervention. The total protein profile of each subject (SalivaPrint) can be integrated with clinical and environmental data to stratify individuals and analyze their health status. This study applies a bioinformatics strategy in order to identify standard protein profiles in healthy sheep. Standard total protein profiles of sheep are compared to human profiles.

Materials and Methods: Saliva samples were collected. Total protein profile of sheep saliva samples was performed using the Experion™ technology. An algorithm which is able to identify common molecular weights ranges in a group of electrophoretic profiles was used. Common molecular weights present in sheep SalivaPrint were then used with OvisOme database to identify proteins. Using AgBase GORetriever tool it was possible to analyze and catalogue their biological processes.

Results: Sheep and Human SalivaPrints are different. The SalivaPrints of individuals from the same flock appear to have a higher degree of similarity than individuals of different flocks. Fourteen proteins were identified using OvisOme database. The most representative biological process found was the response to stress with the proteins: Cathelicidin-1, Beta 2 microglobulin, Cathelin, Kallirein and Chloride intracellular channel protein.

Conclusion: Different species have different salivary protein profiles. The total protein profile seems to be highly conserved between flocks. By using SalivaPrint profiles with bioinformatic tools like OvisOme it was possible to find proteins which are common to healthy sheep populations. Furthermore, it’s possible to infer novel biomarkers and indicators of animal health.


Poster 76: CEMENTOMA CAUSING AN ORONASAL FISTULA IN A HORSE: A RARITY

J. Molín*, J. Asín*, R. de Miguel*, M. Solana*, F. Vázquez*, P. Zalaya*, H. E. Jensen and L. Luján*

*Department of Animal Pathology, University of Zaragoza, Spain; and Section of Pathology, University of Copenhagen, Denmark

Introduction: Cementomas are very rare, benign, slow-growing, expansive, mesenchymal odontogenic tumors arising from proliferation of cementoblasts and characterized by extensive deposition of cemental matrix associated to the tooth root, with occasional destruction of alveolar cortical bone. In humans, they are classified in three categories: periapical cemental dysplasia, benign cementoblastoma (true cementoma), and gigantiform cementoma. Cementomas in domestic animals are infrequent, with just six reports in horses and single cases in feline, bovine, monogastric herbivores and rodents.

Materials and Methods: An eight-years-old, male, Spanish purebred horse presented with a history of chronic unilateral rhinitis refractory to therapy for 3 weeks. Radiologically, a radiopaque mass extending from and surrounding the roots of the P2 maxillary premolar was demonstrated. Excised premolar and mass were routinely processed, decalcified and stained with hematoxylin-eosin, Grocott, and immunostained with polyclonal antibodies against Aspergillus fumigatus.

Results: After excision, a severe oronasal fistula was observed. Grossly the mass showed hard consistence, greyish-whitish color and irregular surface that, on the ventral aspects, fitted with the apical surface of the involved premolar. Histologically the mass consisted of an abundant deposit of eosinophilic irregularly mineralized cementum-like substance with frequent basophilic reversal lines surrounding empty lacunae and intermixed with fibrovascular tissue. Surround this matrix there were numerous fugal hyphae positive for Aspergillus fumigatus immunostaining.

Conclusions: Cementomas should be considered as a differential diagnosis for masses in the oral cavity of the horse. Although benign, they can be associated with sequelae  such as  fistula, as was observed in this case.


Poster 77: CHARACTERISATION OF MACROPHAGES IN EQUINE MULTINODULAR PULMONARY FIBROSIS

E. Holland*, A. Kipar†,‡ and G. H. Leeming *,

*Department of Veterinary Pathology and Public Health, Institute of Veterinary Science and Department of Infectious Biology, Institute of Infection and Global Health, University of Liverpool, UK and Institute of Veterinary Pathology, Vetsuisse Faculty, University of Zurich, Switzerland

Introduction: Equine multinodular pulmonary fibrosis (EMPF) is an emerging disease in the USA and Europe, characterised mainly by a loss of functional pulmonary parenchyma due to extensive interstitial fibrosis and is strongly associated with equine herpesvirus 5 (EHV-5), a gammaherpesvirus (γ-HV).

Materials and Methods: Using archived formalin-fixed paraffin embedded tissue from EHV-5 positive cases of EMPF and control lung tissue from unaffected horses, we examined the macrophage population, using immunohistology to detect Myeloid/histiocyte antigen (clone MAC387), lysozyme and Major histocompatibility complex class II (MHCII). The number of macrophages per high power field was quantified both within nodular lesions and extra-nodular lung in EMPF cases and compared to unaffected controls.

Results: The number of macrophages was significantly higher in the nodular lesions compared to either extra-nodular lung of EMPF cases, or to unaffected control tissue, regardless of the antibody used. Furthermore, the number of MAC387 and MHCII-positive macrophages was significantly higher in the extra-nodular lung of EMPF cases compared to unaffected controls.

Conclusions: EMPF is characterised by a loss of functional pulmonary parenchyma due to extensive multifocal to coalescing interstitial fibrosis; remaining alveoli are lined by cuboidal epithelium which are filled with inflammatory cells. In these horses there are increased numbers of macrophages in both the nodular lesions and the extra-nodular lung, compared to unaffected controls. The use of different antibodies to identify different subsets of macrophages could further our understanding of the pathogenesis of this disease in horses.


Poster 78: COLLABORATIVE PATHOLOGY IN A CASE OF HUMAN POX VIRUS DERMATITIS

E. Karlstam*, M. Tarjan, E. Sundell  and A. Scheltdorf

*Department of Pathology and Wildlife Diseases, National Veterinary Institute, Sweden, Department of Pathology and Cytology, Falu Hospital, Sweden and Department for ENT, Head and Neck Surgery, Falu hospital, Sweden

Introduction: A 50-year old female goat owner presented with an ulcerative, crusting lesion of the skin, non-responsive to antibiotics. Human orf or neoplasia were considered as clinical differentials. Skin biopsies were somewhat inconclusive, and sent for a second opinion to a veterinary diagnostic pathology department.

Materials and Methods: Two formalin-fixed, paraffin- embedded skin biopsies, stained with HE, taken approximately 10 days and 50 days after initial presentation, were examined. A sample for pox virus PCR was sent along with the second biopsy.

Results: The initial biopsy revealed a hyperplastic, necrotizing, intraepidermal vesicular dermatitis with intracytoplasmic, eosinophilic inclusion bodies, typical of pox virus infection, in keratinocytes. In the second biopsy, a hyperplastic, multinodular, deep lymphohistiocytic dermatitis without inclusion bodies was seen. PCR for pox virus was negative.

Conclusions: Collaboration between human and veterinary diagnostic pathologists rendered a histological diagnosis of a pox virus dermatitis, most probable zoonotic orf/contagious ecthyma, in a human patient. PCR for pox virus was probably negative due to late stage sampling, when inclusion bodies were no longer present in the biopsy.


Poster 79: DIAGNOSTIC TEST COMPARISON ON TUBERCULOUS LESIONS FOUND IN SLAUGHTERED CATTLE LYMPH NODES

A. Morey-Matamalas*, E. Vidal, B. Pérez de Val, A. Marco* and J. Martínez*

* Departament de Sanitat i Anatomia Animals, Universitat Autònoma de Barcelona, Spain, and Centre de Recerca en Sanitat Animal (CReSA, IRTA-UAB), Campus de la Universitat Autònoma de Barcelona, Bellaterra, Spain

Introduction:  Bovine tuberculosis (bTB) is a chronic bacterial infectious disease caused by several species of Mycobacterium tuberculosis complex (MTBC). The characteristic tuberculous lesion is the granuloma. For the confirmation of a bTB diagnosis at slaughter, histopathological analysis, PCR and mycobacterial culture are required. The objectives of this retrospective study were to analyze and classify histologically bTB lesions in lymph nodes of 100 animals from slaughterhouse and to correlate the histopathological analysis of these lesions with other diagnostic tests: Ziehl-Neelsen (ZN), PCR and culture.

Materials and Methods: Histopathological analysis consisted of a semi-quantitative assessment of different microscopic characteristics of bTB lesions (neutrophils, lymphocytes, multinucleated giant cells, macrophages, fibrosis, necrosis, hemorrhage and mineral) and classification of granulomas in four developmental stages. PCR and mycobacterial culture were carried out following previously published protocols.

Results: In the histopathological evaluation, most lymph node granulomatous lesions (96 %) were classified as grade III or IV. Regarding the results of the ZN (102 available), PCR (90 available) and culture (92 available); 71, 58 and 72 animals were positive to the tests, respectively. The sensitivity results compared to mycobacterial culture (C) were 78.8 % for the PCR and 74.3 % for the ZN. The Cohen’s kappa index showed minimal to weak agreement between PCR-ZN (32.5 %), C-ZN (23 %) and PCR-C (53.3 %); suggesting the complementarity of these techniques for the bTB diagnosis.

Conclusions: Statistical analysis did not show any significant association between microscopic characteristics of bTB lesions and ZN, PCR and culture


Poster 80: PATHOLOGICAL EVALUATION OF THE EFFECTIVENESS OF EXPERIMENTAL VACCINES AGAINST FASCIOLA HEPATICA IN SHEEP

E. Sierra1, V. Molina-Hernández1, I.L. Pacheco1, R. Zafra2, A. Escamilla1, M.T. Ruiz1, M.J. Bautista1, F.J. Martínez-Moreno2 and J. Pérez1

1Department of Comparative Anatomy and Pathology.2Department of Animal Health (Parasitology). University of Córdoba. Edificio Sanidad animal. Campus Universitario de Rabanales. Carretera Madrid-Córdoba, Km 396. 14071, Córdoba.

Introduction: We present the preliminary results obtained in sheep vaccine trials with a cocktail of recombinant Fasciola hepatica proteins.

Materials and Methods: A total of 46 seven-month-old Merino male sheep’s distributed in 5 groups were used. Groups 1 (n = 10) and 3 (n = 10) were vaccinated with a cocktail of 4 recombinant Fasciola hepatica proteins (CL1, HDM, Prx, LAP) with Montanide and Aluminum as adjuvant, respectively; Group 2 (n = 10) were vaccinated with the proteins (HDM and CL1) in Montanide; Group 4 (n = 10) were used as control of infection without receiving vaccine doses and group 5 (n = 5) were the negative control without vaccination or infection. Groups 1, 2, 3 and 4 were infected orally with 150 metacercariae of F. hepatica and were euthanized at 15 weeks post infection. The adult parasites were counted and a macroscopic and histopathological evaluation of the hepatic lesions were undertaken. 

Results: The most relevant finding was the presence of degenerate parasites within the bile ducts of one of the vaccinated groups. This finding was exclusively observed in group 1 vaccinated with the cocktail of the 4 recombinant proteins and Montanide as adjuvant, and was coincident with a reduction in parasite load of 37% compared to infected control group 4. This group also showed more discrete macroscopic hepatic lesions compared to group 4.

Conclusions: These results suggest that the protective response to the vaccine could be developed, at least in part, when the parasites reached the biliary system.


Poster 81: A CASE OF STREPTOCOCCUS EQUI SUBSPECIES ZOOEPIDEMICUS INFECTION IN A THOROUGHBRED HORSE

JW. Kim, JY. Jung, K.Lee, H. Lee, HY. Kim, SS. Yoon, BJ. So and E. Choi

Animal Disease Diagnostic Research Division, Animal and Plant Quarantine Agency, Gimcheon 39660, Republic of Korea

Introduction: Streptococcus(S) equi subsp. zooepidemicus belongs to the β-haemolytic group C streptococci, which cause systemic disease in humans and animals. Meningitis and septicemia have been rarely reported in horses. This report describes a case of S. equi subsp. zooepidemicus infection in a 6-year-old Thoroughbred mare.

Materials and Methods: A 6-year-old Thoroughbred mare suddenly showed hind limb paresis. Despite medical treatment, the condition became more severe in a very short time. Finally, the animal died, and was submitted for postmortem examination. Brain, spinal cord, hip joint swabs and visceral organs were collected for bacterial culture. To detect the viral genome (Japanese encephalitis, influenza, herpesvirus -1, 4, West Nile) polymerase chain reaction was performed.

Results: Grossly, there were yellowish turbid materials in the left hip joint and extensive muscular hemorrhage and edema of the hind limb. Histopathologically, the brain revealed moderate fibrinosuppurative inflammation of the meninges. Thrombosis in various organs and bacterial colonies in subarachnoidal blood vessels of the spinal cord were observed. The lymph node showed severe purulent, hemorrhagic and necrotizing lymphadenitis with intralesional gram-positive cocci. S. equi subsp. zooepidemicus was isolated from the brain, spinal cord, and hip joint. Viral genome for Japanese encephalitis, influenza, herpesvirus -1, 4, West Nile was not detected by PCR.

Conclusions: Based on the histopathological feature and bacterial culture, this case was diagnosed as S. equi subsp. zooepidemicus infection of horse showing meningitis, arthritis and septicemia. Meningitis and septicemia caused by this organism can occur in more severe cases, but are rare in horse and human beings.


Poster 82: HISTOLOGICAL PATTERNS RELATED TO MYCOPLASMA AGALACTIAE MASTITIS.

R. Puleio, A. Tamburello, F. Messina, A. Parco, S. Tumino, G. Macaluso and G.R. Loria

Istituto Zooprofilattico Sperimentale della Sicilia, Italy

Introduction: Contagious agalactia can be caused by different mycoplasmas, which share mammary, articular or ocular tropism. This study describes histological and immunohistochemical findings in mastitis caused by Mycoplasma agalactiae in sheep with confirmed diagnosis.

Materials and Methods: Laboratory investigations on milk were used to confirm the disease. Udders and supra-mammary lymph nodes were collected for histological and immunohistochemical examination. The expression of M.agalactiae antigen, MHC-II, CD3 and CD79 lymphocytes was investigated by immunohistochemistry.

Results: Histological findings showed an interstitial monocytic infiltrate in the acute phase, while in advanced stages, we found a persistence of monocytic infiltrations with a slight reduction of secretory tissue; in severe cases, a marked atrophy of secretory tissue associated with monocyte infiltrations and interstitial fibrosis was observed. Interestingly, we detected, in spite of negative culture, rare focal monocytic infiltrations within tissues with recovered secretory activity in the udder of some convalescent sheep. Immunohistochemistry for M.agalactiae was strongly positive in the lumen of the acini and ducts, while interstitial monocytic cells were positive for MHC-II and CD3 antigen. CD3 positive cells, in particular, were located in the interstitial tissue around alveoli and ducts, while MHC-II was more reactive in the central areas of interstitial infiltrates.

Conclusions: These findings describe different histopathological patterns; particularly in subclinical mastitis, with a recovery of the daily amount of milk, histology showed rare lymphocytic infiltrates in udder, establishing a chronic, persistent infection. In this case, some immunodeficiency conditions (e.g. internal parasites) might induce a new virulence of pathogen.


Poster 83: EPIDIDYMITIS BY BRUCELLA OVIS IN SMALL RUMINANTS: HISTOPATHOLOGY AND IMMUNOHISTOCHEMISTRY

R. Puleio, G. Chiarenza, D. Vicari, V. Currò, C. Piraino, F. Sciurba, A. Tamburello, F. Messina and G.R. Loria

Istituto Zooprofilattico Sperimentale della Sicilia, Italy

Introduction: Brucella ovis, a gram-negative coccobacillus infecting domestic sheep produces male reproductive tract lesions including epididymitis, testicular atrophy, adhesions of the tunica vaginalis and decrease in semen quality.

Materials and Methods: Diagnosis was based on serology, culture tests, histopathology and immunohistochemistry. Lesions were submitted in 10 % neutral buffered formalin and stained with hematoxylin and eosin (HE). Immunohistochemistry for Brucella ovis, CD3, MHC-II was performed.

Results: Out of a total of 61 Brucella strains, 5 samples were identified according to OIE procedures, as B. ovis strains. Strains were isolated from two different Sicilian districts: Palermo and Trapani. Epididymal lesions, including swelling and abscesses, were grossly observed. Microscopic examination of reproductive tract tissues revealed epididymitis, ampullitis and seminal vesiculitis. Epididymitis was focal and ranged from mild to severe. Lesions were characterized by mild to moderate, focal accumulations of lymphocytes and plasma cells scattered or perivascularly located in interstitial connective tissue. Focal accumulations of lymphocytes, plasma cells, and neutrophils with necrotic debris were also located in ductal lumina.

Conclusions:  B. ovis is occasionally reported in Mediterranean countries, while B. melitensis biovar 3 is the main Brucella species to be isolated from sheep and goats. These findings suggest the need for further investigation also for its implication in hypo fertility of Sicilian flocks, when antibody is detected.


Poster 84: IMMUNOHISTOCHEMICAL EVALUATION OF TLR-4 RESPONSE IN THE MEDIASTINAL ADIPOSE TISSUE OF PIGS NATURALLY INFECTED WITH MYCOPLASMA HYOPNEUMONIAE

B. Passeri, N. Martinelli, L.G. Alborali, A.M. Cantoni, R. Di Lecce, V. Bertani and A. Corradi

Department of Veterinary Science, University of Parma, Parma- Italy

Introduction: Mycoplasma hyopneumoniae (MH) is the agent of enzootic pneumonia of pigs. Toll-like receptors 4 (TLR4) binds lipopolysaccharides (LPS) of gram- bacteria. In the adipose tissue TLR4s seem to be involved in activation and support of inflammation.

Materials and Methods:  Mediastinal adipose tissue and lungs were sampled from 49 pigs at slaughter. Each lung lesion was MH RT-PCR evaluated. Frozen 5 µm sections of lungs were histologically evaluated with scores ranging from 0 to 4, for BALT, bronchi lumen, bronchiolar disepithelization and subacute inflammation. Frozen 5 µm sections of corresponding mediastinal adipose tissues were ABC immunohistochemically stained using commercial anti-TLR4 antibodies. The average histological lung score ranged from 0.14 to 1.98. 32 samples were RT-PCR positive and 17 negative.10 showed subacute inflammation and 22 showed MH lesions. 20 samples, 14 MH positive and 6 MH negative, were evaluated for TLR4s, using a semi-quantitative classification for spread and intensity of staining on four levels: absent, mild, medium, intensive on four, 4 magnifications fields.

Results: 3 samples (2 positive), with severe pulmonary inflammation, showed an intense immunostain for TLR4 in the adipose tissue (3). 6 samples (3 positive), with mild/severe inflammation, showed medium TRL4 stain. 8 adipose tissue samples, with mild alveolar-bronchial inflammation (6 positive), showed TLR4 0 to 1. 3 adipose tissue samples were negative with lung score 1.

Conclusions: Semi-quantitative analysis of the presence of TLR4s in mediastinal adipose tissue, might confirm that a more severe pulmonary inflammation shows a higher concentration of TRL4 than in mild inflammation.


Poster 85: METASTASIZING PERIPHERAL NEUROBLASTOMA IN AN ADULT SAANEN GOAT

J. M. Schoening and H. Jahns

Pathobiology Section, School of Veterinary Medicine, University College Dublin, Dublin, Ireland

Introduction: Peripheral neuroblastomas are embryonal tumours of neuroectodermal origin, that exhibit poor neuronal differentiation. Common primary locations are the adrenal medulla and sympathetic ganglia. Peripheral neuroblastoma is one of the most frequent tumours in children, but has only rarely been reported in domestic animals, mainly in dogs and calves.

Materials and Methods: An adult female Saanen goat presented with a history of coughing and nasal discharge. On clinical examination a large firm swelling in the right parotid region and a markedly enlarged right prescapular lymph node were observed. Caseous lymphadenitis was suspected and the goat was euthanized.

Results: On post mortem examination, a large firm solid mass was present in each of the following locations; nasopharynx, right parotid and prescapular lymph node, and at the thoracic inlet compressing the trachea. Multiple small metastases to the lungs and liver were observed. Histologically, masses consisted of small hyperchromatic polygonal cells arranged in nests and packets separated by prominent dissecting bands of large spindle shaped cells. Mitoses varied between 5-10/10 HPFs. On IHC, >80% of small polygonal cells were diffusely positive for synaptophysin (neuronal progenitor cells). The dissecting spindle cell stroma was positive for S100 (neural crest cells, Schwann cells).

Conclusions: This is the first report of a neuroblastoma in an adult goat and the first documentation of pulmonary metastases of this tumour in the veterinary literature. This highly malignant neoplasm likely arose in sympathetic ganglia of the nasopharynx. A characteristic IHC pattern as reported in the human literature was observed.


Poster 86: PATHOLOGY AND ADVANCED IMAGING, TWO VIEWS OF ONE HEART – CHARACTERISATION OF RARE CONGENITAL CARDIAC DEFECTS IN A PIG

A. Malbon*, G. Pellegrini*, J. Busch, M. Lipiski, M. Sauer, A. Gotschy, M. Fuetterer,                     C. von Deuster, S. Kozerke, CT. Stoeck and N. Cesarovic

*Institute of Veterinary Pathology, Vetsuisse Faculty, University of Zurich and Institute for Biomedical Engineering, University of Zurich and ETH Zurich, and Division of Surgical Research, University Hospital Zurich, Switzerland

Introduction: 4D-Flow MRI is an increasingly important imaging modality in human cardiovascular medicine, however with high risk patients its application is challenging, and correlation with pathological findings is difficult in the majority of cases. We describe the pathological findings and their manifestation in cardiac blood flow pattern in a porcine case of complex congenital cardiac defect.

Materials and Methods:  A female pig (30kg, Edelschwein) underwent functional and 4D-Flow MRI as a preliminary to being part of a study group.  Owing to abnormal findings, the animal was euthanised and the heart submitted to pathology where macroscopical and histological examination was carried out including immunohistology and special staining.

Results: 4D-Flow MRI indicated an active left-to-right atrial-septal shunt as well as mitral and tricuspid valve insufficiency, indicated by regurgitant jets, and disturbed diastolic intra-cardiac blood flow patterns. Post mortem examination of the heart revealed an ostium primum atrial septal defect (ASD), also known as incomplete atrioventricular SD (AVSD) with a tri-leaflet left AV valve, together with a serous valvular cyst partially occluding the defect.

Conclusions:  Pathology examination confirmed the septal defect observed in 4D-Flow MR imaging as well as the suspected AV valve malformation and identified a valvular cyst. The correlation of pathology findings with those of advanced MR imaging modalities brings a greater understanding of the in vivo lesion significance to all parties. AVSDs account for ~3% of human congenital cardiac defects, and are additionally highly associated with both Noonan and Down syndrome, making them of interest from a comparative pathology sense.


Poster 87: ULTRASTRUCTURE OF TRYPANOSOMA EQUIPERDUM ADAPTED TO IN VITRO CULTURE GROWTH

E. M. Liebler-Tenorio, B. Bassarak, I. Moser and C. Menge

Ferderal Research Institute for Animal Health, Friedrich-Loeffler Institut, Germany

Introduction: Trypanosoma (T.) equiperdum, the cause of equine dourine, is a kinetoplastid flagellate of the subgenus Trypanozoon. T. equiperdum antigen is needed for the complement fixation test prescribed by OIE for serological diagnosis of dourine. At the NRL for dourine in Germany, an efficient protocol for growing T. equiperdum in vitro was developed to replace passaging in rats. Ultrastructure of culture-adapted T. equiperdum was investigated to determine a potential influence of this propagation on the phenotype.

Materials and Methods: T. equiperdum strain OVI propagated in liquid culture was adjusted to 108 parasites per ml. Thirty microliters were used for negative contrast preparation to determine size, shape and origin of the flagellum. The remaining microorganisms were fixed in glutaraldehyde, embedded in resin, ultrathin sectioned and contrasted to investigate subcellular structures. Preparations and sections were examined by transmission electron microscopy (TECNAI 12, FEI).

Results: The contrast medium outlined the shape of the slender trypanosomes (29 ± 3 µm long). The flagella originated from the posterior end and wrapped around the cell with a free end at the anterior end. Filopodia were present in a few trypanosomes. A subpellicular array of microtubules enclosed the trypanosomes. Organelles essential for motility and division, e.g. flagellum with axoneme and paraflagellar rod, basal body and kinetoplast, were identified. ER, Golgi and vesicles next to and within the flagellar pocket indicated endo- and exocytosis. Mitochondrium, glycosomes and lysosome represented metabolically active organelles.

Conclusions: T. equiperdum OVI maintains the characteristic trypanozoon morphology when propagated by in vitro culture.


Poster 88: CHANGES IN THE EXPRESSION OF NFKAPPA B AND NRF2 GENES IN THE COLON OF PIGS SUFFERING FROM SWINE DYSENTERIA

K. Wasowicz, P. Podlasz, A. Jakimiuk, M. Chmielewska and J. Szarek

 Department of Pathophysiology, Forensic Veterinary Medicine and Administration, Faculty of Veterinary Medicine, University of Warmia and Mazury in Olsztyn, Poland

Introduction: Oxidative stress may induce inflammatory reaction, but also induces the reaction of antioxidative systems. The induction of inflammation is usually mediated by NFkappaB transcription factor (coded by two genes NFKB1 and NFKB2) and the antioxidant systems by Nrf2 transcription factor (coded by NFE2L2 gene). The study was aimed at finding the changes in the expression of NFKB1, NFKB2 and NFE2L2 genes in pigs suffering from enteritis caused by Brachyspira hyodysenteriae infection.

Materials and Methods: The study was performed on 8 pigs weighting ca. 80, of which 4 were a control group and 4 were experimentally infected with the bacteria suspension delivered via a gastric tube. After the symptoms of the disease were fully developed the animals were sacrificed with a pentobarbital overdose and the samples of ascending colon were dissected out. The mucosa and submucosa were scraped and total RNA was isolated form the scraped mucosa and the muscular membrane. The expression of NFKB1, NFKB2 and NFE2L2 was studied with Real Time PCR.

Results: Expression of NFKB1 changed only slightly both in the mucosa/submucosa and in the muscular membrane, while more significant changes were visible in case of NFKB2 expression which dropped in mucosa/submucosa but increased almost twice in muscular membrane. In case of NFE2L2 gene its expression dropped significantly both in mucosa/submucosa and muscular membrane.

Conclusions: Inflammation associated with enteritis influenced both the expression of transcription factors associated with pro-inflammatory (NFkappaB) and anti-inflammatory (Nrf2) reactions.


Poster 89: IMMUNOHISTOCHEMICAL STUDY OF CYTOKINE PROFILE PRESENT IN LOCAL IMMUNE RESPONSE AGAINST HYDATID CYST.

M. Jiménez*, F. Corrêa*, C. Stoore*, C. Hidalgo*, C. González*and R. Paredes*.

*Laboratorio de Medicina Veterinaria, Facultad de Ecología y Recursos Naturales, Universidad Andres Bello, Santiago, Chile.

Introduction: Cystic Echinococcosis (CE) is a zoonotic infection with high prevalence in part of Eurasia, Africa, Australia, and South America. Chronic infection coexists with detectable humoral and cellular responses against the parasite, but this response regarding the hydatid cyst fertility is scarcely studied. The objective of the present work is to evaluate locally response of lymphocytes T and B with the cytokine profiles Th1 / Th2 present in adventitial layer (AL) of hydatid cysts.

Materials and Methods: Samples from animals with either fertile or infertile hydatid cysts were included in this study, using 5 samples per condition. Histological samples were evaluated by immunohistochemistry with a panel of antibodies for CD3, CD79a, INFγ, TNFα, IL4 and IL10 molecular markers. Digital images were obtained using an Olympus FSX100 Microscope and analyzed with software for morphometric analysis (Image J).       

Results: Immunohistochemical analysis of lymphocyte populations in AL of bovine hydatid cysts showed a predominance of CD3+ T cells compared to CD79+ B cells. With respect to fertility, infertile cysts had a statistical significant increase of CD3+ T cells and CD79+ B cells (p <0.01) in AL. The expression of cytokines showed a statistically significant increase of INFγ and TNFα (p < 0.02) in infertile cysts, unlike the fertile cysts that present an increase of IL4 (p=0.02) and IL10 (p=0.2).

Conclusions: These findings suggest a predominant Th1 polarized local immune response, and a high CD3+ T cell population could contribute to infertility condition of bovine hydatid cysts.


Poster 90: INFLUENCE OF PREVIOUS ORAL SENSITIZATION WITH MYCOBACTERIUM AVIUM SUBSP HOMINISUIS OR CORYNEBACTERIUM PSEUDOTUBERCULOSIS ON THE OUTCOME OF MYCOBACTERIUM AVIUM SUBSP PARATUBERCULOSIS EXPERIMENTAL INFECTION IN A CAPRINE MODEL.

M. Royo*, M. Fernández*, I. A. Sevilla, N. Arteche*, R. Canga*, M. Fuertes*, M. C. Ferreras*, J. Benavides*, N. Elguezabaland V. Pérez*

*Departamento de Sanidad Animal, Instituto de Ganadería de Montaña (CSIC-ULE), Facultad de Veterinaria, Universidad de León, Spain and Departamento de Sanidad Animal, NEIKER-Tecnalia, Derio (Bizkaia), Spain

Introduction: Previous sensitizations with related bacteria has been hypothesized as the explanation for the different outcomes of Mycobacterium avium subsp paratuberculosis infections. In this sense, Corynebacterium pseudotuberculosis (Cptb) and Mycobacterium avium subsp hominisuis (Mah), that share common antigenic properties with Map, are commonly present in ruminant herds.

Materials and Methods: An experimental infection was carried out in 23, 1.5 month-old goat kids that were orally inoculated with a 5x103 cfu of Mah (7 animals) or Cptb (7 kids). Two months after, 5 animals from each treated group, and other 5 not sensitized, were challenged with 1.8x1010 cfu of Map. Peripheral cellular (IFN-γ release assay) and humoral immune responses were analyzed. At 5 months post-infection, pathological examination was carried out. Location and severity of granulomatous lesions were assessed through granuloma quantification in the intestine and related lymph nodes.

Results: No significant differences were found in the IFN-γ or antibody production between the three Map infected groups. Granulomatous microscopic lesions were observed in the three groups; in Map-infected and non-sensitized kids, lesions were seen at more locations, including jejunum with no lymphoid tissue, whereas in animals previously sensitized, either with Mah or Cptb, granulomas were restricted to the lymphoid tissue. However, the highest number of lesions were seen in the Cptb+Map group while the lowest in kids from Mah+Map group.

Conclusions: In the conditions of this study, previous oral sensitization with Mah or Cptb influences the development of lesions at the early stages of Map infection in goats.


Poster 91: PATHOLOGICAL AND ETIOLOGICAL STUDY OF BOVINE RESPIRATORY DISEASE (BRD) IN SLAUGHTERED BEEF CATTLE

M. Fernández*, J. Benavides*, M.C. Ferreras*and V. Pérez*

* Departamento de Sanidad Animal, Instituto de Ganadería de Montaña (CSIC-ULE), Facultad de Veterinaria, Universidad de León, Spain

Introduction: Bovine respiratory disease (BRD) has a significant negative impact in feedlot cattle due to the associated clinical costs, loss of production and high mortality rates. Prevalence varies among countries and diagnostic methods employed. The objective of this study was to evaluate the main types of pneumonia encountered and associated etiologies, in slaughtered beef cattle, together with the impact on the principal productive parameters.

Materials and Methods: 1120 cattle were examined at the slaughterhouse. The presence of gross lesions was recorded and samples taken for histopathological analysis. Different bacterial and viral etiologies were assessed by PCR in frozen tissue samples.

Results: Gross lung lesions were present in 17.7% of the cases and were predominant in veal calves. Chronic suppurative bronchopneumonia was the most prevalent. Acute fibrinous pneumonia was only found in extensively reared cattle. Most of the affected lungs (59.2%) showed lesions in less than 10% of the parenchyma. The presence of lesions was associated with a lower weight of the carcass in male veal from intensive production systems, but not in yearlings or females. Bacteria were the predominant infectious agent associated with these lesions, and the only found in those of fibrinous pneumonia. Mixed bacterial infections (mostly including Mycoplasma bovis) occurred in 50% of the cases. The identification of viruses was scarce and only found in chronic lesions.

Conclusions: This study shows the importance of BRD in Spanish beef feedlots upon productive values and delves into the types of pneumonia and associated infectious etiologies.


Poster 92: VALIDATION OF ANTIGEN RETRIEVAL IS IMPORTANT IN IMMUNOHISTOCHEMICAL PROTOCOLS FOR DETECTION OF PRIONS IN PARAFFIN TISSUE SECTIONS

P. Juntes* and J. Zabavnik Piano

*Institute of Pathology, Wild Animals, Fishes and Bees and Institute of Preclinical Sciences, University of Ljubljana, Veterinary Faculty, Ljubljana, Slovenia

Introduction: Immunohistochemistry is one of the officially approved methods for confirmation of transmissible spongiform encephalopathies in animals. Laboratories practice different immunostaining protocols in which heat-mediated antigen retrieval is an essential part, and for prions, they mostly use wet autoclaving. Autoclave located in our laboratory did not reach the set working temperature during the calibration process, and our aim was to evaluate the quality of immunostaining after different times and temperatures of wet autoclaving to get optimal immunostaining of prions.

Materials and Methods: Paraffin tissue sections of bovine brain positive for bovine spongiform encephalopathy (BSE), and ovine brains positive for classical (cSc) and atypical (atSc) scrapie were deparaffinized and rehydrated. Antigen retrieval was performed in citrate buffer pH 6.0 in a vertical steam autoclave at temperatures 120oC, 121oC and 122oC for 20 or 30 minutes, respectively at each temperature. Immunostaining was made using routine protocol applied for prions in the laboratory.

Results: Immunostaining of cSc tissue sections was stronger at higher temperatures; such differences were less obvious in atSc and BSE slides. Longer incubation gave better staining in cSc and atSc, however that was not so clear for BSE tissue sections.

Conclusions: Autoclaves are instruments with controlled temperature and have to be validated for the use, in this case for optimal antigen retrieval in immunohistochemical protocols for prions in paraffin tissue sections. It is important to define optimal combination of time and temperature for each instrument type and sample type, to control the instrument periodically and adjust settings if necessary.


Poster 93: INFLUENCE OF DIFFERENT FIXATIVES ON QUALITY OF STAINING AND MORPHOLOGY OF FISH OVARY

K. Naumowicz*, J. Pajdak, J. Szarek*, P. Schulz, E. Terech-Majewska and  M.Z. Felsmann #

*Department of Pathophysiology, Forensic Veterinary Medicine and Administration, Department of Epizootiology and Department of Microbiology and Clinical Immunology, Faculty of Veterinary Medicine, University of Warmia and Mazury in Olsztyn, Poland,  #Veterinary Institute, Poznań University of Life Sciences, Poznań, Poland

Introduction:  Fixation is the first and most important step inhistopathological tissue processing. Use of improper solutions can result in a misdiagnosis of histopathologic findings. The most popular fixative in veterinary medicine is neutral buffered formalin (NBF), which is widely available and inexpensive. However, some tissues, especially those rich in fat, such as fish ovary, require fixatives other than NBF to obtain best results.

Materials and Methods:  Five samples of the ovary were obtained during necropsy of healthy female rainbow trout (Oncorhynchus mykiss). Samples were fixed immediately in five fixatives: NBF, 70% alcohol, Dietrich’s, Davidson’s and Bouin’s solutions respectively. Subsequently, samples were dehydrated, embedded in paraffin and stained with haematoxylin-eosin (HE). Afterwards, specimens were analysed by light microscopy and evaluated.

Results:  Analysis of microphotographs revealed that the best quality of staining was obtained in samples fixed in Bouin’s, as well as in Davidson’s solution. Moreover, both fixatives ensured good preservation of cells and tissue, as they naturally occur - oocytes of all stages of development were regularly shape, with clearly visible structures. However, in samples fixated in NBF and alcohol, we observed notable cell shrinking resulting in the irregular shape of oocytes and general tissue distortion.

Conclusions: Ovary tissue should not be fixed in NBF or in alcohol, because it greatly influences morphology of the tissue and results in a poor quality of staining. For the best results, Bouin’s solution should be used. Comparable results can be obtained using Davidson’s fixative, with the additional advantage of avoidance of picric acid usage.


Poster 94: A NOVEL ALPHAHERPESVIRUS ASSOCIATED WITH CASES OF FATAL PENGUIN DIPHTHERIA - LIKE DISEASES IN BANDED PENGUIN CHICKS (SPHENISCUS (S.) HUMBOLDTI AND S. DEMERSUS) FROM TWO GERMAN ZOOLOGICAL GARDENS

C. Schulze*, F. Pfaff, P. König, K. Franzke, S. Bock*, A. Hlinak*, J. Kämmerling, A. Ochs§, A. Schüle§, T.C. Mettenleiter, D. Höper, D. Denk and M. Beer

*Berlin-Brandenburg State Laboratory, Frankfurt (Oder), Germany, Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Greifswald Insel – Riems, Germany, Tierpark Cottbus, Germany, §Zoological Garden Berlin, Germany and IZVG Pathology, Leeds, United Kingdom

Introduction: Penguin diphtheria, a disease of wild and captive juvenile penguins worldwide, causes high mortality in affected birds due to diphtheroid stomatitis and necrotizing gastroenteritis. The primary cause of the disease is yet unknown.

Materials and Methods: Necropsy was performed on three 5 to 6 week old female Humboldt penguins (Spheniscus (S.) humboldti) from the Tierpark Cottbus and one 4 week old African penguin (S. demersus) from the Berlin Zoo. Formalin-fixed and paraffin-embedded tissues were investigated by HE-staining. Native tissues were screened for herpesviruses by PCR. In addition, virus isolation in cell culture and further characterization of the isolates by molecular methods was performed.

Results: Main finding at necropsy in the Humboldt penguins was a severe necrotizing gastroenteritis and in the African penguin a diphtheroid and necrotizing oropharyngitis and laryngotracheitis. Intranuclear amphophilic inclusion bodies indicative of a herpesvirus infection were present in all cases and a pan-herpesvirus PCR was positive in affected tissues. A novel alphaherpesvirus preliminarily designated sphenicid alphaherpesvirus 1 (SpAHV-1) was isolated, its complete genome determined by high-throughput sequencing and its taxonomic association to the genus Mardivirus established by gene content clustering and phylogenetic analysis of herpesvirus core genes.

Conclusions: The presented results imply that SpAHV-1 may be a primary causative agent of penguin-diphtheria in banded penguins. However, future studies will have to show if SpAHV-1 is uniquely responsible for the development of the lesions or one contributing factor to a multifactorial syndrome.


Poster 95: NIDOVIRUS-ASSOCIATED PROLIFERATIVE PNEUMONIA: AN EMERGING DISEASE IN PYTHONS

E. Dervas*, J. Hepojoki*,, P. Palamides*, K. Windbichler*, S. Keller*, A. Kipar*and U. Hetzel*

The BIBD Group, *Institute of Veterinary Pathology, Vetsuisse Faculty, University of Zurich, Switzerland and Medicum, Department of Virology, University of Helsinki, Finland

Introduction: Nidoviruses have recently become evident as potential causative agents of fatal pneumonia in some python species. Here we report that nidoviruses associate with proliferative pneumonia in a wider range of python species.

Materials and Methods: Nidovirus infection was confirmed by NGS and/or PCR in carpet pythons (Morelia spilota), green tree pythons (Morelia viridis), an Indian (Python molurus), a Woma (Aspidites ramsayi) and an Angola (Python anchietae) python with clinical and gross evidence of mucoid pneumonia. In all animals, a detailed histological examination was performed on the respiratory tract and lungs, and nidovirus nucleoprotein was detected by immunohistology.     

Results: In all cases, the air conducting space was filled with mucus. This was accompanied by hyperplasia of the airway epithelium, the multilayered epithelium covering the pulmonary trabeculae and, in the faveolae, of type II pneumocytes. Secretory cells were increased in number, and nidovirus antigen was detected in epithelial cells in all locations, with evidence of a negative correlation between the number of positive cells and the degree of epithelial hyperplasia.

Conclusions: Together with anecdotal published reports since 2014, these results indicate that nidoviruses are an increasingly relevant pulmonary pathogen in pythons and induce a proliferative pneumonia with excessive mucus production. While type II pneumocyte hyperplasia represents a non-specific response of the ophidian lung to damage of the pulmonary epithelium, the distinct epithelial hyperplasia might represent a specific nidovirus-induced process. Affected animals mainly die with no or minimal prior clinical signs; we are therefore currently investigating appropriate intra vitam diagnostic approaches.


Poster 96: BONE MARROW IN THE HYATID BONES AND NASAL TURBINES OF CAPTIVE, SHORT-TAILED FRUIT BATS

D. Wiederkehr*, M. Schediwyand N. Stokar-Regenscheit

*School of Agricultural, Forest and Food Sciences HAFL,  Bern University of Applied Sciences, Switzerland and Institute of Animal Pathology, Vetsuisse Faculty, University of Bern, Switzerland

Introduction: Bats are known to be host of many pathogens without developing clinical disease. It is known that leucocytes numbers in the blood of bats are higher than in other mammals. Hematopoetic tissue in hyatid bones and nasal turbines during routine diagnostic of bats was detected, which is remarkable. Here, the amount of hematopoetic tissue in hyatid bones and nasal turbines bone marrow in bats was compared to known numbers of bone marrow in laboratory mice.

Materials and methods: 96 healthy, captive, short-tailed fruit bats (Carollia perspicillata) from a Swiss zoo were investigated by routine screening histopathology due to reduction of the colony. All organs including tongue and skull bone were analyzed histologically with H E staining. Immunohistochemistry for CD3, CD20 and MPO to evaluate the hematopoietic tissue was performed.

Results: Thymus was still visible in all of the animals. In the hydatid bones as well as in nasal turbines active bone marrow could be seen. The immunohistochemistry of the bone marrow revealed ~ 30% of the cells in the bone marrow CD20, ~ 6% CD3 and ~ 20% MPO positive. In the same areas in the bone marrow around 10 megaloblasts could be seen, respectively.

Conclusions:  The high amount of active bone marrow in hydatid bones and the additional bone marrow in nasal turbines support the hypothesis that bats do have a more effective immune system than other mammals. In comparison to mice, bats seem to have less myeloid precursors and more lymphocytes in the bone marrow


Poster 97: COINFECTION OF ADENOVIRUS AND PARAMYXOVIRUS IN A TROPICAL GIRDLED LIZARD (CORDYLUS TROPIDOSTERNUM)

G.H. Woo

Department of Clinical Laboratory Science, Semyung University, Jecheon 27136, Republic of Korea

Introduction: Parvoviral infections, herpesviral infections, adenoviral infections, and paramyxoviral infections have been most frequently documented in reptiles. The present study reports a co-infection with adenovirus and paramyxovirus in a tropical girdled lizard.

Materials and Methods: Histopathology of all tissues and organs was performed. Nasal swabs, and intestine and liver samples were cultured for bacterial isolation. Viral examination was done by PCR  and edited sequences were compared with database sequences (GenBank, NCBI) and identified by BLAST searching.

Results: At necropsy, redness was seen in intestinal mucosa. 258-bp PCR products of adenovirus and 152-bp PCR products of paramyxovirus from the intestine and liver were detected. When the paramyxovirus sequence identified in the intestine of a lizard was compared with lizard paramyxovirus isolate Igu-GER00 (324/00) hemagglutinin neuraminidase (HN) gene (accession no. GQ277619.1) and snake ATCC-VR-1409 paramyxovirus HN protein (HN) gene (accession no. AF286044.1), the sequence identity was 98%. An adenoviral partial DNA-polymerase gene was compared with partial DNA polymerase genes of other adenoviruses. A partial polymerase gene showed 78% identity to snake adenovirus 3 DNA-dependent DNA polymerase (pol) gene (accession no. FJ012164.1), 79% identity to the eublepharid adenovirus 1 DNA-dependent DNA polymerase (pol) gene (accession no. AY576677.1), 75% identity to the death adder adenovirus 7 DNA polymerase (pol) gene (accession no. JN418933.1), and 75% identity to the lizard adenovirus 2 isolate 7 DNA polymerase (pol) gene (accession no. EU914207.1), indicating as a new gene of adenovirus.

Conclusions: Partial sequencing results from the adenoviral DNA polymerase gene in the lizard indicated that it was a novel adenovirus (accession no. KY347916)


Poster 98: COMPARATIVE OVERVIEW OF HISTOPATHOLOGICAL FINDINGS IN PANCREAS OF SWANS AND HENS INFECTED WITH AVIAN INFLUENZA VIRUS H5N8

I. Vučićević1, S. Nešić1, V. Kukolj1, M. Vučićević1, B. Božić2, V. Polaček2 and T. Petrović2

1Faculty of Veterinary Medicine, University in Belgrade, Serbia and 2Scientific Veterinary Institute Novi Sad, Novi Sad, Serbia

Introduction: First cases of highly pathogenic avian influenza (HPAI) subtype H5N8 in the Republic of Serbia were recorded in December 2016 in swans (Cygnus olor). Control measures included removal of poultry flocks in the restriction zone and presence of HPAI H5N8 was shown in two flocks of hens.

Materials and Methods: To determine the causes of death, 17 swan and 15 hen carcasses were necropsied and organs were examined histopathologically. To characterize the virus-infected cells monoclonal antibody against Avian Influenza nucleoprotein antigen (InfA-NP) were used.

Results: In all examined mute swans, pancreatic tissue was characterised by multifocal areas of necrosis, and petechial haemorrhages. Necrotic fields were scattered, from 1 to 5 mm in diameter. In contrast to swans, there were no macroscopic visible lesions in pancreatic tissue of infected hens. Histopathologically, both the swans and hens developed acute pancreatitis with multifocal acinar necrosis. Necrotic foci were lined by inflammatory infiltration composed of macrophages, lymphocytes and plasma cells. Viral antigen was detected in the cytoplasm and nucleus of necrotic cells as well as in the macrophages.

Conclusions: The only organ that was consistently affected in both avian species was pancreas suggesting that the epithelial cells of exocrine pancreas could be a potential target of HPAI H5N8 infection. Acute pancreatitis could be due to the fact that exogenous trypsin appears to enhance viral replication in pancreatic epithelial cells or influenza virus replication damages the acinar cells and the release digestive enzymes results in pancreatic hemorrhages and necrosis.


Poster 99: DOLPHIN MORBILLIVIRUS INFECTIONS IN STENELLA COERULEOALBA ALONG SICILY COAST.

R. Puleio, D. Vicari, V. Currò, G. Purpari, A. Tamburello, F. Messina, F. Mira, S. Di Bella, P. Di Marco, G.R. Loria, A. Guercio and S. Caracappa

Istituto Zooprofilattico Sperimentale della Sicilia, Italy

Introduction: Dolphin morbillivirus (DMV) is one of the three strains of cetacean morbillivirus (CeMV), which includes porpoise morbillivirus and pilot whale morbillivirus. Dolphin morbillivirus is the main agent described in cases of non suppurative meningoencephalitis in striped dolphins during the last Marine Mammal Unusual Mortality Event occurred along Italy coast in 2013.

Materials and Methods: In 2016, 24 Stenella Coeruleoalba were necropsied according to protocols by National Reference Center for diagnostic activities on dead stranded cetaceans (C.Re.Di.Ma). Location of stranding, body length and sex were recorded. Tissue samples were collected from lungs, heart, liver, kidneys, spleen and brain for virology, bacteriology, histopathology and immunohistochemistry.

Results: Histopathological findings of brain consisted of multifocal, non-suppurative meningoencephalitis of variable intensity, with mononuclear perivascular cuffs, gliosis and glial nodules with neuronophagia and spongiosis. Syncytial cells were sometimes observed. RT-PCR and immunohistochemistry identified DMV in 8 out of 24 dolphins. The obtained sequences were aligned with the related ones of Genbank. Sequence analysis showed a high nucleotide identity with CeMV strains of cetaceans stranded in atlantic coasts of Germany (2007), Spain and Portugal (2012-2013).  

Conclusions: Histological comparison among 2013 and 2016 DMV cases showed a more severe histological pattern in brain and the analysis of DMV sequences obtained from dolphins stranded along Sicily coast has shown high identity with Atlantic strains


Poster 100: Cancelled


Poster 101: WIDESPREAD SKIN LESIONS IN TANZANIAN HORSES CAUSED BY MOLLUSCUM CONTAGIOSUM VIRUS-LIKE POXVIRUS

K. Brandes1, K. von Schlippenbach2, E. Stegmaier3, M. Antwerpen4, M. Walter4, S. Essbauer4, J. Bugert4, J. P. Teifke5 and H. Meyer4

1Animal Pathology Augsburg, Germany, 2Veterinarian Practice, Zusamaltheim, Germany, 3Makoa Farm, Tanzania, 4Bundeswehr Institute of Microbiology, Munich, Germany, 5Federal Research Institute for Animal Health, Greifswald - Insel Riems, Germany

Introduction: Several horses used for horseback safaris in Tanzania showed papular or verrucous masses in variable number distributed across the body. Lesions in sick or stressed horses disappeared over time when horses recovered. This case report describes the skin lesions and the investigation of the causative agent in one of these horses.

Materials and Methods: A10-year-old, female horse presented clinically with numerous papular and verrucous lesions. Before disease blossomed the mare showed signs of colic and neurologic deficits. One of these masses was surgically removed and processed for histopathology, electron microscopy, PCR, in-situ-hybridisation and next generation sequencing.

Results: Histopathology revealed a sharply delimited epidermal hyperplasia of the stratum spinosum with numerous large, eosinophilic, intracytoplasmic inclusion bodies. Electron microscopy demonstrated brick-shaped virions with core structures characteristic for Molluscum contagiosum virus (MCV). PCR followed by sequencing showed sequence identity of 99% with a MCV–like poxvirus derived from a horse and a lower identity (92%) with MCV derived from humans. In-situ-hybridisation revealed signals within the intracytoplasmic inclusions. Cross hybridisation was assessed with samples of human Molluscum contagiosum material which led to an identical staining pattern. Whole genome sequencing with de novo assembly showed 91% nucleotide sequence homology to published human MCV genome sequences over a single 163.400 bp contig mainly in the conserved genome region.

Conclusions: Based on our findings a MCV–like poxvirus dermatitis was diagnosed. Transmission to humans was not observed. Sequencing revealed sufficient nucleotide sequence homology to suggest that this virus is a new member of the genus Molluscipoxvirus


Poster 102: FEASIBILITY OF MIRNA EXTRACTION AND AMPLIFICATION FROM CHICKEN CAECAL CONTENT IN EIMERIA TENELLA INFECTION

S. Fernando,* D.P. Blake,* and J.M. Williams *

* Pathobiology and Population Sciences, Royal Veterinary College, Hawkshead Lane ,North Mymms, Hatfield, Hertfordshire AL9 7TA, United Kingdom

 

Introduction: MicroRNAs (miRNAs) are small, non-coding RNAs that are dysregulated in a range of diseases. They have also been shown to regulate the gut microbiota, and to be relatively stable in faeces; suggesting potential use as biomarkers of intestinal disease. We hypothesised extraction and amplification of miRNAs would be possible from frozen chicken caecal content in the context of coccidiosis due to Eimeria tenella.   

Materials and Methods: Caecal contents were collected 7-days post-infection from Eimeria tenella infected (n=3) and uninfected (n=3) specific pathogen-free (SPF) chickens at post-mortem. Samples were stored at -80˚C for ~16 months and RNA extracted via a Norgen Stool RNA Purification Kit, or Ambion MirVana Isolation Kit. RNA was reverse-transcribed, and Mir19A, Mir19B, Mir155, and Mir1416 amplified using a C1000 thermocycler (Bio-rad) with miRNA qRT-PCR Kit (Takara)

Results: The Norgen kit yielded less total RNA per sample (mean 98.7±17µg) than the Ambion Kit (mean 121.3±48.5 µg) but with greater purity (mean 260/280nm absorbance of 1.95 versus 1.73). RNA extracted by both methods was highly degraded, but Mir19A, Mir19B, Mir155, and Mir1416 were successfully amplified, with Mir155 exhibiting a ~2.6 fold up-regulation in infected versus uninfected birds.

Conclusions: This small-scale study demonstrates that poor quality and degraded RNA may be utilised for successful extraction, reverse-transcription, and amplification of miRNAs. Mir155 is an inflammation associated miRNA and may be correlated to caecal inflammation due to E. tenella infection in this study. However, further work including sequencing and miRNA mapping is required to further elucidate potential biological associations and significance.


Poster 103: FIRST REPORT OF HISTOPATHOLOGICAL CHANGES IN A STONE MARTEN (MARTES FOINA) WITH MOLECULARLY AND MICROSCOPIC CONFIRMED HEPATOZOON SP. INFECTION

A. Beck*, D. Huber*, D. Jurković, V. Mrljak, G. Baneth§ and R. Beck

*Department of Veterinary Pathology, Faculty of Veterinary Medicine, University of Zagreb, Croatia,Department for Bacteriology and Parasitology, Croatian Veterinary Institute, Zagreb, Croatia,Internal Diseases Clinic, Faculty of Veterinary Medicine, University of Zagreb, Croatia, and §Koret School of Veterinary Medicine, The Hebrew University of Jerusalem, Rehovot, Israel

Introduction: Apicomplexan hemogregarines of the genus Hepatozoon, described in more than 300 animal species, are transmitted by various hematophagous arthropods. In the current report we provide the first morphological description, pathological changes and molecular characterization of a Hepatozoon sp. from a stone marten.

Materials and Methods: A paralyzed young adult wild female stone marten from Western Croatia was euthanized for determination of cause of illness. During necropsy representative tissue samples were collected for histology and molecular analyses. Abscess content was sampled for bacterial culture and ectoparasites for morphological determination.

Results: Necropsy revealed cachexia, head and neck abscesses at the site of bite wounds and severe ectoparasitic infestation. Severe emaciation was caused by massive concurrent bacterial and parasitic infections. Cultivation and histology results confirmed β-hemolytic Streptococcus pyogenes and E. coli within multifocal to coalescing subcutaneous purulent lesions. Microscopic examination revealed numerous Hepatozoon meronts and released merozoites from ruptured meronts associated with granulomatous inflammation. Macrophages often contained intracytoplasmatic merozoites. Meronts were sporadically found in the liver, spleen and kidneys without an apparent local inflammation. Sequences of 666 bp fragment of 18S rRNA from the spleen, liver, heart and cervical muscle were identical to Hepatozoon “Badger type” (KU198330). Identical Hepatozoon DNA was confirmed with sequencing in Ixodes ricinus ticks from the stone marten, while fleas from the animal were negative.

Conclusions: Infection with Hepatozoon sp. from a stone marten showed similar lesions and organ distribution to those described from different Hepatozoon sp. in other marten species.


Poster 104: FIRST OUTBREAK OF RABBIT HAEMORRHAGIC DISEASE (RHD) IN RABBITS IN FINLAND

M. Isomursu*, A. Neimanis, V. Karkamo, R. Holopainen§, T. Nokireki§ and T. Gadd§

*Section of Wild and Aquatic Animal Pathology and Companion Animal Pathology and §Virology research Unit, Finnish Food Safety Authority Evira, Oulu and Helsinki, Finland and National Veterinary Institute, Department of Pathology and Wildlife Diseases, Uppsala, Sweden

Introduction: Rabbit Haemorrhagic Disease (RHD) is a highly contagious hepatitis of the European rabbit (Oryctolagus cuniculus) caused by a lagovirus (family Caliciviridae). Endemic infections are controlled by vaccinations. The feral rabbit population in Finland originates from release of domesticated rabbits in southern Finland in the 1980’s. This is the first description of RHD in this population.

Outbreak: Reports of dead, mostly feral, rabbits in the capital city Helsinki began to increase from April 2016 onwards. The animals were examined by necropsy at Finnish Food Safety Authority Evira. The first samples from feral rabbits were sent to National Veterinary Institute (SVA) in Sweden for RHD virus detection. Because the epidemic progressed, a RT-PCR method was set up at Evira.

Results: Feral rabbit mortality peaked in May and became rare by July 2016. The total number of feral carcasses reported was 134 and only one case of a pet rabbit with lesions suggestive of RHD. The main finding in all animals was acute, massive necrotizing hepatitis. The presence of RHD virus in affected liver tissue was confirmed.

Discussion: This is the first documentation of RHD in Finland. Reports likely represent only a small proportion of actual number of dead feral rabbits. No regular monitoring of the rabbit population is done, which makes it impossible to estimate the actual mortality but field observations suggest a significant reduction in the feral population. Only one confirmed case in a pet rabbit was observed, presumably due to prompt reporting of the feral rabbits’ investigations.


Poster 105: HISTOPATHOLOGICAL LESIONS DURING SINGLE AND CO-INFECTION BY TETRACAPSULOIDES BRYOSALMONAE AND MYXOBOLUS CEREBRALIS IN RAINBOW TROUT

M. H. Kotob1,2, B. Gorgoglione1,3, G. Kumar1, M. Abdelzaher2, M. Saleh1 and M. El-Matbouli1

1Clinical Division of Fish Medicine, University of Veterinary Medicine, Veterinärplatz 1, Vienna, Austri.     2Department of Pathology, Faculty of Veterinary Medicine, Assiut University, Assiut, Egypt.   3Department of Biological Sciences, University of Toledo, Toledo, Ohio, USA

Introduction: Co-infections are common in nature and affect the host susceptibility to pathogens. Tetracapsuloides bryosalmonae and Myxobolus cerebralis are two myxozoan parasites causing serious economic losses. This study investigated the impact of single and co-infection by both pathogens on rainbow trout.

Materials and Methods: Rainbow trout were divided into three groups, first group (n = 96) were primarily infected by T. bryosalmonae, 30 days later half of fish were secondary infected by M. cerebralis while the rest remained as single infection. The second group (n = 96) was initially infected by M. cerebralis then secondary infected by T. bryosalmonae, third group (n = 60) was kept as non-infected control. Mortalities and clinical signs were recorded during 120 days of the experiment. Histolopathology and immunohistochemistry were performed to compare between groups.

Results: Fish co-infected initially by M. cerebralis then T. bryosalmonae exhibited a severe form of both diseases with high mortalities. Kidneys showed chronic diffuse nephritis with higher parasite loads and the cranial cartilages showed severe granulomatous lesions compared to single T. bryosalmonae and M. cerebralis infected fish. On the opposite, fish co-infected initially by T. bryosalmonae then M. cerebralis showed less mortalities and mild lesions of whirling disease. However, kidney swelling index was similar to single T. bryosalmonae infected group.

Conclusions: The impact of co-infections depends on the primary pathogens which could affect the outcomes of the secondary pathogens. Co-infection primary by M. cerebralis then secondary by T. brysalmonae had synergistic effect; however the opposite resulted in antagonistic interaction.


Poster 106: HOST MICRO-RNA EXPRESSION IN THE COURSE OF KHV INFECTION IN CARPS

M. Reichert*, M. Matras*, A. Łukasik†, E. Borzym*, J. Maj-Paluch * and M. Stachnik *

*Department of Fish Diseases, National Veterinary Research Institute, Pulawy, Poland and Department of Bioinformatics, Institute of Biochemistry and Biophysics PAS, Warsaw, Poland

Introduction: Koi herpesvirus ( KHV, CyHV-3) shows ability to establish life-long infection in immunocompetent hosts. To get more knowledge on the immune evasion strategy exploited by KHV, we infected carp fry with KHV and measured the expression of host micro-RNAs in blood WBCs of fish.

Materials and Methods: Blood samples were collected from the caudal vein of healthy (before infection), clinically affected and convalescent fish (2 months after challenge) and immediately transferred into EDTA tubes. Clinically affected fish showed also histopathological changes. White blood cells (WBCs) were purified by mixing blood samples with red blood cells lysis buffer. WBCs micro-RNA was purified by using miRNeasy kit (Qiagen, Germany). Expression of host micro-RNAs in healthy, clinically affected and convalescent carps was measured using Illumina deep sequencing.

Results: Changed expression of 27 miRNAs was found in blood WBCs from clinically affected fish as compare to healthy ones. In convalescent fish only 5 miRNAs were expressed differentially in clinical samples versus healthy ones. Most of the differentially expressed miRNAs were up-regulated. Functional analysis revealed that differentially expressed miRNAs are involved inter alia in  PI3K-Akt, Epstein-Barr virus, MAPK and viral carcinogenesis signaling pathways. Differentially expressed miRNAs regulate many important biological processes such as viral gene expression, host mRNA splicing, T-cell activation, metabolism, response to certain stimuli and immune response.

Conclusions: miRNA composition and amounts seems to be very similar in samples collected from healthy and convalescent fish and such results suggest that the virus entered the latency phase.


Poster 107: IMMUNOHISTOCHEMICAL CHARACTERISATION AND PROLIFERATION ACTIVITY OF CUTANEOUS LEIMYOSARCOMA WITH MULTIPLE VISCERAL METASTASES IN A FANTAIL PIGEON

M. Gesek*, T. Stenzel, I. Otrocka-Domagała*, K. Paździor-Czapula*, M. Mikiewicz*, K. Michalska* and A. Koncicki

*Department of Pathological Anatomy and Department of Poultry Diseases, Faculty of Veterinary Medicine, University of Warmia and Mazury in Olsztyn, Poland

Introduction: Cutaneous leiomyosarcomas, reported frequently in mammals and sporadically in avian species (pigeons, budgerigars), occur as solitary, nodular to multinodular, solid masses. Cutaneous leiomyosarcomas recur locally, but metastases (a definite criterion for malignancy) have not been previously reported.

Materials and Methods:  A 4-year-old female fantail pigeon presented with a large (5.5 cm), solid, multinodular cutaneous tumour of the left wing, infiltrating deeply to the humerus. The pigeon was euthanized. Necropsy revealed multiple small (3-6 mm) nodules in the liver, spleen, pancreas and kidneys. Tumours and all internal organs were sampled for histopathology and immunohistochemistry (vimentin, SMA, pan-keratin, desmin, MyoD1 and PCNA).

Results: Cutaneous and visceral tumours were composed of polygonal, round, oval and spindle cells of high anisocytosis and anisokaryosis, pleomorphic nuclei with coarse chromatin, a moderate amount of cytoplasm, and moderate mitotic activity. Similar nodules were also found in the lungs, despite a lack of gross lesions. Neoplastic cells showed expression of SMA and vimentin, and were MyoD1, pan-keratin and desmin negative. In the cutaneous tumour, 30-40% of neoplastic cells expressed PCNA, while in the visceral metastases - 85-95%. Based on the results, leiomyosarcoma was diagnosed. The tumor of the wing was identified as a primary lesion, while the multiple tumours in viscera seemed to be metastatic due to their small size and evidently perivascular localization.

Conclusions: The possible origin of the neoplastic cells was the dermal feather smooth muscles. This case is the first report of cutaneous leiomyosarcoma with multiple visceral metastases in an animal.


Poster 108: IBD ASSOCIATED NECROTIZING VASCULAR LESIONS IN A BOA CONSTRICTOR IMPERATOR.

 

M. Tecilla* and P. Roccabianca*

*DiMeVet, University of Milan, Via Celoria 10, 20133, Milan, Italy

Introduction: Inclusion body disease (IBD) is a common viral disease of captive Boids. IBD is caused by an arenavirus transmitted by the mite Ophionyssus natrics. IBD signs are frequently mild to unapparent until the onset of CNS lesions that result in inability to feed. 

Materials and Methods: A male, adult, Boa constrictor imperator introduced into a collection of seven boids two years earlier, developed depression, dysphagia and dysecdysis rapidly extending to 90% of the body with multifocal ulceration. Because of the poor prognosis euthanasia was elected.

Results: Grossly, ulcerations covered 60% of the lateral axial skin and extended to oral and gastric mucosa. Subcutaneous, pulmonary, serosal haemorrhages and haemocoelom were observed.  Microscopically, necrotizing and ulcerative dermatitis and gastritis with bacteria were confirmed. Cutaneous, gastric, and serosal vessels revealed endothelial cell swelling and necrosis, with numerous intracytoplasmic eosinophilic inclusion bodies consistent with IBD. Elevated numbers of viral inclusions were observed in livers, kidneys but also in the retina, skin keratinocytes and stomach. Following euthanasia of all 7 animals, 4/7 boids had IBD inclusions.

Conclusions: IBD lesions in this boid were severe and unusual (retinal, vascular inclusions). This seems to be the first reported IBD necrotizing vascular lesions. IBD is associated with long incubation and mild lesions including cytoplasmic inclusions in liver, kidneys, airways, intestines and the terminal development of lymphoplasmacytic encephalitis. Noteworthy, mammalian arenaviruses (Junin virus and Machupo virus) cause necro-hemorrhagic lesions. Possibly, necrotizing vascular lesions resulted from direct cytolytic effects of a more aggressive IBD arenavirus strain in an immunocompromised host. 


Poster 109: INFLAMMATORY RESPONSE IN CHRONIC DORSAL DERMATITIS IN MOOSE (ALCES ALCES) IN SWEDEN

E.O. Ågren*, C. Bröjer*, J. Malmsten and T. Mörner

*Department of Pathology and Wildlife Diseases and Department of Epidemiology and Disease Control, National Veterinary Institute, and Wildlife consultant Ltd, Kohagen, Lindhagen, 186 94 Vallentuna, Sweden

Introduction: An unusual outbreak of cases with extensive skin lesions over the dorsal lumbar and sacral regions of male moose (Alces alces) was reported during the summer and autumn of 2015. Single cases have been documented previously, but never as an outbreak.

Materials and Methods: Information on sampling and a questionnaire was sent to all moose hunting districts before the moose hunt 2015. Regular general wildlife disease surveillance networks also contributed with reports and samples for pathology and bacteriology.

Results: 149 cases of skin lesions were reported from the southern half of Sweden within six months, from October 2015. Skin samples or carcasses from 58 moose with skin lesions were investigated. Skin lesions often measured over 1 meter in size and were typically located over the lumbar and sacral areas of the back. Grossly, lesions were dried crusts covering extensive areas of suppurative, ulcerated, and thickened skin. Bacteriology showed consistent findings of Staphylococcus aureus. Abundant deer ked (Lipoptena cervi) were present and occasionally ear mites (Chorioptes sp.). The inflammatory response in early stages was dominated by dermal perivascular plasma cells, with some eosinophils and few lymphocytes. In chronic lesions, there was a massive superficial suppurative, hyperplastic, and ulcerative dermatitis containing bacterial colonies, covering a severely thickened dermis, caused by granulation tissue.

Conclusions: The histopathology changes were unspecific, but resemble chronic pyotraumatic dermatitis. The suspected cause is self-trauma following pruritus of possible parasitic, infectious, or allergic etiology, leading to an itch-scratch cycle, with secondary ulceration and bacterial infection.


Poster 110: INFLUENCE OF EM-PROBIOTIC ON THE MORPHOLOGY OF THE SPLEEN IN ZANDER (SANDER LUCIOPERCA)

E. Terech-Majewska, Z. Zakęś*, A. Kowalska*, A. K. Siwicki†‡, J. Szarek§ and K. Naumowicz§

Department of Epizootiology and §Department of Pathophysiology, Forensic Veterinary Medicine and Administration University of Warmia and Mazury in Olsztyn, Poland, and *Department of Aquaculture and †‡Department of Fish Immunology and Pathology, The Stanisław Sakowicz Inland Fisheries Institute in Olsztyn, Poland

Introduction: The technology of effective microorganisms (EM) uses microorganisms to cooperate with the surrounding environment. Many research centres conduct a study in order to reveal EM effects on aquatic organisms. The aim of the study was to evaluate effects of the EM-Probiotic (produced by Greenland) on the morphology of the spleen in zander (Sander lucioperca) reared in controlled conditions.

Materials and Methods: Zander were reared in recirculating aquaculture systems (RAS). The experimental feeds (Aller) were administered to three groups of fish for 28 days - the addition of EM was: 0% (control - C), 2% and 4% of the feed weight. Spleen samples were collected for histopathological examination after 28 days of feeding with the EM-feed and two weeks later. Samples were fixed in 6% neutral buffered formalin, embedded in paraffin and stained with HE.

Results: During microscopic examination of the spleen specimens haemosiderin pigment and melanomacrophage centres (MMC) were evident in all groups. The average density and size of MMC were highest in the 2% group. In the C group splenic congestion (mild to moderate) and fatty degeneration in the ellipsoids area was the highest. In the group 4%, foamy ellipsoid cells with karyomegaly and nuclear vacuolation occurred.

Conclusions: The results indicate that EM can influence the morphology of the spleen in zander reared in RAS, depending on their amount in the feed. There were also differences in the fish condition during the adaptation period, in favour of fish from the Group 4% EM.


Poster 111: LEIOMYOSARCOMA IN A DALMATIAN PELICAN (PELECANUS CRISPUS)

A. Oliver-Guimeraa,b, L. Lobatoa,b, H. Fernandez-Bellonb and A. Ramisa

aDepartament de Sanitat i Anatomia Animals, Facultat de Veterinària, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, Spain. bParc Zoològic de Barcelona, Parc de la Ciutadella s/n, Barcelona, Spain

Introduction: Leiomyosarcomas are invasive malignant tumors of smooth muscle cells. Their metastatic potential in mammals is difficult to assess and normally affects liver and lungs. They have been rarely described in avian species, including  poultry, pigeons, cranes and psittacines.

Materials and Methods: An adult (> 26 yo) female dalmatian pelican (Pelecanus crispus) died at the Parc Zoologic de Barcelona without reported previous clinical signs. Necropsy was performed in the zoo facilities and selected tissues were submitted to the Servei Diagnòstic de Patologia Veterinària for histopathology examination, where they were routinely stained with HE. Immunohistochemical staining for CD3, CD20, cytokeratin, vimentin, S-100, smooth muscle actin (SMA), desmin and C-KIT were also performed.

Results: On gross examination, multiple nodular whitish masses up to 15 cm in diameter were observed in several organs and tissues, including subcutaneous tissue of the forelimbs, pectoral muscle, proventriculus, spleen, liver, lung and myocardium. Upon microscopic examination, all these masses corresponded to nonencapsulated neoplastic proliferations that infiltrated the surrounding tissues. Neoplastic growths were constituted by anaplastic sarcomatous cells. Despite not being detected grossly, metastatic masses were detected in brain and brain stem. Immunostaining exclusively showed positivity for SMA, which led us to diagnose a leiomyosarcoma with multiple metastases.

Conclusions: To our knowledge, this is the first report of leiomyosarcoma in Pelecaniformes order. A proventricular origin is hypothesized in this case in accordance to histopathological features. Also it is interesting to remark that it is a rare case of CNS metastasis.


Poster 112: LETHAL ULCERATION IN THE BLOWHOLE OF A COMMON BOTTLENOSE DOLPHIN (TURSIOPS TRUNCATUS)

N. van de Velde*, L. Vanbrantegem* and K. Chiers*

*Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium

Introduction: A 32 year-old captive dolphin died after a brief period of quick deterioration with blood and blood clots coming out of the blowhole.

Materials and Methods: A full necropsy and histologic examination was performed.

Results:  At necropsy, a circular ulcerative lesion was found within the dorsal air sac with multiple blood clots. Histologically, the blood clots contained large groups of filamentous organisms with a ‘cloud-like’ appearance, which stained Periodic acid–Schiff positive and variable Grocott's methenamine silver positive. Multiple PCR analyses could not reveal the exact etiology.

Conclusions:  This is a very rare case of lethal ulceration of the blowhole in a common bottlenose dolphin caused by unknown filamentous bacteria.


Poster 113: METASTATIC TUBULOPAPILLARY MAMMARY CARCINOMA IN A LEOPARD (PANTHERA PARDUS)

I.C. Šoštarić-Zuckermann*, B. Artuković*, O. Smolec†, A. Gudan Kurilj*, D. Huber*, Ž. Grabarević* and M. Hohšteter*

*Department of Veterinary Pathology and  †Surgery, Orthopedics and Ophtalmology Clinic, Veterinary Faculty, University of Zagreb, Zagreb, Croatia

Introduction: Mammary tumors are common, usually malignant tumors of domestic cats and also wild felids. This report presents a case of metastatic mammary carcinoma in a leopard.

Materials and Methods: A multilobulated mammary mass was excised from a 16 year, female leopard from Zagreb Zoo and submitted for evaluation. About a year later, the animal died and necropsy was performed. A routine HE and IHC staining with Ki-67 and E-cadherin were used on the initial tumor sample and also on tumor masses collected at necropsy.

Results: Microscopic examination of the initial tumor revealed an infiltrative tubulopapillary mass that invaded the lymph vessels. Mitotic index was moderate (15 per 10 HPF). The tumor was classified as tubulopapillary carcinoma of the mammary gland. Macroscopically, the animal was emaciated. Numerous nodular metastases were evident in the lymph nodes, liver, uterus, and spleen. Smaller microscopic metastases were found in the lung. Histological appearance of the metastases was comparable to the primary tumor. In the primary tumor, Ki-67 proliferation index was 28.7%, and E–cadherin expression was 56.8%. In the metastases, Ki-67 and E-cadherin expression averaged 17.1% and 41.9%, respectively.

Conclusions: This case depicts an otherwise common feline tumor in a large felid. Expression of E-cadherin shows a small drop in the metastases suggesting additional anaplasia of the metastatic tumor cells. The slight drop in the Ki-67 expression could be due to more widespread necrosis in the metastases. This case postulates that mammary tumors in wild felids have similar clinicopathological features as in domestic cats.


Poster 114: RENAL NEPHROBLASTOMA IN A GREEN ANACONDA (EUNECTES MURINUS)

M. Tecilla*, W. Magnone, C. Sandri and P. Roccabianca*

*DiMeVet, University of Milan, Via Celoria 10, 20133, Milan, Italy.Parco Natura Viva - Garda Zoological Park srl, Località Figara 40, 37012 Bussolengo, Italy

Introduction: Nephroblastoma is a renal tumor reported in higher and lower vertebrates. In higher vertebrates, nephroblastoma is more often a pediatric tumor arising from metanephric rests. On the contrary, in lower vertebrates, nephroblastoma derives from the opistonephros (mature kidney). Nephroblastomas seem extremely rare in snakes. The aim of this report is to document the second case of nephroblastoma in an anaconda.

Materials and Methods: A 20 year-old green, male anaconda was ultrasonographically evaluated for severe coelomic distension caused by a bilateral renal mass in 2012. The animal was monitored once a year and due to the progressive increase of tumor size and development of anorexia euthanasia was elected in 2017.

Results: The left kidney was replaced by a 3 Kg, multilobulated tan-white neoplasm and was surrounded by a large hematoma. Histologically a triphasic tumor composed of embryonic mesenchyme, primitive glomeruli and nests of polygonal cells (blastema) were observed. A definitive diagnosis of nephroblastoma was granted.

Conclusions: Nephroblastomas have been anecdotally reported in adult snakes and described only in one anaconda. However, nephroblastoma in snakes is a senile tumor since, contrary to mammals and birds, postembryonic nephrogenesis occurs in reptiles (including Corn snake). WT1 pathway is involved in renal development and increased WT1 expression has been associated with onset of renal neoplasia such as nephroblastoma in several mammalians and in renal cell carcinoma only in man.


Poster 115: NUTRITIONAL MUSCULAR DYSTROPHY IN CHINCHILLA LANIGERA

T.B. Lagoa*, E.F. Bondan*,** and S.V. Santos*

*Department of Veterinary Medicine, University Cruzeiro do Sul, São Paulo, Brazil, and **Department of Environmental and Experimental Pathology, University Paulista (UNIP), São Paulo, Brazil

Introduction: Nutritional muscular dystrophy (NMD), also known as white muscle disease, is a subacute or acute disease of the cardiac and/or skeletal striated musculature of multifactorial origin, although frequently associated with the deficiency of selenium and vitamin E in dietary intake. Deficiencies of these nutrients can lead to oxidative cell damage as they are important as reactive oxygen species scavengers. Chinchillas include two species of crepuscular rodents that are native to the Andes mountain range in South America. The two living species of chinchilla are Chinchilla chinchilla and Chinchilla lanigeraC. lanigera has longer tail and ears and thinner neck than C. chinchilla. Domesticated chinchillas are thought to have come from the C. lanigera species. Little is known about the minimum nutritional requirements for these rodents and, sometimes, nutrient composition is not even discriminated in commercial food rations for the species.

Materials and Methods: A 10-month-old, male specimen of Chinchilla lanigera raised in captivity was submitted to necropsy after presenting signs of cardiorespiratory decompensation, muscular weakness and permanent decubitus. Tissue samples were collected for histopathological examination.

Results: Microscopic analysis revealed segmental rhabdomyolysis (myonecrosis, inflammation and increased endomysial connective tissue) and dilated cardiomyopathy, severe pulmonary edema and moderate renal tubular necrosis. Histopathological findings and the history of poor nutritional management were fundamental to establish the diagnosis for NMD.

Conclusions: To our knowledge this is the first description of the disease in chinchillas and it aims to alert clinicians of exotic animals about the susceptibility of these rodents to NMD.


Poster 116: PATHOLOGICAL STUDY ON PROLIFERATIVE ENDOMETRIAL LESIONS IN 50 FOUR-TOED HEDGEHOGS (ATELERIX ALBIVENTRIS)

J. K. Chambers*, T. Shiga*, H. Takimoto, A. Dohata*, Y. Miwa, H. Nakayama* and K. Uchida *

*Laboratory of Veterinary Pathology, The University of Tokyo and Miwa Exotic Animal Hospital, Japan

Introduction: In recent years, the four-toed hedgehog (Atelerix albiventris) has become a popular household pet in Japan. Uterine tumours are commonly encountered in this animal species. Clinical manifestation, histopathological findings and immunohistochemical features of the endometrial cells were examined.

Materials and Methods: Uteri from 50 four-toed hedgehogs (5 to 51 months old) with clinical signs of uterine disease were histopathologically and immunohistochemically examined. Radiographic, ultrasonographic and haematologic findings were reviewed. Outcome of treatment (ovariohysterectomy) was evaluated.

Results: Sixteen animals were diagnosed with endometrial hyperplasia, and 34 were diagnosed with endometrial neoplasia. The neoplasms were classified into endometrial mixed tumours (n=7), endometrial stromal nodules (n=20), and endometrial stromal sarcomas (n=7).  However, the endometrial stromal nodules and endometrial stromal sarcomas always developed within the mixed tumour lesion.  Interestingly, the stromal nodules and the stromal components of the mixed tumours displayed extraendometrial differentiation, e.g., adipocytes, granular cells, smooth muscle cells. Endometrial stromal sarcomas exhibited severe cellular atypia and invaded the subendometrial tissue. Two animals that were diagnosed with endometrial stromal sarcomas developed recurrent tumours.  Survival rates at 180 days after ovariohysterectomy were 66.6 % in endometetrial hyperplasia; 100.0% in endometrial mixed tumour; 66.6% in endometrial stromal nodule; 50.0% in endometrial stromal sarcoma.  Immunohistochemical examinations demonstrated that the stromal cells of the hyperplastic endometrium as well as the tumours were positive for CD10, progesterone receptor, and Wilms’ tumour 1.

Conclusions: The four-toed hedgehog develops unique uterine neoplasms that are mainly composed of endometrial stromal cells and probably arise from endometrial mixed tumours.


Poster 117: REPEATABILITY OF MYODEGENERATION SCORING IN BROILER WOODEN BREAST MYOPATHY

H.-K. Sihvo*, N. Airas*, O. Simolaand D.K. Meyerholz

*Department of Veterinary Biosciences, University of Helsinki, Finland, Orion Corporation Orion Pharma, Finland, and Department of Pathology, University of Iowa, Iowa City, Iowa USA

Introduction: Myodegeneration is the main histologic finding in wooden breast myopathy (WB), which is macroscopically denoted by hardened muscle consistency and pale color of the Pectoralis major muscle in broiler chickens. A recent publication suggested a four-tier scoring of myodegeneration in the WB diagnosis. The aim of our study was to evaluate the interobserver repeatability of the scoring and the correlation of the re-scored values with the macroscopic WB classification.

Materials and Methods: Histological sections of broiler chicken Pectoralis major muscles (n = 25) were retrieved from the original scoring study material published previously. The samples were chosen by stratified random fashion, representing macroscopically unaffected and WB cases and all histological scores (0-3). Two veterinary pathologists scored the samples based on the previously published scoring criteria. The pathologists were comprehensively masked to the macroscopic WB classification.

Results: The interobserver repeatability of the scoring between the original scoring and pathologist A was good, Cohen’s κ = 0.67, P < .0005 and moderate for pathologist B, κ = 0.53, P < .0005. Spearman correlation between the histologic scoring and the macroscopic WB classification was > 0.8, P < .0005 for all pathologists (original and re-scoring).

Conclusions: The WB myodegeneration scoring criteria yields moderate to good repeatable results between pathologists and the degeneration scores strongly correlate to the macroscopic appearance of the muscle. Unlike in other species, minimal myodegeneration typically occurs also in macroscopically unaffected broiler chickens and the evaluation of repeatability highlights the importance of a detailed scoring criteria description in publications.


Poster 118: RETROVIRAL INFECTIONS IN TWO ALBINO BURMESE PYTHONS (PYTHON MOLURUS BIVITTATUS)

G. H.  Woo

Department of Clinical Laboratory Science, Semyung University, Jecheon 27136, Republic of Korea

Introduction: Parvoviral infections, iridoviral infection, adenoviral infections, and paramyxoviral infections have been most frequently documented in snakes. Two albino Burmese pythons were submitted for diagnostic investigations from private zoos and pet shops. In the present study, we provide information about the molecular epidemiology, clinical signs, gross findings, and histopathological findings of virus.

Materials and Methods: Histopathology of all tissues and organs was performed. Nasal swabs, and intestine and liver samples were cultured for bacterial isolation. Viral examination was done by PCR  and edited sequences were compared with database sequences (GenBank, NCBI) and identified by BLAST searching.

Results: Grossly, the affected animals had respiratory problems and digestive problems. At necropsy, the heart was enlarged and straw-color fluid filled the pericardium. Ulcers were scattered through the intestinal mucosa and whitish-yellow foci were scattered on the liver. Histopathologically, pneumonia was observed and multifocal necrotic foci and fatty changes were seen in the liver. Multifocal ulceration was observed in the intestine. A retroviral gene was amplified in all organs and tissues of two animals.

Conclusions: This polymerase gene of the retrovirus showed high identity to python molurus endogenous retrovirus (accession no. AF500296.1) and python curtus endogenous retrovirus (accession no. AF500299.1).

Poster 119

SINGLE LEFT CORONARY ARTERY IN A COMMON HIPPOPOTAMUS (HIPPOPOTAMUS AMPHIBIUS)

D. Marinković1, M. Aničić1, Ј. Özvegy2, O. Lozanče3 and S. Aleksić-Kovačević1

1 Department of Pathology,3 Department of Anatomy, Faculty of Veterinary Medicine, University of Belgrade, Serbia and 2 Belgrade Zoo Garden, Belgrade, Serbia

Introduction: Single left coronary artery (LCA) is a rare congenital anomaly of the coronary circulation, and often the right coronary artery (RCA) arises as one of the branches of the LCA. This report describes findings of a congenital single LCA with RCA arising as one of its branches in a common hippopotamus (Hippopotamus amphibius).

Materials and Methods: One year old, female inbred common hippopotamus suddenly died without any prior clinical symptoms. A complete necropsy and histopathological examination was performed.

Results: Macroscopically, in the aneurismatic aortic sinus a single widened ostium of the LCA was present. Also the absence of the RCA with blind fossa was noted. This single, 9 mm wide LCA arose above the caudosinistral cusp and reached the coronary groove by passing between the left auricle and the pulmonary trunk and it then divided to form three dilated contortuous branches; the left (paraconal) interventricular branch, left circumflex branch that followed the coronary groove toward the caudal aspect of the heart, and the third branch which represents the RCA. The RCA in turn had a right circumflex branch that followed the coronary groove which divided to form the right (subsinuosal) interventricular branch and coronary branch. Microscopically, degenerative changes of myocardium, pulmonary congestion, oedema and emphysema, as well as passive congestion of the liver were noted.

Conclusions: According to available data this is the first described case report of congenital single left coronary artery in a common hippopotamus (Hippopotamus amphibius).


Poster 120: THE INFLUENCE OF TRT ON THE PROLIFERATION OF LYMPHOCYTES ISOLATED FROM THE BLOOD AND BLOOD-FORMING ORGANS OF RAINBOW TROUT (ONCORHYNCHUS MYKISS)

E. Terech-Majewska*, A.K. Siwicki,  E.Kaczorek, J. Pajdak* and P. Schulz

* Department of Epizootiology, Faculty of Veterinary Medicine, University of Warmia and Mazury, Poland and Department of Fish Pathology and Immunology, Inland Fisheries Institute, Poland and Department of Microbiology and Clinical Immunology, Faculty of Veterinary Medicine, University of Warmia and Mazury, Poland

Introduction: Trans-resveratrol is a potent antioxidant with anti-inflammatory and chemopreventive properties, naturally occurring in many fruits and plants. The aim of this study was to determine the in vitro influence of different concentrations of trans-resveratrol  (TRT) on  lymphocyte activity isolated from the blood and blood-forming organs of rainbow trout (Oncorhynchus mykiss).

Materials and Methods: A  stock solution of trans-resveratrol (Sigma-Aldrich®) with a concentration of 0.001M in 1M dimethyl sulfoxide – DMSO (POCH S.A.) was diluted to final concentrations of 1.25, 2.5, 10 and 20 μM with the RPMI-1640 medium (Sigma-Aldrich) on microtitre plates. The experiment was performed on cells isolated from whole blood and blood-forming organs (head kidney and the spleen) of four rainbow trout (Oncorhynchus mykiss) weighing 1-1.5 kg each. Proliferative activity of lymphocytes was studied by MTT assay.

Results: An analysis of TRT's effect on the proliferation of rainbow trout lymphocytes revealed that the examined compound is a weak immunomodulator for T cells and suppressed the proliferation of B cells isolated from the blood of rainbow trout. MTT of Con A-stimulated lymphocytes from the spleen and head kidney of rainbow trout decreased significantly in response to all five concentrations of TRT. The compound also inhibited proliferation of B lymphocytes isolated from the organs in 5µM and 10µM dose.

Conclusions: TRT did not induce clear dose-dependent response in the immunocompetent cells of rainbow trout.


Poster 121: TRAUMATIC DEATH DUE TO FORCED FEEDING IN A BURMESE PYTHON

R.I. Rizac, E. Ciobotaru, G. Dinescu, T. Soare and M. Militaru

Faculty of Veterinary Medicine, University of Agronomic Sciences and Veterinary Medicine of Bucharest, Romania

Introduction: Snakes naturally feed on large prey, compared to their body size, due to infrequent meals, anatomic characteristics of cranial and trunk bones and an expansible skin in the anterior region. Forced feeding of the captive reptiles – although recommended only in emergency cases and under specific conditions – is sometimes found among owners, and may lead to injuries or death of the animal. Clinical aspects on this matter are known, but without picturing the underlying morphology.

Materials and Methods: One subadult female Python bivittatus was submitted for postmortem investigations due to sudden death. Classic histopathology was performed.

Results: The exterior examination revealed numerous Ophionyssus mites. The gross evaluation of the structures in the coelomic cavity revealed the heart with both atria and ventricle being dark red and having a flaccid consistency, the lung with the anterior parenchyma – corresponding to the heart – exhibiting dark red areas, and the esophagus distended in the precardiac segment by a large amount of raw meat. The liver was slightly discolored, while the other organs showed no gross lesions. Histopathology revealed a severely altered myocardial structure, with large hemorrhagic areas among muscle fibers, edema and hemorrhages within the lung parenchyma, micro and macrovesicular hepatic lipidosis, uric acid deposits, with a granulomatous reaction and a mononuclear infiltrate within the kidneys and lymphocyte depletion within the white splenic pulp. 

Conclusions: The death of the animal was caused by failed forced feeding maneuvers, inducing the obstruction of the airways and the destruction of the myocardium, suggesting a traumatic mechanism.   


Poster 122: Cancelled


Poster 123: ENCEPHALITIS OF UNKNOWN ORIGIN IN HORSES FROM BRAZIL

G. S. Boos *, D. Driemeier † , E. M. Colodel ‡, M. B. Castro § , J. D. Barbosa Neto ¥ , M. Eickmann ¦ and C. Herden **

*and ** Institute for Veterinary-Pathology, Justus-Liebig-University, Gießen, Germany, Department of Veterinary Pathology, Federal University of Rio Grande do Sul, Porto Alegre, Brazil, Veterinary Pathology Laboratory, Federal University of Mato Grosso, Cuiabá, Brazil, University of Brasília, Brasília, Brazil, Federal University of Pará, Castanhal, Brazil and ¦ Institute for Virology, Philips-University, Marburg, Germany

Introduction: Equine encephalitis of unknown etiology from Brazil was further investigated by establishing tissuebased diagnostic tools.

Materials and Methods: Paraffin-embedded CNS from equines with neurological signs were investigated with IHC for rabies virus, Borna disease virus (BDV), EHV-1, Listeria sp., Toxoplasma gondii, Neospora caninum, Sarcocystis neurona, double-stranded RNA (dsRNA) and for astroglial and microglial activation by GFAP and Iba1, respectively. RNA isolation was modified to minimize cross-linking damages, and nucleic acid quality was verified with equid- GAPDH PCRs of 64-517bp. Pan-PCRs were carried out for viruses and coccidia.

Results: Out of 35 CNS samples 5 brains displayed non-inflammatory lesions, while 27 cases presented lymphocytic and 3 granulomatous infiltration. This was associated with microglial activation (mild in 20/35, moderate in 9/35, and severe in 5/35 horses). Astroglial activation was mild in 9/35 brains, moderate in 22/35 and severe in 4/35 cases. A GAPDH 64bp or 170bp product was amplified in 26 or 23 samples, respectively. No specific agent was determined with pan-PCRs lengthening ≥ 200bp. Rabies virus antigen and RNA were demonstrated in 1/35 horses and S. neurona schizonts in 4/35 brains. Arboviruses RNA (flavivirus, alphavirus) were detected in 2/35 samples, and EHV-1 DNA in 3/35 brains.

Conclusions: The inflammatory lesions and glial reaction pattern point to an infectious etiology albeit agents were detected only in 10/35 horses. For FFPE material special conditions had to be used which allowed the first identification of Flavivirus in a horse from Brazil. This study raises awareness to new emergent and re-emergent diseases in Brazil.


Poster 124: NEOPLASMS IN CAPTIVE SNAKES

S.V. Santos1,2, P.S. Quessada2, V.C. Garcia3, P.L. Ramos3, C.F.H. Takakura4, E.F. Bondan2 and V.P. Andrade1

1A.C. Camargo Cancer Center, São Paulo, Brazil, 2Department of Veterinary Medicine, University Cruzeiro do Sul, São Paulo, Brazil, 3University of São Paulo (USP), Brazil, and  4Zoological Park Foundation of São Paulo, Brazil

Introduction: Brazil has more than 10% of the serpent species in the world. Neoplastic diseases were once thought to be rare in reptiles, although it is now known that snakes are frequently affected by malignant neoplasms. Nevertheless, the etiopathogenesis of these tumors is still poorly known.

Materials and Methods: Forty-one snakes (27 females; 14 males) kept in captivity in the Zoological Park Foundation of São Paulo (Brazil) were investigated for tumor detection. Tumor samples were collected for histopathological investigation and microbiological and molecular analyses were performed for identifying possible infectious etiologies.

Results: Of the 41 snakes included in the study, 22 (53.66%) presented neoplasms- one benign neoplasia (4.55% from 22) and 21 malignant neoplasms (95.45%). The latter included 9 sarcomas (40.9%), 5 carcinomas (22.73%), 4 hemo-lymphoproliferative neoplasms (18.18%) and 3 round cell tumors (13.64%). The most affected genera were Bothrops sp. (5/22.73%), Spilotes sp. (4/18.18%) and Oxyrhopus sp. (3/13.64%). Body sites affected included the skin, the gastrontestinal tract, the liver and biliary tract, lymphoid and haematopoietic tissues, followed by oviducts and bones. The most commonly reported clinical signs were dehydration (59.09%) and anorexia (45.45%). All 22 snakes underwent surgical excision and conservative therapy. Mortality rate reached 100% and two snakes (9.9%) presented bone and lung metastases. The presence of viral particles compatible with A-like retroviral particles was found in most cases.

Conclusions: Although literature data on oncology of reptiles consist mainly of single-case reports, our study confirms the neoplastic disease as an important cause of morbidity and mortality in snakes.