Oral A – 1 : T. REGULATORY AND PROINFLAMMATORY CYTOKINES EXPRESSION IN SHEEP VACCINATED WITH CATHEPSINE L1 AND CHALLENGE WITH FASCIOLA HEPATICA.

I. Pacheco- Luque1, R. Zafra2, N. Abril3, N. Morales-Prieto3, M.T. Ruiz1, M.J. Bautista1, V. Molina1, A. Martínez-Moreno2 and J. Pérez 1

1 Department of Anatomy and Comparative Pathology. Veterinary Faculty. University of Córdoba, Spain.2 Department of Animal Health (Parasitology). Veterinary Faculty. University of Córdoba, Spain.3Department of Biochemistry and Molecular Biology. Faculty of Sciences. University of Córdoba, Spain.

Introduction: The aim of this study was to evaluate by qRT-PCR the expression of interleukin-10 (IL-10), transforming growth factor-β (TGF β), Foxp3, interleukin-1β (IL-1 β) and tumor necrosis factor alpha (TNF α) in liver of sheep (immunized and infected) at early stages of infection with F. hepatica.

Material and methods: Forty-four 7-month old female Merino-breed sheep were distributed in three groups: group 1 (n=20) was immunized subcutaneously with recombinant CL1 from F. hepatica in Montanide adjuvant; group 2 (n=20) was used as non-immunized group. Group 3 (n=4) was used as uninfected and unimmunized group. Groups 1 and 2 were orally infected with 200 metarcercariae of F. hepatica and divided into four subgroups each (n=5) which were sacrificed in pairs at 1 day post infection (dpi), 3 dpi, 9 dpi and 18 dpi.

Results: Compared with uninfected group, FoxP3 expression showed a significant increase (P<0.0001) at 9 and 18 dpi. The RNA expression of IL10 showed a slight increase during the experience. Gene expression level for TGFβ showed a significant and progressive increase in both infected and immunized groups (P<0.05 and P<0.001 respectively). The expression of TNFα and IL-1β increased significantly and progressively.

Conclusions: The results of the present work suggest that F. hepatica induces and increased expression of Treg (Foxp3) and IL10, which may be a mechanism of immune evasion during the early stages of hepatic migration. The increase of proinflammatory cytokines may be related to the severe inflammatory response induced by necrosis caused by larval hepatic migration.


Oral A – 2 : IMPROVED MEASUREMENT OF LESIONS FOR ASSESSMENT OF EFFICACY OF TUBERCULOSIS VACCINES IN SMALL RUMINANT MODELS: FROM SCORING TO IMAGING AND VOLUMETRIC QUANTIFICATION

M. Domingo*†, E. Vidal, Y. Espada, X. Moll, A. Balseiro§, A. Canturri* and B. Pérez de Val

*Servei de Diagnòstic Patologia Veterinària, Departament de Sanitat I Anatomia Animal, Universitat Autònoma de Barcelona (UAB), Bellaterra, Barcelona, Catalonia (Spain), IRTA, Centre de Recerca en Sanitat Animal (CReSA, IRTA-UAB), Campus UAB, Bellaterra, Barcelona, Catalonia, Spain, Departament de Medicina i Cirugia Animals, UAB, Bellaterra, Barcelona, Catalonia, Spain, §SERIDA, Servicio Regional de Investigación y Desarrollo Agroalimentario, Centro de Biotecnología Animal, Gijón, Spain.

Introduction: Interest in tuberculosis (TB) vaccines for humans and animals is renewed, and small ruminants are suitable models for TB vaccine efficacy assessment. Goats and sheep are natural hosts for Mycobacterium bovis and M. caprae, and are easier to be held in laboratory settings than cattle. The reduction of macroscopic lesions and mycobacterial burden are considered main indicators of protection. Therefore, there is a need to accurately quantify both parameters.

Materials and Methods: Visual assessment followed by semi-quantitative scoring of lesions has been substituted by computed tomography and quantitative volumetric assessment of lung lesions in an experimental model of tuberculosis both in goats and sheep. Animals are inoculated with M. caprae under anesthesia (1,000-1,500 cfu.) by the endobronchial route. After 12-14 weeks, trachea and lungs are carefully removed at necropsy without excising the pleural surface, and respiratory lymph nodes are removed for quantification of total mycobacterial burden. Lungs are fully inflated by intratracheal infusion of 10% formalin by gravity, the trachea is then tied, and lungs are immersed in 10% formalin for 3-4 weeks. CT scan is then performed to quantify the volume of TB lesions.

Results: This methodological approach allows bacteriological (respiratory lymph nodes) and pathological (whole lungs) assessment of vaccine efficacy in challenge experiments of BCG vaccinated small ruminants. Mycobacterial burden is expressed as whole bacterial burden in lymph nodes, and lesions as absolute volume or as a ratio of total lung volume.

Conclusions: BCG-vaccination reduces significantly lesions and mycobacterial burden in small ruminants. CT-scan allows quantitative pathological assessment.


Oral A – 3 : STUDIES ON INJECTION SITE REACTIONS CAUSED BY ALUMINIUM-CONTAINING PRODUCTS IN SHEEP

J. Asín*, J. Molín*, M. Pérez*, P. Pinczowski*, M. Gimeno*, N. Navascués*, A. Muniesa*, I. de Blas*, D. Lacasta*, A. Fernández*, L. de Pablo, M. Mold, C. Exley, D. de Andrés, R. Reina and L. Luján*

*University of Zaragoza, Spain; Institute of Agrobiotechnology, CSIC-UPNA, Spain; and The Birchall Centre, Keele University, United Kingdom.

Introduction: Aluminium (Al) containing adjuvants are widely used in sheep vaccines to promote an effective immune reaction against antigens but induce local injection-site reactions. This work aims to characterize these reactions and to determine the role of the Al in its genesis.

Materials and Methods: 84 lambs divided into 3 groups (n=28 each) were inoculated with a different substance: A) Vaccines containing aluminium hydroxide; B) Aluminium hydroxide only; C) PBS. Animals received 19 subcutaneous inoculations along 15 months. Injection-site reactions and the regional lymph node (LN) were studied by gross and microscopic pathology, microbiology, fluorescence microscopy with lumogallion, Transmission Electron Microscopy (TEM), Energy Dispersive X-Ray Spectroscopy (EDS) and Graphite Furnace Atomic Absorption Spectroscopy (GFAAS).

Results: Injection-site reactions consisted ofgranulomas that were more numerous (p<0.001) and with more severe central necrosis (p=0.021) in group A than in group B. Most of the vaccinated lambs (76.9%) showed more than 7 granulomas and all granulomas could be recovered in certain cases. Macrophages in the granulomas showed an orange fluorescence emission (590 nm), typical of Al. Similar groups of macrophages were observed in the regional LN. By TEM, macrophages in the granulomas contained aggregates of a spiculated electrondense material identified as Al by EDS. Group A showed longer Al particles than group B (p<0.001). By GFAAS, group A showed higher Al concentration in the regional LN than group B (p<0.001).

Conclusions: Al induces persistent, immunomediated subcutaneous granulomas and the reactions are more severe in vaccinated animals. Al can reach the regional lymph node.


Oral A – 4 : SHEEP AND BLUETONGUE VIRUS: AN ANIMAL MODEL FOR STUDYING THE PATHOGENESIS OF HYPOFERTILITY CAUSED BY ARBOVIRUSES

D. Pintus*, G. Puggioni*, E. Melzi, G. Meloni*, A.M. Rocchigiani*, D. Manunta*, M. Palmarini, G. Savini, A. Oggiano* and C. Ligios*

*Istituto Zooprofilattico Sperimentale della Sardegna, Sassari, Italy, MRC-Centre of Virus Research, University of Glasgow, United Kingdom, and Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise, Teramo, Italy

Introduction: Testicular degeneration with destruction of the tubular germinative cells has been often described in human and animals as a result of viral infections. However, the pathogenesis leading to the oligospermia/azoospermia condition is little known and studies have only referred to a murine model. Herein, Bluetongue virus (BTV) in rams was used as an animal model for studying the pathological consequences of Arbovirus infection on male reproductive tract.

Materials and Methods: By using blood samples collected from naturally BTV serotype 1-infected sheep, two groups of rams were experimentally infected and serially euthanized. At necropsy tissues were collected for virological, immuno-histopathological and confocal immunofluorescent examinations.

Results: Real Time RT-PCR confirmed the presence of BTV in the testis, which histologyda severe degeneration of the germinative epithelium leading to azoospermia was noted. NS2 BTV protein was immunolocalized in the endothelial cells of the intertubular blood vessels in the testicle between 5 and 11 days post infection. Vimentin, interferon-induced MX-1 protein, α-inhibin and 3β-hydroxysteroid dehydrogenase expression profiles demonstrate that infertility was due to impaired testosterone synthesis in the Leydig cells.

Conclusions: Our findings suggest that, although BTV did not directly infect germ cells, Sertoli cells or Leydig cells, its localization in the endothelium of the vessels causes testicular degeneration by a similar pathogenetic mechanism observed in Zika and Mumps viruses infections. In addition, this is the first experimental evidence of infertility caused by Arbovirus in natural male hosts and underlines the pathological effect that endotheliotropic RNA viruses might have on male fertility.


Oral B - 1 : CORNEAL EPITHELIAL HYPERPLASIA COMBINED WITH A SUPERFICIAL CORNEAL STROMAL COLLAGEN DISORDER AS THE MAIN UNDERLYING CHANGES IN TROPICAL KERATOPATHY (FLORIDA SPOTS) IN CATS

S. J. Kelly*, P. J. Kelly, H. C. Wells*, K. H. Sizeland, R. G. Haverkamp*, A. Armien^ and P. Bolfa#

*School of Engineering and Advanced Technology, Massey University, New Zealand,  Australian Synchrotron, Clayton, Australia, ^Veterinary Diagnostic Laboratory, College of Veterinary Medicine, University of Minnesota, St. Paul, USA, Department of Clinical Sciences and #Department of Biomedical Sciences, Ross University School of Veterinary Medicine, St. Kitts

Introduction: Small, inactive, corneal opacities (tropical keratopathy; TK) are common in animals and people in tropical to subtropical areas. To study the anatomical changes in TK we sampled affected and normal cats from St Kitts, West Indies, for examination by light and electron (TEM) microscopy and synchrotron-based small angle X-ray scattering (SAXS) analysis.

Materials and Methods: Punch biopsies (8) of typical TK lesions from 5 affected cats and from normal eyes  of 5 unaffected cats were stored in 10% phosphate buffered formalin, 2.5% gluteraldehyde, and 0.25% Triton X before routine processing.

Results: HE staining showed no inflammation, neovascularization or fibrosis in lesions which were characterized by progressive corneal epithelial hyperplasia towards their centers revealing 13-15 cell layers versus 6 -9 layers in normal eyes. The corneal stroma beneath the lesions was denser and also became progressively thicker towards the lesion center. Besides epithelial hyperplasia, TEM revealed increased thickness of the Bowman membrane and disorganization of the anterior stroma with a more undulating arrangement of the collagen. SAXS imaging revealed the collagen in the lesion was more regularly arranged than in the normal cornea with collagen fibril diameter (~500A) and size distribution (~350A) being greater in the lesion (normal cornea ~300A and ~110A).

Conclusions: There is no evidence TK is caused by toxic or infectious agents with anatomical changes more consistent with UV light induced epithelial hyperplasia and associated collagen realignment. The latter is most likely the cause of the opacification of the lesions


Oral B - 2 : FELINE HYPERTROPHIC CARDIOMYOPATHY (HCM): MORPHOLOGICAL CHARACTERIZATION OF CARDIAC REMODELLING PROCESSES

s. Kitz*, 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: Hypertrophic cardiomyopathy (HCM) is the most commonly diagnosed myocardial disease in cats. Despite some studies into the underlying mechanisms, the complex pathophysiology of HCM is still far from clear. Myocardial remodelling is certainly a key process, and cardiomyocyte disarray, interstitial fibrosis, leukocyte infiltration and vascular dysplasia are described histopathologic features. The aim of the present study was to further investigate the pathological processes.

Materials and Methods: A detailed investigation was performed on 22 HCM cases and hearts from 9 cats without cardiac disease (controls), using histology, immunohistology and digital image analysis to assess the morphological changes, alongside quantitative reverse transcriptase PCR (qRT-PCR) for remodelling mediators in selected cats.

Results:  In HCM, the myocardium exhibited a significantly higher amount of interstitial fibrous tissue and interstitial space, and significantly more interstitial cells (almost exclusively Iba1-positive macrophages). In contrast, the overall cellularity and the amount of small to medium-sized vessels was significantly lower than in control myocardia, whereas the space occupied by cardiomyocytes was unaltered. In addition, focal lesions of fibrosis with neovascularisation and mononuclear infiltration were frequently observed. In HCM, the transcription of inflammatory (IL-1, IL-6, IL-18, TNF-α), and profibrotic (TGF-ß, MMP13, TIMP1) mediators was significantly increased, whereas IL-8 mRNA levels were significantly lower.

Conclusions: Our results suggest that cardiac remodelling in HCM is a cardiomyocyte- and macrophage-driven process in a pro-inflammatory and pro-fibrotic environment, and confirm that thickening of the cardiac wall is rather the consequence of expansion of the interstitial space than of cardiomyocyte hypertrophy.


Oral B - 3 : GENETIC VARIANTS IN KCNJ10 AND ATP1B2 IN MALINOIS PUPPIES WITH SPONGY DEGENERATION AND CEREBELLAR ATAXIA

S. Högler*, N. Mauri, M. Kleiter, M. Leschnik, A. Oevermann§, D. Henke**, E. Dietschi, M. Wiedmer, J. Dietrich, F. Steffen††, S. Schuller‡‡, C. Gurtner§§, N. Stokar-Regenscheit§§, D. O’Toole***, T. Bilzer†††, C. Herden‡‡‡, V. Jagannathan and T. Leeb

*Institute of Pathology and Forensic Veterinary Medicine and University Clinic for Small Animals, University of Veterinary Medicine Vienna, Austria, Institute of Genetics, §Division of Neurological Sciences, **Division of Clinical Neurology, ††Section of Neurology, ‡‡Division of Small Animal Internal Medicine, §§Institute of Animal Pathology, Vetsuisse Faculty, University of Bern, Switzerland, ***Wyoming State Veterinary Laboratory, University of Wyoming, United States of America, †††Institute of Neuropathology, University Hospital Düsseldorf, Germany and ‡‡‡Institute of Veterinary Pathology, Justus-Liebig-University Gießen, Germany

Introduction: Severe ataxia with an onset at 4-8 weeks has been previously reported in the Malinois variety of Belgian Shepherd dogs. Ataxia was associated with histologically confirmed spongy degeneration of the cerebellar nuclei.

Materials and Methods: We performed a genetic investigation in six closely related families with ataxic puppies and seven additional isolated cases. In 12 puppies belonging to these six families neurological examination and in nine of these 12 puppies necropsy and histological analysis were performed.      

Results: The affected dogs from four families and one isolated case shared a missense variant on chromosome 38 in the KCNJ10 gene encoding a potassium channel. In all four affected puppies from another family and another one of the isolated cases we detected a structural variant in the ATP1B2 gene encoding a subunit of the Na+/K+-ATPase. Neurological examination of the affected puppies revealed generalized ataxic gait in all animals. Puppies with the ATP1B2 variant additionally showed seizures, pacing, circling, and central blindness. Moreover, clinical signs progressed rapidly. Histologically a bilateral symmetrical vacuolar degeneration of the neuropil targeting the cerebellar nuclei was present in both variants. However, in the KCNJ10 variant further degenerative lesions were present in the vestibulocochlear tract, while the ATP1B2 variant showed neuronal necrosis in spinal cord, hippocampus, caudate nucleus and cortex.

Conclusions: Neurological as well as histological examination showed differences between both variants. However, we recommend genetic testing for discrimination of the genetic variants. Future studies including immunohistochemistry are required to further characterize these two genetic variants by histopathology.


Oral B - 4 : PATHOLOGICAL AND CLINICAL FEATURES OF CANINE CLITORAL CARCINOMA

R. Verin1, F. Cian2, J. Stewart3, D. Binanti4, A.L. MacNeill5, M. Piviani6, P. Monti7, G. J. Baroni8, S. Le Calzez9, T. J. Scase10 and R. Finotello6

1 Department of Veterinary Pathology and Public Health, Institute of Veterinary Science, University of Liverpool, United Kingdom 2 Batt Laboratories Ltd, Coventry, UK 3Centre of Preventative Medicine, Animal Health Trust, Newmarket, UK  4AbLab, Veterinary Diagnostic Laboratory, Sarzana (La Spezia), Italy 5Departments of Microbiology, Immunology & Pathology, College of Veterinary Medicine and Biomedical Science, Colorado State University, Fort Collins, CO, USA 6Small Animal Teaching Hospital, Institute of Veterinary Sciences, University of Liverpool, Liverpool, UK 7DWR Diagnostics, Dick White Referrals, Newmarket, UK  8Division of Pathological Anatomy, Department of Surgery and Translational Medicine, University of Florence, Florence, Italy 9IDEXX Laboratories, Wetherby, West Yorkshire, UK 10Bridge Pathology Ltd, Bristol, UK

Introduction: Female genital tumours are uncommon in dogs and knowledge on clitoral cancer is restricted to few carcinoma cases. In those, clinical and pathological similarities with apocrine gland anal sac adenocarcinoma (AGASACA) have been suggested.     

Materials and Methods: Cases of primary canine clitoral carcinomas (CCCs) were included if pathological samples and detailed clinical records were available for review. Cases were investigated with cytology, histopathology, immunohistochemistry for pan-cytokeratin and neuroendocrine markers including Chromogranin A (CGA), Synaptophysin (SYN), Neuron Specific Enolase (NSE), S-100 and Transmission Electron Microscopy (TEM).

Results: Six cases were retrieved. Locoregional metastases were documented in 3/6 cases and paraneoplastic hypercalcemia was present in 2/6 dogs. Cytology suggested an epithelial neoplasm with apocrine differentiation in all cases.  Histologically, there were tubular, solid, rosette and/or mixed patterns. Pan-cytokeratin was positive in all cases; CGA and SYN were both mildly expressed in 2/6 tumours, while NSE was consistently expressed in all cases. S-100 was always negative. TEM revealed electrondense cytoplasmic granules, compatible with neuroendocrine granules in all cases. Three dogs were euthanised because of the tumour (7, 150, 180 days) and 3 remained alive: 1 in stable disease (420 days) and 2 in complete remission (100, 500 days).

Conclusions: CCCs present clinico-pathological features resembling AGASACA and 2/6 neoplasms were considered as carcinomas with neuroendocrine differentiation being positive to three neuroendocrine markers. CCCs can often present with HM and long-term outcome is likely poor. CCC seems to be a rare tumour but it might be underestimated due to the overlapping features with AGASACA.


Oral C - 1 : THE ROLE OF THE PORCINE EPIDEMIC DIARRHOEA VIRUS SPIKE PROTEIN IN VIRAL PATHOGENICITY

C. B. Kristen*, P. Rogger, J. Rappe, I. B. Veiga, H. Stalder, H. Posthaus, N. Ruggli, G. Tekes*, and V. Thiel

*Institute of Virology, Giessen University, Germany, Institute for Animal Pathology Bern and Institute for Virology and Immunology Bern and Mittelhäusern, Department of Infectious Diseases and Pathobiology, University of Bern, Switzerland

Introduction: Porcine epidemic diarrhoea virus (PEDV) causes malabsorptive diarrhoea following massive enterocyte destruction with villus atrophy and fusion. To further unveil the role of the spike (S) protein in viral pathogenicity, a synthetic PEDV infectious clone and an S gene mutant were generated and tested in vivo.

Materials and Methods: Four groups (n = 6) of 7-day-old piglets were infected with a cell culture-adapted and low pathogenic strain (LPV), a high pathogenic wild-type virus (WT-HPV), a synthetic infectious clone generated from WT-HPV via reverse genetics with an intact S protein (SV), and a synthetic infectious clone mutant where the WT S protein was replaced by the S protein from the cell culture-adapted strain (SV-deltaS). Blood, oro-nasal and rectal swabs collection and clinical score determination were performed throughout the experiment. Samples for histopathological and immunhistochemical analysis and viral RNA quantification were collected following euthanasia at day 2, 4 and 7 post infection (pi).

Results: WT-HPV and the synthetic infectious clone (SV) induced severe clinical disease with marked jejunal and ileal villus atrophy and fusion and viremia starting from day 2 pi. The cell culture-adapted LPV strain produced only very mild clinical disease and mild villus atrophy without concurrent viremia starting from day 4 pi. The SV-deltaS synthetic virus caused nonsignificant villus length changes and irregular viral loads in examined organs.

Conclusions: The S protein plays an important role in PEDV pathogenicity and the synthetic PEDV infectious clone generated on the genetic backbone of a high pathogenic wild-type strain is highly pathogenic.


Oral C - 2 : GAMMAHERPESVIRUS-5 REPLICATES ONLY IN PULMONARY ALVEOLAR MACROPHAGES AND IS ASSOCIATED WITH A MAC387+ HISTIOCYTIC RESPONSE IN EQUINE MULTINODULAR PULMONARY FIBROSIS

B. Cossic,1 M.R. Pennington,2 A.L. Glaser,3,4 T. Rathbone,5 G. Van de Walle2 and G.E. Duhamel1,3,4†

Department of Biomedical Sciences,1 Baker Institute for Animal Health,2 Department of Population Medicine and Diagnostic Sciences,3 and New York Animal Health Diagnostic Center,4 College of Veterinary Medicine, Cornell University, Ithaca, NY, USA. 5Balanced Horse Veterinary Service, PLLC, PO Box 3134, Redmond, WA, USA 98073

Introduction:  An association between equine multinodular pulmonary fibrosis (EMPF) and equine gammaherpesvirus-5 (EHV-5) is well-established; however, the cellular target of EHV-5 within the lung parenchyma and associated host inflammatory and adaptive immune cellular responses are incompletely characterized. Therefore, we sought to identify the cellular target of EHV-5 replication in the lungs of horses with naturally-occurring EMPF. Additionally, we compared the degree and distribution of collagen deposition, myofibroblast activation and type II pneumocyte hyperplasia in the lungs of horses with EMPF, equine idiopathic pulmonary fibrosis (EIPF) and healthy horses.

Materials and Methods: Infection with EHV-5 was confirmed by PCR assays, while EHV-5 replication in tissues was demonstrated by using a virus-specific in situ hybridization (ISH) assay. Key inflammatory and immune cells, and activation of myofibroblasts and type II pneumocytes were characterized by immunohistochemical staining, while collagen deposition was determined by Masson’s trichrome histochemical staining.

Results: When compared with healthy controls, horses with EMPF had prominent myofibroblast activation and type II pneumocyte hyperplasia together with a predominantly CD3+ T lymphocyte and monocyte/macrophage (Iba-1, pan-macrophage/dendritic cell; MAC387, recently infiltrating M1-like pro-inflammatory monocyte/macrophage) cellular infiltrate. When compared with EIPF, horses with EMPF had a two to three folds increase in MAC387 and EHV-5 replicating exclusively within MAC387- pulmonary alveolar macrophages (PAM), presumably M2-like anti-inflammatory macrophages.

Conclusions: These findings delineate EHV-5 tropism for long-lived, self-renewal PAM accompanied with a T lymphocyte-driven histiocytic response in horses with EMPF that will assist in the development of improved protocols for the diagnosis and control of this disease.


Oral C - 3 : INTRAMURAL CORONARY ARTERIOSCLEROSIS IN BEEF CATTLE REARED UNDER DIFFERENT HOUSING CONDITIONS

I. Biasato*, E. Biasibetti*, Dellarole S.*, Miniscalco B.*, Mioletti S.*, Tarantola M.*, Biagini D. , Capra P. , Leporati M. #, Vincenti M.#@, F. Guarda* and M. T. Capucchio*

*Department of Veterinary Sciences, University of Turin, Italy, Department of Agricultural, Forestry and Food Sciences, University of Turin, Italy,  Istituto Zooprofilattico Sperimentale di Piemonte, Liguria e Valle d’Aosta, Italy,  #Centro Regionale Antidoping e di Tossicologia “A. Bertinaria”, Italy, and @Department of Chemistry, University of Turin, Italy

Introduction:Intramural coronary arteriosclerosis has been reported in several species; no systematic studies are available in bovine. Rearing factors could influence the disease development. This study described coronary arteriosclerosis in regularly slaughtered beef cattle reared in different conditions.

Materials and Methods: A total of 15 Piedmontese breed bulls reared in a tied stall housing system (G1), 15 Piedmontese (G2), 15 Blonde d’Aquitaine (from France) (G3) and 15 cross-breed (from Ireland) (G4) reared in pen under different housing conditions and management practice were considered.  Blood parameters and dROMs were determined 3 weeks after the arrival of the animals and after 5 months. Hair 20β-diidrocortisol was evaluated at the slaughter. Samples of interventricular septum, papillary muscles, ventricular free walls and atria were submitted to histological evaluation (Haematoxylin & Eosin, Weigert VanGieson and AlcianPAS stains). Arteriosclerotic changes were classified using a semi-quantitative scoring system. GraphPad Prism® software was used to perform statistical analysis (P < 0.05).

Results: Hematochemical parameters, dROMs and 20β-dihydrocortisol revealed that G2 and G3 groups were less stressed than G1 and G4. Likewise, interventricular septum and papillary muscles were significantly more affected (P< 0.001). G4 showed more severe arteriosclerotic changes than others (P<0.001). A statistically significant association between G1 and G4 and the total number of affected vessels was observed (P < 0.05). Independently of the group, Anitschkow cells were detected in the 83% of the pathological vessels. 

Conclusions:  Based on these findings, a potential relationship between the development of intramural coronary arteriosclerosis and housing conditions-induced stress cannot be excluded.


Oral C - 4 : NEW INSIGHTS INTO THE PATHOGENESIS OF VASCULITIS IN malignant catarrhal fever

H. Saura*, M. Al-Saadi †, J.P. Stewart and A. Kipar *†

*Institute of Veterinary Pathology, Vetsuisse Faculty, University of Zurich and Department of Infection Biology, Institute of Infection and Global Health, University of Liverpool, United Kingdom

Introduction: Lymphoproliferative vasculitis has been described as the main histological feature of malignant catarrhal fever (MCF), with virus infected T lymphocytes as the dominant cells. Deregulation of uninfected cells through infected lymphocytes, and autoreactive immune responses have been speculated as the underlying pathomechanisms.

Materials and Methods: The study was performed on 42 routinely necropsied confirmed MCF cases (32 cattle, 8 water buffalos, 2 bisons). The carotid rete was histologically re-examined and the presence of vasculitis confirmed. Immunohistology identified infiltrating leukocytes, proliferating cells, and ovine herpesvirus 2 (OvHV-2)-infected cells using a novel antibody to the latency-associated nuclear antigen (LANA).

Results: Arteries of the carotid rete exhibited acute to chronic changes of variable intensity. Beside infiltration of the tunica adventitia by macrophages and fewer T cells, a patchy or diffuse similar infiltration was often seen stretching from the vascular lumen, generally together with obvious activation of the endothelial cells (EC). Chronic changes comprised intimal thickening, with expansion by smooth muscle cells (SMC) and macrophages and disruption of the internal elastic lamina. OvHV-2 LANA was mainly expressed in EC, SMC and infiltrating macrophages and lymphocytes. Both types of leukocytes were also found to proliferate.

Conclusions: Our results indicate that MCF vasculitis is macrophage driven rather than lymphoproliferative, with initial macrophage infiltration of the adventitial tunica via the vasa vasorum and direct leukocyte recruitment from the blood as well as remodelling processes at later stages. Considering that both infiltrating leukocytes and endothelial cells are virus infected, we speculate that the vasculitis is virus driven.


Oral C - 5 : TESCHOVIRUS AS THE CAUSATIVE AGENT OF ENCEPHALOMYELITIS ON TWO SWINE FARMS IN THE NETHERLANDS

S. Vreman*, N. Caliskan**, K. Peperkamp J. Kortekaas*and B. Kolpa

*Wageningen Bioveterinary Research, Lelystad, the Netherlands, **Department of Pathobiology, Utrecht University, the Netherlands † GD Animal Health, Deventer, the Netherlands and ‡Gelre dierenartsen,Groenlo, the Netherlands.

Introduction: Porcine teschovirus (PTV) belongs to the genus Teschovirus in the family Picornaviridae. This virus circulates among wild and domesticated pig populations without causing any concerning health problems. However, a pathogenic variant like PTV1 (Teschen disease), or coinfection with immunosuppressive viruses like porcine reproductive and respiratory syndrome virus (PRSSV) and porcine circo virus (PCV), could result in serious economic losses in swine industry. We examined weanling pigs with neurological symptoms on two swine farms in the Netherlands to identify the underlying cause.

Materials and Methods: Pathological examination was performed on pigs from both farms (n=5). After histological evaluation, brain and spinal cord samples were used for PCR, virus isolation and whole genome sequencing to identify the serotype of PTV and exclude possible immunosuppressive viruses.

Results: Histology of brain and spinal cord revealed clear non-suppurative encephalomyelitis in all the pigs. The other organs did not display any relevant histological changes. Additional immunoperoxidase staining was negative for pathogenic PTV1. However, after PCR and virus sequencing we identified for each farm a new different PTV isolate. The pigs were negative in PCR for PRRSV and PCV2.

Conclusions: New pathogenic variants of PTV still reemerge in domesticated pig populations causing clear neurologic symptoms in a substantial number of pigs. This disease is often underdiagnosed, because histological examination in combination with PCR are needed for determination. Awareness by local veterinarian and pathologist is needed to investigate the incidence of pathogenic PTV in wild and domestic pig populations.


Oral C - 6 : CELL TROPISM OF MIDDLE EAST RESPIRATORY SYNDROME CORONAVIRUS IN EXPERIMENTALLY INFECTED DROMEDARIES

A. Lehmbecker*, A.-K. Uhde*, I. Spitzbarth*, P. Wohlsein*, J. van den Brand†, V. Raj†, S. L. Smits†, D. Schippers†, T. M. Bestebroer†, N. Okba†, T. Kuiken†, A. Bensaid‡, D. Solanes Foz‡, J. Segales‡, A. Volz♯, G. Sutter♯, A. D. M. E. Osterhaus◊, B. L. Haagmans† and W. Baumgärtner*

*Department of Pathology, University of Veterinary Medicine Hannover, Foundation, †Department of Viroscience, Erasmus Medical Center, Rotterdam, Netherlands; ‡Centre de Recerca en Sanidat Animal, Barcelona, Spain, ♯German Centre for Infection Research (DZIF), Institute for Infectious Diseases and Zoonoses, Ludwig-Maximilians-Universität München, Munich, ◊Germany Research Center for Emerging Infections and Zoonoses, University of Veterinary Medicine Hannover, Foundation

Introduction: Middle East Respiratory Syndrome Coronavirus (MERS-CoV) caused hundreds of deaths in humans since 2012. The dromedary (Camelus dromedarius) is suggested to play an important role in the epidemiology of this emerging infectious disease and develops only transient and mild clinical signs upon infection. However, both the basic pathomorphology of the disease and the viral cell tropism in this species are still elusive.

Materials and Methods: Dromedaries were experimentally infected with MERS-CoV. The respiratory tract was investigated histologically, immunohistologically, and ultrastructurally. Double immunofluorescence was representatively performed in order to identify the target cells of MERS-CoV in dromedaries. Ciliary pathology was evaluated by scanning electron microscopy and immunofluorescence.

Results: The animals showed inflammatory alterations within the upper respiratory tract. MERS-CoV was predominantly found within cytokeratin-positive apical epithelial cells and rarely within macrophages. Additionally, marked ciliary loss was evident within the respiratory epithelium of the nasal cavity and trachea as demonstrated by scanning electron microscopy and immunofluorescence for acetylated alpha-tubulin.

Conclusions: Administration of MERS-CoV leads to an infection nearly restricted to cytokeratin-positive respiratory epithelial cells of the upper respiratory tract in dromedaries, which is in contrast to the marked pulmonary disease in humans. Furthermore, the infection is accompanied by impressive ciliary loss in the upper respiratory tract of dromedaries.


Oral D - 1 : CHARACTERIZATION OF INFLAMMATORY INFILTRATE OF ULCERATIVE DERMATITIS IN MICE

D. De Biase*, F. Esposito, G. Piegari*, V. Iovane*, G. Palma, C. Arra, S. Papparella*, A. Fusco and O. Paciello*

*Department of Veterinary Medicine and Animal Production, University of Naples Federico II, Naples, Italy.   Institute of Experimental Endocrinology and Oncology (IEOS), National Research Council (CNR), Naples, Italy.  National Cancer Institute "Fondazione Giovanni Pascale", IRCCS Naples, Italy

Introduction:  Ulcerative dermatitis (UD) is an idiopathic, spontaneous and progressive disease typically affecting aged mice on a C57BL/6 background with an unknown etiopathogenesis.

Materials and Methods: We evaluated twenty cases of ulcerative dermatitis in transgenic mice with a C57BL/6 background. Formalin-fixed, paraffin embedded skin samples were analyzed for morphology. Immunohistochemistry was performed for anti-CD3, CD45, CD4, CD8, IL-17 and MHC II antibody in order to characterize and quantify lymphocytic infiltrate. Moreover, cryopreserved skin samples were processed for molecular analysis. Ten unaffected skin biopsies were used as negative controls.

Results: Affected skin showed extensive areas of ulceration and a diffuse, severe and mixed inflammatory infiltrate. No relevant changes were observed in controls. UD skin showed an increased number of mast cells compared to controls, a higher number of CD3 and CD4 immunopositive lymphocytes compared to CD45 positive cells and IL-17 positive lymphocytes and mast cells. No immunoreaction was observed for CD8 antibody. MHC class II immunoreaction of dermal and subcutaneous endothelial cells as well as inflammatory cells was also detected. Gene expression array of affected mice showed an upregulation of ST2 gene.

Conclusions:  ST2 gene plays a crucial role in allergic Th2 response, while little is known about the interaction between ST2 gene products and Th17 cells. Recently it has been suggested that also Th17 cells, together with Th2 cells, may be involved in the pathogenesis of allergic airway disease in mice. Overall, our preliminary data lead to further investigate the hypothesis that UD in mice have an allergic etiopathogenesis


Oral D - 2 : EVALUATION OF BOTULINUM NEUROTOXIN TYPE A EFFECTS ON THE DISTRIBUTION OF CLEAVED-SNAP25 IN MUSCLES AND SPINAL CORD OF RATS INJECTED IN THE GASTROCNEMIUS MUSCLE OVER A 75-DAY PERIOD

S. Lezmi*, F. Sebal*, C. Favre-Guilmard, D. Carre, V. Martin*, M. Kalinichev, F. Schmidlin and C. George*

*Non-Clinical Drug Safety, †Neurology and Biomarker Departments, Ipsen innovation, France

Introduction: Botulinum neurotoxin type A (BoNT/A) is used for the treatment of muscle hypertonicity in various medical indications, and induces a long-lasting muscle relaxation by cleaving the synaptic protein SNAP25. Little is known about the distribution of BoNT/A after a single intramuscular injection.

Materials and Methods: Twenty-four rats received 125 pg/kg of natural BoNT/A (List) in the left gastrocnemius (highest tolerated dose without body weight effect in this species). The digit abduction score (DAS) was evaluated, and rats sacrificed 1 hour post administration and at different timepoints up to Day 75. Muscles and spinal cord were sampled for morphological analyses and detection of cleaved-SNAP25 (C-SNAP25) by immunohistochemistry.

Results: Treated rats presented with a maximum DAS on D3 and 6, which progressively decreased up to D27, suggesting a diffusion of the toxin from the gastrocnemius to the extensor digit muscles in which levels of C-SNAP25 strongly correlated with the digit abduction inhibition. In the injected gastrocnemius, high levels of C-SNAP25 were gradually detected at neuromuscular junctions from H1 up to D27, and progressively decreased. The distribution of C-SNAP25 and morphological changes were also evaluated in multiple muscles. In the lumbar spinal cord, a strong C-SNAP25 staining was progressively observed from D1 to up to D27 and gradually decreased.

Conclusions: The detection of C-SNAP25 appears to closely reflect the action of BoNT/A in muscles at early stages. However, it’s detection at later stages may not reflect the direct activity of BoNT/A, but rather a persistence of the cleaved protein in various tissues.


Oral D - 3 : EXTENSIVE CHARACTERIZATION OF THE NEWLY DEVELOPED DMDMDX RAT MODEL: A UNIQUE ANIMAL MODEL FOR DMD

T. Larcher*, C. Huchet$, G. Toumaniantz°, B. Fraysse, A. Lafoux$, S. Remy, D. Caudal$, M. Allais, E. Amosse°, I. Anegon and C. Le Guiner

* APEX UMR 703, INRA-ONIRIS, Nantes, France; $ Therassay Platform, Université de Nantes, France; ° Institut du Thorax, Université de Nantes, France; INSERM UMR 1089, Université de Nantes, France; INSERM UMR 1064, ITUN, Nantes, France

Introduction: Duchenne Muscular Dystrophy (DMD) is a severe muscle-wasting disorder caused by mutations in the gene encoding dystrophin. The evaluation of potential therapeutic products requires relevant animal models but the two former described animal models deficient for dystrophin (in mouse and dog) present some limitations including absence or very delayed development of cardiomyopathy.

Materials and Methods: We recently generated a line of dmd mutated-rats (Dmdmdx) using TALENs. To complete the initial characterization, we performed a large natural history study, during which different groups of Dmdmdx rats and littermate wild-type controls were evaluated at different time points (1.5, 3, 4.5, 7, 10 and 12 months). One group of DmDmdx rats was also added to document the lifespan, as well as the causes of death.

Results: We showed that life span of DmDmdx rats was significantly reduced. Weight, blood biomarkers concentrations, muscle strength and fatigue measured by grip force test, muscle calcium homeostasis and histology (including fibrosis) in skeletal muscles, diaphragm and heart, were all significantly impaired as soon as the age of 1.5 months. Importantly, echo- and electro-cardiography as well as necropsy identified cardiac muscle alteration to be systematic and one of the major cause of death.

Conclusions: With a progressive alteration of cardiac and skeletal muscle function and tissue, similar to what occurs in DMD patients, this Dmdmdx rat model is of particular interest for preclinical evaluations of treatments for DMD.


Oral D - 4 : CHARACTERIZATION OF INFLAMMATORY CHANGES, VIRAL SPREAD AND DEMYELINATION AFTER INTRASPINAL INOCULATION OF THEILER’S MURINE ENCEPHALOMYELITIS VIRUS IN A RESISTANT MOUSE STRAIN

E. Leitzen*,, B. Raddatz*,, W. Jin*,, R. Ulrich*,†,$, S. Goebbels, KA. Nave, W. Baumgärtner*, and F. Hansmann*,

*Department of Pathology, Center for Systems Neuroscience, University of Veterinary Medicine, Hanover, Germany, $Department of Experimental Animal Facilities and Biorisk Management, Friedrich-Loeffler-Institute, Greifswald – Insel Riems, Germany and Department of Neurogenetics, Max Planck Institute of Experimental Medicine, Goettingen, Germany

Introduction: Intracranial infection of mice with Theiler’s murine encephalomyelitis virus (TMEV) induces a chronic demyelinating leukomyelitis in susceptible but not in resistant strains. The aim of this study was to investigate, whether TMEV-resistant C57Bl/6 (B6) mice develop a demyelinating leukomyelitis similar to susceptible mice following intraspinal infection.

Materials and Methods: Groups of 6-8 female B6 mice were inoculated intraspinally with the BeAn strain of TMEV or vehicle. Clinical investigation included a scoring system and a rotarod-test. Necropsies were performed at 3, 7, 14 and 28 days post inoculation (dpi). Spinal cord and peripheral nerves were studied using HE staining and immunohistochemistry targeting myelin basic protein (demyelination), TMEV, CD3 (T-lymphocytes), CD45R (B-lymphocytes) and CD107b (microglia/macrophages).

Results: TMEV infected mice showed a progressive clinical change, characterized by paresis of hind limbs, starting at 11dpi. Mononuclear inflammation was present at the injection site after 3dpi, followed by a more widespread distribution at later time points. A demyelination with maximum degree of severity at 14dpi as well as viral antigen was detected immunohistochemically at all investigated time points. Numbers of inflammatory cells (lymphocytes, microglia/macrophages) and TMEV-labelled cells decreased until 28dpi. Peripheral nerves showed degenerative alterations at 14 and 28dpi.

Conclusions: Intraspinal TMEV infection of B6 mice caused significant clinical signs associated with inflammatory and demyelinating lesions in the spinal cord as well as degenerative changes in the peripheral nerves, mimicking features of Guillain-Barré syndrome. This model offers the opportunity to investigate TMEV-induced de- and remyelination as well as peripheral nerve damage in “resistant” B6 mice.


Oral E - 1 : BLINDNESS AND SUDDEN DEATH IN TURKEYS

A. Lehmbecker*, L. Zind*, P. Wolf†, P. Bahrenberg‡, C. Mandischer‡, E. van der Vries♯, W. K. Jó Lei♯, A. Osterhaus♯ and W. Baumgärtner*

*Department of Pathology, University of Veterinary Medicine Hannover, Foundation, †Chair of Nutrition Physiology and Animal Nutrition, University of Rostock; ‡Veterinary practice Güterstrasse, Hamminkeln, ♯Research Center for Emerging Infections and Zoonoses, University of Veterinary Medicine Hannover, Foundation

Introduction: Several cases of poultry exhibiting blindness and signs affecting the central nervous system are reported. The etiology of this disease remains undetermined so far. Various etiologies like infections, intoxications or deficiencies are discussed.

Materials and Methods: Six animals were clinically, macroscopically, histologically and immunohistologically investigated. The diet underwent a detailed analysis with respect to minerals and heavy metals, methanol, vitamin A, coccidiostats, pesticides and amino acids.

Results: Affected animals showed clinically a disturbed balance and gait abnormalities. Macroscopically bilateral symmetric malacia was evident predominantly within the cerebrum. Furthermore, perivascular lympho-histiocytic inflammation accompanied by neuronal necrosis, and astrogliosis was evident histologically. Eyes were macroscopically and histologically unremarkable. The analyzed methionine level was severely increased compared to the declared content.

Conclusions: The observed clinical signs and bilateral malacia in the affected turkeys was mostly likely caused by the severely elevated methionine content within the diet. Methionine is an essential amino acid, which is often used to improve growth curves in turkeys. However, accidental or intentional oversupply of methionine can lead to severe or even fatal cases of encephalopathy in turkeys.


Oral E - 2 : TISSUE DISTRIBUTION OF THE MERS-CORONAVIRUS RECEPTOR IN BATS

A. Widagdo*, L. Begeman*, D. Schipper*, P.R. van Run*, A.A. Cunningham, N. Kley, C.B. Reusken*, B.L. Haagmans* and J.M.A. van den Brand*

*Department of Viroscience, Erasmus Medical Center, Rotterdam, the Netherlands, Institute of Zoology, Zoological Society of London, London, United Kingdom and Institute for Novel and Emerging Infectious Diseases, Friedrich Loeffler Institute, Greifswald, Germany

Introduction: Middle East respiratory syndrome coronavirus (MERS-CoV) has been shown to infect both humans and dromedary camels using dipeptidyl peptidase-4 (DPP4) as its receptor. The distribution of DPP4 in the respiratory tract tissues of humans and camels reflects MERS-CoV tropism. Apart from dromedary camels, insectivorous bats are suggested as another natural reservoir for MERS-like-CoVs.

Materials and Methods: In order to gain insight on the tropism of these viruses in bats, we studied the DPP4 distribution in the respiratory and extra-respiratory tissues of two frugivorous bat species (Epomophorus gambianus and Rousettus aegyptiacus) and two insectivorous bat species (Pipistrellus pipistrellus and Eptesicus serotinus).

Results: In the frugivorous bats, DPP4 was present in epithelial cells of both the respiratory and the intestinal tract, similar to what has been reported for camels and humans. In the insectivorous bats, however, DPP4 expression in epithelial cells of the respiratory tract was almost absent.

Conclusions: The preferential expression of DPP4 in the intestinal tract of insectivorous bats, suggests that transmission of MERS-like-CoVs mainly occurs via the fecal-oral route. Our results highlight differences in the distribution of DPP4 expression among MERS-CoV susceptible species, which might influence variability in virus tropism, pathogenesis and transmission route.


Oral E - 3 : Cancelled


Oral E - 4 : MODULATION OF INTESTINAL MICROBIOTA, MORPHOLOGY AND MUCIN COMPOSITION BY DIETARY INSECT MEAL INCLUSION IN FREE-RANGE CHICKENS

I. Biasato*, I. Ferrocino, E. Biasibetti*, E. Grego*, A. Schiavone*,, L. Gasco†,‡, F. Gai, L. S. Cocolinand M. T. Capucchio*

*Department of Veterinary Sciences, University of Turin, Italy, Department of Agricultural, Forestry and Food Sciences, University of Turin, Italy and  Institute of Science of Food Production, National Research Council, Italy

Introduction: Dietary modifications have been reported to influence intestinal microbiota, morphology and mucin dynamics in poultry. The present study evaluated the effects of dietary Tenebrio molitor (TM) meal inclusion on gut microbiota, morphology and mucin composition in free-range chickens.

Materials and Methods: 100 female medium-growing hybrids were divided into 2 dietary treatments (control feed and 7.5% TM inclusion) and slaughtered at 97 days of age (10 birds/diet). The gut microbiota was assessed on cecal content samples by 16S rRNA amplicon based sequencing. Intestinal morphology was evaluated through morphometric measurements of villus height, crypt depth and villus height/crypt depth ratio on duodenum, jejunum and ileum. Small intestine and caecum were also stained with PAS, Alcian Blue pH 2.5 and HID to discriminate among neutral, acidic sialylated and acidic sulfated mucins.

Results: β-diversity calculation showed a clear separation of the microbiota composition depending on diet (P < 0.01). TM meal inclusion increased the relative abundance of Ruminococcus, Oscillospira and Clostridium (P < 0.05). Gut morphology and mucin composition were not influenced by dietary TM meal inclusion. Duodenum showed the highest morphometric indices in both dietary treatments (P < 0.05). Independently of TM meal inclusion, crypt mucins were predominantly acidic sialylated, lower in caecum and higher in base fragment (P < 0.001), while villus mucins were higher in ileum and base fragment (P < 0.001 and P < 0.01).

Conclusions: Dietary TM meal inclusion positively modulates gut microbiota of free-range chickens with no negative effects on the physiological intestinal development and mucin dynamics.


Oral F - 1 : ANALYSIS OF GENE EXPRESSION SIGNATURES IN CANCER-ASSOCIATED STROMA FROM CANINE MAMMARY TUMOURS REVEALS MOLECULAR HOMOLOGY TO HUMAN BREAST CARCINOMAS

J. Ettlin*, E. Clementi*, P. Amini*, A. Malbon and E. Markkanen*

* Institute of Veterinary Pharmacology and Toxicology and † Institute of Veterinary Pathology, University of Zurich, Switzerland

Introduction: Cancer-associated stroma (CAS) plays a key role in cancer initiation and progression. Spontaneously occurring canine mammary carcinomas are viewed as an excellent model of human breast carcinomas; considering the importance of CAS for human cancer, it likely plays a central role in canine tumours as well. So far however, canine CAS lacks characterisation, and it remains unclear whether the biology is comparable between CAS from canine and human tumours.

Materials and Methods: In this proof-of-principle study, we used laser-capture microdissection to isolate CAS and normal stroma from 13 formalin-fixed paraffin embedded canine simple mammary carcinomas, analysed the expression of seven known human CAS markers by RT-qPCR, and validated some of these targets by immunohistochemistry.

Results: We found that collagen type I alpha I chain, alpha smooth muscle actin, fibroblast activation protein alpha, platelet derived growth factor receptor beta, and Caveolin-1 were significantly upregulated in canine CAS, expression of CXCL12 significantly decreased whereas matrix metalloproteinase 2 and interleukin 6 did not change.

Conclusions: Our results suggest strong similarities in CAS biology in canine and human mammary carcinoma, but also reveal some differences. To the best of our knowledge, this is the first report to provide a comprehensive expression analysis of the most important CAS markers in canine simple mammary carcinomas, and further supports the validity of the dog as a model for human cancer.


Oral F - 2 : MOLECULAR AND IMMUNOHISTOCHEMICAL ANALYSES IDENTIFY THE PRESENCE OF MOUSE MAMMARY TUMOR VIRUS-LIKE SEQUENCES IN FELINE MAMMARY CARCINOMAS

P. Civita*, C. Scopelliti*, M. Menicagli*, F. Lessi*, F. Millanta, S. Borsacchi, G. Freer, M. Pistello, C.M. Mazzanti* and A. Poli

* Fondazione Pisana per la Scienza, Pisa, Italy, Dipartimento di Ricerca Traslazionale e delle Nuove Tecnologie in Medicina e Chirurgia, Università di Pisa, Italy and  Dipartimento di Scienze veterinarie, Università

Introduction: A mouse mammary tumour virus-like (MMTV-like) is suspected to be involved in human breast cancer, but the role that similar viruses play in canine or feline mammary tumorigenesis is not understood  We investigated the presence of MMTV-like nucleotide sequences and viral protein in a larger number of feline mammary carcinomas (FMCs).

Materials and Methods: Neoplastic and normal epithelial cells were isolated using laser capture dissection technique from 86 FMCs and six normal mammary tissue controls. Extracted DNA was analyzed by nested fluorescence-PCR using primer sets specific for MMTV env sequence. For immunohistochemistry, a polyclonal antibody against MMTV-p14 signal peptide of the envelop precursor protein was used as primary antibody, followed by a biotin-streptavidin system.

Results: MMTV-like sequences were detected in seven out of the 86 neoplastic tissues (8.1%). Six of these were tubulopapillary FMCs (four grade I FMCs and two grade II FMCs) and one was a solid grade II FMC. Only PCR-positive tumors scored positive for MMTV p14 protein. Normal mammary gland tissues scored negative for both PCR and MMTV-p14. Multiple nucleotide alignment of feline MMTV-like env gene sequences showed similarity for 97% and 99% homology to HMTV and MMTV respectively.

Conclusions: The results of our study showed the presence of MMTV-like sequences and viral protein in some FMCs. Further studies are needed to understand whether this virus plays a role in the development of FMCs. Furthermore if, as these data suggest, MMTV-like is an exogenous virus, then a further question is from whom this virus is acquired.


Oral F - 3 : CHEMORESISTANCE MARKERS PGP AND BCRP IN CANINE INFLAMMATORY AND GRADE 3 MAMMARY CARCINOMA

M. Levi*, L. Peña, B. Brunetti*, A. Alonso-Díez, L.V. Muscatello*, M.D. Pérez-Alenza, C. Benazzi* and G. Sarli*

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

Introduction:  Multidrug resistance of neoplastic cells is frequently related to the expression of P-glycoprotein (PGP) and Breast Cancer Resistance Protein (BCRP). Canine inflammatory mammary carcinoma (IC) and grade 3 carcinoma (C3) are biologically aggressive and they could benefit from chemotherapy. Our study describes the expression of PGP and BCRP in these tumours.

Materials and Methods:  Samples included 18 C3s and 20 ICs from dogs that had not received chemotherapy before biopsy. Primary carcinoma was available in 15 IC cases. Tumours were classified into histological subtypes. IHC for PGP and BCRP was considered   positive when >20% and >10% of cells were labelled for PGP and BCRP, respectively.

Results:   Immunolabelling was mainly membranous for PGP showing a strong reaction in emboli while membranous and cytoplasmic stain was mainly seen for BCRP. PGP was highly expressed in the different tumours, but significantly higher in emboli of IC vs C3 (PGP P=0.006) and in primary IC vs C3 (PGP P=0.032). There was no significant difference in BCRP expression among groups, being expressed in 78.95% of emboli of CI, 80% of primary IC and 66.67% of C3.

Conclusions:  Chemoresistance is a phenomenon present in dogs with C3 and IC.  Our results indicate the need of a combined therapy rather than chemotherapy alone. The high expression of  PGP in ICs  compared with non-inflammatory mammary carcinomas  is an interesting finding that can explain a higher resistance to chemotherapy in this type of cancer and could be related to the specific pathogenic mechanisms that this disease exhibits.


Oral F - 4 : EXTRACELLULAR VESICLES DERIVED FROM CANINE AND FELINE MAMMARY CARCINOMA: A PRELIMINARY CHARACTERIZATION

G. Finesso*, A. Sammarco*, S. Ferro*, F. Caicci§, R. Zanetti*, R. Sacchetto*, L. Cavicchioli* and V. Zappulli*

*G.F., A.S., S.F., R.Z., L.C., V.Z. from Dept. BCA, University of Padua, Italy and §F.C. from Dept. of Biology, University of Padua, Italy

Introduction: Extracellular Vesicles (EVs) have received considerable attention as novel membrane-bound cell-derived cargo systems involved in intercellular communication and microenvironment modulation in many physiological and pathological processes. They exert pleiotropic biological functions via the horizontal transfer of bioactive molecules (proteins, lipids, DNA, and RNAs). In human cancer EVs raise a large interest both as mediators of pathways that lead to tumor progression and as potential novel cancer biomarkers. In veterinary medicine very little is known on cancer-derived EVs.

Materials and Methods: In this study three established mammary cancer cell lines (human, feline, and canine) were used to isolate, identify, and characterize cancer cell-derived EVs. An ultracentrifugation-based protocol was used to obtain EVs-enriched pellets from cell culture medium. Transmission electron microscopy (TEM), immunogold labeling, and western blot analysis were performed to identify EVs. NanoSight technology was applied for Nanoparticles Tracking Analysis.

Results: EVs were isolated and identified from human, canine, and feline mammary cancer cell lines. Membrane-bound structures were observed by TEM and immunogold labeling detected a membrane-associated positive signal for specific EVs antigens (e.g. CD63 and Alix). Moreover, western blot analysis showed the EVs-specific antigens versus cell-specific markers, confirming the identification of EVs-associated proteins. Finally, the NanoSight technology tracked cell medium-derived nanoparticles of the EVs size range (30nm to 1-2mm) in the variable amount ranging 1-5 x 10^9 per mL of medium.

Conclusions: This preliminary study allowed the identification and characterization of canine and feline mammary cancer cells-derived EVs, opening a new unexplored field of study in veterinary medicine.


Oral F - 5 : FOXP3+ REGULATORY T CELLS IN AND AROUND FELINE INVASIVE MAMMARY CARCINOMAS ARE ASSOCIATED WITH AGGRESSIVENESS

E. Dagher*, L. Simbault*, D. Loussouarn†,w, J. Abadie*,w, M. Campone‡,w, D. Fanuel* and F. Nguyen*,w

*AMaROC, Oniris (Nantes Atlantic College of Veterinary Medicine, Food Science and Engineering), France, Department of Pathology, University Hospital, Nantes, France, w Team 8 “Stress Adaptation and Tumor Escape”, CRCINA, INSERM, Université de Nantes, Université d’Angers, Nantes, France, and Integrated Center for Oncology Nantes/Angers, France

Introduction: FoxP3 is a marker for immunosuppressive regulatory T cells that may play a role in inducing immune tolerance and may be selectively recruited by carcinomas. The aim of this study was to determine the amount and prognostic value of FoxP3-positive peritumoral and intratumoral lymphocytes in feline invasive mammary carcinomas (FMCs).

Materials and Methods: Retrospective study of 180 FMCs, diagnosed in female cats treated with surgery alone, with 2-year follow-up. FoxP3 (clone SP97), ER (clone C311), PR (clone 10A9), Ki-67 (clone MIB1) and HER2 (clone 4B5) expressions were determined by automated immunohistochemistry. FoxP3+ lymphocytes were quantitated in peritumoral and intratumoral locations.

Results: FMCs were considered rich in regulatory T cells when FoxP3+ peritumoral lymphocytes exceeded 40 in a representative x400 field, or when intratumoral FoxP3+ lymphocytes exceeded 21 in ten x400 fields. FoxP3+ peritumoral and intratumoral lymphocytes negatively influenced the disease-free interval, overall survival, and specific survival. For example, FMCs with low FoxP3+ peritumoral lymphocytes (54/180; 30%) were associated with longer overall survival (HR=0.66) with the following covariates: pathologic tumor size (HR=1.58 for pT larger than 20mm), pathologic nodal stage (HR=1.54 for pN1), distant metastasis (HR=2.71 for M1), and the ER index (HR=1.0148; P<0.0001; Cox proportional-hazards regression). The negative prognostic value of FoxP3+ lymphocytes was confirmed in both luminal (ER+ and/or PR+) FMCs (57/180; 32%), and triple-negative (ER–, PR–, HER2–) FMCs (123/180; 68%).

Conclusions: As previously reported in human breast cancer, FMCs enriched in FoxP3+ regulatory T cells are associated with a worse outcome.


Oral F - 6 : PROPOSAL FOR A NEW HISTOLOGICAL STAGING SYSTEM FOR CANINE MAMMARY CARCINOMAS

F. Chocteau*, F. Nguyen*, and J. Abadie*,

*AMaROC (Animal cancers, Models for Research in Comparative Oncology), Oniris (Nantes Atlantic College of Veterinary Medicine, Food Science and Engineering), Nantes, France. Team 8 “Stress Adaptation and Tumor Escape”, CRCINA, INSERM, Université d’Angers, Université de Nantes, Nantes, France

 Introduction: Precise staging is an important step in the management of canine mammary carcinomas (CMCs) since it provides prognostic information and helps in determining appropriate therapy. Here we propose to define the stage of CMCs using histopathological rather than clinical criteria.

Materials and Methods: 433 female dogs with CMC, without distant metastasis at diagnosis, were staged according to pathologic tumor size (pT, greatest diameter in millimeters on histological slides), lymphovascular invasion (LVI), and pathologic nodal stage (pN, confirmed by cytokeratin AE1/AE3 immunohistochemistry). Mammary carcinomas in situ (surrounded by a continuous layer of p63+ myoepithelial cells by immunohistochemistry) were Stage 0 whereas invasive mammary carcinoma were defined as: Stage 1 (pT≤20mm, LVI-, pN0/pNX, where pNX refers to the absence of lymph node sample), Stage 2 (pT>20mm, LVI-, pN0/pNX), Stage 3a (pT≤20mm, LVI+ and/or pN1), and Stage 3b (pT>20mm, LVI+ and/or pN1). Follow-up of the dogs was available over a 24-month period.

Results: Local recurrence, distant metastasis, disease-free-interval, overall survival and specific survival significantly differed by histological stage. For specific survival, median survival times and hazard ratios (HR) by Cox proportional hazards regression (p<0.0001) were: Stage 0 (median survival not reached; HR=1.00; N=89; 21% of the dogs), Stage 1 (1720 days; HR=3.05; p=0.0018; N=81; 19%), Stage 2 (1181 days; HR=4.39; p<0.0001; N=79; 18%), Stage 3a (348 days; HR=10.59; p<0.0001; N=79; 18%), and Stage 3b (163 days; HR=16.59; p<0.0001; N=105; 24%).

Conclusions: The proposed histological staging system (pT, LVI, pN) for canine mammary carcinomas is a new, powerful and easily applicable prognostic indicator.