Enteritis Caused by Type 2c Canine Parvovirus in a 5-Year-Old Dog. Cláudio Wageck Canal 3 & David Driemeier 1

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1 EXAMPLES - EXEMPLOS CASE REPORT Enteritis Caused by Type 2c Canine Parvovirus in a 5-Year-Old Dog Veronica Machado Rolim 1, Luciana Sonne 1, Renata Assis Casagrande 2, Suyene Oltramari Souza 1, Luciane Dubina Pinto 3, Angelica Terezinha Barth Wouters 4, Flademir Wouters 4, Cláudio Wageck Canal 3 & David Driemeier 1 1 Setor de Patologia Veterinária (SPV), Faculdade de Veterinária (FaVet), Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil. 2 Patologia Veterinária, Universidade do Estado de Santa Catarina (UDESC), Lages, SC, Brazil. 3 Setor de Virologia, FaVet, UFRGS, Porto Alegre, RS. 4 Setor de Patologia Veterinária, Universidade Federal de Lavras (UFL), Lavras, MG, Brazil. CORRESPONDENCE: D. Driemeier [davetpat@ufrgs.br - Tel.: +55 (51) ]. Faculdade de Veterinária - UFRGS. Av. Bento Gonçalves n. 9090, Bairro Agronomia. CEP Porto Alegre, RS, Brazil. ABSTRACT Background: Canine parvovirosis, caused by canine parvovirus type 2 (CPV-2), emerged in the 1970s as an important disease affecting dogs, causing severe hemorrhagic gastroenteritis and death. It can occur in any breed, gender, and age; however, puppies of 4 to 12 weeks of age are most commonly afflicted. In 2000 a new variant of the virus, called CPV-2c, was discovered, and has been related to hemorrhagic gastroenteritis in dogs with up to 2 years of age, although some cases have been described in older animals with a full vaccination history. This paper reports a case of enteritis by canine parvovirus type 2c (CPV-2c) in a 5-year-old dog. Case: At necropsy a pallid oral and conjunctival mucosae were observed. The small intestine showed a very reddish and wrinkled serosa, the wall was thickened, the mucosae was diffusely wrinkled and yellowed with evidenced Peyer plaques and there was no content in the final portion of the intestine. The mesenteric lymph nodes were enlarged and reddish. Multiple suffusions on the serosa of the stomach, and petechiae and subepicardial suffusions

2 in the heart were observed. The histological findings were, collapse of the lamina propria of the small intestine, and fusion of the villi, necrosis of enterocytes, atrophy and the disappearance of crypts, with dilation of remaining crypts showing large rounded nuclei with one or two evident nucleoli, exhibiting accentuated cellular pleomorphism in some cases forming syncytia. In addition, there were bacterial colonies and fibrin adhered to the mucosae. The serosa showed diffuse congestion, marked transmural multifocal hemorrhage, thrombosis and fibrin deposition on the serosa surface. Necrosis of the germinative centers with moderate lymphoid depletion was observed in the lymphoid aggregates of the large intestine. In the bone marrow, spleen and mesenteric lymph node there were accentuated lymphoid depletion, hemorrhage and moderate hemosiderosis. The remaining tissue of the thymus showed accentuated multifocal to coalescent hemorrhage. The anti-parvovirus IHC showed intense immunostaining of the cytoplasm of epithelial cells, mainly in the crypts of the small intestine. In the spleen and lymph node there was intense immunostaining in the lymphocytes of follicular centers. The PCR and sequencing techniques applied to the sample allowed the identification of CPV-2c. Discussion: Diarrhea in dogs has been associated with a wide variety of viral agents; the canine parvovirus, rotavirus and coronavirus being the main primary pathogens involved. Since CPV-2 emerged at the end of the 1970s this pathogen has gained great importance in the care of dogs and is probably the most common infectious disease of canine species. Shortly after appearing in the canine population, CPV-2 underwent alterations in some of its amino acids, which resulted in new and better adapted viral strains. Studies in which circulating viral strains have been identified have demonstrated the importance of CPV-2c in outbreaks of parvovirosis in previously vaccinated puppies. There are few reports of the detection of CPV-2c in adult dogs. The majority of cases described relate to dogs up to 2 ½ years of age, one exception being a case involving a 12-year-old dog This new variant of

3 CPV-2 should be considered as an important pathogen in the diagnosis of causes of sanguinolent diarrhea in adult dogs. Keywords: CPV-2c, adult dogs, parvovirus, enteric disease INTRODUCTION Canine parvovirosis, caused by canine parvovirus type 2 (CPV-2), emerged in the 1970s as an important disease affecting dogs, causing severe hemorrhagic gastroenteritis and death [1]. It can occur in any breed, gender, and age; however, puppies of 4 to 12 weeks of age are most commonly afflicted [5,11]. CPV-2 was rapidly replaced with the new antigenic variants CPV-2a and CPV-2b [7]. In 2000 a new variant of the virus, called CPV-2c [4], was discovered, and has since been reported in several parts of the world, including Brazil [13,14]. This variant has been related to hemorrhagic gastroenteritis in dogs with up to 2 years of age [4,7,8], although some cases have been described in older animals with a full vaccination history [6,7]. The aim of this study was to describe a case of parvovirosis caused by variant 2c (CPV-2c) in a 5-year-old dog, with immunohistochemical and molecular characterization CASE A 5-year-old male mixed breed dog was submitted to necropsy with a history of intense sanguinolent diarrhea over three days, vomiting, dehydration, marked apathy and anorexia. It was reported that the dog had received three doses of commercial vaccine against parvovirus, canine distemper, adenovirus type 1, adenovirus type 2 and parainfluenza at 45, 66 and 87 days of age, however, it received no yearly revaccination. At necropsy a good physical condition, pallid oral and conjunctival mucosae were observed. The small intestine showed a very reddish and wrinkled serosa (Figure 1a), the wall was thickened, the mucosae was diffusely wrinkled and yellowed (Figure 1b) with evidenced

4 Peyer plaques and there was no content in the final portion of the intestine. The mesenteric lymph nodes were enlarged and reddish. Multiple suffusions on the serosa of the stomach, and petechiae and subepicardial suffusions in the heart were observed. Several organs were collected, fixed in 10% formalin, routinely processed for histological examination and stained with hematoxylin and eosin. Feces samples were collected from the rectum for viral detection. The tissue samples were submitted to the immunohistochemical (IHC) technique with anti-parvovirus monoclonal antibody (MCA 2064) by the peroxidase-bound streptavidin-biotin method [12]. The extraction of viral DNA from the fecal sample for the detection of CPV was performed as described previously [2]. The PCR was carried out for the amplification of 583 bp (base pairs) of the VP2 gene (position ), using a previously described protocol [4]. The histological findings were, collapse of the lamina propria of the small intestine, and fusion of the villi, necrosis of enterocytes, atrophy and the disappearance of crypts, with dilation of remaining crypts showing large rounded nuclei with one or two evident nucleoli, exhibiting accentuated cellular pleomorphism in some cases forming syncytia (Figure 1c). In addition, there were bacterial colonies and fibrin adhered to the mucosae. The serosa showed diffuse congestion, marked transmural multifocal hemorrhage, thrombosis and fibrin deposition on the serosa surface. Necrosis of the germinative centers with moderate lymphoid depletion was observed in the lymphoid aggregates of the large intestine. In the bone marrow, spleen and mesenteric lymph node there were accentuated lymphoid depletion, hemorrhage and moderate hemosiderosis. The remaining tissue of the thymus showed accentuated multifocal to coalescent hemorrhage. The anti-parvovirus IHC showed intense immunostaining of the cytoplasm of epithelial cells, mainly in the crypts of the small intestine (Figure 1d). In the spleen and lymph node there was intense immunostaining in the lymphocytes of follicular centers (Figure 1e).

5 In the liver, staining was observed in the cytoplasm of Kupffer cells. In the bone marrow there was staining mainly in the cytoplasm of a few cells (Figure 1f). The application of the PCR and sequencing techniques detected CPV-2c. The partial amplification of the VP2 gene revealed a single band with the expected size of 583bp. DISCUSSION Diarrhea in dogs has been associated with a wide variety of viral agents; the canine parvovirus, rotavirus and coronavirus being the main primary pathogens involved [11]. Since CPV-2 emerged at the end of the 1970s this pathogen has gained great importance in the care of dogs and is probably the most common infectious disease of canine species [11]. Shortly after appearing in the canine population, CPV-2 underwent alterations in some of its amino acids, which resulted in new and better adapted viral strains [4,7]. CPV-2c was detected for the first time in 2000 and rapidly spread to diverse regions of the world [4,8,13,14]. Although cases of parvovirosis are typically associated with puppies of 4 to 12 weeks of age, the period in which a decrease in the maternal antibodies occurs, a case of parvovirosis in a 5-year-old dog is reported herein. The clinical signs, as well as the macroscopic and microscopic findings for this dog are similar to those described for other fatal cases of canine parvovirosis [3,12]. Studies in which circulating viral strains have been identified have demonstrated the importance of CPV-2c in outbreaks of parvovirosis in previously vaccinated puppies [8,13,14]. There are few reports of the detection of CPV-2c in adult dogs [6,7]. The majority of cases described relate to dogs up to 2 ½ years of age, one exception being a case involving a 12-year-old dog [7]. Hemorrhagic gastroenteritis in dogs can sporadically be associated with intestinal infections by type A Clostridium perfringens, parasitism by Ancylostoma caninum, [3] and Cryptosporidium parvum [9]. Recently a new canine circovirus (DogCV) was described in the

6 liver of a dog with severe hemorrhagic gastroenteritis, vasculitis, and granulomatous lymphadenitis [10]. DogCV also could be a complicating factor in other canine infectious diseases, as with PCV2 [10]. In conclusion, CPV-2, besides being a principal infectious agent in young dogs, has evolved the new variant 2c which should be considered an important pathogen in the differential diagnosis of the causes of sanguinolent diarrhea Declaration of interest. The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper REFERENCES 1 Appel M.J.G., Scott F.W. & Carmichael L.E Isolation and immunisation studies of a canine parvo-like virus from dogs with haemorrhagic enteritis. Veterinary Record. 105: Boom R., Sol C.J.A., Salimans M.M.M., Jansen C.L., Wertheim-van Dillen P.M.E. & Noordaa V.D Rapid and simple method for purification of nucleic acids. Journal of Clinical Microbiology. 28: Brown C.C., Baker D.C. & Barker I.K Alimentary system. In: Maxie M.G. (Ed.). Jubb, Kennedy, and Palmer s Pathology of Domestic Animals. 5th edn. v.2. Philadelphia: Saunders Elsevier, pp Buonavoglia C., Martella V., Pratelli A., Tempesta M., Cavalli A., Buonavoglia D., Bozzo G., Elia G., Decaro N. & Carmichael L Evidence for evolution of canine parvovirus type 2 in Italy. Journal of General Virology. 82(12): Decaro N. & Buonavoglia C Canine parvovirus - A review of epidemiological and diagnostic aspects, with emphasis on type 2c. Veterinary Microbiology. 155: 1-12.

7 Decaro N., Cirone F., Desario C., Elia G., Lorusso E., Colaianni M.L., Martella V. & Buonavoglia C Severe parvovirus in a 12-year-old dog that had been repeatedly vaccinated. Veterinary Record. 164: Decaro N., Desario C., Elia G., Martella V., Mari V., Lavazza A., Nardi M. & Buonavoglia C Evidence for immunisation failure in vaccinated adult dogs infected with canine parvovirus type 2c. New Microbiologica. 31: Decaro N., Martella V., Desario C., Bellacicco A.L., Camero M., Manna L., D Aloja D. & Buonavoglia C First detection of canine parvovirus type 2c in pups with haemorrhagic enteritis in Spain. Journal of Veterinary Medical Science. 53(10): Hall E.J. & German A.J Diseases of the small intestine. In: Ettinger S.J. & Feldman E.C (Eds). Veterinary Internal Medicine. 6th edn. v.2. St. Louis: Elsevier Saunders, p Li L., McGraw S., Zhu K., Leutenegger C.M., Marks S.L., Kubiski S., Gaffney P., Dela Cruz Jr. F.N., Wang C., Delwart E. & Pesavento P.A Circovirus in tissues of dogs with vasculitis and hemorrhage. Emerging Infectious Diseases. 1994): Mccaw D.L. & Hoskins J.D Canine viral enteritis. In: Greene C.E. (Ed). Infectious diseases of the dog and cat. Cap.8. St. Louis: Elsevier Saunders, pp Oliveira E.C., Pescador C.A., Sonne L., Pavarini S.P., Santos A.S., Corbellini L.G. & Driemeier D Análise imuno-histoquímica de cães naturalmente infectados pelo parvovírus canino. Pesquisa Veterinária Brasileira. 29(2): Pinto L.D., Streck A.F., Gonçalves K.R., Souza C.K., Corbellini A.O., Corbellini L.G. & Canal C.W Typing of canine parvovirus strains circulating in Brazil between 2008 and Virus Research. 165(1):

8 Streck A.F., Souza C.K., Gonçalves K.R., Zang L., Pinto D.B. & Canal C.W First detection of canine parvovirus type 2c in Brazil. Brazilian Journal of Microbiology. 40(3): Figure 1. Canine parvovirosis in dog. Small intestine: A: very reddish and wrinkled serosa. B: thickened intestinal wall and mucosae diffusely wrinkled and yellowed. C: collapsed intestinal lamina propria, with fusion of the villi, atrophy and disappearance of crypts, and dilation of the remaining crypts (HE, Obj. 10). Immunohistochemistry for CPV-2, Biotinstreptavidin peroxidase method: D: small intestine with staining of enterocytes, mainly of crypts (Obj. 10). E: lymph node with immunostaining in lymphocytes of follicular centers (Obj. 20). F: bone marrow with staining in few cells (Obj. 40). CASE REPORT Aspectos histopatológicos e imuno-histoquímicos da raiva em raposas Cerdocyon thous Histopathological and Immunohistochemical Aspects of Rabies in Foxes Cerdocyon thous Jeann Leal de Araújo 1, Antônio Flavio Medeiros Dantas 1, Glauco José Nogueira de Galiza 2, Pedro Miguel Ocampos Pedroso 3, Maria Luana Cristiny Rodrigues Silva 1, Luciano da Anunciação Pimentel 4 & Franklin Riet-Correa Hospital Veterinário, Laboratório de Patologia Animal, CSTR, UFCG, Campus de Patos, PB, Brazil. 2 Laboratório de Patologia Veterinária, Departamento de Patologia, CCS, UFSM, Santa Maria, RS, Brazil. 3 Laboratório de Patologia Veterinária, CCA, UFRB, Cruz das Almas, BA, Brazil. 4 Universidade de Cuiabá, Cuiabá, MT, Brazil. CORRESPONDENCE: A.F.M. Dantas [dantas.af@uol.com.br - Tel.: +55 (83) ]. Hospital Veterinário, Laboratório de Patologia Animal, CSTR, UFCG, Campus de Patos. Avenida Universitária S/N, Santa Cecilia. CEP Patos, PB, Brazil.

9 ABSTRACT Background: Several wild canids are considered reservoirs of rabies virus in the Northeast of Brazil, two wild canids have been reported as reservoirs of rabies virus Cerdocyon thous (crab-eating fox) and Pseudalopex vetulus (hoary fox) (previously called Dusicyon vetulus). The diagnosis of rabies in foxes is usually performed through fluorescent antibody test (FAT) and mouse inoculation test (MIT). However, until the moment, there are no detailed histopathological and immunohistochemical (IHC) description studies in foxes affected by this disease studies. Therefore, the aim of this work was the characterization of pathological and IHC findings of foxes with rabies sent to the Laboratory of Animal Pathology (LPA) of the Federal University of Campina Grande (UFCG) in Patos, semiarid region of Paraiba, Brazil. Case: Two foxes were sent to the LPA, phenotypic species identification through analysis of morphological aspects was performed and posteriorly necropsied. Fragments of organs of the thoracic and abdominal cavities, salivary glands, eye and Gasser ganglia were collected in addition to the central nervous system (CNS) that was collected integer and fixed at 10% buffered formalin. Later, serial sections of the 16 fragments of the CNS were performed, measuring about 0.5 cm thick and cleaved. Fragments of cerebrum, cerebellum, brainstem, spinal cord and salivary glands were sent to performing FAT and MIT. Paraffin blocks with fragments of the hippocampus were selected and submitted to IHC. Macroscopically, the two foxes had multiple skin lacerations, bone fractures and ruptures of abdominal organs from injuries. The vessels of the meninges were slightly congested. Histologically, the CNS had diffuse non-suppurative encephalitis, with inflammatory infiltrate composed primarily by lymphocytes and plasma cells, forming mononuclear perivascular cuffing, and gliosis associated with mild eosinophilic intracytoplasmic inclusion corpuscles primarily in neurons

10 of the cortex, basal ganglia, hippocampus, thalamus, colliculi, bridge, obex and cerebellum. Meningitis and mild myelitis non-suppurative were also observed in both cases with rare viral inclusions in spinal cord neurons. Similar inflammation was also observed in the Gasser ganglion and in others peripheral nerve ganglia. Adrenal and salivary glands showed multifocal areas of moderate mononuclear inflammatory infiltrate composed mainly by macrophages and plasma cells. Strong positive IHC labeling was observed for rabies in the neurons in different brain regions, especially in the cerebral cortex and in Purkinje cells of the cerebellum. In both cases the diagnosis of rabies was confirmed by immunofluorescence and mouse inoculation. Discussion: The diagnosis of rabies in foxes wasconducted through the characteristic histopathologic findings of the disease observed in the CNS and confirmed by the FAT, MIT and IHC. Although the histopathological findings in foxes are similar to what is observed in other species, the severity of inflammatory lesions and the large amount of inclusion bodies in the nervous tissue is an outstanding feature, regardless of the inflammatory response. The diagnosis of rabies in foxes can be achieved by characteristic histopathologic findings of the CNS, supported by evaluating peripheral nerve ganglia, salivary glands and adrenal which may also present similar microscopic lesions. Auxiliary Laboratory tests must be performed, such as FAT, MIT and IHC for confirmation of the disease. Keywords: wild animals, canids, Lyssavirus. Descritores: animais selvagens, canídeos, Lyssavirus INTRODUÇÃO Vários canídeos silvestres são considerados reservatórios do vírus rábico, a exemplo da raposa vermelha (Vulpes vulpes), distribuída mundialmente na Europa, América do Norte, norte da África e na Austrália [4].

11 No Brasil, o ciclo silvestre terrestre da raiva é representado principalmente por saguis (Callithrix jacchus) ou raposas (Cerdocyon thous). Na região Nordeste, dois canídeos silvestres já foram relatados como reservatórios do vírus rábico: Cerdocyon thous (crab-eating fox, cachorro-do-mato) e Pseudalopex vetulus (hoary fox, raposa cinzenta) (previamente denominada Dusicyon vetulus) [3,6]. Existem relatos de casos de raposas com raiva e transmissão para humanos nos estados do Ceará, Paraíba, Pernambuco, Bahia e Minas Gerais [1,2] e dos 329 casos de raiva notificados no período de 2002 a 2009, cerca de 88% eram de canídeos silvestres, todos ocorridos na região Nordeste, onde muitos desses eram mantidos como animais de estimação. Segundo dados da Secretaria de Vigilância em Saúde (SVS/MS) [13], no Brasil, os canídeos silvestres foram responsáveis por 7,9% dos 165 óbitos de humanos com raiva, no período de No Estado da Paraíba entre os anos de 2007 e 2010, foram notificados sete casos de raiva em cães e gatos, e cinco casos em canídeos silvestres [13]. Até o momento, não existem estudos detalhados de descrição histopatológica e imunohistoquímica em raposas acometidas por essa doença, portanto, o objetivo do presente trabalho é a caracterização dos achados patológicos e imuno-histoquímicos de raposas com raiva encaminhadas ao Laboratório de Patologia Animal (LPA) da Universidade Federal de Campina Grande (UFCG) em Patos, semiárido da Paraíba, Brasil CASOS As raposas utilizadas foram encaminhas mortas para o LPA da UFCG, realizada a identificação fenotípica da espécie através da análise de aspectos morfológicos do animal com base no Guia de Identificação de Canídeos Brasileiros [10] e posteriormente necropsiadas. Foram coletados fragmentos de órgãos das cavidades torácica e abdominal, glândulas salivares, globo ocular e gânglio de Gasser, além do sistema nervoso central (SNC) que foi coletado e fixado em formol tamponado a 10%. Posteriormente foram realizados cortes

12 seriados do encéfalo, medindo aproximadamente 0,5 cm de espessura e clivados 16 fragmentos do SNC identificados: 1) córtex frontal, 2) córtex parietal, 3) córtex temporal, 4) córtex occipital, 5) núcleos da base, 6) hipocampo, 7) tálamo, 8) colículo rostral, 9) colículo caudal, 10) pedúnculos cerebelar, 11) ponte, 12) óbex, 13) cerebelo, 14) medula cervical, 15) medula torácica e 16) medula lombar. Para a confecção das lâminas histológicas, os fragmentos clivados foram processados rotineiramente e coradas pela técnica de hematoxilina e eosina (HE). Seguindo protocolos estabelecidos previamente [5,8] foram realizadas as técnicas de imunofluorescência direta (IFD) e inoculação intracerebral em camundongos (ICC), respectivamente, em fragmentos do cérebro, cerebelo, tronco encefálico, medula espinhal e glândulas salivares das raposas foram enviados para a execução dessas técnicas. Blocos de parafina com fragmentos do hipocampo foram selecionados e submetidos à técnica de imuno-histoquímica para a detecção do vírus da raiva. Após desparafinizanação e reidratação dos tecidos, foi realizada recuperação antigênica com solução de citrato (ph 6,0) em forno micro-ondas, em potência máxima, por dez min. O anticorpo primário utilizado era policlonal para raiva produzido em cabras marcado com FITC (anticorpo conjugado de isotiocianato de fluorescência - Chemicon #5199) 1, diluído 1:1000 em solução tamponada fosfato salina (PBST) com Tween 20 (Sigma P2287) 2, e incubado por 60 min a 37Cº. O anticorpo secundário biotilinilado e o complexo estreptavidina-biotina-peroxidase (LSAB+System HRP) 3 foram utilizados consecutivamente, incubados à temperatura ambiente por 30 min e marcados através da adição do DAB + Substrate - Choromogen System 3 e contra corados com hematoxilina de Harris. Como controle positivo foi utilizado secções histológicas de casos confirmados de raiva em bovinos. Como controle negativo, as mesmas secções foram utilizadas, com substituição do anticorpo primário por PBST.

13 Esses casos ocorreram em anos distintos, sendo o primeiro em maio de 2010 e o segundo em abril de As duas raposas enviadas foram identificadas como pertencentes à espécie Cerdocyon thous. Segundo informações obtidas dos moradores da zona rural do Município de São José de Espinharas - PB, local onde os animais foram encontrados, as raposas foram atropeladas após terem sido vistas com sinais nervosos caracterizados por incoordenação, perda de equilíbrio, acentuado balançar compulsivo da cabeça e aparente debilidade muscular. Macroscopicamente as duas raposas apresentavam múltiplas lacerações cutâneas, fraturas ósseas e rupturas de órgãos abdominais provenientes de traumatismos. Os vasos das leptomeninges estavam levemente congestos. Histologicamente verificou-se reação inflamatória mononuclear, principalmente no SNC, variando no grau de intensidade e sua localização (Tabela 1). Havia encefalite não supurativa, caracterizada pela presença de infiltrado inflamatório constituído principalmente por linfócitos e plasmócitos, formando manguitos perivasculares (Figura 1A), associada a discreta gliose e corpúsculos de inclusões eosinofílicos intracitoplasmáticos principalmente em neurônios dos córtices, núcleos da base, hipocampo (Figura 1B), tálamo, colículos, ponte, óbex e cerebelo. Meningomielite linfoplasmocitária discreta com raras inclusões virais em neurônios da medula espinhal também foram observadas nos dois casos. Inflamação semelhante também foi observada nos gânglios nervosos periféricos, glândulas salivares e adrenais dos dois casos. Havia infiltrado inflamatório principalmente de linfócitos e plasmócitos entre os feixes nervosos do gânglio de Gasser, caracterizando ganglioneurite não supurativa (Figura 1C), associada a raras inclusões virais intracitoplasmáticas em neurônios. Ganglioneurite não supurativa também foi observada no gânglio ciliar do primeiro caso. Infiltrado linfoplasmocitário também foi encontrado nas glândulas salivares (Figura 1D) e nas adrenais dos dois casos, característicos de adenite e

14 adrenalite não supurativa. Corpúsculos de inclusões raramente foram verificados em agregados de neurônios distribuídos perifericamente a essas estruturas glandulares. No exame de IFD e na ICC o resultado foi positivo em todos os fragmentos testados para o vírus da raiva. Pela imuno-histoquímica os dois casos marcaram fortemente com anticorpos para o vírus rábico, demonstrando múltiplos agregados de grânulos distribuídos no pericário, como também na forma de corpúsculos grandes, únicos ou múltiplos no citoplasma de neurônios do córtex (Figura 2A) e ponte (Figura 2B). No controle negativo não foram observadas nenhum tipo de imunomarcação (Figura 2C) DISCUSSÃO O diagnóstico da raiva em raposas foi realizado através dos achados histopatológicos característicos da doença, observados no SNC e confirmados pela IFD, ICC e IHQ. Apesar dos achados histopatológicos encontrados nas raposas serem semelhantes ao que são observados em outras espécies [14], a severidade das lesões inflamatórias e a grande quantidade de corpúsculos de inclusão no tecido nervoso é uma característica marcante, independente da resposta inflamatória. A inflamação não supurativa e a presença de inclusões observadas nos gânglios nervosos periféricos encontrados ao redor ou dentro do tecido das glândulas salivares e das adrenais, semelhantemente a reação inflamatória observada no SNC, podem auxiliar no diagnóstico dessa patologia, principalmente nos casos em que não são observadas inclusões no SNC. As glândulas salivares também tem sido um importante órgão para a realização do isolamento viral do Lyssavirus, já tendo sido encontrada positividade para raiva em glândulas salivares avaliadas por outros autores [11]. Diferentemente dos herbívoros, onde geralmente as principais lesões são cerebelares, os carnívoros tendem a apresentar lesões mais intensas na região de hipocampo, entretanto, de

15 forma semelhante aos bovinos, pode haver áreas com pouca ou ausente reação inflamatória [14]. Um aspecto importante da análise histopatológica é a realização de cortes seriados do sistema nervoso central, uma vez que a localização e intensidade das lesões podem variar entre as regiões do SNC, não havendo uma uniformidade. A forte marcação nos neurônios do hipocampo no presente trabalho através da imunohistoquímica, difere dos achados de Stein et al. [12] que encontraram uma marcação moderada no hipocampo de raposas da espécie Urocyon cinereoargenteus e Vulpes vulpes. Apesar das implicações legais, muitas pessoas tem o hábito de criar raposas e outros animais silvestres no Nordeste brasileiro. Essa tradição aumenta significativamente o risco de transmissão da raiva para humanos e outros animais. No ano de 2012, duas pessoas morreram no Nordeste vítimas de raiva transmitidas por animais silvestres [13]. No Estado da Paraíba, a raposa tem sido o animal silvestre com maior número de agressões contra humanos, havendo no período de 2000 a 2003, cerca de 24 casos de agressões em humanos por raposas [7]. Propõe-se que a ocorrência de raiva em herbívoros nos Estados da Paraíba e Pernambuco é causada principalmente por uma variante do vírus chamada de RABV, relacionada a morcegos hematófagos e que ela está circulante nessa área do Nordeste por pelo menos sete anos, isolada por barreiras geográficas [9]. Sugere-se ainda, a presença de duas ramificações de Lyssavirus na região da Paraíba, sendo uma associada com quirópteros e outra com carnívoros. Entretanto, existe uma variabilidade genética nessas ramificações, subdividindo o grupo de quirópteros em morcegos insetívoros e morcegos hematófagos, e o grupo dos carnívoros em cão, raposa 1 e raposa 2 (está mais próxima do grupo cão ) [7]. Essa existência de variabilidade entre as ramificações das variantes do vírus rábico sugere que pelo menos dois grupos de vírus coexistem na mesma região e apesar de somente a variante de morcegos hematófagos ter sido incriminada como causadora da raiva em herbívoros, a criação de animais silvestres como animais de estimação no Nordeste favorece o

16 risco de transmissão da doença para os animais de produção, sugerindo um papel importante das raposas nesse cenário. A presença da variabilidade genética das variantes de raposas, sugerem que estes animais tem um papel muito mais importante na manutenção e disseminação da raiva no Brasil do que antes se pensava, tendo uma implicação de saúde pública significante já que nessa região o monitoramento desses animais é muitas vezes ineficiente ou ausente, e a prática da vacinação de animais silvestres não tem sido empregada no país [2]. O diagnóstico da raiva em raposas pode ser realizado pelos achados histopatológicos característicos do SNC, auxiliado pela avaliação dos gânglios nervosos periféricos, glândulas salivares e adrenais que também podem apresentar lesões microscópicas semelhantes. Em adição os exames laboratoriais auxiliares devem ser realizados, como IFD, ICC e IHQ para a confirmação da doença. MANUFACTURERS 1 Chemicon International Inc. Temecula, CA, USA. 2 Sigma-Aldrich Corp. St. Louis, MO, USA. 3 Dako Cytomation. Carpinteria, CA, USA. Declaration of interest. The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. REFERENCES 1 Araújo F.A.A Raiva humana no Brasil: f. Belo Horizonte, MG. Dissertação (Mestrado em Medicina Veterinária) - Programa de Pós-graduação em Medicina Veterinária, Universidade Federal de Minas Gerais. 2 Bernardi F., Nadin-Davis S.A., Wandeler A.I., Armstrong J., Gomes, A.A.B., Lima F.S., Nogueira F.R.B. & Ito F.H Antigenic and genetic characterization of rabies

17 viruses isolated from domestic and wild animals of Brazil identifies the hoary fox as a rabies reservoir. Journal of General Virology. 86(11): Carnieli Jr. P., Brandão, P.E., Carrieri M.L., Castilho J.G., Macedo C.I., Machado L.M., Rangel N., Carvalho R.C., Carvalho V.A., Montebello L., Wada M. & Kotait I Characterization of rabies virus isolated from canids and identification of the main wild canid host in Northeastern Brazil. Virus Research. 131(1): Childs J.E. & Real L.A Epidemiology. In: Jackson A.C. & Wunner W.H. (Eds). Rabies. San Diego: Academic Press, pp Dean D.J., Abelseth M.K. & Atanasiu P The fluorescent antibody test. In: Meslin F.X., Kaplan M.M. & Koprowski H. (Eds). Laboratory Techniques in Rabies. 4th edn. Geneva: World Health Organization, pp Gomes A.A.B Epidemiologia da raiva: caracterização de vírus isolados de animais domésticos e silvestres do semi-árido paraibano da região de Patos, Nordeste do Brasil. 107f. São Paulo, SP. Tese (Doutorado em Medicina Veterinária) - Programa de Pós-graduação em Epidemiologia Experimental Aplicada às Zoonoses, Universidade de São Paulo. 7 Gomes A.A.B., Silva M.L.C.R., Bernardi F., Sakai T., Itou T. & Ito F.H Molecular epidemiology of animal rabies in the semiarid region of Paraíba, Northeastern Brazil. Arquivos do Instituto Biológico. 79(4): Koprowski H The mouse inoculation test. In: Meslin F.X., Kaplan M.M. & Koprowski H. (Eds). Laboratory Techniques in Rabies. 4th edn. Geneva: World Health Organization, pp Mochizuki N., Kawasaki H., Silva M.L.C.R., Afonso J.A.B., Itou T., Fumio H. & Sakai T Molecular epidemiology of livestock rabies viruses isolated in the northeastern Brazilian states of Paraíba and Pernambuco from BMC Research Notes. 5(32): 1-7.

18 Ramos Jr. V.A., Pessutti C. & Chieregatto C.A.F.S Guia de Identificação dos Canídeos Silvestres Brasileiros. Sorocaba: JoyJoy Studio Ltda. - Comunicação Ambiental, 35p. 11 Silva M.L.C.R, Lima F.S., Gomes A.A.B., Azevedo S.S., Alves C.J., Bernardi F. & Ito F.H Isolation of rabies virus from the parotid salivary glands of foxes (Pseudalopex vetulus) from Paraíba State, Northeastern Brazil. Brazilian Journal of Microbiology. 40(3): Stein L.T., Rech R.R., Harrison L. & Brown C.C Immunohistochemical study of rabies virus within the central nervous system of domestic and wildlife species. Veterinary Pathology. 47(4): SVS - Ministério da Saúde. Fundação Nacional de Saúde Mapas da Raiva no Brasil. Brasília: Organização Pan-Americana da Saúde; Organização Mundial da Saúde; Ministério da Saúde. 14 Zachary J.F Sistema Nervoso. In: McGavin M.D. & Zachary J.F. (Eds). Bases da Patologia em Veterinária. 4.ed. Rio de Janeiro: Elsevier, pp Tabela 1. Distribuição das lesões no SNC de raposas (Cerdocyon thous) com raiva de acordo com a intensidade da resposta inflamatória e presença de corpúsculos de Negri. Figura 1. Lesões histológicas de raposas (Cerdocyon thous) com raiva. (A) Córtex occipital mostrando manguitos mononucleares perivasculares e corpúsculos de Negri (seta) [HE, 10x]. (B) Hipocampo com múltiplos corpúsculos de Negri (setas) [HE, 20x]. (C) Gânglio trigêmeo com infiltrado inflamatório mononuclear [HE, 20x]. (D) Glândula salivar com inflamação não supurativa [HE, 20x]. Figura 2. Seções do encéfalo de raposas (Cerdocyon thous) com raiva, submetidas à técnica de imuno-histoquímica. (A) Córtex e (B) Tronco encefálico (ponte) mostrando múltiplos agregados de grânulos amarronzados distribuídos no pericário e prolongamentos citoplasmáticos de neurônios, caracterizando imunomarcação positiva para raiva. (C) Córtex. Controle negativo (sem anticorpo). Técnica de imuno-histoquímica método da estreptavidinabiotina-peroxidase (LSAB+System HRP) contracorados com Hematoxilina de Harris [Barra = 20 µm].

19 CASE REPORT Classical Scrapie Diagnosis in ARR/ARR Sheep in Brazil Juliano Souza Leal 1,2, Caroline Pinto de Andrade 2, Gabriel Laizola Frainer Correa 2, Gisele Silva Boos 2, Matheus Viezzer Bianchi 2, Sergio Ceroni da Silva 2,Rui Fernando Felix Lopes 3 & David Driemeier Programa de Pós-graduação em Ciências Veterinárias (PPGCV), Faculdade de Veterinária (FaVet), Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil. 2 Setor de Patologia Veterinária (SPV), Departamento de Patologia Clínica Veterinária (DPCV), FAVET, UFRGS, Porto Alegre, RS, Brazil. 3 Departamento de Ciências Morfológicas, Instituto de Ciências Básicas da Saúde (ICBS), UFRGS, Porto Alegre, RS. CORRESPONDENCE: J.S. Leal [julianoob@gmail.com - Tel.: +55 (51) ]. Setor de Patologia Veterinária, FAVET, UFRGS. Av. Bento Gonçalves n. 9090, Bairro Agronomia. CEP Porto Alegre, RS, Brazil.

20 ABSTRACT Background: Scrapie is a transmissible spongiform encephalopathy (TSE) that affects sheep flocks and goat herds. The transfer of animals or groups of these between sheep farms is associated with increased numbers of infected animals and with the susceptibility or the resistance to natural or classical scrapie form. Although several aspects linked to the etiology of the natural form of this infection remain unclarified, the role of an important genetic control in scrapie incidence has been proposed. Polymorphisms of the PrP gene (prion protein, or simply prion), mainly in codons 136, 154, and 171, have been associated with the risk of scrapie. Case: One animal from a group of 292 sheep was diagnosed positive for scrapie in the municipality of Valparaíso, state of São Paulo, Brazil. The group was part of a flock of 811 free-range, mixed-breed Suffolk sheep of the two genders and ages between 2 and 7 years from different Brazilian regions. Blood was collected for genotyping (for codons 136, 141, 154 and 171), and the third lid and rectal mucosa were sampled for immunohistochemistry (IHC) for scrapie, from all 292 animals of the group. IHC revealed that seven (2.4%) animals were positive for the disease. Collection of samples was repeated for 90 animals, among which the seven individuals diagnosed positive and 83 other animals that had some degree of kinship with those. These 90 sheep were sacrificed and necropsied, when samples of brain (obex), cerebellum, third eyelid, rectal mucosa, mesenteric lymph node, palatine tonsil, and spleen were collected for IHC. The results of IHC analyses carried out after necropsy of the seven positive animals submitted to the second collection of lymphoreticular tissue and of the 83 animals with some degree of kinship with them confirmed the positive diagnosis obtained in the first analysis, and revealed that three other sheep were also positive for scrapie. Samples of 80 animals (89%) were negative for the disease in all organs and tissues analyzed. In turn, 10 sheep (11%) were positive, presenting immunoreactivity in one or more tissues. 20

21 Genotyping revealed the presence of four of the five alleles of the PrP gene commonly detected in sheep: ARR, ARQ, VRQ and ARH. These allele combinations formed six haplotypes: ARR/ARR, ARR/ARQ, ARH/ARH, ARQ/ARH, ARQ/ARQ and ARQ/VRQ. Animals were classified according to susceptibility to scrapie, when 8.9% of the genotyped sheep were classified into risk group R1 (more resistant, with no restriction to breeding). In turn, 40% of the animals tested ranked in groups R4 and R5 (genetically very susceptible, cannot be used for breeding purposes). Discussion: The susceptibility of sheep flocks depends on the genetic pattern of animals and is determined by the sequence of the gene that codifies protein PrP. Additionally, numerous prion strains are differentiated based on pathological and biochemical characteristics, and may affect animals differently, depending on each individual s genotype. Most epidemiologic data published to date indicate that animals that carry the ARR/ARR genotype are less susceptible to classical scrapie. However, in the present study, the fact that two scrapie-positive sheep presented the haplotype ARR/ARR indicates that this genotype cannot always be considered an indicator of resistance to the causal agent of the classical manifestation of the disease. The coexistence in the same environment of several crossbred animals from different flocks and farms, which characterizes a new heterogeneous flock, may have promoted a favorable scenario to spread the disease, infecting animals in the most resistant group. Keywords: biopsy, scrapie, TSEs, immunohistochemistry. Descritores: biopsia, scrapie clássico, EETs, imuno-histoquímica INTRODUCTION Scrapie, also called epizootic tremor, is a transmissible spongiform encephalopathy (TSE) that affects sheep flocks and goat herds [44]. The relocation of animals to and from sheep farms has been associated with increased numbers of infected animals [28,39]. Once it 21

22 is introduced in a flock, the disease may be transmitted both vertically, from ewe to lamb, and horizontally, across animals [15,39,49]. Many aspects surrounding the etiology of the natural form of this infection remain to be clarified, though the existence of an important genetic control has been proposed to explain the disease s incidence [24]. The analysis of the gene PrP (prion protein, or simply prion) in ovine of different breeds has drawn attention to the interaction between host genotype polymorphisms and susceptibility to the infectious agent of scrapie [10,21-23,31]. Single nucleotide polymorphisms (SNT) have been linked to susceptibility or resistance to classical scrapie. These polymorphisms occur at codons 136 (A or V, alanine or valine), 154 (R or H, arginine or histidine) and 171 (R, Q or H, arginine, glutamine or histidine) [16]. The diagnosis of the classical form in sheep with haplotype A136R154R171 is rare [24]. Under natural exposure conditions, this genotype (ARR/ARR) has been acknowledged as having the lowest risk for the classical form [16]. This case report describes the occurrence of an outbreak in a flock of mixed Suffolk sheep of varied origins in the state of São Paulo, southeastern Brazil, when the disease was diagnosed in two animals carrying the genotype ARR/ARR, compatible with classical scrapie CASE In 2011, one ovine head from a group of 292 animals was diagnosed with the classical form of scrapie. These sheep were part of a larger flock of 811 free-range animals of both genders and between 2 and 7 years of age that were brought from southern, southeastern and midwestern Brazil. Since the animal died, and diagnosis was carried out after the death, a decision was made to collect blood samples from all 292 animals of the group, for sequencing and genotyping (for codons 136, 141, 154 and 171). In addition, the third eyelid and the rectal mucosa of all 292 animals were biopsied for immunohistochemistry (IHC). After IHC, a new 22

23 collection was conducted in 90 animals (approximately 30% of the original group). These included the animals with positive diagnosis in the first collection, and those that had some degree of kinship with scrapie-positive sheep in the original group. These animals were sacrificed and necropsied to collect brain tissue (obex), cerebellum, third eyelid, rectal mucosa, mesenteric lymph node, palatine tonsil, and spleen used in the IHC analyses. Tissue samples were collected and processed for histology and IHC for PrP Sc following the methodology proposed by O Rourke et al. [43]. Rectal biopsy samples were collected and processed according to Espenes et al. [17]. Anti-prion 1 monoclonal antibodies F89/ and F99/ were diluted to a 1:500 solution and added to samples, which were then incubated in a humid chamber at 4ºC for 12 h [34]. Blood was collected by punction of the jugular vein using EDTA as anticoagulant and stored at -20ºC for subsequent processing. Genomic DNA of sheep was extracted using 500 μl whole blood and the QIAmp DNA Blood Kit 2 according to the manufacturer s instructions. PCR was carried out using the DNA sample, 15 pmol each primer, 1X PCR buffer (Tris-HCl ph 8.4, 50 mm KCl) 3, MgCl2 1.5 mm, dntp μm, and 1U Platinum enzyme Taq DNA Polymerase 3 according to the following cycles: 95ºC for 5 min, 35 cycles at 95ºC for 30 s and at 58ºC for 30 s, and 72ºC for 30 s. PCR was performed using a forward primer flanking the 136 codon position (5 -ATGAAGCATGTGGCAGGAGC-3 ) and a reverse primer flanking the 171 codon position (5 -GGTGACTGTGTGTTGCTTGACTG-3 ). A 245-bp fragment was generated, which contains the regions of the main codons analyzed for susceptibility to scrapie [36]. The PCR product was purified and quantified using the commercial products Purelin 5 and Qubit 5, respectively, following the manufacturers instructions. Sequencing was performed with 3 ng DNA and 3.2 pmol each primer, using the BigDye Terminator v.1.1 Cycle Sequencing kit 6 in the ABI PRISM 3110 Genetic Analyzer 6. 23

24 Of the 292 mixed Suffolk sheep whose lymphoreticular tissues of the third eyelid were analyzed by IHC, seven (2.4%) were positive for scrapie in the first sample collection. The IHC results of the second samples collected from these seven sheep after necropsy and of the samples collected from the other 83 animals with some degree of kinship with them confirmed the positive diagnosis obtained initially, and revealed that three other animals were also positive for the scrapie. The samples of all organs and tissues of 80 animals (89%) were negative, while those of 10 sheep (11%) were positive, with immunoreactivity in one or more tissues. At least three lymphoid follicles were analyzed by IHC in all samples obtained from necropsied animals. No animal was positive in all samples collected, but different organs and tissues showed immunoreactivity. The third eyelid (Figure 1) and the palatine tonsil were the tissues with the highest percentage of immunoreactive samples (90%, 9/10). The lymphoid tissue of the rectal mucosa (Figure 2) showed immunoreactivity in only one animal (10%, 1/10). No immunoreactivity was observed in mesenteric lymph node, spleen and obex samples. Genotyping of codon 141 showed homozygosis for lysine (L141L or L/L) in all 90 animals investigated. The genotypes and frequencies of alleles for codons 136, 154 and 171 of these sheep (10 positive and 80 related) are shown in Table 1. Four of the five alleles of the PrP gene commonly detected in ovine were found: ARR, ARQ, VRQ and ARH. The allele AHQ was not detected in any sample. Of the 15 possibilities, these allele combinations formed six haplotypes: ARR/ARR, ARR/ARQ, ARH/ARH, ARQ/ARH, ARQ/ARQ and ARQ/VRQ. The haplotype ARR/ARQ was detected in 39 samples (43.3%) and was the most frequent, followed by haplotypes ARQ/ARQ, detected in 34 (37.7%), ARR/ARR, present in eight (8.9%), and ARQ/ARH, observed in five samples (5.6%). Haplotypes ARH/ARH and 24

25 ARQ/VRQ were detected in two samples each (2.2%). The classification of animals according to the susceptibility criteria described by Dawson et al. [13] placed 8.9% of the total number of genotyped animals in scrapie risk group R1, which includes more resistant animals that are not subject to reproduction restrictions. A significant percentage of animals (43.3%) was in risk group R2, which requires careful selection for breeding. In addition, 7.8% of animals were in group R3 (intermediate risk), while 40% were in groups R4 and R5 (highly susceptible animals that should not be included in reproduction programs) DISCUSSION The susceptibility of sheep flocks to scrapie depends largely on the genetic pattern of the animal, and is determined mainly by the sequence of the gene that codifies the PrP protein, since there are several polymorphisms that affect the conversion of the cell protein PrP C to its pathological form, PrP Sc [8,9]. Nevertheless, it is not possible to consider the occurrence of only one form of ovine prion, since there are numerous prion strains with different pathological and biochemical characteristics that may affect animals distinctively, depending on their genotypes [1,30]. In the present study, the frequency of codon VRQ was very low (2.2%), confirming previous findings, which revealed that the alleles ARR and ARQ prevail in Suffolk sheep, and that the allele ARH sometimes is detected [12,32]. The high sensitivity of homozygous VRQ carriers or of individuals with ARQ haplotypes has also been reported in the literature [24]. This condition raises concerns about susceptibility from the epidemiological perspective, since the allele VRQ, which is rare or absent in breeds like Suffolk, was present in two animals, one of which was positive for scrapie. Most epidemiological and genetic data published indicate that sheep carrying the haplotype ARR/ARR are less susceptible to classical form, while animals with the haplotype 25