EXAMPLES - EXEMPLOS 1
CASE REPORT 2
3
Enteritis Caused by Type 2c Canine Parvovirus in a 5-Year-Old Dog 4
Veronica Machado Rolim1, Luciana Sonne1, Renata Assis Casagrande2, Suyene Oltramari 5
Souza1, Luciane Dubina Pinto3, Angelica Terezinha Barth Wouters4, Flademir Wouters4, 6
Cláudio Wageck Canal3 & David Driemeier1 7
8
1Setor de Patologia Veterinária (SPV), Faculdade de Veterinária (FaVet), Universidade 9
Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil. 2Patologia Veterinária, 10
Universidade do Estado de Santa Catarina (UDESC), Lages, SC, Brazil. 3Setor de Virologia, 11
FaVet, UFRGS, Porto Alegre, RS. 4 Setor de Patologia Veterinária, Universidade Federal de 12
Lavras (UFL), Lavras, MG, Brazil. CORRESPONDENCE: D. Driemeier 13
[davetpat@ufrgs.br - Tel.: +55 (51) 3308-6107]. Faculdade de Veterinária - UFRGS. Av. 14
Bento Gonçalves n. 9090, Bairro Agronomia. CEP 91540-000 Porto Alegre, RS, Brazil. 15
16 17
ABSTRACT 18
Background: Canine parvovirosis, caused by canine parvovirus type 2 (CPV-2), emerged in 19
the 1970s as an important disease affecting dogs, causing severe hemorrhagic gastroenteritis 20
and death. It can occur in any breed, gender, and age; however, puppies of 4 to 12 weeks of 21
age are most commonly afflicted. In 2000 a new variant of the virus, called CPV-2c, was 22
discovered, and has been related to hemorrhagic gastroenteritis in dogs with up to 2 years of 23
age, although some cases have been described in older animals with a full vaccination history. 24
This paper reports a case of enteritis by canine parvovirus type 2c (CPV-2c) in a 5-year-old 25
dog. 26
Case: At necropsy a pallid oral and conjunctival mucosae were observed. The small intestine 27
showed a very reddish and wrinkled serosa, the wall was thickened, the mucosae was 28
diffusely wrinkled and yellowed with evidenced Peyer plaques and there was no content in 29
the final portion of the intestine. The mesenteric lymph nodes were enlarged and reddish. 30
Multiple suffusions on the serosa of the stomach, and petechiae and subepicardial suffusions 31
in the heart were observed. The histological findings were, collapse of the lamina propria of 32
the small intestine, and fusion of the villi, necrosis of enterocytes, atrophy and the 33
disappearance of crypts, with dilation of remaining crypts showing large rounded nuclei with 34
one or two evident nucleoli, exhibiting accentuated cellular pleomorphism in some cases 35
forming syncytia. In addition, there were bacterial colonies and fibrin adhered to the mucosae. 36
The serosa showed diffuse congestion, marked transmural multifocal hemorrhage, thrombosis 37
and fibrin deposition on the serosa surface. Necrosis of the germinative centers with moderate 38
lymphoid depletion was observed in the lymphoid aggregates of the large intestine. In the 39
bone marrow, spleen and mesenteric lymph node there were accentuated lymphoid depletion, 40
hemorrhage and moderate hemosiderosis. The remaining tissue of the thymus showed 41
accentuated multifocal to coalescent hemorrhage. The anti-parvovirus IHC showed intense 42
immunostaining of the cytoplasm of epithelial cells, mainly in the crypts of the small 43
intestine. In the spleen and lymph node there was intense immunostaining in the lymphocytes 44
of follicular centers. The PCR and sequencing techniques applied to the sample allowed the 45
identification of CPV-2c. 46
Discussion: Diarrhea in dogs has been associated with a wide variety of viral agents; the 47
canine parvovirus, rotavirus and coronavirus being the main primary pathogens involved. 48
Since CPV-2 emerged at the end of the 1970s this pathogen has gained great importance in 49
the care of dogs and is probably the most common infectious disease of canine species. 50
Shortly after appearing in the canine population, CPV-2 underwent alterations in some of its 51
amino acids, which resulted in new and better adapted viral strains. Studies in which 52
circulating viral strains have been identified have demonstrated the importance of CPV-2c in 53
outbreaks of parvovirosis in previously vaccinated puppies. There are few reports of the 54
detection of CPV-2c in adult dogs. The majority of cases described relate to dogs up to 2 ½ 55
years of age, one exception being a case involving a 12-year-old dog This new variant of 56
CPV-2 should be considered as an important pathogen in the diagnosis of causes of 57
sanguinolent diarrhea in adult dogs. 58
Keywords: CPV-2c, adult dogs, parvovirus, enteric disease. 59
60
INTRODUCTION 61
Canine parvovirosis, caused by canine parvovirus type 2 (CPV-2), emerged in the 62
1970s as an important disease affecting dogs, causing severe hemorrhagic gastroenteritis and 63
death [1]. It can occur in any breed, gender, and age; however, puppies of 4 to 12 weeks of 64
age are most commonly afflicted [5,11]. CPV-2 was rapidly replaced with the new antigenic 65
variants CPV-2a and CPV-2b [7]. In 2000 a new variant of the virus, called CPV-2c [4],was 66
discovered, and has since been reported in several parts of the world, including Brazil [13,14]. 67
This variant has been related to hemorrhagic gastroenteritis in dogs with up to 2 years of age 68
[4,7,8], although some cases have been described in older animals with a full vaccination 69
history [6,7]. The aim of this study was to describe a case of parvovirosis caused by variant 2c 70
(CPV-2c) in a 5-year-old dog, with immunohistochemical and molecular characterization. 71
72
CASE 73
A 5-year-old male mixed breed dog was submitted to necropsy with a history of 74
intense sanguinolent diarrhea over three days, vomiting, dehydration, marked apathy and 75
anorexia. It was reported that the dog had received three doses of commercial vaccine against 76
parvovirus, canine distemper, adenovirus type 1, adenovirus type 2 and parainfluenza at 45, 77
66 and 87 days of age, however, it received no yearly revaccination. 78
At necropsy a good physical condition, pallid oral and conjunctival mucosae were 79
observed. The small intestine showed a very reddish and wrinkled serosa (Figure 1a), the wall 80
was thickened, the mucosae was diffusely wrinkled and yellowed (Figure 1b) with evidenced 81
Peyer plaques and there was no content in the final portion of the intestine. The mesenteric 82
lymph nodes were enlarged and reddish. Multiple suffusions on the serosa of the stomach, and 83
petechiae and subepicardial suffusions in the heart were observed. 84
Several organs were collected, fixed in 10% formalin, routinely processed for 85
histological examination and stained with hematoxylin and eosin. Feces samples were 86
collected from the rectum for viral detection. The tissue samples were submitted to the 87
immunohistochemical (IHC) technique with anti-parvovirus monoclonal antibody (MCA 88
2064) by the peroxidase-bound streptavidin-biotin method [12]. The extraction of viral DNA 89
from the fecal sample for the detection of CPV was performed as described previously [2]. 90
The PCR was carried out for the amplification of 583 bp (base pairs) of the VP2 gene 91
(position 4003-4585), using a previously described protocol [4]. 92
The histological findings were, collapse of the lamina propria of the small intestine, 93
and fusion of the villi, necrosis of enterocytes, atrophy and the disappearance of crypts, with 94
dilation of remaining crypts showing large rounded nuclei with one or two evident nucleoli, 95
exhibiting accentuated cellular pleomorphism in some cases forming syncytia (Figure 1c). In 96
addition, there were bacterial colonies and fibrin adhered to the mucosae. The serosa showed 97
diffuse congestion, marked transmural multifocal hemorrhage, thrombosis and fibrin 98
deposition on the serosa surface. Necrosis of the germinative centers with moderate lymphoid 99
depletion was observed in the lymphoid aggregates of the large intestine. In the bone marrow, 100
spleen and mesenteric lymph node there were accentuated lymphoid depletion, hemorrhage 101
and moderate hemosiderosis. The remaining tissue of the thymus showed accentuated 102
multifocal to coalescent hemorrhage. 103
The anti-parvovirus IHC showed intense immunostaining of the cytoplasm of 104
epithelial cells, mainly in the crypts of the small intestine (Figure 1d). In the spleen and lymph 105
node there was intense immunostaining in the lymphocytes of follicular centers (Figure 1e). 106
In the liver, staining was observed in the cytoplasm of Kupffer cells. In the bone marrow there 107
was staining mainly in the cytoplasm of a few cells (Figure 1f). 108
The application of the PCR and sequencing techniques detected CPV-2c. The partial 109
amplification of the VP2 gene revealed a single band with the expected size of 583bp. 110
DISCUSSION 111
Diarrhea in dogs has been associated with a wide variety of viral agents; the canine 112
parvovirus, rotavirus and coronavirus being the main primary pathogens involved [11]. Since 113
CPV-2 emerged at the end of the 1970s this pathogen has gained great importance in the care 114
of dogs and is probably the most common infectious disease of canine species [11].Shortly 115
after appearing in the canine population, CPV-2 underwent alterations in some of its amino 116
acids, which resulted in new and better adapted viral strains [4,7]. 117
CPV-2c was detected for the first time in 2000 and rapidly spread to diverse regions of 118
the world [4,8,13,14]. Although cases of parvovirosis are typically associated with puppies of 119
4 to 12 weeks of age, the period in which a decrease in the maternal antibodies occurs, a case 120
of parvovirosis in a 5-year-old dog is reported herein. The clinical signs, as well as the 121
macroscopic and microscopic findings for this dog are similar to those described for other 122
fatal cases of canine parvovirosis [3,12]. 123
Studies in which circulating viral strains have been identified have demonstrated the 124
importance of CPV-2c in outbreaks of parvovirosis in previously vaccinated puppies 125
[8,13,14]. There are few reports of the detection of CPV-2c in adult dogs [6,7]. The majority 126
of cases described relate to dogs up to 2 ½ years of age, one exception being a case involving 127
a 12-year-old dog [7]. 128
Hemorrhagic gastroenteritis in dogs can sporadically be associated with intestinal 129
infections by type A Clostridium perfringens, parasitism by Ancylostoma caninum, [3] and 130
Cryptosporidium parvum [9]. Recently a new canine circovirus (DogCV) was described in the
liver of a dog with severe hemorrhagic gastroenteritis, vasculitis, and granulomatous 132
lymphadenitis [10]. DogCV also could be a complicating factor in other canine infectious 133
diseases, as with PCV2 [10]. 134
In conclusion, CPV-2, besides being a principal infectious agent in young dogs, has 135
evolved the new variant 2c which should be considered an important pathogen in the 136
differential diagnosis of the causes of sanguinolent diarrhea. 137
138
Declaration of interest. The authors report no conflicts of interest. The authors alone are 139
responsible for the content and writing of the paper. 140
141
REFERENCES 142
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of a canine parvo-like virus from dogs with haemorrhagic enteritis. Veterinary Record. 144
105: 156-159. 145
2 Boom R., Sol C.J.A., Salimans M.M.M., Jansen C.L., Wertheim-van Dillen P.M.E. & 146
Noordaa V.D. 1990. Rapid and simple method for purification of nucleic acids. Journal of 147
Clinical Microbiology. 28: 495-503.
148
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Jubb, Kennedy, and Palmer’s Pathology of Domestic Animals. 5th edn. v.2. Philadelphia:
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Saunders Elsevier, pp.3-293. 151
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parvovirus type 2 in Italy. Journal of General Virology. 82(12): 3021-3025. 154
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6 Decaro N., Cirone F., Desario C., Elia G., Lorusso E., Colaianni M.L., Martella V. &
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Buonavoglia C. 2009. Severe parvovirus in a 12-year-old dog that had been repeatedly 158
vaccinated. Veterinary Record. 164: 593-595. 159
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Buonavoglia C. 2008. Evidence for immunisation failure in vaccinated adult dogs infected 161
with canine parvovirus type 2c. New Microbiologica. 31: 125-130. 162
8 Decaro N., Martella V., Desario C., Bellacicco A.L., Camero M., Manna L., D’Aloja 163
D. & Buonavoglia C. 2006. First detection of canine parvovirus type 2c in pups with 164
haemorrhagic enteritis in Spain. Journal of Veterinary Medical Science. 53(10): 468-472. 165
9 Hall E.J. & German A.J. 2005. Diseases of the small intestine. In: Ettinger S.J. & 166
Feldman E.C (Eds). Veterinary Internal Medicine. 6th edn. v.2. St. Louis: Elsevier 167
Saunders, p.1332-1377. 168
10 Li L., McGraw S., Zhu K., Leutenegger C.M., Marks S.L., Kubiski S., Gaffney P., 169
Dela Cruz Jr. F.N., Wang C., Delwart E. & Pesavento P.A. 2013. Circovirus in tissues 170
of dogs with vasculitis and hemorrhage. Emerging Infectious Diseases. 1994): 534-541. 171
11 Mccaw D.L. & Hoskins J.D. 2006. Canine viral enteritis. In: Greene C.E. (Ed). Infectious 172
diseases of the dog and cat. Cap.8. St. Louis: Elsevier Saunders, pp.63-73.
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12 Oliveira E.C., Pescador C.A., Sonne L., Pavarini S.P., Santos A.S., Corbellini L.G. & 174
Driemeier D. 2009. Análise imuno-histoquímica de cães naturalmente infectados pelo 175
parvovírus canino. Pesquisa Veterinária Brasileira. 29(2): 131-136. 176
13 Pinto L.D., Streck A.F., Gonçalves K.R., Souza C.K., Corbellini A.O., Corbellini L.G. 177
& Canal C.W. 2010. Typing of canine parvovirus strains circulating in Brazil between 178
2008 and 2010. Virus Research. 165(1): 29-33. 179
14 Streck A.F., Souza C.K., Gonçalves K.R., Zang L., Pinto D.B. & Canal C.W. 2009. 180
First detection of canine parvovirus type 2c in Brazil. Brazilian Journal of Microbiology. 181
40(3): 465-469. 182
183
Figure 1. Canine parvovirosis in dog. Small intestine: A: very reddish and wrinkled serosa. 184
B: thickened intestinal wall and mucosae diffusely wrinkled and yellowed. C: collapsed 185
intestinal lamina propria, with fusion of the villi, atrophy and disappearance of crypts, and 186
dilation of the remaining crypts (HE, Obj. 10). Immunohistochemistry for CPV-2, Biotin-187
streptavidin peroxidase method: D: small intestine with staining of enterocytes, mainly of 188
crypts (Obj. 10). E: lymph node with immunostaining in lymphocytes of follicular centers 189
(Obj. 20). F: bone marrow with staining in few cells (Obj. 40). 190
191 192
CASE REPORT 193
Aspectos histopatológicos e imuno-histoquímicos da raiva em raposas
194Cerdocyon thous
195H
istopathological and
I
mmunohistochemical
A
spects of
R
abies in
F
oxes
196
Cerdocyon thous
197198
Jeann Leal de Araújo1, Antônio Flavio Medeiros Dantas1, Glauco José Nogueira de 199
Galiza2, Pedro Miguel Ocampos Pedroso3, Maria Luana Cristiny Rodrigues Silva1, 200
Luciano da Anunciação Pimentel4& Franklin Riet-Correa1 201
202
1Hospital Veterinário, Laboratório de Patologia Animal, CSTR, UFCG, Campus de Patos, PB, 203
Brazil. 2Laboratório de Patologia Veterinária, Departamento de Patologia, CCS, UFSM, Santa 204
Maria, RS, Brazil. 3Laboratório de Patologia Veterinária, CCA, UFRB, Cruz das Almas, BA, 205
Brazil. 4Universidade de Cuiabá, Cuiabá, MT, Brazil. CORRESPONDENCE: A.F.M. Dantas 206
[dantas.af@uol.com.br - Tel.: +55 (83) 9929-8064]. Hospital Veterinário, Laboratório de 207
Patologia Animal, CSTR, UFCG, Campus de Patos. Avenida Universitária S/N, Santa 208
Cecilia. CEP 58708-110 Patos, PB, Brazil. 209
210
ABSTRACT 211
Background: Several wild canids are considered reservoirs of rabies virus in the Northeast of 212
Brazil, two wild canids have been reported as reservoirs of rabies virus Cerdocyon thous 213
(crab-eating fox) and Pseudalopex vetulus (hoary fox) (previously called Dusicyon vetulus). 214
The diagnosis of rabies in foxes is usually performed through fluorescent antibody test (FAT) 215
and mouse inoculation test (MIT). However, until the moment, there are no detailed 216
histopathological and immunohistochemical (IHC) description studies in foxes affected by 217
this disease studies. Therefore, the aim of this work was the characterization of pathological 218
and IHC findings of foxes with rabies sent to the Laboratory of Animal Pathology (LPA) of 219
the Federal University of Campina Grande (UFCG) in Patos, semiarid region of Paraiba, 220
Brazil. 221
Case: Two foxes were sent to the LPA, phenotypic species identification through analysis of 222
morphological aspects was performed and posteriorly necropsied. Fragments of organs of the 223
thoracic and abdominal cavities, salivary glands, eye and Gasser ganglia were collected in 224
addition to the central nervous system (CNS) that was collected integer and fixed at 10% 225
buffered formalin. Later, serial sections of the 16 fragments of the CNS were performed, 226
measuring about 0.5 cm thick and cleaved. Fragments of cerebrum, cerebellum, brainstem, 227
spinal cord and salivary glands were sent to performing FAT and MIT. Paraffin blocks with 228
fragments of the hippocampus were selected and submitted to IHC. Macroscopically, the two 229
foxes had multiple skin lacerations, bone fractures and ruptures of abdominal organs from 230
injuries. The vessels of the meninges were slightly congested. Histologically, the CNS had 231
diffuse non-suppurative encephalitis, with inflammatory infiltrate composed primarily by 232
lymphocytes and plasma cells, forming mononuclear perivascular cuffing, and gliosis 233
associated with mild eosinophilic intracytoplasmic inclusion corpuscles primarily in neurons 234
of the cortex, basal ganglia, hippocampus, thalamus, colliculi, bridge, obex and cerebellum. 235
Meningitis and mild myelitis non-suppurative were also observed in both cases with rare viral 236
inclusions in spinal cord neurons. Similar inflammation was also observed in the Gasser 237
ganglion and in others peripheral nerve ganglia. Adrenal and salivary glands showed 238
multifocal areas of moderate mononuclear inflammatory infiltrate composed mainly by 239
macrophages and plasma cells. Strong positive IHC labeling was observed for rabies in the 240
neurons in different brain regions, especially in the cerebral cortex and in Purkinje cells of the 241
cerebellum. In both cases the diagnosis of rabies was confirmed by immunofluorescence and 242
mouse inoculation. 243
Discussion: The diagnosis of rabies in foxes wasconducted through the characteristic 244
histopathologic findings of the disease observed in the CNS and confirmed by the FAT, MIT 245
and IHC. Although the histopathological findings in foxes are similar to what is observed in 246
other species, the severity of inflammatory lesions and the large amount of inclusion bodies in 247
the nervous tissue is an outstanding feature, regardless of the inflammatory response. The 248
diagnosis of rabies in foxes can be achieved by characteristic histopathologic findings of the 249
CNS, supported by evaluating peripheral nerve ganglia, salivary glands and adrenal which 250
may also present similar microscopic lesions. Auxiliary Laboratory tests must be performed, 251
such as FAT, MIT and IHC for confirmation of the disease. 252
Keywords: wild animals, canids, Lyssavirus. 253
Descritores: animais selvagens, canídeos, Lyssavirus. 254
255
INTRODUÇÃO
256Vários canídeos silvestres são considerados reservatórios do vírus rábico, a exemplo 257
da raposa vermelha (Vulpes vulpes), distribuída mundialmente na Europa, América do Norte, 258
norte da África e na Austrália [4]. 259
No Brasil, o ciclo silvestre terrestre da raiva é representado principalmente por saguis 260
(Callithrix jacchus) ou raposas (Cerdocyon thous). Na região Nordeste, dois canídeos 261
silvestres já foram relatados como reservatórios do vírus rábico: Cerdocyon thous (crab-eating
262
fox, cachorro-do-mato) e Pseudalopex vetulus (hoary fox, raposa cinzenta) (previamente 263
denominada Dusicyon vetulus) [3,6]. Existem relatos de casos de raposas com raiva e 264
transmissão para humanos nos estados do Ceará, Paraíba, Pernambuco, Bahia e Minas Gerais 265
[1,2] e dos 329 casos de raiva notificados no período de 2002 a 2009, cerca de 88% eram de 266
canídeos silvestres, todos ocorridos na região Nordeste, onde muitos desses eram mantidos 267
como animais de estimação. Segundo dados da Secretaria de Vigilância em Saúde (SVS/MS) 268
[13], no Brasil, os canídeos silvestres foram responsáveis por 7,9% dos 165 óbitos de 269
humanos com raiva, no período de 1986-2006. No Estado da Paraíba entre os anos de 2007 e 270
2010, foram notificados sete casos de raiva em cães e gatos, e cinco casos em canídeos 271
silvestres [13]. 272
Até o momento, não existem estudos detalhados de descrição histopatológica e imuno-273
histoquímica em raposas acometidas por essa doença, portanto, o objetivo do presente 274
trabalho é a caracterização dos achados patológicos e imuno-histoquímicos de raposas com 275
raiva encaminhadas ao Laboratório de Patologia Animal (LPA) da Universidade Federal de 276
Campina Grande (UFCG) em Patos, semiárido da Paraíba, Brasil. 277
CASOS 278
As raposas utilizadas foram encaminhas mortas para o LPA da UFCG, realizada a 279
identificação fenotípica da espécie através da análise de aspectos morfológicos do animal com 280
base no Guia de Identificação de Canídeos Brasileiros [10] e posteriormente necropsiadas. 281
Foram coletados fragmentos de órgãos das cavidades torácica e abdominal, glândulas 282
salivares, globo ocular e gânglio de Gasser, além do sistema nervoso central (SNC) que foi 283
coletado e fixado em formol tamponado a 10%. Posteriormente foram realizados cortes 284
seriados do encéfalo, medindo aproximadamente 0,5 cm de espessura e clivados 16 285
fragmentos do SNC identificados: 1) córtex frontal, 2) córtex parietal, 3) córtex temporal, 4) 286
córtex occipital, 5) núcleos da base, 6) hipocampo, 7) tálamo, 8) colículo rostral, 9) colículo 287
caudal, 10) pedúnculos cerebelar, 11) ponte, 12) óbex, 13) cerebelo, 14) medula cervical, 15) 288
medula torácica e 16) medula lombar. Para a confecção das lâminas histológicas, os 289
fragmentos clivados foram processados rotineiramente e coradas pela técnica de hematoxilina 290
e eosina (HE). 291
Seguindo protocolos estabelecidos previamente [5,8] foram realizadas as técnicas de 292
imunofluorescência direta (IFD) e inoculação intracerebral em camundongos (ICC), 293
respectivamente, em fragmentos do cérebro, cerebelo, tronco encefálico, medula espinhal e 294
glândulas salivares das raposas foram enviados para a execução dessas técnicas. 295
Blocos de parafina com fragmentos do hipocampo foram selecionados e submetidos à 296
técnica de imuno-histoquímica para a detecção do vírus da raiva. Após desparafinizanação e 297
reidratação dos tecidos, foi realizada recuperação antigênica com solução de citrato (pH 6,0) 298
em forno micro-ondas, em potência máxima, por dez min. O anticorpo primário utilizado era 299
policlonal para raiva produzido em cabras marcado com FITC (anticorpo conjugado de 300
isotiocianato de fluorescência - Chemicon #5199)1, diluído 1:1000 em solução tamponada 301
fosfato salina (PBST) com Tween® 20 (Sigma P2287)2, e incubado por 60 min a 37Cº. O 302
anticorpo secundário biotilinilado e o complexo estreptavidina-biotina-peroxidase 303
(LSAB+System HRP)3 foram utilizados consecutivamente, incubados à temperatura ambiente 304
por 30 min e marcados através da adição do DAB + Substrate - Choromogen System3 e contra 305
corados com hematoxilina de Harris. Como controle positivo foi utilizado secções 306
histológicas de casos confirmados de raiva em bovinos. Como controle negativo, as mesmas 307
secções foram utilizadas, com substituição do anticorpo primário por PBST. 308
Esses casos ocorreram em anos distintos, sendo o primeiro em maio de 2010 e o 309
segundo em abril de 2013. As duas raposas enviadas foram identificadas como pertencentes à 310
espécie Cerdocyon thous. Segundo informações obtidas dos moradores da zona rural do 311
Município de São José de Espinharas - PB, local onde os animais foram encontrados, as 312
raposas foram atropeladas após terem sido vistas com sinais nervosos caracterizados por 313
incoordenação, perda de equilíbrio, acentuado balançar compulsivo da cabeça e aparente 314
debilidade muscular. 315
Macroscopicamente as duas raposas apresentavam múltiplas lacerações cutâneas, 316
fraturas ósseas e rupturas de órgãos abdominais provenientes de traumatismos. Os vasos das 317
leptomeninges estavam levemente congestos. 318
Histologicamente verificou-se reação inflamatória mononuclear, principalmente no 319
SNC, variando no grau de intensidade e sua localização (Tabela 1). Havia encefalite não 320
supurativa, caracterizada pela presença de infiltrado inflamatório constituído principalmente 321
por linfócitos e plasmócitos, formando manguitos perivasculares (Figura 1A), associada a 322
discreta gliose e corpúsculos de inclusões eosinofílicos intracitoplasmáticos principalmente 323
em neurônios dos córtices, núcleos da base, hipocampo (Figura 1B), tálamo, colículos, ponte, 324
óbex e cerebelo. Meningomielite linfoplasmocitária discreta com raras inclusões virais em 325
neurônios da medula espinhal também foram observadas nos dois casos. 326
Inflamação semelhante também foi observada nos gânglios nervosos periféricos, 327
glândulas salivares e adrenais dos dois casos. Havia infiltrado inflamatório principalmente de 328
linfócitos e plasmócitos entre os feixes nervosos do gânglio de Gasser, caracterizando 329
ganglioneurite não supurativa (Figura 1C), associada a raras inclusões virais 330
intracitoplasmáticas em neurônios. Ganglioneurite não supurativa também foi observada no 331
gânglio ciliar do primeiro caso. Infiltrado linfoplasmocitário também foi encontrado nas 332
glândulas salivares (Figura 1D) e nas adrenais dos dois casos, característicos de adenite e 333
adrenalite não supurativa. Corpúsculos de inclusões raramente foram verificados em 334
agregados de neurônios distribuídos perifericamente a essas estruturas glandulares. 335
No exame de IFD e na ICC o resultado foi positivo em todos os fragmentos testados 336
para o vírus da raiva. 337
Pela imuno-histoquímica os dois casos marcaram fortemente com anticorpos para o 338
vírus rábico, demonstrando múltiplos agregados de grânulos distribuídos no pericário, como 339
também na forma de corpúsculos grandes, únicos ou múltiplos no citoplasma de neurônios do 340
córtex (Figura 2A) e ponte (Figura 2B). No controle negativo não foram observadas nenhum 341
tipo de imunomarcação (Figura 2C). 342
343
DISCUSSÃO 344
O diagnóstico da raiva em raposas foi realizado através dos achados histopatológicos 345
característicos da doença, observados no SNC e confirmados pela IFD, ICC e IHQ. 346
Apesar dos achados histopatológicos encontrados nas raposas serem semelhantes ao 347
que são observados em outras espécies [14], a severidade das lesões inflamatórias e a grande 348
quantidade de corpúsculos de inclusão no tecido nervoso é uma característica marcante, 349
independente da resposta inflamatória. A inflamação não supurativa e a presença de inclusões 350
observadas nos gânglios nervosos periféricos encontrados ao redor ou dentro do tecido das 351
glândulas salivares e das adrenais, semelhantemente a reação inflamatória observada no SNC, 352
podem auxiliar no diagnóstico dessa patologia, principalmente nos casos em que não são 353
observadas inclusões no SNC. As glândulas salivares também tem sido um importante órgão 354
para a realização do isolamento viral do Lyssavirus, já tendo sido encontrada positividade 355
para raiva em glândulas salivares avaliadas por outros autores [11]. 356
Diferentemente dos herbívoros, onde geralmente as principais lesões são cerebelares, 357
os carnívoros tendem a apresentar lesões mais intensas na região de hipocampo, entretanto, de 358
forma semelhante aos bovinos, pode haver áreas com pouca ou ausente reação inflamatória 359
[14]. Um aspecto importante da análise histopatológica é a realização de cortes seriados do 360
sistema nervoso central, uma vez que a localização e intensidade das lesões podem variar 361
entre as regiões do SNC, não havendo uma uniformidade. 362
A forte marcação nos neurônios do hipocampo no presente trabalho através da imuno-363
histoquímica, difere dos achados de Stein et al. [12] que encontraram uma marcação 364
moderada no hipocampo de raposas da espécie Urocyon cinereoargenteus e Vulpes vulpes. 365
Apesar das implicações legais, muitas pessoas tem o hábito de criar raposas e outros 366
animais silvestres no Nordeste brasileiro. Essa tradição aumenta significativamente o risco de 367
transmissão da raiva para humanos e outros animais. No ano de 2012, duas pessoas morreram 368
no Nordeste vítimas de raiva transmitidas por animais silvestres [13]. No Estado da Paraíba, a 369
raposa tem sido o animal silvestre com maior número de agressões contra humanos, havendo 370
no período de 2000 a 2003, cerca de 24 casos de agressões em humanos por raposas [7]. 371
Propõe-se que a ocorrência de raiva em herbívoros nos Estados da Paraíba e 372
Pernambuco é causada principalmente por uma variante do vírus chamada de RABV, 373
relacionada a morcegos hematófagos e que ela está circulante nessa área do Nordeste por pelo 374
menos sete anos, isolada por barreiras geográficas [9]. Sugere-se ainda, a presença de duas 375
ramificações de Lyssavirus na região da Paraíba, sendo uma associada com quirópteros e 376
outra com carnívoros. Entretanto, existe uma variabilidade genética nessas ramificações, 377
subdividindo o grupo de quirópteros em “morcegos insetívoros” e “morcegos hematófagos”, e 378
o grupo dos carnívoros em “cão”, “raposa 1” e “raposa 2” (está mais próxima do grupo “cão”) 379
[7]. Essa existência de variabilidade entre as ramificações das variantes do vírus rábico sugere 380
que pelo menos dois grupos de vírus coexistem na mesma região e apesar de somente a 381
variante de morcegos hematófagos ter sido incriminada como causadora da raiva em 382
herbívoros, a criação de animais silvestres como animais de estimação no Nordeste favorece o 383
risco de transmissão da doença para os animais de produção, sugerindo um papel importante 384
das raposas nesse cenário. A presença da variabilidade genética das variantes de raposas, 385
sugerem que estes animais tem um papel muito mais importante na manutenção e 386
disseminação da raiva no Brasil do que antes se pensava, tendo uma implicação de saúde 387
pública significante já que nessa região o monitoramento desses animais é muitas vezes 388
ineficiente ou ausente, e a prática da vacinação de animais silvestres não tem sido empregada 389
no país [2]. 390
O diagnóstico da raiva em raposas pode ser realizado pelos achados histopatológicos 391
característicos do SNC, auxiliado pela avaliação dos gânglios nervosos periféricos, glândulas 392
salivares e adrenais que também podem apresentar lesões microscópicas semelhantes. Em 393
adição os exames laboratoriais auxiliares devem ser realizados, como IFD, ICC e IHQ para a 394
confirmação da doença. 395
MANUFACTURERS 396
1Chemicon International Inc. Temecula, CA, USA. 397
2Sigma-Aldrich Corp. St. Louis, MO, USA. 398
3Dako Cytomation. Carpinteria, CA, USA. 399
400
Declaration of interest. The authors report no conflicts of interest. The authors alone are 401
responsible for the content and writing of the paper. 402
REFERENCES 403
1 Araújo F.A.A. 2002. Raiva humana no Brasil: 1992-2001. 90f. Belo Horizonte, MG. 404
Dissertação (Mestrado em Medicina Veterinária) - Programa de Pós-graduação em Medicina 405
Veterinária, Universidade Federal de Minas Gerais. 406
2 Bernardi F., Nadin-Davis S.A., Wandeler A.I., Armstrong J., Gomes, A.A.B., Lima 407
F.S., Nogueira F.R.B. & Ito F.H. 2005. Antigenic and genetic characterization of rabies 408
viruses isolated from domestic and wild animals of Brazil identifies the hoary fox as a rabies 409
reservoir. Journal of General Virology.86(11): 3153-3162. 410
3 Carnieli Jr. P., Brandão, P.E., Carrieri M.L., Castilho J.G., Macedo C.I., Machado 411
L.M., Rangel N., Carvalho R.C., Carvalho V.A., Montebello L., Wada M. & Kotait I. 412
2008. Characterization of rabies virus isolated from canids and identification of the main wild 413
canid host in Northeastern Brazil. Virus Research. 131(1): 33-46. 414
4 Childs J.E. & Real L.A. 2007. Epidemiology. In: Jackson A.C. & Wunner W.H. (Eds). 415
Rabies. San Diego: Academic Press, pp.123-199.
416
5 Dean D.J., Abelseth M.K. & Atanasiu P. 1996. The fluorescent antibody test. In: Meslin 417
F.X., Kaplan M.M. & Koprowski H. (Eds). Laboratory Techniques in Rabies. 4th edn. 418
Geneva: World Health Organization, pp.88-93. 419
6 Gomes A.A.B. 2004. Epidemiologia da raiva: caracterização de vírus isolados de animais 420
domésticos e silvestres do semi-árido paraibano da região de Patos, Nordeste do Brasil. 107f. 421
São Paulo, SP. Tese (Doutorado em Medicina Veterinária) - Programa de Pós-graduação em 422
Epidemiologia Experimental Aplicada às Zoonoses, Universidade de São Paulo. 423
7 Gomes A.A.B., Silva M.L.C.R., Bernardi F., Sakai T., Itou T. & Ito F.H. 2012. 424
Molecular epidemiology of animal rabies in the semiarid region of Paraíba, Northeastern 425
Brazil. Arquivos do Instituto Biológico. 79(4): 611-615. 426
8 Koprowski H. 1996. The mouse inoculation test. In: Meslin F.X., Kaplan M.M. & 427
Koprowski H. (Eds). Laboratory Techniques in Rabies. 4th edn. Geneva: World Health 428
Organization, pp.80-86. 429
9 Mochizuki N., Kawasaki H., Silva M.L.C.R., Afonso J.A.B., Itou T., Fumio H. & Sakai 430
T. 2012. Molecular epidemiology of livestock rabies viruses isolated in the northeastern 431
Brazilian states of Paraíba and Pernambuco from 2003-2009. BMC Research Notes. 5(32): 1-432
7. 433
10 Ramos Jr. V.A., Pessutti C. & Chieregatto C.A.F.S. 2003. Guia de Identificação dos 434
Canídeos Silvestres Brasileiros. Sorocaba: JoyJoy Studio Ltda. - Comunicação Ambiental,
435
35p. 436
11 Silva M.L.C.R, Lima F.S., Gomes A.A.B., Azevedo S.S., Alves C.J., Bernardi F. & Ito 437
F.H. 2009. Isolation of rabies virus from the parotid salivary glands of foxes (Pseudalopex 438
vetulus) from Paraíba State, Northeastern Brazil. Brazilian Journal of Microbiology. 40(3):
439
446-449. 440
12 Stein L.T., Rech R.R., Harrison L. & Brown C.C. 2010. Immunohistochemical study of 441
rabies virus within the central nervous system of domestic and wildlife species. Veterinary 442
Pathology. 47(4): 630-633.
443
13 SVS - Ministério da Saúde. Fundação Nacional de Saúde. 2012. Mapas da Raiva no 444
Brasil. Brasília: Organização Pan-Americana da Saúde; Organização Mundial da Saúde;
445
Ministério da Saúde. 446
14 Zachary J.F. 2009. Sistema Nervoso. In: McGavin M.D. & Zachary J.F. (Eds). Bases da 447
Patologia em Veterinária. 4.ed. Rio de Janeiro: Elsevier, pp.833-971.
448
Tabela 1. Distribuição das lesões no SNC de raposas (Cerdocyon thous) com raiva de acordo 449
com a intensidade da resposta inflamatória e presença de corpúsculos de Negri. 450
Figura 1. Lesões histológicas de raposas (Cerdocyon thous) com raiva. (A) Córtex occipital 451
mostrando manguitos mononucleares perivasculares e corpúsculos de Negri (seta) [HE, 10x]. 452
(B) Hipocampo com múltiplos corpúsculos de Negri (setas) [HE, 20x]. (C) Gânglio trigêmeo 453
com infiltrado inflamatório mononuclear [HE, 20x]. (D) Glândula salivar com inflamação não 454
supurativa [HE, 20x]. 455
Figura 2. Seções do encéfalo de raposas (Cerdocyon thous) com raiva, submetidas à técnica 456
de imuno-histoquímica. (A) Córtex e (B) Tronco encefálico (ponte) mostrando múltiplos 457
agregados de grânulos amarronzados distribuídos no pericário e prolongamentos 458
citoplasmáticos de neurônios, caracterizando imunomarcação positiva para raiva. (C) Córtex. 459
Controle negativo (sem anticorpo). Técnica de imuno-histoquímica método da estreptavidina-460
biotina-peroxidase (LSAB+System HRP) contracorados com Hematoxilina de Harris [Barra = 461
20 µm]. 462
463
CASE REPORT
464
Classical Scrapie Diagnosis in ARR/ARR Sheep in Brazil 465
Juliano Souza Leal1,2, Caroline Pinto de Andrade2, Gabriel Laizola Frainer 466
Correa2, Gisele Silva Boos2, Matheus Viezzer Bianchi2, Sergio Ceroni da 467
Silva2 ,Rui Fernando Felix Lopes3 & David Driemeier2 468
469 470
1Programa de Pós-graduação em Ciências Veterinárias (PPGCV), Faculdade de Veterinária 471
(FaVet), Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil. 472
2Setor de Patologia Veterinária (SPV), Departamento de Patologia Clínica Veterinária 473
(DPCV), FAVET, UFRGS, Porto Alegre, RS, Brazil. 3Departamento de Ciências 474
Morfológicas, Instituto de Ciências Básicas da Saúde (ICBS), UFRGS, Porto Alegre, RS. 475
CORRESPONDENCE: J.S. Leal [julianoob@gmail.com - Tel.: +55 (51) 3308 3631]. Setor 476
de Patologia Veterinária, FAVET, UFRGS. Av. Bento Gonçalves n. 9090, Bairro Agronomia. 477
CEP 91540-000 Porto Alegre, RS, Brazil. 478
20 ABSTRACT
480
Background: Scrapie is a transmissible spongiform encephalopathy (TSE) that affects sheep 481
flocks and goat herds. The transfer of animals or groups of these between sheep farms is 482
associated with increased numbers of infected animals and with the susceptibility or the 483
resistance to natural or classical scrapie form. Although several aspects linked to the etiology 484
of the natural form of this infection remain unclarified, the role of an important genetic
485
control in scrapie incidence has been proposed. Polymorphisms of the PrP gene (prion 486
protein, or simply prion), mainly in codons 136, 154, and 171, have been associated with the 487
risk of scrapie. 488
Case: One animal from a group of 292 sheep was diagnosed positive for scrapie in the 489
municipality of Valparaíso, state of São Paulo, Brazil. The group was part of a flock of 811 490
free-range, mixed-breed Suffolk sheep of the two genders and ages between 2 and 7 years 491
from different Brazilian regions. Blood was collected for genotyping (for codons 136, 141, 492
154 and 171), and the third lid and rectal mucosa were sampled for immunohistochemistry 493
(IHC) for scrapie, from all 292 animals of the group. IHC revealed that seven (2.4%) animals 494
were positive for the disease. Collection of samples was repeated for 90 animals, among 495
which the seven individuals diagnosed positive and 83 other animals that had some degree of 496
kinship with those. These 90 sheep were sacrificed and necropsied, when samples of brain 497
(obex), cerebellum, third eyelid, rectal mucosa, mesenteric lymph node, palatine tonsil, and 498
spleen were collected for IHC. The results of IHC analyses carried out after necropsy of the 499
seven positive animals submitted to the second collection of lymphoreticular tissueand of the 500
83 animals with some degree of kinship with them confirmed the positive diagnosis obtained 501
in the first analysis, and revealed that three other sheep were also positive for scrapie. 502
Samples of 80 animals (89%) were negative for the disease in all organs and tissues analyzed. 503
In turn, 10 sheep (11%) were positive, presenting immunoreactivity in one or more tissues. 504
21 Genotyping revealed the presence of four of the five alleles of the PrP gene commonly 505
detected in sheep: ARR, ARQ, VRQ and ARH. These allele combinations formed six 506
haplotypes: ARR/ARR, ARR/ARQ, ARH/ARH, ARQ/ARH, ARQ/ARQ and ARQ/VRQ. 507
Animals were classified according to susceptibility to scrapie, when 8.9% of the genotyped 508
sheep were classified into risk group R1 (more resistant, with no restriction to breeding). In 509
turn, 40% of the animals tested ranked in groups R4 and R5 (genetically very susceptible, 510
cannot be used for breeding purposes). 511
Discussion: The susceptibility of sheep flocks depends on the genetic pattern of animals and 512
is determined by the sequence of the gene that codifies protein PrP. Additionally, numerous 513
prion strains are differentiated based on pathological and biochemical characteristics, and may 514
affect animals differently, depending on each individual’s genotype. Most epidemiologic data 515
published to date indicate that animals that carry the ARR/ARR genotype are less susceptible 516
to classical scrapie. However, in the present study, the fact that two scrapie-positive sheep 517
presented the haplotype ARR/ARR indicates that this genotype cannot always be considered 518
an indicator of resistance to the causal agent of the classical manifestation of the disease. The 519
coexistence in the same environment of several crossbred animals from different flocks and 520
farms, which characterizes a new heterogeneous flock, may have promoted a favorable 521
scenario to spread the disease, infecting animals in the most resistant group. 522
Keywords: biopsy, scrapie, TSEs, immunohistochemistry. 523
Descritores: biopsia, scrapie clássico, EETs, imuno-histoquímica. 524
525
INTRODUCTION 526
Scrapie, also called epizootic tremor, is a transmissible spongiform encephalopathy 527
(TSE) that affects sheep flocks and goat herds [44]. The relocation of animals to and from 528
sheep farms has been associated with increased numbers of infected animals [28,39]. Once it 529
22 is introduced in a flock, the disease may be transmitted both vertically, from ewe to lamb, and 530
horizontally, across animals [15,39,49]. Many aspects surrounding the etiology of the natural 531
form of this infection remain to be clarified, though the existence of an important genetic 532
control has been proposed to explain the disease’s incidence [24]. The analysis of the gene 533
PrP (prion protein, or simply prion) in ovine of different breeds has drawn attention to the 534
interaction between host genotype polymorphisms and susceptibility to the infectious agent of 535
scrapie [10,21-23,31]. 536
Single nucleotide polymorphisms (SNT) have been linked to susceptibility or 537
resistance to classical scrapie. These polymorphisms occur at codons 136 (A or V, alanine or 538
valine), 154 (R or H, arginine or histidine) and 171 (R, Q or H, arginine, glutamine or 539
histidine) [16]. The diagnosis of the classical form in sheep with haplotype A136R154R171 is 540
rare [24]. Under natural exposure conditions, this genotype (ARR/ARR) has been 541
acknowledged as having the lowest risk for the classical form [16]. This case report describes
542
the occurrence of an outbreak in a flock of mixed Suffolk sheep of varied origins in the state 543
of São Paulo, southeastern Brazil, when the disease was diagnosed in two animals carrying 544
the genotype ARR/ARR, compatible with classical scrapie. 545
546
CASE 547
In 2011, one ovine head from a group of 292 animals was diagnosed with the classical 548
form of scrapie. These sheep were part of a larger flock of 811 free-range animals of both 549
genders and between 2 and 7 years of age that were brought from southern, southeastern and 550
midwestern Brazil. Since the animal died, and diagnosis was carried out after the death, a 551
decision was made to collect blood samples from all 292 animals of the group, for sequencing 552
and genotyping (for codons 136, 141, 154 and 171). In addition, the third eyelid and the rectal 553
mucosa of all 292 animals were biopsied for immunohistochemistry (IHC). After IHC, a new 554
23 collection was conducted in 90 animals (approximately 30% of the original group). These 555
included the animals with positive diagnosis in the first collection, and those that had some 556
degree of kinship with scrapie-positive sheep in the original group. These animals were 557
sacrificed and necropsied to collect brain tissue (obex), cerebellum, third eyelid, rectal 558
mucosa, mesenteric lymph node, palatine tonsil, and spleen used in the IHC analyses. 559
Tissue samples were collected and processed for histology and IHC for PrPSc 560
following the methodology proposed by O’Rourke et al. [43]. Rectal biopsy samples were 561
collected and processed according to Espenes et al. [17]. Anti-prion1 monoclonal antibodies 562
F89/160.1.5 and F99/97.6.1 were diluted to a 1:500 solution and added to samples, which 563
were then incubated in a humid chamber at 4ºC for 12 h [34]. 564
Blood was collected by punction of the jugular vein using EDTA as anticoagulant and 565
stored at -20ºC for subsequent processing. Genomic DNA of sheep was extracted using 500 566
μL whole blood and the QIAmp™ DNA Blood Kit2 according to the manufacturer’s 567
instructions. PCR was carried out using the DNA sample, 15 pmol each primer, 1X PCR 568
buffer (Tris-HCl pH 8.4, 50 mM KCl)3, MgCl
2 1.5 mM, dNTP4 200 μM, and 1U Platinum™ 569
enzyme Taq DNA Polymerase3 according to the following cycles: 95ºC for 5 min, 35 cycles 570
at 95ºC for 30 s and at 58ºC for 30 s, and 72ºC for 30 s. PCR was performed using a forward 571
primer flanking the 136 codon position (5’-ATGAAGCATGTGGCAGGAGC-3’) and a 572
reverse primer flanking the 171 codon position (5’-GGTGACTGTGTGTTGCTTGACTG-3’). 573
A 245-bp fragment was generated, which contains the regions of the main codons analyzed 574
for susceptibility to scrapie [36]. 575
The PCR product was purified and quantified using the commercial products Purelin5 576
and Qubit5, respectively, following the manufacturers’ instructions. Sequencing was 577
performed with 3 ng DNA and 3.2 pmol each primer, using the BigDye Terminator v.1.1 578
Cycle Sequencing kit6 in the ABI PRISM 3110 Genetic Analyzer6. 579
24 Of the 292 mixed Suffolk sheep whose lymphoreticular tissues of the third eyelid were 580
analyzed by IHC, seven (2.4%) were positive for scrapie in the first sample collection. 581
The IHC results of the second samples collected from these seven sheep after necropsy 582
and of the samples collected from the other 83 animals with some degree of kinship with them 583
confirmed the positive diagnosis obtained initially, and revealed that three other animals were 584
also positive for the scrapie. The samples of all organs and tissues of 80 animals (89%) were 585
negative, while those of 10 sheep (11%) were positive, with immunoreactivity in one or more 586
tissues. 587
At least three lymphoid follicles were analyzed by IHC in all samples obtained from 588
necropsied animals. No animal was positive in all samples collected, but different organs and 589
tissues showed immunoreactivity. The third eyelid (Figure 1) and the palatine tonsil were the 590
tissues with the highest percentage of immunoreactive samples (90%, 9/10). The lymphoid 591
tissue of the rectal mucosa (Figure 2) showed immunoreactivity in only one animal (10%, 592
1/10). No immunoreactivity was observed in mesenteric lymph node, spleen and obex 593
samples. 594
Genotyping of codon 141 showed homozygosis for lysine (L141L or L/L) in all 90 595
animals investigated. The genotypes and frequencies of alleles for codons 136, 154 and 171 of 596
these sheep (10 positive and 80 related) are shown in Table 1.
597
Four of the five alleles of the PrP gene commonly detected in ovine were found: ARR, 598
ARQ, VRQ and ARH. The allele AHQ was not detected in any sample. Of the 15 599
possibilities, these allele combinations formed six haplotypes: ARR/ARR, ARR/ARQ, 600
ARH/ARH, ARQ/ARH, ARQ/ARQ and ARQ/VRQ. 601
The haplotype ARR/ARQ was detected in 39 samples (43.3%) and was the most 602
frequent, followed by haplotypes ARQ/ARQ, detected in 34 (37.7%), ARR/ARR, present in 603
eight (8.9%), and ARQ/ARH, observed in five samples (5.6%). Haplotypes ARH/ARH and 604
25 ARQ/VRQ were detected in two samples each (2.2%). The classification of animals 605
according to the susceptibility criteria described by Dawson et al. [13] placed 8.9% of the 606
total number of genotyped animals in scrapie risk group R1, which includes more resistant 607
animals that are not subject to reproduction restrictions. A significant percentage of animals 608
(43.3%) was in risk group R2, which requires careful selection for breeding. In addition, 7.8% 609
of animals were in group R3 (intermediate risk), while 40% were in groups R4 and R5 (highly 610
susceptible animals that should not be included in reproduction programs). 611
612
DISCUSSION 613
The susceptibility of sheep flocks to scrapie depends largely on the genetic pattern of 614
the animal, and is determined mainly by the sequence of the gene that codifies the PrP 615
protein, since there are several polymorphisms that affect the conversion of the cell protein 616
PrPC to its pathological form, PrPSc [8,9]. Nevertheless, it is not possible to consider the 617
occurrence of only one form of ovine prion, since there are numerous prion strains with 618
different pathological and biochemical characteristics that may affect animals distinctively, 619
depending on their genotypes [1,30]. 620
In the present study, the frequency of codon VRQ was very low (2.2%), confirming 621
previous findings, which revealed that the alleles ARR and ARQ prevail in Suffolk sheep, and 622
that the allele ARH sometimes is detected [12,32]. The high sensitivity of homozygous VRQ 623
carriers or of individuals with ARQ haplotypes has also been reported in the literature [24]. 624
This condition raises concerns about susceptibility from the epidemiological perspective, 625
since the allele VRQ, which is rare or absent in breeds like Suffolk, was present in two 626
animals, one of which was positive for scrapie. 627
Most epidemiological and genetic data published indicate that sheep carrying the 628
haplotype ARR/ARR are less susceptible to classical form, while animals with the haplotype 629
26 VRQ in homozygosis or with ARQ haplotypes are highly susceptible [24]. This hypothesis is 630
supported by genotyping data for thousands of sheep with the disease around the world. For 631
example, a study carried out in Japan described a classical scrapie case in one ARR/ARR 632
sheep [16]. Sensitivity of ARR/ARR sheep in a scenario of oral exposure to the disease has 633
also been reported [3]. Atypical cases were observed in ARR/ARR animals [11,42]. 634
Polymorphisms at codon positions 136, 154 and 171 are not the only ones associated 635
with resistance or susceptibility to scrapie [33]. An analysis of the variation of codon 636
positions 136 and 171, for instance, showed that each has several adjacent polymorphic sites 637
and may codify up to four amino acids [7,50]. The atypical scrapie form, characterized by 638
strain Nor98 [6], is more frequently detected in AHQ animals that carry a polymorphism in 639
codon 141, and has not been described in Suffolk sheep in Brazil [2]. This atypical form 640
expresses phenylalanine (F), instead of leucine (L) in the form L141F [6,37,46]. 641
However, although it is generally acceptable that classical scrapie is an infectious and 642
contagious disease [14], contagion with the atypical form is questionable in light of the fact 643
that the specific marker for the atypical manifestation of the disease is detected outside the 644
central nervous system [5,20,29], even in cases experimentally transmitted to transgenicmice 645
[35] and sheep [47]. Several studies have demonstrated that susceptibility to the atypical form 646
is consistently associated with PrP codons 141 (L/F) and 154 (R/H) [6,42]. In fact, studies 647
have proposed the hypothesis that this form may evolve when the animal is not exposed to the 648
infectious agent [5,18,29,48], given the limited knowledge of the physiopathology of this 649
manifestation of the disease [19]. 650
In the present study, two (2/8) positive animals presented the haplotype ARR/ARR, 651
which is considered to be the least susceptible and therefore responsible for the lowest risk of 652
scrapie. However, like all sheep that were genotyped, these animals did not present any 653
change in lysine in codon position 141. This change (that is, when lysine is replaced by 654
27 phenylalanine) has been associated with atypical scrapie in Suffolk sheep [6]. Therefore, these 655
two ARR/ARR sheep do not fit in the genotypic characteristics of sheep that may commonly 656
present the atypical form. It is possible that the presence of several crossbred animals of 657
different flocks and farms in the same environment, which characterizes an heterogeneous 658
flock, has created the favorable conditions for the disease to evolve and spread, infecting the 659
more susceptible animals. 660
The variation in the frequency of the PrP genotype between flocks has been identified 661
as a real risk factor for the disease [4]. The introduction of adult sheep free of scrapie in 662
contaminated flocks is believed to allow lateral transmission, even between adult animals 663
with less susceptible genotypes [40,45], although young sheep are more predisposed [43]. 664
Other reasons behind differences in occurrence include the stress caused during husbandry 665
and large population numbers [26]. Additionally, the lack of a defined epidemiological pattern 666
and the different strains of the causal agent play an important role in inter-flock variability 667
[40]. Several models were based on the assumption that outbreak duration is influenced by 668
flock size and by the frequency of the PrP genotype in one flock [25,26,38,51]. Commercial 669
flocks with high genetic diversity, mainly in codons other than 136, 154 and 171, are more 670
consistently affected. In these animals, the onset of clinical manifestations occurs at 671
significantly different ages, with means varying from 2 to 5.7 years, due to noteworthy 672
dissimilarities in age and PrP genotype profiles [40]. The purchase of infected animals has 673
been pointed out as the main scrapie infection mechanism in flocks [27, 41]. 674
675
CONCLUSION 676
The diagnosis of scrapie in two homozygous ARR/ARR sheep indicates that the 677
resistance of this genotype to the classical form of the disease is debatable. Although scrapie 678
in these animals is rare, the cases presented in this case report lend strength to the notion that 679
28 its occurrence depends on a combination of infectious factors, including differences in 680
biological and biochemical properties in the natural hosts to this prion. 681
MANUFACTURERS 682
1DAKO Corp. Carpinteria, CA, USA. 683
2Qiagen. Hilden, Germany. 684
3InvitrogenTM, São Paulo, Brazil. 685
4Life TechnologiesTM. Gaithersburg, MD, USA. 686
5InvitrogenTM. Carlsbad, CA, USA. 687
6Applied Biosystems Inc. Foster City, CA, USA. 688
689
Declaration of interest. The authors report no conflicts of interest. The authors alone are 690
responsible for the content and writing of the paper. 691
692
REFERENCES 693
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