PESQUISA VETERINÁRIA BRASILEIRA

Abstract in English:

Our objective was the characterization and staging of histological lesions in different anatomical sites of the central nervous system (CNS) of rabid cattle. The severity of the lesions was compared with the clinical stages of the disease, the variants of viral isolates, and with the load of virus. Thirty-one spontaneously affected rabid cattle the state of Santa Catarina underwent clinical follow-up and were eventually necropsied. CNS tissues were sampled and submitted to direct fluorescent antibody technique (DFAT), immunohistochemistry (IHC), routine histopathology with hematoxylin and eosin stain (HE), reverse transcriptase polymerase chain reaction (RT-PCR), and polymerase chain reaction in quantitative reverse transcriptase in real time (qRT-PCR). Affected cattle were allotted in four groups according to their clinical stage when euthanized: G1, euthanized while standing; G2, euthanized when in sternal recumbence; G3, euthanized when in lateral recumbence; and G4, affected cattle with natural death. In order to evaluate the degree of severity of the lesions and the presence of Negri bodies (NBs), the brain was sectioned at 9 sites. Additionally, spinal cord and trigeminal ganglion sections were examined. The intensity of the lesions was graded as either absent, mild, moderate, or marked, and the presence or absence of the NBs was noted. Histological lesions were characterized by lymphocytic and monocytic meningoencephalitis with NBs in 28 cases. In all analyzed groups, intensities of histological lesions ranging from mild to severe were observed. Brain regions with the highest inflammatory lesion intensity were the medulla at the level of obex, followed by the colliculus and thalamus. NBs were observed in a higher percentage in the cerebellum, followed by medulla at the obex level, striatum complex, and frontal telencephalon. The duration of the clinical course of the disease did not influence the intensity of the inflammatory lesion, but it did influence the presence of NBs, with a higher percentage of these inclusions in cattle that died naturally than in euthanized cattle. All isolated rhabdovirus included in this study were genetically compatible with samples from hematophagous bats Desmodus rotundus. The evaluation by qRT-PCR did not demonstrate a correlation between lesion intensity and the amount of virus.

• Cattle rabies clinical course virus genetic lineage histology lesion severity RT-PCR.

Abstract in Portuguese:

Nosso objetivo foi a caracterização e estadiamento de lesões histológicas em diferentes locais anatômicos do sistema nervoso central (SNC) de bovinos raivosos. A gravidade das lesões foi comparada com os estágios clínicos da doença, as variantes dos isolados virais e com a quantidade de vírus. Trinta e um bovinos do estado de Santa Catarina, afetados naturalmente por raiva, foram acompanhados clinicalmente e, ao final, necropsiados. Os tecidos do SNC foram amostrados e submetidos a imunofluorescência direta, imunohistoquímica, histopatologia de rotina, reação em cadeia da polimerase via transcriptase reversa (RT-PCR) e reação em cadeia da polimerase em transcriptase reversa quantitativa em tempo real (qRT-PCR). Os bovinos afetados foram distribuídos em quatro grupos, de acordo com sua fase clínica: G1, eutanasiados quando ainda se mantinham em pé; G2, eutanasiados quando em decúbito esternal; G3, eutanasiados quando em decúbito lateral; e G4, bovinos afetados com morte natural. Para avaliar o grau de gravidade das lesões e a presença de corpúsculos de Negri (CNs), o cérebro foi seccionado em 9 locais. Além disso, seções da medula espinhal e do gânglio trigêmeo foram examinadas. A intensidade das lesões foi graduada como ausente, leve, moderada ou acentuada, e a presença ou ausência dos CNs foi anotada. Lesões histológicas foram caracterizadas por meningoencefalite linfocítica e monocítica com CNs em 28 casos. Em todos os grupos analisados foram observadas intensidades de lesões histológicas variando de leve a grave. As regiões cerebrais com maior intensidade de lesão inflamatória foram o bulbo no nível do obex, seguido do colículo e tálamo. CNs foram mais prevalentes no cerebelo, seguido pelo bulbo ao nível do óbex, corpo estriado e telencéfalo frontal. A duração do curso clínico da raiva não influenciou a intensidade da lesão inflamatória, mas influenciou a presença de CNs, com maior porcentagem dessas inclusões em bovinos que morreram naturalmente do que em bovinos sacrificados. Todos os isolados rabdovírus obtidos neste estudo eram geneticamente compatíveis com amostras provenientes de morcegos hematófagos Desmodus rotundus.

• Raiva bovina evolução clínica linhagem genética vírus histologia intensidade das lesões RT-PCR bovinos

Abstract in English:

Abstract.- Brocchi M., Ferreira A., Lancellotti M., Stehling E.G., Campos T.A., Nakazato G., Pestana de Castro A.F. & Silveira W.D. 2006. Typing of avian pathogenic Escherichia coli strains by REP-PCR. Pesquisa Veterinária Brasileira 26(2):69-73. Departamento de Microbiologia e Imunologia, Instituto de Biologia, Universidade de Campinas, Cx. Postal 6109, Campinas, SP 13081-862, Brazil. E-mail: wds@unicamp.br In the present study the repetitive extragenic palindromic (REP) polymerase chain reaction (PCR) technique was used to establish the clonal variability of 49 avian Escherichia coli (APEC) strains isolated from different outbreak cases of septicemia (n=24), swollen head syndrome (n=14) and omphalitis (n=11). Thirty commensal strains isolated from poultry with no signs of these illnesses were used as control strains. The purified DNA of these strains produced electrophoretic profiles ranging from 0 to 15 bands with molecular sizes varying from 100 bp to 6.1 kb, allowing the grouping of the 79 strains into a dendrogram containing 49 REP-types. Although REP-PCR showed good discriminating power it was not able to group the strains either into specific pathogenic classes or to differentiate between pathogenic and non-pathogenic strains. On the contrary, we recently demonstrated that other techniques such as ERIC-PCR and isoenzyme profiles are appropriate to discriminate between commensal and APEC strains and also to group these strains into specific pathogenic classes. In conclusion, REP-PCR seems to be a technique neither efficient nor universal for APEC strains discrimination. However, the population clonal structure obtained with the use of REP-PCR must not be ignored particularly if one takes into account that the APEC pathogenic mechanisms are not completely understood yet.

• Avian Escherichia coli typing REP-PCR.

Abstract in Portuguese:

Abstract.- Brocchi M., Ferreira A., Lancellotti M., Stehling E.G., Campos T.A., Nakazato G., Pestana de Castro A.F. & Silveira W.D. 2006. Typing of avian pathogenic Escherichia coli strains by REP-PCR. Pesquisa Veterinária Brasileira 26(2):69-73. Departamento de Microbiologia e Imunologia, Instituto de Biologia, Universidade de Campinas, Cx. Postal 6109, Campinas, SP 13081-862, Brazil. E-mail: wds@unicamp.br In the present study the repetitive extragenic palindromic (REP) polymerase chain reaction (PCR) technique was used to establish the clonal variability of 49 avian Escherichia coli (APEC) strains isolated from different outbreak cases of septicemia (n=24), swollen head syndrome (n=14) and omphalitis (n=11). Thirty commensal strains isolated from poultry with no signs of these illnesses were used as control strains. The purified DNA of these strains produced electrophoretic profiles ranging from 0 to 15 bands with molecular sizes varying from 100 bp to 6.1 kb, allowing the grouping of the 79 strains into a dendrogram containing 49 REP-types. Although REP-PCR showed good discriminating power it was not able to group the strains either into specific pathogenic classes or to differentiate between pathogenic and non-pathogenic strains. On the contrary, we recently demonstrated that other techniques such as ERIC-PCR and isoenzyme profiles are appropriate to discriminate between commensal and APEC strains and also to group these strains into specific pathogenic classes. In conclusion, REP-PCR seems to be a technique neither efficient nor universal for APEC strains discrimination. However, the population clonal structure obtained with the use of REP-PCR must not be ignored particularly if one takes into account that the APEC pathogenic mechanisms are not completely understood yet.

Abstract in English:

Simionatto S., Lima-Rosa C.A.V., Rubin L.L. & Canal C.W. 2005. [A nested-PCR protocol for detection of the chicken anemia virus.] Um protocolo de “nested-PCR” para detecção do virus da anemia das galinhas. Pesquisa Veterinária Brasileira 25(2):106-110. Laboratório de Virologia, Faculdade de Veterinária, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves 9090, Porto Alegre, RS 91540-000, Brazil. E-mail: claudio.canal@ufrgs.br This paper reports a nested polymerase chain reaction (nested-PCR) protocol for detection of chicken anemia virus (CAV), the causal agent of infectious chicken anemia. For DNA extraction from clinical samples, a method based on guanidine thiocyanate was found more sensitive and practical than other extraction protocols tested. The pair of primers used in the initial PCR targeted a 664 bp fragment on the VP1 gene. The primers for the internal PCR targeted a fragment of 520 bp. The specificity of the primers was evaluated on samples of CAV controlled flocks. Thirty different viruses and bacteria isolated from chickens did not give rise to any amplification product in the assay. The sensitivity of the nested-PCR was determined on serial dilutions of a CAV vaccine. The nested-PCR was more sensitive than a one step PCR and was able to detect at least 0.16 TCID50 of the vaccine strain. In addition, the protocol employed here detected viral DNA from tissues, sera and litter from flocks with or without clinical signs of disease. It is concluded that the nested-PCR protocol described here is more sensitive, faster and less cumbersome than virus isolation in cell culture as a diagnostic technique for detection of CAV.

• Chicken anemia virus CAV molecular diagnosis nested-PCR.

Abstract in Portuguese:

Simionatto S., Lima-Rosa C.A.V., Rubin L.L. & Canal C.W. 2005. [A nested-PCR protocol for detection of the chicken anemia virus.] Um protocolo de “nested-PCR” para detecção do virus da anemia das galinhas. Pesquisa Veterinária Brasileira 25(2):106-110. Laboratório de Virologia, Faculdade de Veterinária, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves 9090, Porto Alegre, RS 91540-000, Brazil. E-mail: claudio.canal@ufrgs.br This paper reports a nested polymerase chain reaction (nested-PCR) protocol for detection of chicken anemia virus (CAV), the causal agent of infectious chicken anemia. For DNA extraction from clinical samples, a method based on guanidine thiocyanate was found more sensitive and practical than other extraction protocols tested. The pair of primers used in the initial PCR targeted a 664 bp fragment on the VP1 gene. The primers for the internal PCR targeted a fragment of 520 bp. The specificity of the primers was evaluated on samples of CAV controlled flocks. Thirty different viruses and bacteria isolated from chickens did not give rise to any amplification product in the assay. The sensitivity of the nested-PCR was determined on serial dilutions of a CAV vaccine. The nested-PCR was more sensitive than a one step PCR and was able to detect at least 0.16 TCID50 of the vaccine strain. In addition, the protocol employed here detected viral DNA from tissues, sera and litter from flocks with or without clinical signs of disease. It is concluded that the nested-PCR protocol described here is more sensitive, faster and less cumbersome than virus isolation in cell culture as a diagnostic technique for detection of CAV.

Abstract in English:

Penatti M.P.A., Silva A.S., Valadares G.F. & Leite D.S. 2005. Occurrence of F42 colonization factor in Escherichia coli strains isolated from piglets with diarrhea. Pesquisa Veterinária Brasileira 25(1):31-33. Depto Microbiologia e Imunologia, Instituto de Biologia, Unicamp, Campinas, SP 13081-970, Brazil. E-mail: domingos@unicamp.br The objective of this study was to determine the presence of the colonization factor F42 in 168 strains of Escherichia coli isolated from diarrheic stools of newborn piglets. The presence of F42 in 12 (7.1%) strains was detected with the agglutination test. Through the Polymerase Chain Reaction (PCR) of F42 positive strains, gene encoding enterotoxins (ST-I, ST-II, LT-I and LT-II) were detected. The finding of ST-I/ST-II genes in 50% of the strains, ST-I (16%) and ST-II (25%) indicates a strong association of FC F42 with heat-stable enterotoxins (91%). In contrast, the thermolabile enterotoxin (LT-I and LT-II) genes were not detected. Serogroups of F42 positive strains were determined, serogroup O8 being the most prevalent (41,7%). Other serogroups, as there are O9, O11, O18, O32, O35, O98 and O101, were also identified. Thus, FC F42 was confirmed as an additional factor of virulence in the pathogenesis of porcine colibacillosis.

• Escherichia coli F42 piglets fimbriae diarrhea PCR.

Abstract in Portuguese:

Penatti M.P.A., Silva A.S., Valadares G.F. & Leite D.S. 2005. Occurrence of F42 colonization factor in Escherichia coli strains isolated from piglets with diarrhea. Pesquisa Veterinária Brasileira 25(1):31-33. Depto Microbiologia e Imunologia, Instituto de Biologia, Unicamp, Campinas, SP 13081-970, Brazil. E-mail: domingos@unicamp.br The objective of this study was to determine the presence of the colonization factor F42 in 168 strains of Escherichia coli isolated from diarrheic stools of newborn piglets. The presence of F42 in 12 (7.1%) strains was detected with the agglutination test. Through the Polymerase Chain Reaction (PCR) of F42 positive strains, gene encoding enterotoxins (ST-I, ST-II, LT-I and LT-II) were detected. The finding of ST-I/ST-II genes in 50% of the strains, ST-I (16%) and ST-II (25%) indicates a strong association of FC F42 with heat-stable enterotoxins (91%). In contrast, the thermolabile enterotoxin (LT-I and LT-II) genes were not detected. Serogroups of F42 positive strains were determined, serogroup O8 being the most prevalent (41,7%). Other serogroups, as there are O9, O11, O18, O32, O35, O98 and O101, were also identified. Thus, FC F42 was confirmed as an additional factor of virulence in the pathogenesis of porcine colibacillosis.

Abstract in English:

França T.N., Peixoto P.V., Brito M.F., Driemeier D., Mores N. & Zanella J. 2005. [Outbreak of Circovirosis (Porcine Postweaning Multisystemic Wasting Syndrome) in the state of Rio de Janeiro, Brazil.] Surto de Circovirose (Síndrome Definhante Multissistêmica de Suínos Desmamados) no estado do Rio de Janeiro. Pesquisa Veterinária Brasileira 25(1):39-53. Universidade Estácio de Sá, Curso de Medicina Veterinária, Disciplina de Anatomia Patológica, Estrada Boca do Mato 850, Vargem Pequena, RJ 22783-320, Brazil. E-mail: ticianaf@uol.com.br The first outbreak of Postweaning Multisystemic Wasting Syndrome (PMWS) in swine, which occurred in southeastern Brazil, in the state of Rio de Janeiro, is described. The disease, which affects mainly weaned about 4 month-old pigs, caused the death of at least 14 animals. The property, where the outbreak occurred, had inadequate sanitary and management conditions. Clinically the disease was characterized by wasting, poor development, cough, tachypnoea, dispnoea, diarrhoea, ataxia, tremors after stimulation, decubitus and convulsions. The course of the disease was acute or subacute. The most important post-mortem findings were enlarged lymphnodes, non-collapsed lungs, with consolidated areas mainly in the cranial lobes. Histological lesions consisted mainly of lymphohistiocytic infiltration with multinucleate giant cells in lymph nodes, spleen, Peyer’s patches, kidney, lung and liver, depletion or lymphoid hyperplasia, as well as lymphohistiocytic interstitial pneumonia and areas of secondary bronchopneumonia. The diagnosis was established through observations of the symptoms and typical lesions, and was confirmed by immunohistochemical examination and PCR. The objective of this study was to characterize the epidemiological, clinical and pathological aspects of the outbreak of PMWS, because of the severe direct or indirect economical losses caused by the disease to the world pig industry.

• Postweaning multisystemic wasting syndrome porcine circovirus type 2 pathology immunohistochemistry PCR.

Abstract in Portuguese:

França T.N., Peixoto P.V., Brito M.F., Driemeier D., Mores N. & Zanella J. 2005. [Outbreak of Circovirosis (Porcine Postweaning Multisystemic Wasting Syndrome) in the state of Rio de Janeiro, Brazil.] Surto de Circovirose (Síndrome Definhante Multissistêmica de Suínos Desmamados) no estado do Rio de Janeiro. Pesquisa Veterinária Brasileira 25(1):39-53. Universidade Estácio de Sá, Curso de Medicina Veterinária, Disciplina de Anatomia Patológica, Estrada Boca do Mato 850, Vargem Pequena, RJ 22783-320, Brazil. E-mail: ticianaf@uol.com.br The first outbreak of Postweaning Multisystemic Wasting Syndrome (PMWS) in swine, which occurred in southeastern Brazil, in the state of Rio de Janeiro, is described. The disease, which affects mainly weaned about 4 month-old pigs, caused the death of at least 14 animals. The property, where the outbreak occurred, had inadequate sanitary and management conditions. Clinically the disease was characterized by wasting, poor development, cough, tachypnoea, dispnoea, diarrhoea, ataxia, tremors after stimulation, decubitus and convulsions. The course of the disease was acute or subacute. The most important post-mortem findings were enlarged lymphnodes, non-collapsed lungs, with consolidated areas mainly in the cranial lobes. Histological lesions consisted mainly of lymphohistiocytic infiltration with multinucleate giant cells in lymph nodes, spleen, Peyer’s patches, kidney, lung and liver, depletion or lymphoid hyperplasia, as well as lymphohistiocytic interstitial pneumonia and areas of secondary bronchopneumonia. The diagnosis was established through observations of the symptoms and typical lesions, and was confirmed by immunohistochemical examination and PCR. The objective of this study was to characterize the epidemiological, clinical and pathological aspects of the outbreak of PMWS, because of the severe direct or indirect economical losses caused by the disease to the world pig industry.