Teste imunoenzimático com base em anticorpo monoclonal para a detecção de anticorpos contra os herpesvírus bovino tipos 1 e 5

Os herpesvírus bovino tipos 1 (BoHV-1) e 5 (BoHV-5) são agentes virais genética e antigenicamente relacionados, associados com diversas manifestações clínicas em bovinos, incluindo doença respiratória, genital, neurológica e abortos. Estudos epidemiológicos indicam que esses vírus estão amplamente disseminados no rebanho bovino brasileiro. O diagnóstico sorológico, que permite identificar animais portadores da infecção latente, se constitui em importante ferramenta para monitoramento individual e de rebanho. O presente artigo relata a padronização de um teste imunoenzimático do tipo ELISA, com base em anticorpo monoclonal (AcM), para a detecção de anticorpos séricos que reagem contra BoHV-1 e/ou BoHV-5. Inicialmente, determinou-se o AcM mais adequado para a sensibilização das placas, as diluições apropriadas do antígeno e dos soros-teste e o ponto de corte do ensaio. Após a padronização, o ensaio foi validado testando-se 506 amostras de soro bovino, previamente testadas para anticorpos neutralizantes contra BoHV-1 e/ou BoHV-5 pela técnica de soroneutralização (SN). Comparando-se com os resultados da SN frente a BoHV-1, o teste de ELISA apresentou sensibilidade e especificidade de 96,6% e 98,3%, respectivamente. Os valores preditivos positivo e negativo foram de 97,6%, a concordância foi de 97,6% e o índice de correlação kappa entre os testes foi de 0,95, o que indica uma excelente concordância. Comparando-se com os resultados da SN frente o BoHV-5, o ELISA apresentou 94,3% de sensibilidade; 97,9% de especificidade; 97,1% de valor preditivo positivo e 95,9% de valor preditivo negativo. Para BoHV-5, a concordância entre os testes foi de 96,4% e o índice de correlação foi de 0,92, também excelente. Esses resultados demonstram que o teste padronizado apresenta sensibilidade e especificidade adequados para o diagnóstico sorológico das infecções por BoHV-1 e BoHV-5 em nível individual e de rebanho. Dessa forma, o ensaio pode se constituir em alternativa para o teste de SN e para os kits de ELISA importados.Bovine herpesviruses 1 (BoHV-1) and 5 (BoHV-5) are antigenic and genetically related viruses associated with different clinical syndromes in cattle, including respiratory, reproductive, neurological disease and abortion. Epidemiological studies indicate the widespread distribution of both viruses among Brazilian cattle. Serological diagnosis, that allows the identification of latently infected animals, represents an important tool for individual and herd monitoring. The present article describes the standardization of a monoclonal antibody (MAb)-based immunoenzymatic test (ELISA) for detection of antibodies to BoHV-1 and/or BoHV-5. The initial steps involved the determination of the most suitable MAb, the appropriate dilutions of viral antigen and serum samples, and the cut-off value of the assay. After standardization, the ELISA was validated by testing 506 cattle serum samples previously tested for neutralizing antibodies to BoHV-1 and BoHV-5 by virus neutralizing assay (VN). Comparing to the VN for BoHV-1 antibodies, the ELISA presented sensitivity and specificity of 96.6% and 98.3%, respectively. Positive and negative predictive values were 97.6%, the concordance between the tests was 97.6% and the coefficient of correlation k (kappa) was 0.95, demonstrating an excellent correlation. Comparing to the VN for BoHV-5 antibodies, the ELISA presented 94.3% of sensitivity, 97.9% of specificity, 97.1% of positive predictive value, 95.9% negative predictive value, concordance of 96.4% and kappa coefficient of 0.92. These results demonstrate that the ELISA presents suitable specificity and sensitivity to be used for individual and herd serological diagnosis of BoHV-1 and BoHV-5, thus, representing an alternative for VN assays and imported ELISA kits

Genetic analysis of the porcine group B rotavirus NSP2 gene from wild-type Brazilian strains

Group B rotaviruses (RV-B) were first identified in piglet feces, being later associated with diarrhea in humans, cattle, lambs, and rats. In human beings, the virus was only described in China, India, and Bangladesh, especially infecting adults. Only a few studies concerning molecular analysis of the RV-B NSP2 gene have been conducted, and porcine RV-B has not been characterized. In the present study, three porcine wild-type RV-B strains from piglet stool samples collected from Brazilian pig herds were used for analysis. PAGE results were inconclusive for those samples, but specific amplicons of the RV-B NSP2 gene (segment 8) were obtained in a semi-nested PCR assay. The three porcine RV-B strains showed the highest nucleotide identity with the human WH1 strain and the alignments with other published sequences resulted in three groups of strains divided according to host species. The group of human strains showed 92.4 to 99.7% nucleotide identity while the porcine strains of the Brazilian RV-B group showed 90.4 to 91.8% identity to each other. The identity of the Brazilian porcine RV-B strains with outer sequences consisting of group A and C rotaviruses was only 35.3 to 38.8%. A dendrogram was also constructed to group the strains into clusters according to host species: human, rat, and a distinct third cluster consisting exclusively of the Brazilian porcine RV-B strains. This is the first study of the porcine RV-B NSP2 gene that contributes to the partial characterization of this virus and demonstrates the relationship among RV-B strains from different host species

Efeito do dimetildioctadecilamônio na resposta imune humoral e celular de cobaios inoculados com o herpesvírus bovino-1 inativado Effect of dimethyl dioctadecyl ammonium chloride on the humoral and cellular response to inactivated bovine herpesvirus-1 in guinea pigs

Avaliou-se o potencial adjuvante do cloreto de dimetildioctadecilamônio (DDA cloreto) em estimular as respostas imune humoral e celular, do tipo hipersensibilidade cutânea tardia (DTH), em cobaios que receberam preparações de antígeno contendo o herpesvírus bovino tipo 1 (BHV-1) inativado. Os animais foram vacinados com o BHV-1 em cinco diferentes formulações, representadas pelos grupos: A- adjuvante completo / incompleto de Freund, B- hidróxido de alumínio (Al(OH)3), C- DDA cloreto, D- associação Al(OH)3 / DDA cloreto e E- BHV-1 em solução aquosa sem adjuvante. Os animais do grupo F, grupo-controle negativo, foram inoculados apenas com meio de cultivo celular. Os maiores títulos de anticorpos neutralizantes do BHV-1, expressos em log2, foram obtidos nos grupos D, A e C, com títulos de 6,25, 6,0 e 5,25, respectivamente. Na avaliação da DTH, os maiores aumentos na espessura da dobra da pele foram observados nos grupos A (2,4mm), C (1,8mm) e D (1,1mm). O DDA cloreto, utilizado tanto de forma isolada quanto em associação, determinou a potencialização das respostas imunológicas humoral e celular de cobaios imunizados com o BHV-1 inativado.<br>The adjuvanticity of dimethyl dioctadecyl ammonium chloride (DDA chloride) to induce humoral and cell mediated (delayed type hypersensitivity - DTH -) immune responses was assessed in guinea pigs that received antigen preparations containing inactivated bovine herpesvirus type 1 (BHV-1). The animals were vaccinated with five different formulations, containing BHV-1 represented by groups: A- complete/incomplete Freund adjuvant, B- aluminum hydroxide (Al(OH)3), C- DDA chloride, D- association Al(OH)3/DDA and E- BHV-1 in aqueous solution without adjuvant. Group F was the negative control group and the animals received only cell culture medium. The higher titers of BHV-1 neutralizing antibodies, expressed in log2, were obtained in groups D, A and C with values of 6.25, 6.0 and 5.25, respectively. In the DTH assessment, the higher increase in skin thickness was observed in group A (2.4mm), C (1.8mm) and D (1.1mm). These results showed that DDA chloride used alone or in association to other substance boosted the humoral and cell mediated immune responses in guinea pigs immunized with inactivated BHV-1

Torque teno sus virus (TTSuV) infection at different stages of pig production cycle

Torque teno sus virus (TTSuV) infection is present in pig herds worldwide. It has been demonstrated that TTSuV might increase the severity of other important viral diseases with economic and public health impacts. At present, there is no information on the age distribution of pigs infected with TTSuV in Brazilian herds. This study evaluated the frequency of TTSuV infection in pigs at different stages of production. Fecal samples (n=190) from pigs at 1 to 24 weeks of age and from breeders at 6 farrow-to-weaning (up to 8 weeks of age) and 9 grower-to-finish (9 weeks of age onwards) farms in the western region of Paraná state, Brazil, were evaluated by PCR. Fragments of the 5' UTRs of TTSuV1 and/or TTSuVk2 DNAs were identified in 126 (66.3%) of the fecal samples. Significant differences were found with the percentages of positive samples for TTSuV1, TTSuVk2, and mixed infections by both genera between and within the different pig production stages. Fecal samples from the grower-to-finish farms had TTSuV detection rates (90.1%; 64/71) that were significantly (p<0.05) higher than those from the farrow-to-weaning farms (52.1%; 62/119). TTSuV detection was significantly (p<0.05) more frequent in finisher pigs than in the animals from the other stages. The UTR nucleotide sequences in this study presented higher similarities to strains from Norway (96%, TTSuV1), and Argentina and China (97.1%, TTSuVk2). These results suggest that TTSuV infection has spread to pigs of all production stages and that the viral infection rate increases with the age of the animals. In the western region of Paraná state, Brazil, TTSuV1 and TTSuVk2-induced infections were more frequently observed in suckling piglets and finisher pigs, respectively. Phylogenetic analysis pointed out the possibility of different strains of TTSuV1 and TTSuVk2 circulating in pig herds of Brazil

Diarréia em bezerros da raça Nelore criados extensivamente: estudo clínico e etiológico Diarrhea in Nelore calves: Clinical and etiologic study

A diarréia é considerada uma das principais causas de morbidade e mortalidade de bezerros neonatos. Foram colhidas 100 amostras fecais diarréicas e 30 amostras não diarréicas (grupo controle), de bezerros Nelore com até nove semanas de idade com o objetivo de detectar os enteropatógenos Salmonella spp., Escherichia coli, rotavírus, coronavírus, Cryptosporidium spp. e ovos de helmintos. Enteropatógenos foram detectados em 79,0% das amostras diarréicas e em 70,0% das amostras não-diarréicas. No grupo de bezerros com diarréia, E. coli (69,0%) foi o agente mais freqüentemente isolado, seguido de Cryptosporidium spp. (30,0%), coronavírus (16,0%) e rotavírus (11,0%). No grupo controle, E. coli, Cryptosporidium spp. e coronavírus foram detectados, respectivamente, em 66,7%, 10,0% e 3,3% das amostras. Salmonella spp. e ovos de estrongilídeos não foram encontrados nos dois grupos avaliados. A fímbria K99 foi identificada exclusivamente nas linhagens de E. coli isoladas de bezerros com diarréia (5,8%). Entre os antimicrobianos avaliados "in vitro" a enrofloxacina, a norfloxacina e a gentamicina foram os mais efetivos. O peso dos bezerros aos 210 dias de idade não apresentou diferença significativa entre os animais com e sem diarréia.<br>Diarrhea is considered as one of the main causes of morbidity and mortality in neonates calves. Fecal samples from 100 diarrheic and 30 non-diarrheic (control group) Nelore calves less than 9 weeks old were collected for Salmonella spp., Escherichia coli, rotavirus, coronavirus, Cryptosporidium spp., and for helminth eggs investigation. Enteropathogens were detected in 79.0% diarrheic samples and 70.0% non-diarrheic samples. Among diarrheic calves, Escherichia coli (69.0%) was the most common agent found, following by Cryptosporidium spp. (30.0%), coronavirus (16.0%), and rotavirus (11.0%). In the control group, E. coli, Cryptosporidium spp. and coronavirus were detected in 66.7%, 10.0% and 3.3% of the samples, respectively. Salmonella spp. and strongylids were not found in any of the calves from either group. The K99 fimbrial only was detected in E. coli strains from diarrheic calves (5.8%). Enrofloxacin, norfloxacin, and gentamicin were the most effective among the antimicrobials tested. The weight of 210-day-old calves did not show statistic differences between diarrheic and non-diarrheic calves