Dynamics of neutralizing antibodies against Bovine respiratory syncytial virus in a dairy herd from Santa Fe Province, Argentina

Abstract Bovine respiratory syncytial virus (BRSV) is one of the most relevant agents responsi-ble for respiratory disease in cattle from both dairy and beef farms. BRSV is spread by horizontalcontact causing a constant presence of seropositive animals that favors viral circulation throughout the year. Moreover, reinfections with BRSV are frequent between animals regardless of theirage as BRSV does not confer long-lasting protective immunity. Several studies have demonstrated the circulation of BRSV in cattle from different regions of the world; however, little isknown about the dynamics of BRSV infection in cows before and after they begin lactation. Theaim of this work was to study the dynamics of BRSV neutralizing antibodies from birth up to36 months of age in a closed dairy herd of Argentina specifically around the lactation period. Passive maternal antibodies against BRSV started to decrease monthly and became almost undetectable at 8 months of age. We detected two potential infection points at months 11 and 27after birth, in which 30% and 45% of the animals showed seroconversion, respectively. Specifically, an increase in the proportion of seropositive cows after the start of lactation suggests thatthey became reinfected around the time they began lactating. We demonstrate the importanceof understanding BRSV dynamics in a closed dairy herd to review the vaccination schedule ofthe animals to achieve protection against BRSV infection.

Resumen El virus respiratorio sincitial bovino (Bovine respiratory syncytial virus, [BRSV]) es uno de los principales agentes responsables de la enfermedad respiratoria en bovinos, tanto de tambos como de cría. El virus se transmite horizontalmente y causa la presencia constante de animales seropositivos, lo cual favorece la circulación viral a lo largo del ano. A su vez, las reinfecciones por BRSV son frecuentes entre animales independientemente de su edad, dado que el virus no confiere inmunidad protectora a largo plazo. Numerosos estudios han demostrado la circulación de BRSV en bovinos de diferentes regiones del mundo, sin embargo, poco se conoce acerca de la dinámica de infección en vacas antes y después del inicio de la fase de lactancia. El objetivo de este trabajo fue estudiar la dinámica de anticuerpos neutralizantes anti- BRSV en vacas lecheras desde el nacimiento hasta los 36 meses de vida en un tambo cerrado de Argentina, específicamente, en el período de lactancia. Los anticuerpos pasivos específicos para BRSV comenzaron a declinar mensualmente hasta ser casi indetectables a los 6 meses. Detectamos dos potenciales puntos de infección a los meses 11 y 27 luego del nacimiento, momentos en los que el 30 y el 45% de los animales mostraron seroconversión, respectivamente. El incremento en la proporción de vacas seropositivas luego del comienzo de la lactancia sugiere que estas se reinfectaron en el inicio de dicha etapa. Demostramos la importancia de entender la dinámica de circulación del BRSV en un tambo cerrado, a fin de revisar el esquema de vacunación de los animales para que estén protegidos frente a la posible infección por este virus.

Establishment and evaluation of DAS-ELISA for detecting bovine respiratory syncytial virus / 中国人兽共患病学报

In order to develop a double antibody sandwich assay (DAS-ELISA) for detecting bovine respiratory syncytial virus (BRSV),New Zealand white rabbits and BALB/c mice were immunized with the purified G protein as an antigen to prepare anti-G protein polyclonal and monoclonal antibodies.The antibody concentration and reaction conditions of DAS-ELISA were optimized by square titration,and its sensitivity,specificity,and coincidence rate were validated.Five hybridoma were stably secreting Mab which subclass belonged to IgG1κ.Western blot and IFA test showed that PcAb and Mab could react specifically with G protein and BRSV.The PcAb and Mab as the capture antibody and detection antibody respectively,and their optimal working concentrations were determined to be 2.5 μg/mL and 10μg/mL,the critical value 0.22 and the detection limit of 1.43 μg/mL,batch,inter-assay coefficient of variation less than 10 ％.The DAS-ELISA had no cross-reaction with several pathogens which often caused bovine respiratory disease.When 45 nasal swabs of clinical samples were simultaneously detected by the DAS-ELISA and RT-PCR,the sensitivity,specificity and coincidence rate were 92.0 ％,100 ％,95.6 ％,respectively.It' s indicated that the established DAS-ELISA detection method can be used to detect a large number of clinical samples.It was the foundation of monitoring and quick diagnosis for BRSV.

Parainfluenza-3 and bovine respiratory syncytial virus: intraherd correlation adjusted for sensitivity and specificity / Parainfluenza-3 y virus respiratorio bovino sincitial: correlación intrahato ajustado por la sensibilidad y especificidad

Objective. The purpose of this study was to compare the intra-class correlation coefficients (ICC) and design effects (D) estimates adjusted or unadjusted for sensibility (Se) and specificity (Sp) of the diagnostic tests using a Bayesian procedure. Materials and methods. Sera from 232 animals from 44 randomly selected herds, to detect antibodies against parainfluenza-3 virus (PIV3) from non-vaccinated dual-purpose cattle from Colima Mexico, were used. Only 176 animals from 33 herds were used to evaluate the presence of the bovine respiratory syncytial virus (BRSV). Results. The ICC and D values adjusted and unadjusted for PIV3 were 0.33, 2.73, 0.32, and 2.71, respectively. For BRSV the values were 0.31, 2.64, 0.28 and 2.49. Conclusions. The adjusted or unadjusted ICC and D estimates were similar because of the high Se and Sp of the diagnostic tests and the relatively high prevalence of the diseases here studied.

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Doenças respiratórias em bezerros na região sul do Rio Grande do Sul: estudo retrospectivo de 33 surtos / Respiratory diseases in calves in southern Rio Grande do Sul: study of 33 outbreaks

Foram estudados 33 surtos de pneumonia em bovinos jovens na área de influência do Laboratório Regional de Diagnóstico (LRD) da Faculdade de Veterinária da Universidade Federal de Pelotas (UFPel) entre os anos de 2000 e 2011. Foram diagnosticados 18 surtos de pneumonia (54,54%) em bovinos de raças leiteiras, Holandês ou Jersey e 13 surtos (39,39%) em gado de corte ou cruzas de gado de corte. A morbidade variou entre 0,06%-100% e a mortalidade foi de 0,06%-34,6%. A doença ocorreu igualmente em todas as estações do ano e foi mais frequente em bezerros de 1-3 meses totalizando 13 surtos. Sete surtos ocorreram em bovinos entre 4-6 meses, sete entre 7-12 meses e seis surtos ocorreram em bezerros de 1-29 dias. Pneumonia enzoótica pela infecção pelo vírus sincicial respiratório bovino (BRSV) com lesões histológicas de broncopneumonia, pneumonia intersticial e presença de células sinciciais foi mais frequente em bovinos de raças de corte com dez surtos (58,8%); seis surtos dessa enfermidade ocorreram em raças de leite (35,2%). O diagnóstico foi confirmado por imuno-histoquímica em sete casos. Os sinais clínicos da maioria dos casos de pneumonia observados caracterizaram-se por dispneia, emagrecimento, apatia, tremores, bruxismo, desidratação, respiração ruidosa, tosse, corrimento nasal seroso ou mucopurulento, decúbito e morte. As lesões macroscópicas caracterizaram-se por presença de áreas de consolidação vermelho-escuras, edema e enfisema nas regiões crânio-ventrais dos lobos pulmonares cardíaco e apical ou pneumonia intersticial com distribuição difusa, edema e enfisema. Histologicamente, as lesões pulmonares eram variáveis. Broncopneumonia necrossupurativa difusa acentuada com hiperplasia de pneumócitos tipo II e edema intersticial e alveolar foi observada em 15 casos. Os resultados deste trabalho demonstram que as pneumonias são importantes causas de perdas econômicas em bovinos jovens na região de influência do LRD. Deve ser destacado que a pneumonia enzoótica devido a infecção pelo BRSV é importante tanto em bovinos de corte como de leite independente da forma de criação.

Thirty-three outbreaks involving pneumonia in young cattle diagnosed in the area of influence of the Regional Diagnostic Laboratory (LRD) of the Veterinary School of the Federal University of Pelotas (UFPel) between 2000 and 2011 were studied. Eighteen outbreaks (54.54%) were diagnosed in dairy breeds and Jersey or Holstein and 13 outbreaks (39.39%) in beef cattle or crossbred beef cattle. Morbidity ranged from 0.06% -100% and mortality was 0.06% to 34.61%. The disease occurred in all seasons of year and was more frequent in calves aged 1-3 months (11 outbreaks). Seven outbreaks occurred in cattle between 4-6 months, seven between 6-12 months and six outbreaks occurred in calves 1-30 days. Bovine respiratory disease due to infection with bovine respiratory syncytial virus (BRSV) was more frequent in beef cattle breeds with eight outbreaks (44.44%); six outbreaks of this disease occurred in calves of dairy breeds (33.33%). The diagnosis was confirmed by immunohistochemistry in seven cases. The main clinical signs were characterized by dyspnea, weight loss, lethargy, tremors, bruxism, dehydration, rapid and noisy breathing, coughing, serous or mucopurulent nasal discharge, recumbency and death. Gross lesions were characterized by dark red areas of consolidation, edema and emphysema in cranio-ventral regions of the apical and cardiac lung lobes or diffuse interstitial pneumonia, edema and emphysema. Histologically, lung lesions were variable. Diffuse bronchopneumonia necrotizing with marked hyperplasia of type II pneumocytes and alveolar and interstitial edema was observed in 15 cases. The results of this study demonstrate that pneumonia is an important cause of economic loss in young cattle in the area of influence of the LRD and that enzootic pneumonia due to BRSV infection is important in both beef cattle and milk cattle regardless of husbandry systems.

Detecção molecular e análise filogenética de vírus respiratório sincicial bovino (BRSV) em swabs e tecido pulmonar de bovinos adultos / Molecular detection and phylogenetic analysis of bovine respiratory syncytial virus (BRSV) in swabs and lung tissues of adult cattle

Bovine respiratory syncytial viruses virus (BRSV) is one of the etiologic agents of pneumonia in young cattle. Few studies have been made aiming detection of the virus in samples collected from adult animals, especially those asymptomatic bovines. However, it is assumed that infections in these groups may occur mostly asymptomatic and this would be an important mechanism for maintaining of BRSV in herds. In this study, the goal was to conduct an analysis of the occurrence of asymptomatic infections by BRSV in lung samples (n=68) and nasal swabs (209) taken from adult animals collected in abattoirs from Southern and Southeastern Brazil respectively, to detect via polymerase chain reaction the occurrence of infected animals in populations of adult cattle. The samples that resulted positive (6) on RT-PCR were subsequently subjected to cutting with restriction enzymes and sequencing for genetic characterization (2 samples). All samples belongs to subgroup B of BRSV, which is reported as the one circulating in Brazil. The results obtained demonstrate that BRSV may be present in samples taken from adult animals, which is in agreement the hypothesis that infections in adults run in a sub-clinical way that may be of importance as a maintenance mechanism of the virus in bovine herds.

O vírus respiratório sincicial bovino (BRSV) é rebanhos. No presente estudo, o objetivo foi realizar uma um dos agentes etiológicos de pneumonias em bovinos jo-análise da prevalência de infecções assintomáticas pelo vens. Poucos estudos foram realizados visando à detecção BRSV em pulmões (n=68) e swabs nasais (209) coletados do agente em amostras coletadas de animais adultos, e em de bovinos adultos coletadas em frigoríficos da região Sul especial de bovinos assintomáticos. No entanto, presume-e Sudeste respectivamente, no sentido de detectar por in-se que as infecções ocorridas nestes grupos possam ocor-termédio de reação da polimerase em cadeia qual a taxa rer em sua maioria de forma assintomática e este seria de animais infectados em populações de animais adultos um mecanismo importante para manutenção do BRSV nos onde não ocorram sinais clínicos da infecção. As amostras positivas à RT-PCR (6) foram posteriormente submetidas ao corte com enzimas de restrição (REA) e sequenciamento para caracterização genética do gene F (2 das amostras). Todas as amostras se enquadram no subgrupo B de BRSV, o grupo circulante no Brasil conforme estudos anteriores. Os resultados obtidos demonstram que o BRSV pode estar presente em amostras obtidas de animais sadios, reforçando a hipótese de que infecções subclínicasfazem parte do mecanismo de manutenção do vírus nos rebanhos.

Clonagem das glicoproteínas transmembrana G e F de um isolado brasileiro do vírus respiratório sincicial bovino em sistema procarioto / Cloning of the transmembrane glycoproteins G and F from a Brazilian isolate of bovine respiratory syncytial virus in a prokaryotic system

The aim of this work was the cloning of those transmembrane glycoproteins G and F from an isolate bovine respiratory syncytial viruses (BRSV) - a Brazilian isolate of BRSV, named BRSV-25-BR in previous studies, in a prokaryotic system to proceed the sequencing of larger genomic fragments. The nucleotide substitutions were confirmed and these clones may also be used in further studies regarding the biological effects of those proteins in vitro and in vivo.

O objetivo deste trabalho foi a clonagem das glicoproteínas transmembrana G e F de um isolado de vírus respiratório sincicial bovino (BRSV) - um isolado brasileiro denominado BRSV-25-BR- que já demonstrou possuir mutações em regiões altamente conservadas do gene da proteína G - em sistema procariótico, com o intuito de sequenciar fragmentos genômicos maiores. As substituições de nucleotídeos foram confirmadas e tais clones podem ser utilizados em futuros estudos sobre os efeitos biológicos destas proteínas tanto in vitro como in vivo.

Antibody responses in mice after immunization with inactivated bovine respiratory syncytial virus using different adjuvants / Respostas de anticorpos em camundongos após imunização com vírus respiratório sincicial bovino inativado utilizando diferentes adjuvantes

Bovine respiratory syncytial virus (BRSV) causes pneumonia in young cattle. Modified-live-virus (MLV) and inactivated vaccines are currently used for the control of clinical effects of BRSV infections in cattle. On the present research, the stimulation of specific anti-BRSV immunoglobulin isotypes was investigated, through the use of different commercially available adjuvants (Water-in-oil emulsion, Quil A, Aluminum-hydroxide) in inbred mice (Balb/C and C57BL/6). BRSV antibodies were measured using an enzyme-linked immunosorbent assay (ELISA) and the results were compared to the antibody levels induced by immunization of animals using live-BRSV-virus. Water-in-oil emulsion and alum- adjuvant preparations induced higher levels of IgG1 immunoglobulins, whereas Quil A favored the production of IgG2 antibodies, this last being a more appropriate response profile for the specific case of BRSV. Not using adjuvants resulted in poor levels of specific antibodies. The isotype profile of specific antibodies obtained varied greatly depending on the adjuvants used. This information may be useful for the formulation of more effective BRSV inactivated vaccines; however, these findings have to be confirmed in cattle.

O vírus respiratório sincicial bovino (BRSV) causa pneumonia em bovinos jovens. Vacinas de vírus vivo modificado (MLV) e vacinas inativadas são atualmente utilizadas para o controle dos efeitos clínicos de infecções pelo BRSV em bovinos. No presente trabalho, investigou-se a estimulação dos isotipos de imunoglobulinas específicas anti-BRSV, através da utilização de diferentes adjuvantes disponíveis comercialmente (água em óleo de emulsão, Quil A, hidróxido de alumínio) em camundongos isogênicos (Balb/C e C57BL/6). Anticorpos contra o BRSV foram medidos usando-se um ensaio imunoenzimático (ELISA), e os resultados foram comparados com os níveis de anticorpos induzidos pela imunização de animais utilizando-se o BRSV vivo. As preparações em que se empregou óleo mineral e alumínio como adjuvantes induziram altos níveis de imunoglobulinas IgG1, enquanto QuilA favoreceu a produção de anticorpos de classe IgG2, sendo este último um perfil de resposta mais desejável para o caso específico de BRSV. A não utilização de adjuvantes resultou em baixa produção de anticorpos específicos. O perfil de isotipos de imunoglobulinas secretados variou bastante conforme o adjuvante utilizado. Esta informação pode ser útil futuramente na formulação de vacinas inativadas mais eficazes contra o BRSV. Todavia, esses achados devem ser confirmados em bovinos.

Expression and Identification Truncated Glycoprotein G of Bovine Respiratory Syncytial Virus in Escherichia coli / 中国生物工程杂志

Two fragments G1 and G2 of the glycoprotein G gene of bovine respiratory syncytial virus(BRSV) were selected for expression in Escherichia coli based on the analysis of glycoprotein G by DNA Star software.Then the two fragments of glycoprotein G were amplified by PCR with synthesized G gene of BRSV as the template.The amplified fragments G1 and G2 are 570bp and 308bp in length,respectively.The PCR products were cloned into pET30a vector and expressed in soluble form in E.coli after induction of cultured E.coli with IPTG.Both of the recombinant proteins G1 and G2 were purified by immobilized Ni ion affinity chromatography under native conditions.Then the purified proteins were analysed by Western blotting.The results showed that the purified recombinant protein G1 retained good antigenicity and specificity.But the purified recombinant protein G2 didn't possess biological activity.Antibodies against BRSV were detected in suspected bovine serum samples in China by using indirect ELISA and Western blotting with the purified recombinant protein G1.The purified recombinant protein G1 might be used as antigen for establishing serological methods for diagnosis of BRSV infection.And the purified recombinant protein G1 might also be used for preparing polyclonal and monoclonal antibodies for research on biological functions of glycoprotein G of BRSV.