Monitoramento Do Vírus Da Raiva Em Diferentes Espécies De Morcegos Em Maringá, Sul Do Brasil / Rabies Virus Monitoring In Different Species Of Bats In Maringá, Southern Brazil / Vigilancia De Virus De La Rabia En Especies Diferentes De Murciélagos En Maringá, Sur De Brasil

A raiva é uma enfermidade infectocontagiosa de caráter zoonótico, causada pelo vírus da raiva (RABV). Os quirópteros juntamente com os canídeos são os principais reservatórios do RABV, sendo responsáveis respectivamente pela manutenção dos ciclos aéreo e terrestre da doença. O presente trabalho teve por objetivo identificar interações entre o vírus e o reservatório no ciclo aéreo da raiva. Para isto, a presença do RABV foi investigada em amostras de saliva de morcegos de diferentes espécies. Foram realizadas trinta e seis capturas de morcegos, na Região de Maringá, Paraná, sul do Brasil, no período de abril a dezembro de 2013. Os morcegos foram capturados com auxílio de redes de nylon e acondicionados em sacos de algodão. Foram registrados os dados biométricos e coletada uma amostra de swab oral de cada exemplar. Para a identificação do RABV, foi realizada a técnica de Semi-Nested RT-PCR (“Reverse transcription polymerase chain reaction”) tendo como alvo o gene N que codifica a nucleoproteína do vírus. A análise de dados foi realizada por estatística descritiva. Ao longo do estudo, foram capturados 444 morcegos, pertencentes a quatro famílias e quinze espécies. O RABV não foi identificado em nenhuma das amostras analisadas. Estes resultados demonstram a ausência de excreção do RABV pela saliva de morcegos saudáveis na região alvo do estudo e salientam para a necessidade de mais estudos sobre a manutenção da raiva nas diferentes espécies de morcegos.(AU)

Rabies is an zoonotic infectious disease, caused by rabies virus (RABV). The bats with canids are the main RABV reservoirs, accounting respectively for maintaining the air cycles and terrestrial of the disease. This study aimed to identify interactions between the virus and the reservoir in air rabies cycle. For this, the presence of RABV was investigated in bat saliva samples from different species. Thirty-six capture bats were held in Maringá Region Parana, southern Brazil, from April to December 2013. The bats were captured with the help of nylon nets and placed in cotton bags. Were registered biometric data and collected a sample of oral swab of each especimen. For the identification of RABV, the semi-nested RT-PCR technique ("Reverse transcription polymerase chain reaction") targeting the N gene encodes the nucleoprotein of the virus was performed. Data analysis was performed using descriptive statistics. Throughout the study, were captured 444 bats belonging to four families and fifteen species. The RABV was not identified in the evaluated samples. These results demonstrate the absence of excretion of RABV in the saliva of healthy bats in the studied area and reinforce the need for more studies about the maintenance of rabies in different species of bats.(AU)

La rabia es una enfermedad infecciosa de zoonótica causada por el virus de la rabia (RABV). Los murciélagos con los cánidos son los principales reservorios RABV, que representan, respectivamente, para el mantenimiento de los ciclos de aire y terrestre de la enfermedad. Este estudio tuvo como objetivo identificar las interacciones entre el virus y el reservatório en el ciclo aéreo de la rabia. Para ello, la presencia de RABV se investigó en muestras de saliva de murciélago de diferentes especies. treinta y seis colecciones de murciélagos de hisopos orales se llevaron a cabo en Maringá Región Paraná, sur de Brasil, de abril a diciembre de 2013. Los murciélagos fueron capturadas con la ayuda de redes de nylon y se colocaron en bolsas de algodón eran registrado los datos biométricos y se recogió una muestra de hisopo bucal de cada individuo. Para la identificación de RABV, la técnica de semi-anidada RT-PCR ( "reacción en cadena de la polimerasa de transcripción inversa") en el gen N se realizó codifica la nucleoproteína del virus. El análisis de datos se realizó mediante estadística descriptiva. Durante todo el estudio, 444 murciélagos fueron capturados pertenecientes a cuatro familias y quince especies. El RABV no fue identificado en ninguna de las muestras. Estos resultados demuestran la ausencia de RABV en excreción de la saliva de los murciélagos sanos en el estudio de la región de destino y subrayan la necesidad de más estudios sobre la ira de mantenimiento en diferentes especies de murciélagos.(AU)

Virus isolation in cell culture for confirmatory diagnostic of rabies in bovine specimens / Isolamento viral em cultivo celular para o diagnóstico confirmatório de raiva em amostras de bovinos

This study investigated the suitability of virus isolation (VI) in mouse neuroblastoma cells (N2A) and baby hamster kidney cells (BHK-21) as a confirmatory test for diagnosis of bovine rabies. Fourty-eight brain samples from cattle suspected of rabies were initially submitted to fluorescent antibody test (FAT) and mouse inoculation test (MIT) for routine diagnostic. Subsequently, these specimens were submitted to three protocols of VI in each cell line: a single 24h or 72h passage (T1, T2), or three 48h passages (T3). The FAT and MIT combined detected 32/48 positive samples, from which MIT detected 32 and FAT 31. The average time required for final MIT results was 12.3 days (8 - 21). VI in BHK-21 cells provided definitive, positive results in 100% of the samples in 72h (T2) and in 96.9% after three 48h passages (T3). VI in N2A cells yielded positive results in 100% in 72h (T2) and in 93.7% of samples after three 48h passages (T3). Sensitivity, specificity, positive and negative predictive values were 100% in T2 in N2A and BHK-21 cells, and the Kappa value was excellent in both cells (k=1). A single 24h passage (T1) in both cell lines performed poorly, detecting less than 40% of the positive samples. Taking together, these results indicate that VI in both cell lines, especially in BHK-21 cells that grow faster and are much easier to maintain, does represent an adequate alternative for MIT as a confirmatory test for rabies diagnostic in bovine specimens, yielding reliable results in reduced time.(AU)

Este estudo investigou a sensibilidade do isolamento viral (VI) em células de neuroblastoma murino (N2A) e células de rim de hamster (BHK-21) como um teste confirmatório para o diagnóstico de raiva bovina. Quarenta e oito amostras de cérebro de bovinos com suspeita de raiva foram inicialmente submetidas aos testes diagnósticos de rotina, imunofluorescência direta (FAT) e inoculação intracerebral em camundongos (MIT). Subsequentemente, essas amostras foram submetidas a três protocolos de VI em cada linhagem celular: uma única passagem de 24h ou 72h (T1, T2), ou três passagens de 48h (T3). Os testes FAT e MIT combinados detectaram 32/48 amostras positivas, das quais o MIT detectou 32 e a FAT 31. O tempo médio requerido para o resultado final no MIT foi 12,3 dias (8-21 dias). O teste de VI em células BHK-21 obteve resultados positivos em 100% das amostras em 72h (T2), e em 96,9% após três passagens de 48h (T3). O teste de VI em células N2A forneceu resultado positivo em 100% em 72h (T2), e em 30 das 32 amostras após três passagens de 48h (T3). Sensibilidade, especificidade, valores preditivos positivos e negativos foram de 100% tanto em N2A quanto em BHK-21 após 72h de incubação (T2). Além disso, o valor Kappa foi excelente em ambas as células (k=1). Uma única passagem de 24h (T1) em ambas as linhagens celulares não apresentou resultados satisfatórios, detectando menos de 40% das amostras positivas. Esses resultados indicam que o isolamento viral em ambas as linhagens celulares, especialmente em BHK-21 - que multiplica mais rápido e é de mais fácil manutenção - representa uma alternativa adequada para a substituição do MIT como um teste confirmatório para o diagnóstico da raiva em amostras de bovinos, fornecendo resultados confiáveis em tempo reduzido.(AU)

Epidemiologia molecular de surto de raiva bovina na região central do Rio Grande do Sul, 2012 / Molecular epidemiology of an outbreak of bovine rabies in central Rio Grande do Sul, Brazil, 2012

A raiva é uma doença infecciosa do sistema nervoso central de mamíferos causada pelo vírus da raiva (RabV), geralmente, transmitido pela mordedura de animais infectados. No Brasil, os morcegos hematófagos Desmodus rotundus são as principais fontes de infecção do RabV para bovinos e equinos. Este artigo descreve uma investigação epidemiológica e molecular de surtos de raiva ocorridos na região central do Rio Grande do Sul, entre maio e agosto de 2012. Nesse período, 45 casos suspeitos de raiva foram relatados em 22 pequenos rebanhos, localizados dentro de um raio de 4,7km, no município de Pinhal Grande. Desses, 32 amostras foram submetidas para diagnóstico da raiva, sendo que o RabV e/ou antígenos virais foram identificados em 27 amostras. Em um segundo momento, 11 amostras foram submetidas à transcrição reversa/reação em cadeia da polimerase (RT-PCR) para o gene da nucleoproteína (N) do RabV, seguido de sequenciamento nucleotídico e análise filogenética. Sete das 11 amostras apresentaram sequências nucleotídicas idênticas e uma apresentou mutação sinônima, não-codificante, indicando uma provável origem comum dos vírus. Por outro lado, três amostras apresentaram mutações que resultaram em alterações de aminoácidos, sugerindo uma origem diferente do vírus. Esses resultados sugerem que RabV de diferentes origens/linhagens co-circulam na região e foram envolvidos nos surtos descritos. Investigações sobre a circulação de ambos os genótipos em morcegos na região estão em andamento.(AU)

Rabies is an infectious disease of the central nervous system of mammals caused by rabies virus (RabV), generally transmitted by the bite of rabid animals. In Brazil, vampire bats Desmodus rotundus are the main reservoirs of RabV for livestock. The present study describes a molecular and epidemiological investigation of outbreaks of bovine rabies occurring in the central region of Rio Grande do Sul state, Brazil, between May and August 2012. In this period, 45 cases suspected of rabies were reported in 22 small herds, located within a 4.7km range, in the county of Pinhal Grande. From these, 32 samples were submitted to rabies diagnosis and RabV and/or viral antigens were identified in 27 samples. Subsequently, 11 brain samples were submitted to reverse transcription/polymerase chain reaction (RT-PCR) for the nucleoprotein gene (N) followed by nucleotide sequencing and phylogenetic analysis. Seven out of 11 samples yielded identical sequences; one presented a synonymous, non-coding mutation, indicating a likely common origin of the virus. However, three other samples presented nucleotide mutations which resulted in amino acid changes, suggesting a different origin of the virus. In summary, these results suggest that RabV strains of different origin/lineages co-circulate in the region and were involved in the outbreaks. Investigations on the circulation of both genotypes in bats in the region are currently underway.(AU)

Bovine herpesviruses do not play a major role in the differential diagnosis of rabies in cattle in southern Brazil

Background: Rabies has long been recognized as the major cause of encephalitis in cattle in Latin American countries. It has been estimated that nearly 50.000 cattle heads per year are lost due to encephalitis in that subcontinent, with a significant economic impact on cattle productive chains. In Brazil only, 2.500 to 3.000 cattle heads are estimated to be lost every year due to rabies. However, it is believed that rabies incidence in cattle is much larger, since usually only a few samples from affected animals in disease outbreaks are submitted to diagnostic laboratories. Rabies encephalitis is promptly and accurately diagnosed; however, particularly when rabies is excluded as causa mortis, the agent responsible for neurological disease of infectious origin often remains undetermined. Two bovine herpesviruses (BoHVs), bovine herpesvirus type 1 (BoHV-1) and bovine herpesvirus type 5 (BoHV-5) are major pathogens of cattle which are widely disseminated in Brazil. As usual in herpesvirus' biology, these tend to infect a large number of hosts and establish lifelong latent infections which may occasionally be reactivated. Both viruses, particularly BoHV-5, are often recovered from cases of neurological disease in cattle. The participation of BoHVs in the differential diagnosis of rabies must be evaluated. Besides, there might be associations between the occurrence of rabies and BoHV infections that deserve investigation. The aim of this study was to investigate whether bovine herpesvirus 1 and 5 would play a significant role in cases of neurological disease where rabies was the presumptive clinical diagnosis. In addition, associations between the occurrence of rabies and BoHV infections were searched for. The approach adopted for conducting such investigations was based on the search for viral nucleic acids as well as classical virus isolation on tissues of cattle submitted to rabies diagnosis over a two-year period, including rabies-positive and rabies-negative specimens. Materials, Methods & Results: Brain tissue samples of 101 cattle originally submitted to rabies diagnosis were collected over a two year period (2009-2010) from various municipalities within the state of Rio Grande do Sul, Brazil. Thirty nine of these samples had the diagnosis of rabies confirmed by standard laboratory diagnostic methods. Aliquots of tissues were submitted to DNA extraction and examined in search for genomes of bovine herpesviruses (BoHV) types 1 (BoHV-1) and 5 (BoHV-5) by as well as for infectious virus. Bovine herpesvirus genomes were detected in 78/101 (77.2%) samples, in which BoHV-1 genomes were detected in 26/78 (25.7%), BoHV-5 genomes in 22/78 (21.8%) and mixed BoHV infections (BoHV-1 and BoHV-5 genomes) were detected in 30/101 (29.7%) samples. In the 39 samples with confirmed rabies diagnosis, BoHV-1 DNA was detected in 9/39 (23%), BoHV-5 DNA in 6/39 (15.4%) and mixed infections with both BoHV types in 16/39 (41%) samples. However, no infectious herpesvirus was recovered from any of the specimens examined. Discussion: The high prevalence of BoHV1 and BoHV-5 infections was evidenced in the sampled population, but the absence of infectious BoHVs indicate that these were not associated to the occurrence of the cases of encephalitis where rabies was the primary suspicion. In addition, no association was detected between occurrence of rabies and detection of BoHVs, since the frequency of detection of herpesvirus genomes did not significantly differ between rabies-positive and rabies-negative samples. The detection of BoHV DNA in scattered areas of the brain with no infectious virus suggests that latency may take place in different regions of the brain.