Diagnóstico e epidemiologia molecular de casos de raiva bovina na região central do Rio Grande do Sul

Abstract

Rabies is an infectious disease of the central nervous system caused by the rabies virus (RABV), which affects all mammals. In Brazil the rabies has caused considerable losses to cattle herds in various regions. The official diagnosis is made by the fluorescent antibody technique (FAT) concomitantly with biological assay, usually the mouse inoculation tests (MIT). The MIT is considered a sensitive, specific and effective technique for rabies diagnosis, but has disadvantages such as long time to obtain the results and the need to use animals. The first paper of this dissertation 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. The second paper describes an evaluation of sensitivity for VI of Rabv in neuroblastoma cells (N2A) and baby hamster kidney cells (BHK-21). For this, thirty-six samples derived bovine brains of suspected rabies cases were initially submitted to the FAT and MIT test and subsequently to three protocols VI in each cell line: a single pass 24h and 72h, and three passes 48h. The average time to obtain final results at MIT was 12.3 days (8-21). The average time required for final MIT results was 12.3 days (8 21). The FAT and MIT combined detected 32/36 positive samples, these MIT detected 32 (100%) and the FAT detected 31 (96.8%). The isolation in BHK-21 cells resulted in 100% (32/32) positivity in the protocol of 72h and 96.9% (31/32) after three passages of 48h. The isolation in N2A cells resulted in 100% (32/32) positive for 72h and 30/32 (93.7%) samples 48h after three passages. A single 24h passage protocol (T1) in both cell lines performed poorly, detecting less than 40% of the positive samples. These results indicate that VI in either cell line, especially in BHK-21 cells that grow faster and are much easier to maintain than N2A cells, does represent an adequate alternative for MIT as a confirmatory test for rabies diagnostic in bovine specimens, yielding reliable results in reduced time.