ABSTRACT
Abstract Bovine pestiviruses are the causative agents of bovine viral diarrhea, a disease thatcauses severe economic losses in cattle. The aim of this study was to improve their diagnosisby developing a RT-qPCR to detect bovine pestiviruses A, B and H; and to set up a protocolfor collecting, shipping and preserving bovine pestiviral RNA on filter papers. The developedRT-qPCR showed high sensitivity in detecting these viruses in different matrices: viral stocks,semen and serum samples. With regard to the possibility of using the technique to test serumpools, it was possible to identify a positive serum sample within a pool containing 30 sera.In addition to evaluating the qPCR from fresh samples, the use of filter papers to sow bovinesamples was analyzed. The sampling method on two different filter papers using bovine blooddrops was a useful alternative for diagnostic purposes and allowed to preserve pestiviral RNAfor up to 12 months under refrigeration.
Resumen Los Pestivirus bovinos son los agentes causales de la diarrea viral bovina, una enfermedad que genera importantes pérdidas económicas en el ganado vacuno. El objetivo de este trabajo fue mejorar su diagnóstico mediante el desarrollo de una RT-qPCR para detectar los Pestivirus bovinos A, B y H y disenar un protocolo de recolección, envío y conservación de ARN viral en papeles de filtro. La RT-qPCR desarrollada demostró alta sensibilidad en la detección de estos virus en diferentes matrices: stock viral, suero y semen. Respecto de la posibilidad de usar la técnica para testear pools de suero, fue posible identificar un suero positivo dentro de un pool compuesto por 30 sueros. Además de evaluar la qPCR en muestras frescas, se analizó el uso de papeles de filtro para sembrar muestras de bovinos. La metodología de toma de muestras en dos tipos de papeles de filtro usando gotas de sangre fue una alternativa útil para el diagnóstico y permitió conservar ARN viral por hasta 12 meses a temperaturas de refrigeración.
ABSTRACT
Bovine pestiviruses are the causative agents of bovine viral diarrhea, a disease that causes severe economic losses in cattle. The aim of this study was to improve their diagnosis by developing a RT-qPCR to detect bovine pestiviruses A, B and H; and to set up a protocol for collecting, shipping and preserving bovine pestiviral RNA on filter papers. The developed RT-qPCR showed high sensitivity in detecting these viruses in different matrices: viral stocks, semen and serum samples. With regard to the possibility of using the technique to test serum pools, it was possible to identify a positive serum sample within a pool containing 30 sera. In addition to evaluating the qPCR from fresh samples, the use of filter papers to sow bovine samples was analyzed. The sampling method on two different filter papers using bovine blood drops was a useful alternative for diagnostic purposes and allowed to preserve pestiviral RNA for up to 12 months under refrigeration.
