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A specific real time reverse transcription polymerase chain reaction (RT-PCRrt) for the detection of foot-and-mouth disease virus was validated using the LightCycler thermocycler 2.0 and its reagents as recommended by the World Organization for Animal Health and was assessed for the detection of the virus in acute infection of cattle experimentally vaccinated and challenged with virus A Argentina/2001 or A24 Cruzeiro. The technique proved to be robust, showing coefficients of variation lower than 4% for different ARN extractions, days or repetitions and was able to detect up to 0,4 TCID 50%, and/or up to 100 RNA molecules. In probang samples, diagnostic sensitivity was 93.1 (95% CI 86.5-96.6) and diagnostic specificity 100 (95% CI 96.3-100). The results of the challenge in vaccinated or multivaccinated bovines showed that although there were high levels of clinical protection in the vaccinated group, FMDV could be detected in all challenged groups. However, detection was 100 times lower in immunized animals.

ResumenSe validó una técnica de RT-PCR en tiempo real utilizando el termociclador y los reactivos del LightCycler 2.0 de Roche, según las recomendaciones de la Organización Mundial de Sanidad Animal, y se evaluó su desempeño para la detección del virus de fiebre aftosa en la infección aguda de bovinos vacunados e infectados experimentalmente con virus A Argentina/2001 o A24 Cruzeiro. La técnica demostró ser robusta, presentó coeficientes de variación menores del 4 % al considerar días, extracciones y repeticiones diferentes y logró detectar hasta 0,4 DICT50%, o hasta 100 moléculas de ARN. La sensibilidad diagnóstica en líquido esófago-faríngeo (LEF) fue 93,1 (IC95%: 86,5–96,6) y la especificidad diagnóstica fue 100 (IC95%: 96,3–100). Se observó protección total en lo referido a aparición de síntomas clínicos tras la infección aguda experimental con virus A Argentina/2001 o A24 Cruzeiro en animales multivacunados o vacunados 7 o 14 días antes de la exposición viral, y se pudo detectar la presencia viral por RT-PCRtr en LEF desde el primer día y hasta por lo menos el día 9 posexposición en todos los animales expuestos. Los títulos virales en estos bovinos fueron de hasta 2 log 10 inferiores que los observados en los animales con signos clínicos.AbstractA specific real time reverse transcription polymerase chain reaction (RT-PCRrt) for the detection of foot-and-mouth disease virus was validated using the LightCycler thermocycler 2.0 and its reagents as recommended by the World Organization for Animal Health and was assessed for the detection of the virus in acute infection of cattle experimentally vaccinated and challenged with virus A Argentina/2001 or A24 Cruzeiro. The technique proved to be robust, showing coefficients of variation lower than 4% for different ARN extractions, days or repetitions and was able to detect up to 0,4 TCID 50%, and/or up to 100 RNA molecules. In probang samples, diagnostic sensitivity was 93.1 (95% CI 86.5–96.6) and diagnostic specificity 100 (95% CI 96.3-100). The results of the challenge in vaccinated or multivaccinated bovines showed that although there were high levels of clinical protection in the vaccinated group, FMDV could be detected in all challenged groups. However, detection was 100 times lower in immunized animals.