Semi-nested PCR for detection and typing of bovine Papillomavirus type 2 in urinary bladder and whole blood from cattle with enzootic haematuria

Bovine Papillomavirus type 2 (BPV-2) and chronic intoxication by bracken fern ingestion were associated with urinary bladder lesions and the clinical signs of enzootic haematuria in adult cattle. Clinically enzootic haematuria is characterized by intermittent haematuria followed by animal death. Enzootic haematuria causes considerable economical impact on extensive cattle breeding worldwide. The demonstration of BPV-2 participation in the etiology of bovine urinary bladder carcinoma by conventional virological methods is not easy and the integrity of epidemiological studies relies on methods that are sensitive and specific for BPV-2 detection and typing. A multiplex-PCR was evaluated for BPV-2 L1 gene and bovine mitochondrial genome ND5 gene (internal control) detection followed by a second round of BPV-2 amplification by a semi-nested PCR (SN-PCR). Six skin papilloma samples were used for PCR technique development. Twenty-two urinary bladder samples from symptomatic (n = 12) and asymptomatic (n = 10, control group) cows and 25 blood samples from cows grazed on enzootic haematuria-endemic (n = 14) and enzootic haematuria-free (n = 11, control group) geographical regions of Parana State, Brazil were analyzed. The SN-PCR detected BPV-2 in seven urinary bladder and 10 whole blood samples collected from cows with enzootic haematuria and in one urinary bladder and one whole blood samples of asymptomatic cows. The specificity of the amplicon was performed by restriction fragment length polymorphism and sequence analysis. The SN-PCR technique developed in this study will make possible the realization of diagnosis and comparative epidemiological studies to evaluate BPV-2 infection rates in cattle, and the association of this infection with bracken fern chronic intoxication in the etiology of enzootic haematuria and opens the possibility of ante mortem studies by lymphocytes analysis.