Highly sensitive method for diagnosis of subclinical B. ovis infection

Babesia ovis is a tick-transmitted protozoa parasite that infects small ruminants causing fever, anaemia, hemoglobinuria, anorexia and, in acute cases, death. Common in tropical and sub-tropical areas, the presence of this parasite in sheep herds has an economic impact on industry and therefore sensitive methods for the diagnosis and disease eradication are required. To achieve this goal, a semi-nested PCR for B. ovis specific identification was developed and consequent reaction conditions and enzymes were optimized and tested with field samples. 300 blood samples from small ruminants and 39 ticks from Rhipicephalus genus were collected from different regions of Portugal. Afterwards, DNA extraction was performed and conventional and semi-nested PCR were accomplished for all samples. The results obtained from both methodologies were compared and the sensitivity was evaluated. Employing the semi-nested PCR it was possible to identify a higher number of positive cases among the evaluated samples than using the conventional PCR, namely 38/300 blood samples and 7/39 ticks. However, fragment amplification was only observed in 5 out of 300 blood samples and in none of the 39 ticks when a conventional PCR was employed. The validation of the results was achieved by sequencing the DNA fragments corresponding to the hypervariable v4 region of the 18S ribosomal RNA gene and performing an alignment with sequences already published on GenBank(®). The ticks collected in this study belong to the Rhipicephalus genus, although other species could be involved as a vector in the Babesia spread. The diagnostic assay here described is presently the most effective and sensitive method for detection of B. ovis in field blood samples and ticks, enabling the detection up to 1 parasite into 10(9) erythrocytes.