Piroplasms in feral and domestic equines in rural areas of the Danube Delta, Romania, with survey of dogs as a possible reservoir

•Investigation of genetic diversity of T. equi and B. caballi in horses and donkeys for the first time in Romania.•One of the few studies dealing with piroplasmids in connection with feral horses.•Evidence of a higher prevalence of T. equi over B. caballi.•Proven limited host specificity of piroplasmids.

Rural areas of Romania, particularly the localities covering Danube Delta, are still not sufficiently explored in terms of epidemiological aspects, despite the large density of domestic animals living in close contact with people and natural environment of the Danube Delta Biosphere Reserve. Between 2010 and 2012, a survey on equine piroplasmids species was performed in this area, using a total of 178 horses, 15 donkeys and 177 dogs from 18 localities inside and outside the Danube Delta. None of the sampled hosts showed any clinical symptoms typical for equine piroplasmoses. A 25.4% overall prevalence for both equine species of piroplasmids was detected by PCR. Detection by “catch-all” primers followed by multiplex PCR revealed 20.3% prevalence of T. equi, beside 2.2% of B. caballi and 3.0% of mixed infections for all examined animals. Based on sequencing of 67 PCR amplicons of 18S rRNA gene of T. equi, four genotypes (A, C, D and E) were detected, whereas four B. caballi sequences clustered within a single genotype (A). A single dog infected with T. equi was found and confirmed by sequencing, clustering within genotype D. Our study further proved limited host specificity of piroplasmid. Higher prevalence was revealed inside the Danube delta, presumably caused by different environmental conditions, different tick densities and lower availability of veterinary services.