Phylogenetic analysis of a novel Hepatozoon species (Hepatozoon sp. SK3) and an additional yet unknown Hepatozoon species (Hepatozoon sp. BV2) besides H. erhardovae in small rodents from Central Europe.

Hepatozoon spp. are tick-borne apicomplexan parasites of terrestrial vertebrates that occur worldwide. Tissue samples from small rodents and their parasitizing fleas were sampled for molecular detection and phylogenetic analysis of Hepatozoon-specific 18S rRNA gene region. After alignment and tree inference the Hepatozoon-sequences retrieved from a yellow-necked mouse (Apodemus flavicollis) placed into a strongly supported single clade demonstrating the presence of a novel species, designated Hepatozoon sp. SK3. The mode of transmission of Hepatozoon sp. SK3 is yet unknown. It is important to note that this isolate may be identical with the previously morphologically described Hepatozoon sylvatici infecting Apodemus spp.; however, no sequences are available for comparison. Furthermore, the previously reported variants Hepatozoon sp. BV1/SK1 and BV2/SK2 were detected in bank voles (Clethrionomys glareolus). It has been suggested that these variants should be identified as Hepatozoon erhardovae leading to the assumption that BV1 and BV2 are paralogous 18S rRNA gene loci of this species. Evidence has also been presented that fleas are vectors of H. erhardovae. In this study, we show with high significance that only the Hepatozoon sp. BV1 variant, but not BV2, infects the studied flea species Ctenophthalmus agyrtes, Ctenophthalmus assimilis, and Megabothris turbidus (p < 0.001). This finding suggests that Hepatozoon sp. BV2 represents an additional species besides H. erhardovae (= Hepatozoon sp. BV1), for which alternative arthropod vectors or non-vectorial modes of transmission remain to be identified. Future studies using alternative molecular markers or genome sequencing are required to demonstrate that BV1/SK1 and BV2/SK2 are different Hepatozoon species.

Serological and molecular detection of Theileria equi infection in horses in Hungary.

The prevalence of Theileria equi infection was studied in 324 healthy horses from 27 farms in Hungary with cELISA and IFAT and the blood samples of 101 horses selected randomly were also examined by PCR. The results indicate that there are many stud farms where one or more horses are infected with T. equi. Among 27 farms 17 (67.9%) were found to have seropositive horses. The seroprevalence of theileriosis among the tested stud farms ranged between 0 and 100%. No marked differences were found in seropositivity between geographical areas. The overall prevalence of positive samples was 32.0% with cELISA as well as with IFAT. The results obtained with cELISA and IFAT in this study had the strongest agreement, except for 9 samples in which the two serological tests gave different results. The prevalence of infection among 101 horses was 49% with PCR. All 14 sequenced samples were found by BLAST analysis to be closest to the T. equi 18S rRNA gene sequences in GenBank with a similarity of ≥ 99%. No significant association was found between the seropositivity and the age of horses. Horses below 5 years of age had three times higher chance to be PCR-positive, than older ones. There was no significant association between the gender and the results of diagnostic tests (cELISA: p=0.40; IFAT: p=0.25; PCR: p=0.41). Based on the findings, the prevalence of equine theileriosis is much higher than expected and it occurs in many regions of the country unlike equine babesiosis. To the authors' knowledge, this is the first report of the serological and molecular survey of T. equi infection in horses in Hungary.

Serological evidence for Babesia canis infection of horses and an endemic focus of B. caballi in Hungary.

In order to evaluate the seroconversion of horses to Babesia caballi and B. canis in Hungary, blood samples were collected from 371 animals on 23 different locations of the country. The presence of antibodies to B. caballi was screened with a competitive ELISA. All 29 positive samples came from one region (the Hortobágy). The prevalence of infection did not show correlation with sexes, and reached 100% in the age group of 2-5 years. Babesia canis-specific antibodies were demonstrated by IFAT in 6.74% of animals kept in 7 regions. The titres were low or medium level (1:40 to 1:160), indicating that the horses had previously been exposed to this piroplasm, but their infection must have been limited. The highest seropositivity rate was observed in the age group of 3-4 years, and males (stallions and geldings) were significantly more frequently infected than females. However, neither B. caballi nor B. canis could be identified in the peripheral blood samples of infected horses by PCR. Since most of the B. caballi-positive horses remained negative in the B. canis IFAT, whereas seroconversion solely to B. canis was detected in several regions of the country, serological cross-reaction between the two species can be discounted. This is the first serological evidence of horses being naturally infected with B. canis, supporting the view that piroplasms are less host specific than previously thought.