Seroprevalence of Babesia caballi and Theileria equi in the Swiss horse population

In Switzerland, the prevalence and incidence of equine piroplasma parasite (EPP) infections are unknown. In order to obtain a first insight into the prevalence, a representative sample of 689 sera of horses from Switzerland was serologically tested for the presence of antibodies directed against T. equi and B. caballi using the Indirect Fluorescence Antibody Test (IFAT). A total of 50 (7.3%) horses were seropositive for EPP: overall, the seroprevalence of T. equi was significantly higher than that of B. caballi (p=0.002). The seropositivities in indigenous horses (animals bred and raised in Switzerland) and in imported horses were 4.8% (11/230) and 8.5% (39/459), respectively. Unlike in indigenous horses, where no significant difference in seroprevalences could be observed between the two parasite species, the seroprevalence of T. equi was significantly higher (p<0.001) than that of B. caballi in imported horses. Horses imported from France, Spain and Portugal exhibited a significantly higher seroprevalence, and horses imported from Germany a significantly lower seroprevalence of EPP compared to indigenous horses. There were no associations between sex, age, weight loss, surgery or blood transfusions with T. equi and B. caballi seroprevalences. The overall seroprevalence of 7.3% clearly shows that infection with EPP is a threat to the health of the horses in Switzerland. With the presumed expansion of permissive tick vectors, EPP infections will potentially increase in importance in the future. Therefore, continuous monitoring is mandatory.

Presence of potentially pathogenic Babesia sp. for human in Ixodes ricinus in Switzerland

We have designed and performed a new PCR method based on the 18S rRNA in order to individuate the presence and the identity of Babesia parasites. Out of 1159 Ixodes ricinus (Acari: Ixodidae) ticks collected in four areas of Switzerland, nine were found to contain Babesia DNA. Sequencing of the short amplicon obtained (411-452 bp) allowed the identification of three human pathogenic species: Babesia microti, B. divergens, for the first time in Switzerland, Babesia sp. EU1. We also report coinfections with B. sp. EU1-Borrelia burgdorferi sensu stricto and Babesia sp. EU1-B. afzelii.

Babesia capreoli infections in alpine chamois (Rupicapra r. Rupicapra), roe deer (Capreolus c. Capreolus) and red deer (Cervus elaphus) from Switzerland

Five cases of fatal babesiosis in free-ranging chamois (Rupicapra r. rupicapra) attributed to infections with Babesia capreoli were recently recorded in two regions of the Swiss Alps. To investigate the ecologic factors that possibly lead to those fatal B. capreoli infections in chamois, blood, ticks, and demographic data of 46 roe deer (Capreolus c. capreolus), 48 chamois, and nine red deer (Cervus elaphus) were collected in 2006 and 2007 in both affected regions. Whereas no parasitic inclusions were found by microscopical examination of blood smears, B. capreoli was identified by polymerase chain reaction/sequencing in blood of 12 roe deer (26%, 95% confidence interval [CI]: 14.3-41.1), one chamois (2%, CI: 0-6.1), and one red deer (11%, CI: 0.3-48.2). Prevalence of B. capreoli was significantly higher in roe deer compared with chamois (P<0.001). All 214 ticks were identified as Ixodes ricinus, and significantly more roe deer (63%, CI: 47.5-76.8) were infested compared with chamois (21%, CI: 10.5-35.0, P<0.001). Overall, prevalences of both tick infestation and Babesia infection increased significantly (P<0.001) with decreasing altitude, and Babesia-positive samples were detected significantly more often from animals with tick infestation compared with animals without ticks (P = 0.040). Our results indicate that roe deer may play an important reservoir role for B. capreoli. It is hypothesized that the expansion of the presumed vector I. ricinus to higher elevations and its increased abundance in overlapping habitats of roe deer and chamois may favor the spillover of B. capreoli from roe deer to chamois.

Questing Dermacentor reticulatus harbouring Babesia canis DNA associated with outbreaks of canine babesiosis in the Swiss Midlands

In 2011 and 2012, outbreaks of clinical canine babesiosis were observed in 2 areas of the Swiss Midlands that had no history of this disease so far. In one area, cases of canine babesiosis occurred over 2 consecutive tick seasons. The outbreaks involved 29 dogs, 4 of which died. All dogs were infected with large Babesia sp. as diagnosed in Giemsa-stained blood smears and/or PCR. These were identified as B. canis (formerly known as B. canis canis) by subsequent partial sequencing of the 18S rRNA gene of Babesia sp. Interestingly, the sequence indicated either a genotype with heterogeneity in the ssrRNA gene copies or double infection with different B. canis isolates. None of the dogs had a recent travel history, but one had frequently travelled to Hungary and had suffered twice from clinical babesiosis 18 and 24 months prior to the outbreak in autumn 2011. Retrospective sequencing of a stored blood DNA sample of this dog revealed B. canis, with an identical sequence to the Babesia involved in the outbreaks. For the first time in Switzerland, the partial 18S rRNA gene of B. canis could be amplified from DNA isolated from 19 out of 23 adult Dermacentor reticulatus ticks flagged in the same area. The sequence was identical to that found in the dogs. Furthermore, one affected dog carried a female D. reticulatus tick harbouring B. canis DNA. Our findings illustrate that, under favourable biogeographic and climatic conditions, the life-cycle of B. canis can relatively rapidly establish itself in previously non-endemic areas. Canine babesiosis should therefore always be a differential diagnosis when dogs with typical clinical signs are presented, regardless of known endemic areas.

Babesia spp. in European wild ruminant species: parasite diversity and risk factors for infection.

Babesia are tick-borne parasites that are increasingly considered as a threat to animal and public health. We aimed to assess the role of European free-ranging wild ruminants as maintenance mammalian hosts for Babesia species and to determine risk factors for infection. EDTA blood was collected from 222 roe deer (Capreolus c. capreolus), 231 red deer (Cervus e. elaphus), 267 Alpine chamois (Rupicapra r. rupicapra) and 264 Alpine ibex (Capra i. ibex) from all over Switzerland and analysed by PCR with pan-Babesia primers targeting the 18S rRNA gene, primers specific for B. capreoli and Babesia sp. EU1, and by sequencing. Babesia species, including B. divergens, B. capreoli, Babesia sp. EU1, Babesia sp. CH1 and B. motasi, were detected in 10.7% of all samples. Five individuals were co-infected with two Babesia species. Infection with specific Babesia varied widely between host species. Cervidae were significantly more infected with Babesia spp. than Caprinae. Babesia capreoli and Babesia sp. EU1 were mostly found in roe deer (prevalences 17.1% and 7.7%, respectively) and B. divergens and Babesia sp. CH1 only in red deer. Factors significantly associated with infection were low altitude and young age. Identification of Babesia sp. CH1 in red deer, co-infection with multiple Babesia species and infection of wild Caprinae with B. motasi and Babesia sp. EU1 are novel findings. We propose wild Caprinae as spillover or accidental hosts for Babesia species but wild Cervidae as mammalian reservoir hosts for B. capreoli, possibly Babesia sp. EU1 and Babesia sp. CH1, whereas their role regarding B. divergens is more elusive.

Identification of two Th1 cell epitopes on the Babesia bovis-encoded 77-kilodalton merozoite protein (Bb-1) by use of truncated recombinant fusion proteins

Previous studies have demonstrated the serologic and T-cell immunogenicity for cattle of a recombinant form of the apical complex-associated 77-kDa merozite protein of Babesia bovis, designated Bb-1. The present study characterizes the immunogenic epitopes of the Bb-1 protein. A series of recombinant truncated fusion proteins spanning the majority of the Bb-1 protein were expressed in Escherichia coli, and their reactivities with bovine peripheral blood mononuclear cells and T-cell clones derived from B. bovis-immune cattle and with rabbit antibodies were determined. Lymphocytes from two immune cattle were preferentially stimulated by the N-terminal half of the Bb-1 protein (amino acids 23 to 266, termed Bb-1A), localizing the T-cell epitopes to the Bb-1A portion of the molecule. CD4+ T-cell clones derived by stimulation with the intact Bb-1 fusion protein were used to identify two T-cell epitopes in the Bb-1A protein, consisting of amino acids SVVLLSAFSGN VWANEAEVSQVVK and FSDVDKTKSTEKT (residues 23 to 46 and 82 to 94). In contrast, rabbit antiserum raised against the intact fusion protein reacted only with the C-terminal half of the protein (amino acids 267 to 499, termed Bb-1B), which contained 28 tandem repeats of the tetrapeptide PAEK or PAET. Biological assays and Northern (RNA) blot analyses for cytokines revealed that following activation with concanavalin A, T-cell clones reactive against the two Bb-1A epitopes produced interleukin-2, gamma interferon, and tumor necrosis factors beta and alpha, but not interleukin-4, suggesting that the Bb-1 antigen preferentially stimulates the Th1 subset of CD4+ T cells in cattle. The studies described here report for the first time the characterization, by cytokine production, of the Th1 subset of bovine T cells and show that, as in mice, protozoal antigens can induce Th1 cells in ruminants. This first demonstration of B. bovis-encoded Th1 cell epitopes provides a rationale for incorporation of all or part of the Bb-1 protein into a recombinant vaccine.

Development of PCR/dot blot assay for specific detection and differentiation of taeniid cestode eggs in canids

We report the development of a colourimetric PCR/dot blot assay targeting the mitochondrial gene NADH dehydrogenase subunit 1 (nad1) for differential diagnosis of taeniid eggs. Partial sequences of the cestode nad1 gene were aligned and new primers were designed based on conserved regions. Species-specific oligonucleotide probes (S-SONP) for canine taeniid cestodes were then designed manually based on the variable region between the conserved primers. Specifically, S-SONP were designed for the Taenia crassiceps, T. hydatigena, T. multiceps, T. ovis, T. taeniaeformis, Echinococcus granulosus (genotype 1), E. multilocularis and E. vogeli. Each probe showed high specificity as no cross-hybridisation with any amplified nad1 fragment was observed. We evaluated the assay using 49 taeniid egg-positive samples collected from dogs in Zambia. DNA from 5 to 10 eggs was extracted in each sample. Using the PCR/dot blot assay, the probes successfully detected PCR products from T. hydatigena in 42 samples, T. multiceps in 3 samples, and both species (mixed infection) in the remaining 4 samples. The results indicate that the PCR/dot blot assay is a reliable alternative for differential diagnosis of taeniid eggs in faecal samples.

Equine piroplasmoses at the reintroduction site of the Przewalski's horse (Equus ferus przewalskii) in Mongolia

Piroplasmosis has been identified as a possible cause of mortality in reintroduced Przewalski's horses (Equus ferus przewalskii) in the Dsungarian Gobi (Mongolia). A cross-sectional and a longitudinal study were conducted in a representative sample (n = 141) of the resident domestic horse population and in 23 Przewalski's horses to assess the prevalence of Theileria equi and Babesia caballi. Piroplasms were detected in blood by light microscopy in 6.7% (95% confidence interval [CI]: 3.6-12.2%) of the domestic horse samples. Antibody prevalence was 88.6% (95% CI: 82.4-92.9%) for T. equi and 75.2% (95% CI: 67.4-81.6%) for B. caballi. Antibody prevalence did not change over time, but antibody prevalence for both piroplasms were significantly lower in animals less than 1 yr of age. For both piroplasms, the prevalence of presumably maternal antibodies (falling titers) in foals was 100%. Only one of 16 foals seroconverted against T. equi during the study period, despite that piroplasms were found in two other individuals. The incidence density (ID) of T. equi in foals was therefore 0.0012 seroconversions per horse day (95% CI: 0.00029-0.0057). In contrast, yearlings had an ID of 0.0080 (95% CI: 0.0049-0.010) for T. equi and 0.0064 (95% CI: 0.0036-0.0093) for B. caballi, and in seven individuals piroplasms were detected. The seroprevalence of both piroplasms rose from 20% in spring to 100% in autumn. Comparison of domestic and Przewalski's horses resulted in a standardized prevalence ratio (SPR) of 0.98 (95% CI: 0.80-1.24, not significant) for B. caballi; in contrast, the prevalence of T. equi in Przewalski's horses was significantly lower than expected (SPR = 0.51, 95% CI: 0.50-0.64).

Babesiosis in free-ranging chamois (Rupicapra r. rupicapra) from Switzerland

Pathological examination of five adult chamois (Rupicapra r. rupicapra) found dead in two different regions from the Swiss Alps revealed pale mucous membranes and musculature, swollen spleen and haemoglobinuria. Histologically, haemosiderosis in the spleen and centrilobular hepatic necrosis were the predominant findings. On blood smears, small (approximately 0.84-1.47 microm), round to pyriform, peripherally located inclusions were present in the erythrocytes. PCR followed by sequencing of DNA extracted from blood or spleen of the infected animals revealed 99-100% identity of the amplified part of the 18S rRNA gene with GenBank entries attributed to Babesia divergens/Babesia capreoli. This is the first report of fatal Babesia infections in chamois raising the question of an emerging disease in this species.

[A descriptive epidemiological study on canine babesiosis in the Lake Geneva region]

A descriptive study was carried out in the district of the Lake Geneva between March 1, 2005 and August 31,2006 to assess the incidence and prevalence of canine babesiosis, to genotype the Babesia species occurring, to assess the most frequently clinical signs found and to address the potential of autochthonous transmission. This included a data assessment on the different tick-populations occurring in the area and on the prevalence of Babesia-DNA in these ticks. A total of 56 veterinary practices participated in the study. By blood smear and PCR, Babesia canis canis was found in 12 out of 21 cases with suspected babesiosis. In an additional 13th case, the parasite could only be detected by PCR. All autochthonous cases originated from the Western part of the Lake Geneva region. Clinical signs in affected dogs included inappetence, apathy, anemia, fever, hemoglobinuria and thrombocytopenia. There were no risk factors with regard to age, sex and breed. Most cases were diagnosed during the spring periods of 2005 and 2006 (11 cases) and two cases in autumn 2005, coinciding with the main activity period of Dermacentor reticulatus, the main vector of B. canis canis. A total of 495 ticks were collected on patients by the veterinarians, 473 were identified as Ixodes sp., 7 as Rhipicephalus sanguineus and 15 as Dermacentor reticulatus. While Ixodes sp. was found in the whole study area, D. reticulatus and R. sanguineus occurred only in the Western part till Lausanne. PCR and sequencing yielded B. canis canis positivity in 3 D. reticulatus specimen, these three ticks were collected from two different dogs both suffering from babesiosis. All R. sanguineus were negative by Babesia-PCR. Global warming, ecological changes in the potential habitat of ticks, increasing host- and vector-populations and increasing mobility of dog owners may be responsible for an emergence situation of infection risk for Babesia spp. by time. E.g., Dermacentor reticulatus has become autochtonously prevalent already till Lausanne in the Lake Geneva region, and further surveillance is suggested to tackle this problem.