First detection and molecular identification of the zoonotic Anaplasma capra in deer in France.

Cervids are known to be reservoirs of zoonotic bacteria transmitted by ticks. This study aimed to identify the Anaplasma species carried by captive red deer and swamp deer in a wild fauna reserve in France. Blood from 59 red deer and 7 swamp deer was collected and analyzed over a period of two years. A semi-nested PCR targeting the 23S rRNA was performed to detect and characterize Anaplasma spp. and determine the presence of zoonotic species. Anaplasma phagocytophilum was identified in 14/59 red deer (23.7%) but it was not identified in any of the swamp deer (7 animals). Three sequences could not be assigned to any particular species based on the 23S rRNA sequences. Complementary nested PCR targeting 16S rRNA, gltA and groEL genes and sequencing analysis then identified these sequences as a recently reported zoonotic species, Anaplasma capra; this species was found in 2 red deer (Cervus elaphus) and 1 swamp deer (Rucervus duvaucelii). This is the first report of the tick-borne zoonotic bacterium A. capra in France, a species otherwise described only in China, Japan, Malaysia and South Korea in goats, sheep, deer, cattle and Japanese serows (Capricornis crispus). While this bacterium may have been introduced into the reserve by infected imported animals, its local epidemiological cycle via tick transmission seems possible as locally born deer were found infected. Diagnostic methods, especially molecular ones, should take into account the potential infection of animals and humans with this species.

Detecting and characterizing mixed infections with genetic variants of Anaplasma phagocytophilum in roe deer (Capreolus capreolus) by developing an ankA cluster-specific nested PCR.

BACKGROUND: Anaplasma phagocytophilum is a tick-transmitted Gram-negative obligate intracellular bacterium able to infect a wide variety of wild and domestic animals worldwide. Based on the genetic diversity observed with different molecular markers, several host-specific lineages have been identified. Roe deer is one of the most important reservoirs of this bacterium and hosts different genetic groups sometimes found on domestic animals. We therefore developed an ankA cluster-specific nested PCR (nPCR) to evaluate the prevalence of the three different ankA genetic groups described in roe deer (clusters II, III and IV) at three locations in France and the level of co-infections. RESULTS: The specificity of the three nPCRs was assessed by partially sequencing 35 amplicons of ankA genes obtained from the different nested PCRs. All three genetic lineages were detected in roe deer from all three geographical locations. Of the infected deer population, 60.7% were co-infected by two or three different genetic variants. Co-infections varied from 42.9 to 70.6% of the infected population depending on the local infection prevalences (from 33.3 to 73.9%). All types of mixed infections occurred, suggesting the absence of a strict variant exclusion by another variant. CONCLUSIONS: Mixed infections by two or three genetic variants of A. phagocytopilum are a common feature in roe deer. Genetic variants (cluster IV) also found in domestic ruminants (cattle and sheep) were present in all the roe deer populations analyzed, suggesting a shared epidemiological cycle.

Isolation and characterization of Babesia pecorum sp. nov. from farmed red deer (Cervus elaphus).

The diversity of Babesia species infecting cervids in parts of central and southern Spain was analyzed by collecting blood from farmed red deer (Cervus elaphus). Babesia sp. was isolated in vitro from two red deer herds in Cádiz and Ciudad Real. The number of Babesia sp. carriers differed between the two herds: 36/77 in Cádiz and 1/35 in Ciudad Real. Hyalomma lusitanicum was the most prevalent tick species identified on the Cádiz farm vegetation and on sampled animals, and is therefore a candidate vector. The molecular characteristics of 21 isolates were determined by complete (8 isolates) or partial (13 isolates) 18S rRNA gene sequencing. The sequences were highly similar (over 99.4% identity) and 6 sequence types were identified at the level of one herd only, demonstrating a rather high genetic diversity. They formed a monophyletic clade, and members of the three main sequence types shared a similar morphology and the same erythrocyte susceptibility pattern. This clade also included Babesia sp. Xinjiang isolated from sheep in China and Babesia sp. identified in giraffe in South Africa, with identities higher than 98.3% and statistically relevant phylogenetic support. None of the biological properties analyzed for both Babesia from red deer and Babesia sp. Xinjiang allowed their differentiation (ability to develop in vitro in erythrocytes from cattle and sheep, as well as in erythrocytes from different cervids, unsuccessful infection of calves). We propose the Babesia isolated from red deer as a new species named B. pecorum. Whether Babesia sp. Xinjiang and the Babesia characterized in South Africa belong to the same species is debated.

Antibody prevalence and molecular identification of Babesia spp. In roe deer in France.

In a region-wide serologic study carried out in 2004 on free-ranging hunted roe deer in various landscapes, we found that 58% of the animals (237 out of 406) were antibody positive for Babesia divergens antigen. Serologic and infection status was also analyzed for 327 roe deer live-trapped in two fenced forest areas over 5 yr (2004-08). For two consecutive years during this period, 92 and 94% of the deer in these closed populations were antibody-positive for B. divergens. Babesia spp. were isolated in autologous red blood cell culture for 131 of the trapped animals (40%). Molecular typing was done on 76 isolates with polymerase chain reaction (PCR)-restriction fragment length polymorphism methods targeted at the 18S ribosomal subunit gene (18 isolates) and the Bd37 gene coding for a merozoïte surface antigen implicated in a protective response (60 isolates). Results indicated continuous cocirculation of B. capreoli and B. venatorum in both forests and possible coinfection of animals with both species. No infection with B. divergens was detected. Fifteen isolates were confirmed to be B. capreoli by sequencing part of the 18S rRNA gene. Using PCR detection of the Bd37 gene, all nine isolates of B. venatorum in this study were negative, whereas the 15 confirmed and 50 putative B. capreoli isolates showed very variable restriction profiles, distinct from those known for Bd37 in B. divergens. Two isolates showed conflicting results, suggestive of mixed infection.

Molecular cloning and genetic polymorphism of Babesia capreoli gene Bcp37/41, an ortholog of Babesia divergens merozoite surface antigen Bd37.

Babesia divergens and Babesia capreoli are closely related species with distinct host ranges, a zoonotic feature being described only for B. divergens. The two species are 99.8% similar in the 18S rDNA gene sequence and indistinguishable by morphological or serological means, leading to confusion as to their species status. The phylogenetic relatedness between the two species, and the frequent involvement of surface components in serological cross-reactions led us to postulate that an ortholog of Bd37, the merozoite surface antigen described for B. divergens, could also exist in B. capreoli. We were able to amplify a single partial PCR product from B. capreoli genomic DNA using primers specific for the B. divergens merozoite surface protein coding gene Bd37, and sequencing confirmed the presence of a Bd37 ortholog in B. capreoli, named Bcp37/41. The full sequences of the Bcp37/41 genes and their intron-exon structures were obtained for two cloned lines of B. capreoli. They suggest functional homologies between Bd37 and Bcp37/41 such as their surface localization, their role in immune escape mechanism and in the initial non-specific attachment to the erythrocyte. Restriction fragment length polymorphism (RFLP) analysis of the amplicons and partial sequencing revealed an extreme polymorphism within B. capreoli, greater than the one observed for its ortholog Bd37. Such a marker could thus be useful in epidemiological as well as phylogenetic studies.

Babesiosis in immunocompetent patients, Europe.

We report 2 cases of babesiosis in immunocompetent patients in France. A severe influenza-like disease developed in both patients 2 weeks after they had been bitten by ticks. Diagnosis was obtained from blood smears, and Babesia divergens was identified by PCR in 1 case. Babesiosis in Europe occurs in healthy patients, not only in splenectomized patients.

Redescription of Babesia capreoli (Enigk and Friedhoff, 1962) from roe deer (Capreolus capreolus): isolation, cultivation, host specificity, molecular characterisation and differentiation from Babesia divergens.

The recent use of the sole molecular identification of Babesia infecting European cervids has led to confusion between the closely related Babesia divergens and Babesia capreoli, and to their grouping together as "B. divergens-like". In order to clarify this taxonomic confusion, Babesia from roe deer, cattle and human blood were isolated, cultured and their biological as well as molecular characteristics compared. On this basis, we conclude that: (i) the parasites isolated from roe deer blood are B. capreoli; (ii) there are no intraspecific variations in the 18S rDNA within B. capreoli and B. divergens spp.; (iii) these two species are closely related as demonstrated by their morphology, serological cross-reactions and 99.83% identity in their 18S rDNA; (iv) these two species are distinct as demonstrated by their different abilities to grow in vitro in cattle, human and sheep erythrocytes, by their infectivity for gerbils, and by a conserved three bases difference at positions 631, 663 and 1637 of their 18S rDNA; (v) B. capreoli does not pose a threat to either humans or livestock. An integrated description is given of the host range, geographical distribution, biological and molecular characterisation of B. capreoli, and reference materials have been deposited at the Museum d'Histoire Naturelle de Paris.

Babesia divergens experimental infection of spleen-intact sheep results in long-lasting parasitemia despite a strong humoral response: preliminary results.

Babesia divergens is an intraerythrocytic Apicomplexa and the main agent of bovine babesiosis in Europe. The infection in cattle develops in 2 phases: an acute phase with hemolytic anemia and a chronic phase with asymptomatic persistence of the parasite for several years. The acute phase of B. divergens infection can be studied using the gerbil (Meriones unguiculatus) as a laboratory model but unlike cattle, this animal rapidly eliminates the parasite. An experimental model to study the chronic phase of infection was therefore developed by our laboratory. Spleen-intact sheep, with a potential full immune response, were inoculated with infected red blood cells (iRBC) or with free merozoites, by several routes (intraperitoneal, intravenous or subcutaneously). No clinical signs were ever observed but the installation of a persistent low level infection was shown in sheep with susceptible erythrocytes (able to sustain B. divergens growth in vitro). Neither feature was observed in sheep with non-susceptible erythrocytes. IgG production, involving both IgG1 and IgG2, was mainly directed against the major merozoite surface antigen Bd37, similar to the humoral immune response described in naturally infected cattle. The use of spleen-intact sheep to study the immune response to B. divergens is discussed.

Experimental in vitro transmission of Babesia sp. (EU1) by Ixodes ricinus.

Babesia sp. (EU1), first characterized in 2003, has been implicated in human cases of babesiosis in Italy, Austria and Germany. It has been identified in roe deer and in its suspected tick vector, Ixodes ricinus, in several European countries. The aim of the present study was to validate the competence of I. ricinus as a vector of Babesia sp. (EU1) via experimental infections. For this purpose, a parasite strain isolated from roe deer was cloned in sheep erythrocytes. After experimental infections, parasite DNA was successfully amplified by PCR in both eggs and larvae originating from infected I. ricinus females and in the salivary glands of females exposed to Babesia sp. (EU1) as nymphs. We also demonstrate that infected females were able to transmit parasite DNA during a new blood meal. Together with previous epidemiological studies, these results validate I. ricinus as a competent vector for Babesia sp. (EU1).

Natural transmission of Zoonotic Babesia spp. by Ixodes ricinus ticks.

To determine characteristics of natural transmission of Babesia sp. EU1 and B. divergens by adult Ixodes ricinus ticks, we examined tick salivary gland contents. We found that I. ricinus is a competent vector for EU1 and that their sporozoites directly invade erythrocytes. We conclude that EU1 is naturally transmitted by I. ricinus.

Individual heterogeneity in erythrocyte susceptibility to Babesia divergens is a critical factor for the outcome of experimental spleen-intact sheep infections.

Susceptibility of sheep erythrocytes to Babesia divergens was investigated in vitro and a high inter-individual variability in their ability to support parasite population development was demonstrated, with some individuals having refractory red blood cells (RBC). As neither changes in growth conditions nor the use of different B. divergens strains influenced the level of susceptibility, the main factor postulated for this variability is the erythrocyte itself. Sheep therefore represent an excellent in vitro model to study the parasite-erythrocyte interaction. In addition, the existence of refractory RBC should help in the identification of the erythrocyte components required for B. divergens development. Experimental infections were carried out on spleen-intact sheep characterized by refractory or fully susceptible erythrocyte types. These differences translated into the successful infection of only those animals with susceptible erythrocytes: infected animals showed no clinical signs, but maintained an asymptomatic persistent infection, as usually observed in the natural bovine host. Sheep therefore represent model organisms that can allow us to study interactions between B. divergens and its vertebrate host at different levels of biological organisation, from the target cell to the intact animal, and represent an experimental infection model of concomitant immunity. Only a low percentage (13%) of the sheep population tested possessed susceptible erythrocytes and the potential role of sheep as a natural host or reservoir of B. divergens is discussed.

Babesia sp. EU1 from roe deer and transmission within Ixodes ricinus.

We report in vitro culture of zoonotic Babesia sp. EU1 from blood samples of roe deer in France. This study provides evidence of transovarial and transstadial transmission of the parasite within Ixodes ricinus, which suggests that this tick could be a vector and reservoir of EU1.