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).

INTRODUCTION

Babesia sp. infections are caused by tick-borne protozoan parasites that infect animal and human erythrocytes. In animals, babesiosis is responsible for major economic losses around the world and, in humans, it is gaining increasing attention as an emerging disease [15]. Since the identification of the etiologic agent of the disease, hundreds of human cases have been reported in the USA, where babesiosis is mostly attributed to the parasite Babesia (Theileria) microti which is transmitted by the tick Ixodes scapularis [14]. In Europe, approximately 30 human cases of babesiosis have been reported over the last fifty years [14, 25]. These infections have been traditionally attributed to the bovine parasite Babesia divergens transmitted by Ixodes ricinus [2, 9]. However, in 2003, Herwaldt et al. described the first molecular characterization of a new Babesia species, Babesia sp. (EU1), provisionally named Babesia venatorum, and isolated from two human cases in Austria and Italy [12]. Since this time, another case of human babesiosis has been attributed to Babesia sp. (EU1) in Germany [11]. Thus, since molecular-based tests were often not performed to confirm infection by B. divergens, we can hypothesize that other cases of human babesiosis may have been caused by Babesia sp. (EU1) and that the prevalence of this parasite in humans has been underestimated. Recently reported serological cross-reactivity between B. divergens and Babesia sp. (EU1) support this hypothesis [8].

Babesia sp. (EU1) was identified from roe deer (Capreolus capreolus) in Slovenia in 2005 [6] and in Italy and France in 2007 [3, 22]. Such studies validate the potential of roe deer as wild reservoirs that could maintain the circulation of the parasite. In addition, the fact that roe deer are often heavily infested with I. ricinus, a tick that also frequently feeds on humans, suggests that this tick could be the vector of Babesia sp. (EU1).

Duh et al. reported the first detection of Babesia sp. (EU1) DNA in I. ricinus in Slovenia in 2005 where, among the 135 ticks analyzed, 2.2% were carrying this parasite [7]. Since then, other studies have revealed by PCR that 1–2% of I. ricinus ticks collected from wild and domestic animals in Switzerland [5, 13] and in the Netherlands [15] harbored Babesia sp. (EU1) DNA. Finally, we have also previously reported some epidemiological evidence that I. ricinus could be a potential vector of Babesia sp. (EU1); parasite DNA was detected in 26% of larval pools from female ticks collected on infected roe deer in France [3]. Finally, some recent data showed that Babesia sp. (EU1) sporozoites isolated from salivary glands (SGs) of naturally infected ticks are able to invade erythrocytes in vitro [1]. Some experimental infection studies are now needed in order to definitively prove the competence of I. ricinus as a vector of Babesia sp. (EU1).

We report here the cloning of Babesia sp. (EU1), as well as a morphological description of the different parasite stages present in vertebrate blood cells and in ticks. Experimental infections of I. ricinus were performed with in vitro infected blood and demonstrate that parasite DNA was transstadialy and transovarialy transmitted between tick life stages, as well as from infected ticks to blood during a re-feeding step on non-infected blood.

MATERIALS AND METHODS

Blood samples used in culture

Roe deer blood and the parasite strain used in the study were obtained from animals captured in the Wild Fauna Reserve of Chizé (Deux-Sèvres, France). Sheep blood samples used for cultivation of B. divergens came from one sheep commonly used as a donor for B. divergens cultures and reared at the National Veterinary School of Nantes (France). The Babesia-free status of this animal was confirmed by in vitro culture. All blood samples were collected in heparin containing Venoject tubes (Terumo), centrifuged 10 min at 800 g and the plasma and buffy coat were discarded. The pellet of red blood cells was then washed and diluted 1:3 with RPMI 1640 containing 50 μg/mL gentamicin and 0.25 μg/mL amphotericin B. This solution was then maintained at 4 °C until use in parasite cultures and for tick feeding. The autologous plasma was maintained at –20 °C until use in tick feeding. All media and antibiotics used in the study were from Cambrex (Charles city, Inc., IA, USA).

Ticks

I. ricinus ticks used for experimental feedings were collected from the forest of Princé (Loire-Atlantique, France) by flagging the vegetation as previously described [24]. Negative controls used for PCR amplification came from I. ricinus collected from Belle-Île-en-Mer, a babesiosis-free island in the Gulf of Morbihan (France). All ticks were reared and maintained in humidity chambers with a relative humidity (r.h.) of 80–90% at 22 °C.

Parasite isolation and cloning

The Babesia sp. (EU1) isolate used in this study, named C201, came from one single roe deer during a study carried out in January 2006 and was isolated as previously described [3, 18]. Briefly, parasites were first isolated from blood samples in autologous erythrocytes (7.5% hematocrit) in RPMI 1640 containing 50 μg/mL gentamicin, 0.25 μg/mL amphotericin B and 20% fetal calf serum (decomplemented 30 min at 56 °C). Cultures were performed at 37 °C in 5% CO2-air with replacement of the medium every 2–3 days during 3 weeks (without disturbance to the erythrocyte layer), and were monitored periodically for parasitaemia by examination of thin blood smears stained with May-Grünwald Giemsa-based (Diff-Quick, Dade Behring, Deerfield, IL, USA) stain. Six subcultures were then carried out by a 10% dilution of parasitized culture with fresh complete culture media containing autologous roe deer erythrocytes (7.5% hematocrit) every 3 or 4 days. Parasites were then adapted to culture in sheep erythrocytes during 30 subculture rounds and cloned by limiting dilution in 96 well microplates (up to 0.25 parasites per well). At last, direct sequencing was performed by Qiagen (Hilden, Germany) in order to obtain the complete 18S ribosomal RNA gene of the parasite. This was done using the amplification product of a 1 727 bp sequence obtained with the PCR primer set CRYPTO F and CRYPTO R and following the amplification conditions described by Herwaldt et al. [12].

Artificial feeding and infection of ticks

Ticks were infected with an in vitro culture of Babesia sp. (EU1) clone C201A as previously described for B. divergens [4]. Cultures were diluted daily with fresh sheep red blood cells to give a mean parasitaemia of 8% infected erythrocytes. Parasitized erythrocytes were pelleted by centrifugation for 10 min at 800 g and mixed with 1/3 (v/v) homologous plasma containing 50 μg/mL gentamicin and 0.25 μg/mL amphotericin B. Three mL of restored blood were put into the glass feeder and presented to the ticks until repletion, with two blood changes per day. Rabbit skins used in artificial feeding were obtained from animals previously used for rearing tick colonies in the laboratory and were treated as previously described [4]. Fifty-two nymphs and 9 adult females were used for artificial infection. Only fully engorged ticks that spontaneously detached were then retained for the study and placed in a humidity chamber. Since mating is required for the completion of female engorgement, an equal number of males and females were used to feed adult female ticks. The rearing was performed in an acclimatized incubator at 22 °C, 80–90% r.h. and an ambient CO2 level.

Babesia sp. (EU1) DNA detection

Parasite DNA extraction from ticks was performed as previously described using the Promega extraction kit (Promega, Charbonnière, France) [4]. DNA was extracted from individual adult (SGs) or from pools of around 50 eggs or larvae. During the final step, the DNA was rehydrated in 50 μL and 300 μL rehydration solution for respectively adult SGs and pools of eggs and larvae, and stored at 4 °C until use. For all analyzed samples, the efficiency of DNA extraction was confirmed by a PCR assay with primers designed to amplify Ixodes spp. 16S rRNA gene [4]. For blood samples removed from the artificial feeder, DNA was extracted from erythrocyte pellets with the Promega extraction kit following the manufacturer’s instructions and suspended in a final volume of 60 μL.

PCR amplification was performed on 10 μL and 20 μL of extracted DNA samples from tick and blood samples, respectively, using the primer pair EU1up and EU1down [13] designed for the Babesia sp. (EU1) 18S rRNA. Reactions were performed in a final volume of 30 μL or 50 μL, for either tick or blood samples, and contained 0.33 mM dNTPs (Eurobio, Lille, France), 2 mM MgCl2, 1× PCR buffer, 1 U Taq polymerase (Eurobio) and 1 μM of each primer. PCR conditions included 94 °C for 5 min, 45 cycles of 30 s at 94 °C, 30 s at 61 °C, 30 s at 72 °C, and a final elongation step of 10 min at 72 °C. The 362 bp amplification product was then visualized on an ethidium bromide-stained 1.5% agarose gel.

Babesia sp. (EU1) detection in ticks after an infectious blood meal

In order to show parasite ingestion by the ticks, 6 engorged nymphs and 2 adult females were dissected the day post repletion. The whole intestine was removed and dissected in sterile PBS in order to detect parasites in the blood meal by microscopic examination of Giemsa-stained smears. Remaining engorged nymphs were then allowed to molt to adults (around 2–3 months p.r.) and at least 1 month after molting, SGs of 3 females infected as nymphs were dissected in order to detect parasite DNA by PCR amplification. This procedure allowed us to detect parasites that could be transmitted by ticks and not simply those that might have persisted in the tick from the previous blood meal. Engorged females were allowed to lay eggs during 10–30 days and 3 broods were analyzed. Part of the eggs was subjected to DNA extraction to test for the presence of parasitic DNA by PCR amplification, and the remaining eggs were allowed to hatch into larvae. These larvae were also subjected to DNA extraction for parasite DNA detection by PCR amplification.

Babesia sp. (EU1) DNA detection after transmission from infected ticks to host blood

Eight female ticks, fed on Babesia sp. (EU1) infected blood as nymphs, were successfully subjected to a partial re-feeding step for 8 days in order (1) to detect parasite DNA in their SG after the stimulus of a re-feeding step, and (2) to evaluate their ability to transmit DNA of the acquired parasite to the blood contained in the artificial feeder. Three milliliters of non-infected restored sheep blood were used in the feeder and changed once a day. At day 3 post fixation, 3 females were detached and dissected in order to perform DNA extraction and PCR amplification on SG, whereas the remaining females were allowed to continue their meal. Each day, from 0 to 9 days post-fixation, a Giemsa-stained blood smear was prepared for microscopic examination using blood removed from the feeder; the remaining blood was centrifuged for 10 min at 800 g and the pellet of red blood cells was maintained at –20 °C until DNA extraction. This blood was tested for parasite presence as described above.

RESULTS

Babesia sp. (EU1) cloning

Subcultures of C201 samples, positive for Babesia sp. (EU1) in homologous erythrocytes, were successfully performed in sheep erythrocytes. Morphological aspects of Babesia sp. (EU1) are typical of a small Babesia species as shown in Figure 1. Two clones were obtained from the 10 wells containing 0.25 parasite per well. From the 30 wells with 0.5 parasite per well, 6 were positive, demonstrating a good cloning efficiency.

Figure 1.

Light micrographs of developmental stages of Babesia sp. (EU1) from in vitro culture (May-Grünwald Giemsa based stain). The first two lines show C201 isolate cultured in roe deer red blood cells. Free merozoites, trophozoites, dividing forms and small geminate merozoites at the border of the erythrocyte membrane are very similar to B. capreoli in roe deer erythrocytes or B. divergens in bovine erythrocytes. The last line shows geminate merozoites of the C201 clone in sheep erythrocytes; morphological aspects are very similar, but geminate forms appear more variable in shape and localization.

Sequencing of the PCR-amplified 18S rRNA gene revealed 100% identity with that of the complete 18S rRNA gene sequence published for the Babesia sp. (EU1) isolate from a human [12].

Babesia sp. (EU1) transmission between tick life stages

Efficient parasite acquisition by ticks was validated by microscopic examination of the gut contents after feeding on Babesia sp. (EU1) C201A infected blood (Fig. 2). Babesia were ingested over a period of time and the development of the parasites was therefore not synchronous. As a result, several different parasite stages lying free of erythrocytes could be observed in the tick midgut. Line 1 of Figure 2 shows a polymorphic population of individual elongated Strahlenkörper with only one nucleus and some protrusions. Line 2 shows examples of two Strahlenkörper cojoined; this close adherence of the two gametes represents the beginning of the fusion, which is illustrated in Line 3, where some bi-nucleated parasites possibly represent zygotes.

Figure 2.

Light micrographs of developmental stages of Babesia sp. (EU1) from the midgut of I. ricinus females after an infectious blood meal with in vitro cultured parasites (May-Grünwald Giemsa based stain). Line 1: individual elongated Strahlenkörper with only one nucleus and some protrusions. Line 2: examples of two Strahlenkörper cojoined with close adherence. Line 3: bi-nucleated parasites, possibly representing zygotes from the fusion of two Strahlenkörper.

Among the 52 nymphs fed on Babesia sp. (EU1)-infected blood, 17 molted to female adults. Three of these were dissected and their SG was analyzed for Babesia sp. (EU1) by PCR: two harbored parasite DNA in their SGs. Eight other females were subjected to the re-feeding step on non-infected sheep blood. Amongst these, 3 were dissected 3 days after the start of the meal. PCR amplification on DNA extracted from their SG revealed that all of them harbored parasite DNA.

Among the 9 adult females fed on Babesia sp. (EU1) infected blood, 3 laid eggs and successive larvae in which parasite DNA was searched for. Two broods, as well as the corresponding pools of larvae harbored Babesia sp. (EU1) DNA.

No positive DNA amplifications were obtained from control ticks from the Babesia-free colony.

Babesia sp. (EU1) transmission to blood

Among the 8 females infected with Babesia sp. (EU1) as nymphs and subjected to a re-feeding step, 5 fixed from day 2 to day 5 and were allowed to feed until day 9 on non-infected sheep blood. PCR amplifications performed each day on the blood removed from the feeder revealed the presence of parasite DNA from day 6 to day 9, indicating that ticks transmitted Babesia sp. (EU1) DNA via their saliva at least 4 days after their attachment to the membrane. No parasite was observed in microscopic examination of the prepared blood smears (in the 5 μL of blood removed each day).

DISCUSSION

Babesia sp. (EU1) was previously identified from human cases, as well as from roe deer and I. ricinus, in different areas in Europe, and represents a potential agent of an emerging zoonotic disease. In order to evaluate this potential, it is essential to define its biology and distribution, including the determination of its vector and vertebrate hosts. The aim of the present study was to validate, via experimental infections, the competence of I. ricinus to transmit Babesia sp. (EU1). For this purpose, a parasite strain isolated from roe deer was cloned in sheep erythrocytes, allowing us to make a morphological description of the parasite in both mammalian cells and in ticks. We then report an experimental demonstration that I. ricinus is able to acquire the parasite, to allow its sexual development, and to transmit the parasite to successive tick life stages and to vertebrate blood via saliva.

The Babesia sp. (EU1) clone used in the present study was isolated from roe deer blood and was successfully cultivated both in autologous roe deer and sheep red blood cells. To our knowledge, this study represents the first cloning of Babesia sp. (EU1) in culture and in heterologous erythrocytes from other ruminants, which may represent a possible range of vertebrate reservoir hosts. Until now, Babesia sp. (EU1) has been detected only in roe deer [3, 6, 22], which appears to be a wild reservoir host of the parasite, and in humans [11, 12]. A postulated strict host and vector specificity is traditionally accepted for Babesia spp., but B. divergens can infect a wide range of animals with or without splenectomy including bovines, gerbils, sheep, some cervid species, various non-human primates and humans (see review by Zintl et al. [25]). Additional vertebrate hosts can therefore not be excluded for Babesia sp. (EU1).

I. ricinus is the most prevalent and widely distributed tick in Western Europe and represents the main vector of several zoonotic diseases including B. divergens which, until now, was believed to be the main etiological agent of human babesiosis on this continent [25]. DNA of Babesia sp. (EU1) has been detected by PCR from I. ricinus ticks in Slovenia [7], Switzerland [5, 13], and in the Netherlands [21]. However, such reports are not necessarily proof of the vectorial capacity of the tick, but only of its infection status. In fact, we have previously demonstrated that some parasite DNA acquired during the last blood meal remains in the carcass of the arthropod, in addition to those present in the SGs, the only location from which Babesia can be transmitted to a vertebrate host [4].

Observations by light microscopy immediately after the tick feeding on infected blood showed various parasitic stages of Babesia sp. (EU1). These different stages are similar to those observed for B. bigemina [10, 20, 23], B. divergens [4], and B. canis [19] and suggest sexual reproduction of the parasites. Even if sexual development of Babesia species in their vector has long been a matter of controversy, direct proof of such an event has been obtained by DNA quantification of B. bigemina, B. canis [17] and B. divergens [16], and it is now a generally accepted phenomenon. Microscopic observations reported here for Babesia sp. (EU1) lead to the same conclusion, as the fusion of the cell membrane and the close proximity of the nuclei reported in Figure 2 (Line 3) clearly suggest a sexual phase in development of the parasite. The apparition of sexual parasite stages was only found to occur in the tick vector, suggesting that some, as of yet, unidentified factors associated with engorged I. ricinus ticks may be required.

In a preceding study, we reported some field evidence of transovarial transmission of Babesia sp. (EU1) in I. ricinus; parasite DNA was detected in eggs and larvae issued from females collected on Babesia sp. (EU1) infected roe deer in France [3]. The results of the present study validate the transovarial transmission of parasite DNA, from artificially infected adult females to eggs and larvae. The existence of transovarial transmission is distinctive of the Babesia genus and means that the ticks themselves can represent a reservoir of the pathogen in the field. In addition, it is noteworthy that some Babesia sp. (EU1) negative eggs and larvae came from adults engorged with infected blood. This demonstrates that transovarial transmission is not a systematic event, as previously reported for naturally infected ticks [3].

In our previous study, we also reported that some Babesia sp. (EU1) positive eggs and larvae could come from adults engorged on non-infected roe deer. This finding suggested that, in addition to vertical transmission, there was transstadial transmission of the parasite, at least from the nymph to the adult. The same conclusion can be drawn from the study of Hilpertshauser et al. since Babesia sp. (EU1) was also detected in one adult male I. ricinus, a stage that is not hematophagous [13]. Here we demonstrate that I. ricinus nymphs and adults are able to acquire Babesia sp. (EU1) and that when nymphs are fed with infected blood, parasite DNA can be recovered in the SG of the succeeding female life stage. These females were then able to transmit it to vertebrate blood during a new meal on non-infected blood. In this case, transmission occurred at least four days after the attachment of the female to the membrane. Such a delay is not surprising given the time it takes for tick attachment, cement synthesis, and probably multiplication of the parasite in the SG. Indeed, two hypotheses are predicated concerning the effect of blood meal stimulus: the blood meal stimulates pathogen multiplication in the SGs or pathogen migration to this area. Our results show that some parasite DNA could be detected in the SGs without a re-feeding step and thus, we assume that parasites were already present in tick SG before the stimulus of the blood meal. Here we report DNA transmission from I. ricinus SGs to blood and not infectious parasites. However, it is more than likely that this DNA was carried by viable parasites to both SGs within ticks and to vertebrate blood during tick feeding. Further investigations are now needed to optimize culture conditions in order to obtain the in vitro development of sporozoites, the parasite stage transmitted by ticks. Alternatively, some laboratory animal models also have to be developed that could be used to test this assumption and to study the life cycle of this parasite in more detail.

In conclusion, Babesia sp. (EU1) is a recently characterized parasite that may be responsible for an unaccounted number of human cases of babesiosis, and therefore needs increased attention and vigilance because of its important potential as an emerging zoonotic pathogen. In vitro cloning of Babesia sp. (EU1) is a very promising method for further studies to analyze the biology of this parasite, along with mechanisms of infection and transmission by ticks.