Ornithodoros Moubata (Acari: Argasidae)

1 Babesia parasites develop and are transmitted by the non-vector soft tick Ornithodoros moubata (Acari: Argasidae)

B. BATTSETSEG1,T.MATSUO2,X.XUAN1,D.BOLDBAATAR1,S.H.CHEE1, R. UMEMIYA1,T.SAKAGUCHI1,T.HATTA1,J.ZHOU1,A.R.VERDIDA1, D. TAYLOR3 and K. FUJISAKI1* 1 National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan 2 Department of Infectious Diseases, Kyorin University, School of Medicine, Mitaka, Tokyo 181-8611, Japan 3 Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8577, Japan

(Received 24 April 2006; revised 30 May 2006; accepted 2 June 2006; ﬁrst published online 18 September 2006)

SUMMARY

Ornithodoros moubata ticks were fed on blood infected with Babesia equi. However, the parasites were quickly cleared as evidenced by the disappearance of B. equi-speciﬁc ribosomal RNA from the ticks. We hypothesized that if the Babesia parasite can escape midgut-associated barriers a non-vector tick can become infected with Babesia. To test this hypothesis, B. equi parasite-infected blood from in vitro culture was injected into the haemocoel of ticks. B. equi-speciﬁc rRNA was surprisingly detected 45 days after injection even in the eggs. Babesia-free dogs were infested with O. moubata ticks that were infected by inoculation with B. gibsoni-infected red blood cells. Parasitaemia and antibody production against Bg-TRAP of B. gibsoni increased gradually. These results indicate that O. moubata may be a useful vector model for Babesia parasites and also a very important tool for studies on tick immunity against Babesia parasites and tick-Babesia interactions.

Key words: Babesia, Ornithodoros moubata, midgut-associated barrier, tick transmission, vertebrate host.

INTRODUCTION glands. Sporozoites invade the salivary glands to be injected into a vertebrate host when the tick ingests Babesia are tick-transmitted protozoa that comprise an infected bloodmeal (Kuttler, 1988). For trans- some of the most ubiquitous and widespread para- mission to occur, therefore, the Babesia parasite must sites of erythrocytes in humans and a wide range complete an elaborate developmental programme in of wild and economically valuable domestic animals the hostile tick environment. However, the precise such as cattle and horses (Kuttler, 1988; Kjemtrup mechanisms by which ticks limit parasite develop- and Conrad, 2000; Homer et al. 2000; Wei et al. ment still remain to be elucidated. 2001). These single-celled organisms invade red In mosquito responses to midgut invasion by blood cells and cause fatigue, aches, fever, chills, malarial parasites, microvillar proteins, peritrophic sweating, dark urine, enlarged spleen and anaemia. matrix and mosquito digestive enzymes appear Infections can range from no serious symptoms to a important as barriers to parasite development fatal disease. The life-cycle of Babesia includes (Shen et al. 1999; Sinden and Billingsley, 2001; asexual and sexual stages in the vertebrate and tick Abraham and Jacobs-Lorena, 2004). Soft ticks also host, respectively. Ticks transfer Babesia sporozoites have a number of midgut barriers such as a peri- during feeding and the sporozoites invade the host trophic membrane (Grandjean, 1983), gut lysozymes red blood cells. When Babesia gametocytes are (Kopacek et al. 1999) and antimicrobial peptides ingested by a suitable tick host they develop into (Nakajima et al. 2002a, b) that limit or prevent gametes, fuse to form a motile zygote that penetrates invasion of parasites in non-vector species. The non- the peritrophic matrix (PM), enters the cells of the vector tick Ornithodoros moubata is easier to maintain intestinal epithelium and divides into kinetes. The and handle in laboratory experiments than vector kinetes break free, enter the body cavity (haemocoel) ticks like Haemaphysalis longicornis because they are by crossing the midgut and migrate to the salivary larger in size and have a short blood-feeding period. Therefore, if the Babesia parasite can develop in O. moubata, it may be useful for elucidating the * Corresponding author: National Research Center for genetic pathways involved in vector competence and Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido clarifying the importance of the midgut barrier for 080-8555, Japan. Tel: +81 155 49 5646. Fax: +81 155 development and transmission of Babesia parasites 49 5643. E-mail: [email protected] in ticks.

Parasitology (2007), 134, 1–8. f 2006 Cambridge University Press doi:10.1017/S0031182006000916 Printed in the United Kingdom

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MATERIALS AND METHODS thiocyanate solution (Chomczynski and Sacchi, Parasites 1987) from injected and artificially fed ticks. Then 1 mg of total RNA from each sample was applied to a The United States Department of Agriculture One Step RNA PCR kit (TAKARA, Otsu, Japan) (USDA) strain of Babesia equi was grown in horse according to the conditions recommended by the erythrocytes in vitro as described by Avarzed et al. manufacturer with the following primers. Primers (1997). A B. gibsoni strain isolated from a hunting designed from 18S ribosomal RNA of B. equi were dog in Hyougo Prefecture Japan, designated strain rE1F 5k-GTTTATTTGATGTTTGTTT-3k and NRCPD (Fukumoto et al. 2001a) was used for rE1R 5k-CCAAGCGCAGTCAACGAAA-3k and experimental infection of beagles. for O. moubata ribosomal protein 18S were rOM 5k-GTTCCTTCC TTGATTGTCATGAG-3k and Dogs rOM 5k-TCGGTTAGATGCACTGCTCGTCT-3k as a loading control. Tick and blood samples col- One-year-old female beagle dogs were confirmed to lected from dogs were lysed in 0.1 M Tris-HCl be free of natural B. gibsoni infection by detection of (pH 8.0) containing 1% SDS, 0.1 M NaCl and 10 mM specific antibody prior to use in the experiments. All EDTA and treated with proteinase K (100 mg/ml) animal experiments were conducted in accordance for 2 h at 55 xC. The DNA was extracted with with Standards for the Care and Management of phenol/chloroform and precipitated with ethanol, Experimental Animals promulgated by the National then dissolved in 25 ml of TE buffer and stored at Research Center for Protozoan Diseases, Obihiro 4 xC. PCR was performed with p18d3 and p18d4 University of Agriculture and Veterinary Medicine, oligonucleotide primers (5k-TCCGTTCCCACAA- Japan. CACCAGC-3k and 5k-CGAATGAGGATGATG- AGGAGGA-3k) as targets for the 182 bp fragment of Ticks B. gibsoni P18 gene (Fukumoto et al. 2001b). DNA products were run on an agarose gel, stained with Soft ticks, Ornithodoros moubata (Acari: Argasidae), ethidium bromide and photographed. were obtained from the National Institute of Animal Health (Tsukuba, Japan) and have been maintained at the National Research Center for Protozoan Transmission electron microscopy (TEM) Diseases, Obihiro University of Agriculture and The salivary glands and ovaries of ticks 7 days after Veterinary Medicine for several generations by injection with Babesia parasites were removed, fixed feeding on rabbits (Oryctolagus cunniculus) and with cold 3% glutaraldehyde in sodium cacodylate maintained at 27¡1 xC, 50–60% RH and total buffer (pH 7.4) overnight at 4 xC, post-fixed with 1%

darkness. Feeding and rearing of ticks were per- OsO4 in the same buffer for 2 h at 4 xC after washing formed as described by Chinzei (1983). thoroughly with the same buffer, dehydrated in an ethanol series and embedded in quetol 651 resin (Nissin EM, Tokyo, Japan). Thin sections (ap- Artificial feeding proximately 80 nm thick) were cut on a Leica An artificial feeding study was used to introduce UCT ultramicrotome using a diamond knife and B. equi into the tick digestive tract. Adult female doubly stained with uranyl acetate and lead citrate O. moubata were fed by an artificial membrane sys- before examination with a Hitachi H-7500 electron tem using Parafilm M (Pechiney plastic packaging microscope. Menasha, WI 54952) similar to the Baudruche membrane described by Waladde et al. (1991) and Immuno-electron microscopy (IEM) Matsuo et al. (2004). The artificial meal consisted of B. equi-infected red blood cells with 12% para- The ovaries were removed from ticks injected with sitaemia suspended in the same volume of PBS. infected RBC of B. equi and B. gibsoni, fixed in 4% paraformaldehyde with 0.1% glutaraldehyde in PBS overnight at 4 xC, washed thoroughly in PBS and Tick injection embedded in 2% agarose. After dehydration with an Ticks were infected under the last coxa with 15 mlof ethanol series, the samples were embedded in LR B. equi-orB. gibsoni-infected RBC suspended in Gold resin (Polysciences Inc., USA). Thin sections PBS with a 27-gauge intravenous needle. (about 80 nm thick) were cut on a Leica UCT ultramicrotome using a diamond knife and placed on nickel grids. Sections were exposed at room tem- Reverse transcriptase-polymerase chain reaction perature (RT) for 30 min to 5% skim milk PBS as a analysis (RT-PCR) and PCR blocking agent then incubated with anti-B. equi Total RNA was extracted with solution D, prepared specific merozoite antigen 1 and EMA-1 (Kapp- by adding 2-mercaptoethanol to a stock guanidinium meyer et al. 1993) or EMA–2 (Knowles et al. 1997)

M12 345678 M123456789PN

357 bp 357 bp

C D

SB DMBS

ER N

2 µm 2 µm

Fig. 1. Detection of Babesia equi in Ornithodoros moubata ticks. (A) RT-PCR for detection of B. equi rRNA in O. moubata tick samples after artiﬁcial feeding with B. equi-infected red blood cells (8% parasitaemia). Lane M, Marker; Lane 1, Day 1; Lane 2, Day 3; Lane 3, Day 5; Lane 4, Day 9; Lanes 5–8, tick sample before feeding. (B) RT-PCR for detection of B. equi rRNA in O. moubata tick samples after injection of B. equi-infected red blood cells into haemocoel of ticks. Lane M, Marker; Lane 1, Day 1; Lane 2, Day 7; Lane 3, Day 15; Lane 4, Day 45; Lanes 5–9 are samples of eggs; Lane P, positive control (RNA from in vitro culture of B. equi); Lane N, negative control (tick injected with normal RBC). (C) Electron micrographs showing B. equi and (D) immuno-electron microscopy using anti-EMA-2 antibody against the parasites in the ovary of O. moubata 1 week after injection with infected RBC. DMBS, double membrane-bounded structure; ER, endoplasmic reticulum; N, nucleus; SB, spherical body.

antibodies overnight at 4 xC, and subsequently in- ELISA cubated with 10 nm gold-labelled goat anti-mouse ELISA was carried out with B. gibsoni-speciﬁc IgG IgM antibody (Amersham Biosciences, UK) + antibodies according to the methods described pre- at RT for 2 h. Normal mouse IgG was used instead viously (Fukumoto et al. 2004). Microplates (96 well) of the primary antibodies as a negative control. were coated with GST-Bg-TRAP (B. gibsoni- Finally, these sections were counter-stained with thrombospondin related adhesive protein) (Zhou uranyl acetate and lead citrate before examination et al. 2006) and GST (negative control) antigens at a with a H-7500 transmission electron microscope concentration of 100 ng per well. The OD value of (HITACHI, Japan). 0.1 was estimated as a cut-oﬀ value expressed as the reciprocal of the maximum dilution. The absorbance Tick transmission challenge on a dog was shown in the distance of OD value of the antigen (GST-Bg-TRAP) well and the control antigen The ears of beagles infected with B. gibsoni by (GST) well. injection of infected blood were infested with 20

fourth instar nymphs (N4) and 20 adult O. moubata ticks. Ticks were recovered from the dogs 3 h after RESULTS infestation when all ticks had dropped off. A O. moubata ticks were fed blood infected with thin blood smear from each dog was fixed with Babesia equi, which causes babesiosis in horses. The methanol for 2 min and stained with Giemsa solution ticks ingested a bloodmeal, as evidenced by changes for 30 min. The slides were then microscopically in tick size and form, but the parasites were quickly examined to calculate the peripheral parasitaemia. cleared as shown by the disappearance of B. equi specific ribosomal RNA (rRNA) from the ticks 5 days after feeding (Fig. 1A). The Babesia parasite rRNA Splenectomy of a dog was not detected in the babesia-fed ticks, suggesting At day 20 after tick infestation the experimental dog that the parasites were passed in the feces or digested. was splenectomized under anaesthesia as described The failure of Babesia parasites fed to O. moubata to earlier by Fukumoto et al. (2000). All surgical pro- establish an infection is likely a consequence of the cedures were conducted in accordance with the parasite’s inability to invade the gut cells or survive Guiding Principles for the Care and Use of Research tick midgut-associated barriers such as the peri- Animals promulgated by Obihiro University of trophic membrane (Grandjean, 1983), and presence Agriculture and Veterinary Medicine. of lysozymes (Kopacek et al. 1999) and defensins

M 1 2 3 4 5 6 7 8 9 10 1112P N M 1 2 3 4 5 6 7 8 9 10 11 12 13 N P

357 bp 357 bp

V SB ER

DMBS ER N

2 µm

C Fig. 2. PCR for detection of Babesia gibsoni DNA in Ornithodoros moubata ticks. (A) Nymph and female samples. Lane

M, Marker; Lanes 1–6, samples of N4 female 7 days after injection with B. gibsoni-infected dog blood. Lanes 1–4, negative; Lanes 5 and 6, positive. Lanes 7–12, samples of adult females 7 days after injection with B. gibsoni-infected dog blood. Lanes 7 and 8, negative; Lanes 9–12, positive. (B) Egg samples. Lane M, Marker; Lanes 1–13 are samples of eggs from females injected with B. gibsoni- infected dog blood. Lanes 6, 9 and 11, positive; Lane N, negative control – tick DNA injected with normal dog blood. Lane P, positive control – B. gibsoni DNA samples. (C) Electron micrographs showing B. gibsoni in the ovary of O. moubata 1 week after injection of infected RBC. DMBS, double membrane-bounded structure; ER, endoplasmic reticulum; N, nucleus; SB, spherical body.

(Nakajima et al. 2002a, b) in the midgut. Therefore, electron microscopy (TEM). Numerous Babesia-like we hypothesized that if we could succeed in structures were ultrastructurally identified in the developing a way for the Babesia parasite to escape/ oocytes of ticks injected with Babesia-infected RBC avoid the midgut-associated immune barrier, a non- (Fig. 1C). The surface and periphery of the oocyte vector tick may become infected with Babesia. cytoplasm of these structures reacted with both anti- Moreover, it is very important to see whether EMA-1 and -2 antibodies confirming the presence of O. moubata can transmit the Babesia parasite to a the Babesia parasites (Fig. 1D). The reactions against vertebrate host under such conditions. We conduc- the anti-EMA-2 antibody were slightly stronger than ted 2 experiments to test this hypothesis. First, against the anti-EMA-1 antibody. No Babesia-like B. equi parasite-infected horse blood from in vitro structures were detected in the oocytes of normal culture was prepared and injected directly into the ticks and no reaction against the antibodies was seen haemocoel of adult female ticks to determine if the in normal ticks and the negative control. The second Babesia parasite can survive in the body of the non- experiment was conducted with B. gibsoni-infected vector tick. Next, infected ticks were allowed to dog red blood cells to determine whether the feed on Babesia-free dogs to determine whether the O. moubata generated sporozoites were infectious to a parasite could be transmitted to the vertebrate host vertebrate host. B. gibsoni-infected dog red blood by the non-vector tick. In the first experiment cells were injected into the haemocoel of O. moubata B. equi-specific rRNA was detected up to 45 days and infection confirmed by PCR and TEM after injection of the parasite (Fig. 1B). Very sur- (Fig. 2A, B, C). No difference in the survival of prisingly, the rRNA was detectable even in eggs O. moubata-injected ticks during babesial infection oviposited by injected ticks. These results suggest was observed. Then Babesia-free dogs were infested

that the parasites can survive in the tick haemocoel with 20 fourth instar nymphs (N4) and 20 adult and develop to be transmitted to the next generation females of O. moubata infected by inoculation with through the egg stage. If the parasite can be trans- B. gibsoni-infected red blood cells. However, the mitted transovarially the parasite oocysts must in- B. gibsoni parasites were not detected by PCR until vade the tick ovary. This was conﬁrmed by observing 2 weeks after infestation and the blood smears the Babesia parasites in the ovary by transmission stained by Giemsa solution showed less than 1%

9 8 7 6 5 Dog 4 3 Parasitemia (%) 2 1 0 0 25 8 1114172125283034374043 46 50 53 57 60 63 66 69 72 75 Days post infestation C

M580 2 11141721252830343740 N P M46505357606366697275NP43

1·8 1·6 1·4 1·2 1 Dog 0·8

OD (415 nm) 0·6 0·4 0·2 0 2 5 10 15 18 25 30 37 40 42 46 48 50 53 57 60 63 66 70 73 Days after infestation E Fig. 3. Transmission of Babesia gibsoni to dogs by Ornithodoros moubata ticks. (A and B) Light microscopy of Giemsa- stained thin blood smears. B. gibsoni infection in an experimental dog infested with B. gibsoni-infected O. moubata ticks. (C) Infectious course of B. gibsoni in an experimental dog infested with B. gibsoni-infected O. moubata ticks. m, Points to day of splenectomy. (D) Detection of B. gibsoni infection by PCR in an experimental dog. Lane numbers show days post-infestation with infected O. moubata ticks. (E) Detection of antibodies against the Bg-TRAP protein by ELISA in a dog experimentally infested with B. gibsoni-infected O. moubata ticks. Dotted line shows 0.1 cut-oﬀ value. The mean values from 3 individual wells are shown.

parasitaemia until 3 weeks after infestation. However, haemocytes participate in both cellular after successful splenectomy of the dogs the para- (Kryuchechnikov, 1991; Johns et al. 1998; Inoue sitaemia increased gradually so that at 75 days the et al. 2001; Ceraul et al. 2002) and humoral (Ceraul parasitaemia was as high as 8% (Fig. 3A, B, C). et al. 2003; Simser et al. 2004) immune responses in Moreover, the antibody production against B. response to various types of challenges. In this study, gibsoni thrombospondin-related adhesive protein the injected Babesia appeared to deal with the (Bg-TRAP) (Zhou et al. 2006) also gradually in- humoral factors present in the haemolymph such creased (Fig. 3E). These results indicate that the as phenoloxidases and plasma (Kadota et al. 2002) in dogs became infected with B. gibsoni after infestation O. moubata as with the host tick because Babesia was with B. gibsoni-infected O. moubata ticks. The dogs successfully transmitted to host dogs by O. moubata showed typical clinical symptoms for Babesia in- after injection. The cellular and humoral factors fection such as fever, progressive haemolytic anaemia important in the immune responses of ticks are still and haemoglobinuria (data not shown). poorly understood. Recently, very interesting molecules such as a–2-macroglobulin-like glycoprotein DISCUSSION (TAM), have been reported from O. moubata haemolymph (Kopacek et al. 2000; Saravanan et al. This is the first report to show that Babesia mero- 2003). Moreover, a significant up-regulation of zoites can develop in a non-vector tick and develop TAM expression was observed in haemocytes, sali- into sporozoites that are infective for vertebrate host vary glands and gut tissues of the ticks 24 h after a animals. The results of this study indicate that the bloodmeal. The mechanisms controlling the tran- midgut may act as a major barrier for prevention of scription of the TAM gene are unclear but one acti- development of Babesia in non-vector ticks. Kopacek vating factor may be an immune response to foreign et al. (1999) have shown that O. moubata ticks have particles (Saravanan et al. 2003). Research with lysozymes in the gut that may function in the O. moubata has numerous advantages available for immune response. Nakajima et al. (2001, 2002a, b) investigation of not only Babesia-tick interactions have shown that defensins are secreted into the but also tick vector immunology. By identifying midgut lumen and have strong antimicrobial activity. factors and molecules that are essential to the survival These immune responses in the gut may play a major of Babesia in the haemocoel of O. moubata, these role in the prevention of Babesia invasion in the experiments may contribute to the elucidation of non-vector tick O. moubata. Defensins have also been factors in the host tick and the development of isolated from various hard tick species (Johns et al. transmission-blocking vaccines against babesial in- 2001; Fogaca et al. 2004; Lai et al. 2004). However, fection. Further studies on the differences between Sonenshine et al. (2005) showed that defensins are Babesia transmission in vector and non-vector ticks not secreted into the midgut of Dermacentor may help to elucidate the mechanisms by which variabilis and concluded that antimicrobial activity in Babesia invades the tick and can be transmitted to D. variabilis is primarily related to proteins derived vertebrate hosts. In addition, Babesia must overcome from the host bloodmeal such as haemoglobin the immune responses of the host tick to be trans- fragments (Fogaca et al. 1999; Nakajima et al. 2003, mitted successfully. However, the understanding of 2005). These studies indicate that there are import- tick innate immunity against pathogens especially ant differences in the role of the midgut in the the Babesia parasite is still in its infancy. These re- immune response of soft ticks and hard ticks. These sults indicate that O. moubata may provide a bio- differences may be important in determining the logical tool with numerous advantages available for microorganisms that can be transmitted by certain investigation of not only Babesia-tick interactions tick species. This study indicates that the Babesia but also tick vector immunology. may be affected by these differences because if the parasite is injected into the haemocoel of the non- We thank Dr Fukumoto S. for kindly providing Babesia gibsoni parasite-infected blood and technical support. vector tick O. moubata, avoiding the midgut immune This study was supported by Grant-in-Aid for Scientific mechanisms, it can develop and be successfully Research from the Japan Society for the Promotion of transmitted to a vertebrate host. Further studies Sciences, by grants from the Bio-oriented Technology comparing midgut immune mechanisms in non- Research Advancement Institution (BRAIN) and by vector and vector ticks of Babesia as well as other the 21st Center of Excellence Program of the Scientific Research from the Ministry of Education, Culture, Sports, infective microorganisms are needed. Science, and Technology of Japan. 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