• View in gallery

    Detection of B. equi DNA in tissue samples of H. longicornis from engorged adults injected with B. equi–infected erythrocytes. Arrowheads indicate the position of the expected 218-bp band. M, 100-bp DNA ladder marker; P, positive control; N, negative control.

  • View in gallery

    Detection of B. equi DNA in tissue samples of H. longicornis from engorged nymphs injected with B. equi–infected erythrocytes. Arrowheads indicate the position of the expected 218-bp band. M, 100-bp DNA ladder marker; P, positive control; N, negative control.

  • View in gallery

    Detection of B. equi DNA in salivary gland and carcass tissue samples of adult H. longicornis injected with B. equi–infected erythrocytes as engorged nymphs. Arrowheads indicate the position of the expected 218-bp band. M, 100-bp DNA ladder marker; P, positive control; N, negative control.

  • View in gallery

    Immunofluorescence detection of B. equi in salivary gland cells. Left, Hoechst staining. Middle, Staining with antibodies against B. equi. Right, Overlay images. Arrowheads indicate cells infected with B. equi. Bar, 50 μm. This figure appears in color at www.ajtmh.org.

  • 1

    Brüning A, Phipps P, Posnett E, Canning EU, 1997. Monoclonal antibodies against Babesia caballi and Babesia equi and their application in serodiagnosis. Vet Parasitol 58 :11–26.

    • Search Google Scholar
    • Export Citation
  • 2

    Friedhoff KT, 1982. The piroplasms of Equidae. Significance for the international equine trade. Berl Munch Tierarztl Wochenschr 95 :368–374.

    • Search Google Scholar
    • Export Citation
  • 3

    Purnell RE, 1981. Babesiosis in various hosts. Ristic M, Krier JP, eds. Babesiosis. New York: Academic Press, 25–64.

  • 4

    Schein E, 1988. Equine babesiosis. Ristic M, ed. Babesiosis of Domestic Animals and Man. Boca Raton, FL: CRC Press, 197–208.

  • 5

    Holbrook AA, 1969. Biology of equine piroplasmosis. J Am Vet Med Assoc 155 :453–454.

  • 6

    Ikadai H, Nagai A, Xuan X, Igarashi I, Tsugihiko K, Tsuji N, Oyamada T, Suzuki N, Fujisaki K, 2002. Seroepidemiologic studies on Babesia caballi and Babesia equi infections in Japan. J Vet Med Sci 64 :325–328.

    • Search Google Scholar
    • Export Citation
  • 7

    Yoshihara T, 1997. Equine piroplasmosis. J Anim Protozool 11 :1–7.

  • 8

    Fujisaki K, 1978. Development of acquired resistance and precipitating antibody in rabbits experimentally infested with females of Haemaphysalis longicornis (Ixodidea: Ixodidae). Nat Inst Anim Health Quart 18 :27–38.

    • Search Google Scholar
    • Export Citation
  • 9

    Fujisaki K, Kawazu S, Kamio T, 1994. The taxonomy of the bovine Theileria spp. Parasitol Today 10 :31–33.

  • 10

    Oliver JH, Herrin CS, 1976. Differential variation of parthenogenetic and bisexual Haemaphysalis longicornis (Acari: Ixodidae). J Parasitol 62 :475–484.

    • Search Google Scholar
    • Export Citation
  • 11

    Rodriguez BJL, Ikadai H, You M, Battsetseg B, Igarashi I, Nagasawa H, Fujisaki K, 2001. Molecular evidence of Babesia caballi (Nuttall and Strickland, 1910) parasite transmission from experimentally-infected SCID mice to the ixodid tick, Haemaphysalis longicornis (Neuman, 1901). Vet Parasitol 102 :185–191.

    • Search Google Scholar
    • Export Citation
  • 12

    Battsetseg B, Lucero S, Xuan X, Claveria FG, Byambaa B, Battur B, Boldbaatar D, Batsukh Z, Khaliunaa T, Battsetseg G, Igarashi I, Nagasawa H, Fujisaki K, 2002. Detection of equine Babesia spp. gene fragments in Dermacentor nuttalli Olenev 1929 infesting Mongolian horses, and their amplification in egg and larval progenies. J Vet Med Sci 64 :727–730.

    • Search Google Scholar
    • Export Citation
  • 13

    Battsetseg B, Lucero S, Xuan X, Claveria FG, Inoue N, Alhassan A, Kanno T, Igarashi I, Nagasawa H, Mikami T, Fujisaki K, 2002. Detection of natural infection of Boophilus microplus with Babesia equi and Babesia caballi in Brazilian horses using nested polymerase chain reaction. Vet Parasitol 107 :375–377.

    • Search Google Scholar
    • Export Citation
  • 14

    Battsetseg B, Xuan X, Ikadai H, Bautista JL, Byambaa B, Boldbaatar D, Battur B, Battsetseg G, Batsukh Z, Igarashi I, Nagasawa H, Mikami T, Fujisaki K, 2001. Detection of Babesia caballi and Babesia equi in Dermacentor nuttalli adult ticks. Int J Parasitol 31 :384–386.

    • Search Google Scholar
    • Export Citation
  • 15

    Friedhoff KT, 1988. Equine babesiosis. Ristic M, ed. Babesiosis of Domestic Animals and Man. Boca Raton, FL: CRC Press, 23–51.

  • 16

    Guimarãs AM, Lima JD, Ribeiro MFB, 1998. Spologony and experimental transmission of Babesia equi by Boophilus microplus. Parasitol Res 84 :323–327.

    • Search Google Scholar
    • Export Citation
  • 17

    Moltmann UG, Mehlhorn H, Schein E, Voigt WP, Friedhoff KT, 1983. Ultrastructural study on the development of Babesia equi (Coccidia: Piroplasmia) in the salivary glands of its vector ticks. J Protozool 30 :218–225.

    • Search Google Scholar
    • Export Citation
  • 18

    Zapf F, Schein E, 1994. The development of Babesia (Theileria) equi (Laveran, 1901) in the gut and the haemolymph of the vector ticks, Hyalomma species. Parasitol Res 80 :297–302.

    • Search Google Scholar
    • Export Citation
  • 19

    Ikadai H, Tanaka T, Shibahara N, Tanaka H, Matsuu A, Kudo N, Shimazaki K, Igarashi I, Oyamada T, 2005. Inhibitory effect of lactoferrin on in vitro growth of Babesia caballi. Am J Trop Med Hyg 73 :710–712.

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    Ueti MW, Palmer GH, Kappmeyer LS, Scoles GA, Knowles DP, 2003. Expression of equi merozoite antigen 2 during development of Babesia equi in the midgut and salivary gland of the vector tick Boophilus microplus. J Clin Microbiol 41 :5803–5809.

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  • 23

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    Ohta M, Kawazu S, Terada Y, Kamio T, Fujisaki K, 1996. Experimental transmission of Babesia ovata oshimensis n. var. of cattle in Japan by Haemaphysalis longicornis. J Vet Med Sci 58 :1153–1155.

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  • 25

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  • 26

    Sparagano OA, Allsopp MT, Mank RA, Rijpkema SG, Figueroa JV, Jongejan F, 1999. Molecular detection of pathogen DNA in ticks (Acari: Ixodidae): a review. Exp Appl Acarol 23 :929–960.

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MOLECULAR EVIDENCE OF BABESIA EQUI TRANSMISSION IN HAEMAPHYSALIS LONGICORNIS

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  • 1 Department of Veterinary Parasitology, School of Veterinary Medicine and Animal Sciences, Kitasato University, Towada, Aomori, Japan; Department of Veterinary Internal Medicine, Faculty of Agriculture, Tottori University, Koyama-Minami, Tottori, Japan; National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, Japan

We studied the tick, Haemaphysalis longicornis, to determine the possibility of both transovarial and transstadial transmission of Babesia equi. We also studied the usefulness of the needle injection method for pathogenic tick-transmitted organisms including Babesia parasites. Erythrocytes infected with B. equi were injected into the midgut of engorged adults or nymphs using a hypodermic needle passed through the integument. DNA of B. equi in ticks was detected using nested polymerase chain reaction (PCR). B. equi DNA was present in adults, eggs, and larvae, indicating that transovarial transmission occurred. B. equi DNA was present in adults that developed from infected nymphs, and the B. equi antigen was present in their salivary glands, indicating that transstadial transmission occurred. These findings suggest that H. longicornis may play a role in the transmission of B. equi.

INTRODUCTION

Equine babesiosis, also known as biliary fever, is an acute, subacute, or chronic tick-borne disease of Equidae caused by infection with the hemoprotozoan parasites Babesia caballi or Babesia equi. The parasites, which are widely distributed in tropical and subtropical areas worldwide, result in significant economic loss for the horse industry. Babesiosis is generally characterized by fever, anemia, jaundice, and edema. It causes death in some cases.14 Infected horses often carry the parasites for long periods and act as sources of tick-borne infection of other horses.5 To date, there have been no reports of clinical cases of equine babesiosis in Japan,6 but there has been a long-term increase in the number of horses imported from foreign countries, including those where equine babesiosis is endemic. The presence of two tick vectors, Dermacentor reticulates and Rhipicephalus sanguineus, in Japan has also been reported.7 These conditions indicate that introduction of Babesia-infected horses poses a significant threat to the horse industry in Japan.

The hard tick, Haemaphysalis longicornis, is distributed mainly in East Asia and Australia, where it is known to transmit Babesia.8,9 H. longicornis is the most widespread tick species on wild and domestic animals, including horses, in Japan.8 Reproduction of this species is thelytokous.10 Moreover, H. longicornis was shown to be a capable biologic vector of equine B. caballi in an experimental study using non-obese, diabetic, and severe combined immune-deficient mice as the host model.11 B. equi is transmitted by tick species of the genera Dermacentor, Hyalomma, Rhipicephalus, and Boophilus.1218 However, whether H. longicornis is a capable biologic vector of B. equi is unknown.

In this study, to elucidate the transmission system of B. equi, we studied transovarial and transstadial transmission of the parasite in H. longicornis by nested polymerase chain reaction (PCR) and show the tick as a potential biologic vector for the parasite.

MATERIALS AND METHODS

Haemaphysalis longicornis.

The parthenogenetic Okayama strain of H. longicornis was obtained from the National Institute of Animal Health, Tsukuba, Japan, and was maintained in rabbits (Japan White, female; Crea Japan, Tokyo, Japan) in our laboratory for several generations.8 Tick colonies were kept in incubators at 25°C and 90–95% relative humidity under continuous darkness.

Parasite.

US Department of Agriculture strains of B. equi were maintained in horse erythrocytes in continuous culture as previously described.19,20 The culture medium contained Medium 199 (M199; Sigma–Aldrich, St. Louis, MO) with 0.1 mmol/L hypoxanthine (Invitrogen, Carlsbad, CA). Horse erythrocytes at a packed cell volume of 10% in M199 supplemented with 40% (vol/vol) horse serum were cultured in 24-well culture plates (1-mL suspension/well) under a humidified atmosphere of 5% CO2 in air at 37°C. The culture medium was changed daily.

Transovarial transmission test.

Twenty-two detached, engorged adult ticks were injected through the integument into the midgut with ~20 μL of a solution containing equal volumes of B. equi–infected erythrocytes with 19.0% parasitemia and M199. A hypodermic needle (MS needle 1/8; Tsuda, Saitama, Japan) was used for injection. The infected ticks were transferred into individual glass flasks and kept in an incubator at 25°C and ~85% relative humidity to allow oviposition. DNA samples were collected from adults after oviposition, from 200 eggs and from larvae hatched from the eggs.

Transstadial transmission test.

Twenty detached, engorged nymphs were injected through the integument into the midgut with ~4 μL of a solution containing equal volumes of B. equi–infected erythrocytes with 10.9% parasitemia and M199. All nymphs were injected using a hypodermic needle (Tsuda). Five nymphs were collected for DNA analysis on Days 0, 10, 20, and 30 after injection.

Migration of the B. equi parasite to adults was monitored as follows. Fifty detached, engorged nymphs were injected through the integument into the midgut with 4 μL of a solution containing equal volumes of B. equi–infected erythrocytes with 8.8% parasitemia and M199. All nymphs were injected using a hypodermic needle (Tsuda). The infected nymphs were allowed to molt. After molting, the ticks were allowed to feed on rabbits for 3 days to develop sporoblasts. Twenty of these adult ticks were dissected, and DNA was collected from salivary glands and carcass tissues for analysis by nested PCR. The remaining 30 adults were used for indirect immunofluorescence antibody tests (IFATs).

DNA preparation and nested PCR assay.

Babesia-free H. longicornis adults were used as negative controls for PCR, and cultured B. equi merozoites were used as positive controls. DNA extraction of samples from ticks was performed using previously reported methods.11 Extracted DNA was stored in 50 μL TE buffer (10 mmol/L Tris-HCl, 1 mmol/L EDTA) at 4°C until use. Four oligonucleotide primers specific for B. equi were used for the PCR and nested PCR. Primers were derived from the equi merozoite antigen-1 (EMA-1) gene of B. equi. Forward primer EMA5 (5′-TCGACTTCCAGTTGGAGTCC-3′) and reverse primer EMA6 (5′-AGCTCGACCCACTTATCAC-3′) were used in the first amplification reaction. Primers EMA7 (5′-ATTGACCACGTCACCATCGA-3′) and EMA8 (5′-GTCCTTCTTGAGAACGAGGT-3′) were used in nested PCR.21 The 50-μL reaction mixture contained 1 μL of template DNA, 5 μL of 10 × PCR buffer containing 15 mmol/L MgCl2 (Applied Biosystems, Foster City, CA), 5 μL of the respective 2 mmol/L dNTP mix (Applied Biosystems), 1.25 U Taq DNA polymerase (Applied Biosystems), and 50 pmol of B. equi–specific primers for PCR. Amplification conditions were as follows: 94°C for 4 minutes, 40 cycles of denaturation at 94°C for 1 minute, primer annealing at 60°C for 1 minute, and amplification at 72°C for 1 minute, followed by final extension at 72°C for 5 minutes. The product was chilled to 4°C. The final PCR products were subjected to electrophoresis in a 2.0% agarose gel with TBE buffer (89 mmol/L Tris-base, 89 mmol/L boric acid, 2 mmol/L EDTA). B. equi DNA produced visible bands at 268 bp in the first PCR and 218 bp in the second. The amplified DNA was cloned into a pCR 2.1-TOPO vector using a TOPO TA cloning kit (Invitrogen). The entire ligation reaction was used to transform Escherichia coli DH5 α-competent cells. Plasmid DNA from two positive transformants was used for DNA sequencing of the insert. The plasmid containing the gene was isolated and subjected to DNA sequence analysis.

Anti-B. equi mouse serum.

The purified B. equi merozoites used to immunize mice were prepared by treating infected erythrocytes from in vitro cultures with lysis solution (10 mmol/L KHCO3, 155 mmol/L NH4Cl, 0.1 mmol/L EDTA) for 5 minutes at room temperature, centrifugation (11,000g, 10 minutes, 4°C), three washes of the resulting pellet with cold phosphate-buffered saline (PBS), and suspension in PBS. Purified B. equi merozoites (1 × 108) in Freud complete adjuvant (Difco Laboratories, Detroit, MI) were injected intraperitoneally into mice (BALB/c mice; age, 7 weeks). The same antigen in Freud incomplete adjuvant (Difco Laboratories) was injected intraperitoneally into the mice on Days 14, 28, and 42. Sera were collected from immunized mice 10 days after the last immunization.

IFAT.

Salivary glands from adult ticks were prepared on slides, dried, fixed in cold methanol–acetone (1:1) for 20 minutes, and incubated in anti-B. equi mouse serum at 37°C for 1 hour. The slides were washed with PBS for 10 minutes and incubated with fluorescein-conjugated goat anti-mouse IgM + IgG + IgA (H+L; Southern Biotechnology, Birmingham, AL) with 5 μg/mL Hoechst 33258 (Polysciences, Warrington, PA) at 37°C for 1 hour. The slides were washed with PBS for 10 minutes and mounted in 90% glycerol for microscopic observation. Fluorescence microscopy and digital image collection were performed using a Nikon Eclipse E600 fluorescence-DIC microscope (Tokyo, Japan) and a Pixera cooled CCD camera (Penguin 600CL) equipped with InStudio software from Pixera (Los Gatos, CA).

RESULTS

Transovarial transmission of B. equi in H. longicornis.

Of the 22 adults that were injected with B. equi–infected erythrocytes, 2 died immediately. The 20 surviving adults laid ~400 or more eggs each, and eggs from 10 of the 20 adults hatched. DNA was extracted from 20 adults after they had laid eggs, from pooled samples of 200 eggs each from the 20 adults, and from pooled samples of larvae hatched from eggs laid by the 10 adults. Nested PCR revealed the presence of 218-bp bands in extracts from adults, eggs, and larvae (Figure 1). B. equi DNA was amplified from 14 of the adults injected with B. equi (14 of 20; 70.0%), from 10 of the pooled egg samples (10 of 20; 50.0%), and from 7 of the samples of pooled larvae (7 of 10; 70.0%; Table 1).

Transstadial transmission of B. equi in H. longicornis.

PCR analysis revealed that all five nymphs tested were positive for B. equi immediately after injection with B. equi–infected erythrocytes. Samples taken on Days 0 and 10 consisted of five nymphs each, and those taken on Days 20 and 30 consisted of five adults each. All 10 nymphs molted in the 20 days after injection and reached adulthood between Days 20 and 30. The results of nested PCR analyses of the samples are shown in Figure 2. B. equi DNA was amplified from samples taken 10 (4 of 5; 80.0%), 20 (3 of 5; 60.0%), and 30 days after injection (1 of 5; 20.0%).

Babesia equi in the salivary glands of H. longicornis.

Babesia equi was injected into nymphs, and its presence in the salivary glands of the adults after molting was studied. Nested PCR revealed the presence of B. equi in the carcass tissues of 6 of 20 adults (30.0%) and in the salivary glands of 5 of 20 adults (25.0%; Figure 3). These results suggest that B. equi was transmitted transstadially in H. longicornis. None of these adults were B. equi–positive for both carcass and salivary glands. Moreover, salivary gland cells were positive for the IFAT with anti-B. equi mouse serum, suggesting that B. equi was present in salivary gland cells (Figure 4).

DISCUSSION

We injected engorged adult or nymph H. longicornis with B. equi–infected erythrocytes and used nested PCR to show the presence of parasite DNA in adults after oviposition in eggs, larvae, and in adults after molting. These findings suggest that transovarial and transstadial transmission of B. equi occurs in H. longicornis.

Babesia species are generally transmitted transovarially through the eggs of adults to their offspring. However, the normal mode of transmission of B. equi in ticks is considered to be transstadial.22 The recent detection of B. equi DNA in eggs and subsequent larvae from field isolates of Dermacentor nuttalli and Boophilus microplus suggests that transovarial transmission of this parasite does occur.12,13 In our study, when engorged adults were injected with B. equi–infected erythrocytes, B. equi DNA was detected in the adults and in the eggs and larvae. After injection of B. equi–infected erythrocytes into detached, engorged nymphs, B. equi DNA was also detected in the nymphs and in the adults after molting. Moreover, the salivary glands of adults that developed from injected nymphs were positive for B. equi DNA, and salivary gland cells reacted to anti-B. equi mouse serum. These results show that B. equi can be transmitted experimentally by trans-ovarial and transstadial routes and suggest that the route of transmission of B. equi differs from that of most other species of Babesia. In addition, B. equi may be transmitted by H. longicornis as well as known vectors (tick species of the genera Dermacentor, Hyalomma, Rhipicephalus, and Boophilus1218). Although no clinical cases of B. equi infection in Japan have been reported to date,6 our results indicate that horses in Japan are at risk of contracting babesiosis through transmission of B. equi from imported horses by H. longicornis.

Ticks are second only to mosquitoes as vectors of disease-causing agents in humans and are the most important arthropod that transmits pathogens to domestic and wild animals (e.g., Babesia and Theileria protozoa, Rickettsia rickettsia, Borrelia bacteria, and tick-borne encephalitis virus as flavivirus).2326 Our injection method is relatively simple to perform. The needle injection method can probably be applied with other pathogenic tick–transmitted organisms, including rickettsia, bacteria, and viruses, which would be useful for studying transmission methods and interactions of pathogenic organisms and ticks.

In conclusion, we developed a method for transovarial and transstadial transmission of B. equi parasites to H. longicornis using engorged adults or nymphs injected with B. equi–infected erythrocytes. Our findings show that B. equi can be transmitted transovarially and transstadially in H. longicornis, indicating that it is not unlikely that H. longicornis will become a transmission vector for B. equi. Further studies are necessary to determine whether this tick species is able to infect horses with B. equi under field conditions.

Table 1

Summation of detected B. equi DNA in 20 adults, its eggs, and larvae of H. longicornis from engorged adults injected with B. equi–infected erythrocytes using nested PCR

No. positive/no. examinedPercent of positive B. equi
Adult14/2070.0
Egg10/2050.0
Larvae7/1070.0
Figure 1.
Figure 1.

Detection of B. equi DNA in tissue samples of H. longicornis from engorged adults injected with B. equi–infected erythrocytes. Arrowheads indicate the position of the expected 218-bp band. M, 100-bp DNA ladder marker; P, positive control; N, negative control.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 76, 4; 10.4269/ajtmh.2007.76.694

Figure 2.
Figure 2.

Detection of B. equi DNA in tissue samples of H. longicornis from engorged nymphs injected with B. equi–infected erythrocytes. Arrowheads indicate the position of the expected 218-bp band. M, 100-bp DNA ladder marker; P, positive control; N, negative control.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 76, 4; 10.4269/ajtmh.2007.76.694

Figure 3.
Figure 3.

Detection of B. equi DNA in salivary gland and carcass tissue samples of adult H. longicornis injected with B. equi–infected erythrocytes as engorged nymphs. Arrowheads indicate the position of the expected 218-bp band. M, 100-bp DNA ladder marker; P, positive control; N, negative control.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 76, 4; 10.4269/ajtmh.2007.76.694

Figure 4.
Figure 4.

Immunofluorescence detection of B. equi in salivary gland cells. Left, Hoechst staining. Middle, Staining with antibodies against B. equi. Right, Overlay images. Arrowheads indicate cells infected with B. equi. Bar, 50 μm. This figure appears in color at www.ajtmh.org.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 76, 4; 10.4269/ajtmh.2007.76.694

*

Address correspondence to Hiromi Ikadai, Department of Veterinary Parasitology, School of Veterinary Medicine and Animal Sciences, Kitasato University, Towada, Aomori 034-8628, Japan. E-mail: ikadai@vmas.kitasato-u.ac.jp

Authors’ addresses: Hiromi Ikadai, Mizuki Sasaki, Hidekazu Ishida, and Takashi Oyamada, Department of Veterinary Parasitology, School of Veterinary Medicine and Animal Sciences, Kitasato University, Towada, Aomori 034-8628, Japan. Aya Matsuu, Department of Veterinary Internal Medicine, Faculty of Agriculture, Tottori University, Koyama-Minami 4-101, Tottori 680-8553, Japan. Ikuo Igarashi and Kozo Fujisaki, National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan.

Financial support: This study was supported by Grants-in-Aid for Scientific Research and Young Scientists from the Ministry of Education, Culture, Sports, Science and Technology of Japan and the Japan Society for the Promotion of Science; the Sasakawa Scientific Research Grant from the Japan Science Society; and the Kitasato University Research Grant for Young Researchers and Encouragement of Young Scientists.

REFERENCES

  • 1

    Brüning A, Phipps P, Posnett E, Canning EU, 1997. Monoclonal antibodies against Babesia caballi and Babesia equi and their application in serodiagnosis. Vet Parasitol 58 :11–26.

    • Search Google Scholar
    • Export Citation
  • 2

    Friedhoff KT, 1982. The piroplasms of Equidae. Significance for the international equine trade. Berl Munch Tierarztl Wochenschr 95 :368–374.

    • Search Google Scholar
    • Export Citation
  • 3

    Purnell RE, 1981. Babesiosis in various hosts. Ristic M, Krier JP, eds. Babesiosis. New York: Academic Press, 25–64.

  • 4

    Schein E, 1988. Equine babesiosis. Ristic M, ed. Babesiosis of Domestic Animals and Man. Boca Raton, FL: CRC Press, 197–208.

  • 5

    Holbrook AA, 1969. Biology of equine piroplasmosis. J Am Vet Med Assoc 155 :453–454.

  • 6

    Ikadai H, Nagai A, Xuan X, Igarashi I, Tsugihiko K, Tsuji N, Oyamada T, Suzuki N, Fujisaki K, 2002. Seroepidemiologic studies on Babesia caballi and Babesia equi infections in Japan. J Vet Med Sci 64 :325–328.

    • Search Google Scholar
    • Export Citation
  • 7

    Yoshihara T, 1997. Equine piroplasmosis. J Anim Protozool 11 :1–7.

  • 8

    Fujisaki K, 1978. Development of acquired resistance and precipitating antibody in rabbits experimentally infested with females of Haemaphysalis longicornis (Ixodidea: Ixodidae). Nat Inst Anim Health Quart 18 :27–38.

    • Search Google Scholar
    • Export Citation
  • 9

    Fujisaki K, Kawazu S, Kamio T, 1994. The taxonomy of the bovine Theileria spp. Parasitol Today 10 :31–33.

  • 10

    Oliver JH, Herrin CS, 1976. Differential variation of parthenogenetic and bisexual Haemaphysalis longicornis (Acari: Ixodidae). J Parasitol 62 :475–484.

    • Search Google Scholar
    • Export Citation
  • 11

    Rodriguez BJL, Ikadai H, You M, Battsetseg B, Igarashi I, Nagasawa H, Fujisaki K, 2001. Molecular evidence of Babesia caballi (Nuttall and Strickland, 1910) parasite transmission from experimentally-infected SCID mice to the ixodid tick, Haemaphysalis longicornis (Neuman, 1901). Vet Parasitol 102 :185–191.

    • Search Google Scholar
    • Export Citation
  • 12

    Battsetseg B, Lucero S, Xuan X, Claveria FG, Byambaa B, Battur B, Boldbaatar D, Batsukh Z, Khaliunaa T, Battsetseg G, Igarashi I, Nagasawa H, Fujisaki K, 2002. Detection of equine Babesia spp. gene fragments in Dermacentor nuttalli Olenev 1929 infesting Mongolian horses, and their amplification in egg and larval progenies. J Vet Med Sci 64 :727–730.

    • Search Google Scholar
    • Export Citation
  • 13

    Battsetseg B, Lucero S, Xuan X, Claveria FG, Inoue N, Alhassan A, Kanno T, Igarashi I, Nagasawa H, Mikami T, Fujisaki K, 2002. Detection of natural infection of Boophilus microplus with Babesia equi and Babesia caballi in Brazilian horses using nested polymerase chain reaction. Vet Parasitol 107 :375–377.

    • Search Google Scholar
    • Export Citation
  • 14

    Battsetseg B, Xuan X, Ikadai H, Bautista JL, Byambaa B, Boldbaatar D, Battur B, Battsetseg G, Batsukh Z, Igarashi I, Nagasawa H, Mikami T, Fujisaki K, 2001. Detection of Babesia caballi and Babesia equi in Dermacentor nuttalli adult ticks. Int J Parasitol 31 :384–386.

    • Search Google Scholar
    • Export Citation
  • 15

    Friedhoff KT, 1988. Equine babesiosis. Ristic M, ed. Babesiosis of Domestic Animals and Man. Boca Raton, FL: CRC Press, 23–51.

  • 16

    Guimarãs AM, Lima JD, Ribeiro MFB, 1998. Spologony and experimental transmission of Babesia equi by Boophilus microplus. Parasitol Res 84 :323–327.

    • Search Google Scholar
    • Export Citation
  • 17

    Moltmann UG, Mehlhorn H, Schein E, Voigt WP, Friedhoff KT, 1983. Ultrastructural study on the development of Babesia equi (Coccidia: Piroplasmia) in the salivary glands of its vector ticks. J Protozool 30 :218–225.

    • Search Google Scholar
    • Export Citation
  • 18

    Zapf F, Schein E, 1994. The development of Babesia (Theileria) equi (Laveran, 1901) in the gut and the haemolymph of the vector ticks, Hyalomma species. Parasitol Res 80 :297–302.

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Author Notes

Reprint requests: Hiromi Ikadai, Department of Veterinary Parasitology, School of Veterinary Medicine and Animal Sciences, Kitasato University, Towada, Aomori 034-8628, Japan. E-mail: ikadai@vmas.kitasato-u.ac.jp.
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