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1 Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University, College Station, Texas 77843, USA
2 Cardigan Veterinary Clinic, Canaan St., Canaan, New Hampshire 03741, USA
3 Animal Disease Research Unit, Agricultural Research Service, US Department of Agriculture, Pullman, Washington 99164, USA
4 Minnesota Zoological Garden, Apple Valley, Minnesota 55124, USA
5 Corresponding author (email: pholman{at}cvm.tamu.edu)
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Babesia odocoilei is a tick-borne, intraerythrocytic, apicomplexan parasite that is known to infect cervids. First isolated as an unknown Babesia sp. from white-tailed deer (Odocoileus virginianus) in Texas, the parasite was later named B. odocoilei (Emerson and Wright, 1968, 1970). The organism was not transmissible to sheep, goats, or splenectomized calves, but it caused severe anemia and, sometimes, death in splenectomized deer (Emerson and Wright, 1968; Perry et al., 1985; Waldrup, 1991). Although B. odocoilei infections may contribute to decreased body condition in free-ranging deer, experimental inoculation of spleen-intact white-tailed deer and red deer (Cervus elaphus elaphus) resulted in benign infections (Emerson and Wright, 1968; Waldrup, 1991; Holman et al., 2000). Babesia odocoilei parasitemias are found in healthy immature deer, indicating that enzootic stability may exist (Perry et al., 1985). To date, B. odocoilei has not been reported in mammalian hosts outside of the family Cervidae.
Experimental transstadial transmission of B. odocoilei in white-tailed deer by Ixodes scapularis ticks has been demonstrated; however, transovarial transmission and competent vector ticks have not been shown (Waldrup et al., 1990). Dermacentor albipictus ticks were found on elk (C. e. canadensis) infected with B. odocoilei (Holman et al., 1994, 2000). This tick species, however, is not a confirmed vector for B. odocoilei. Moreover, this outbreak occurred within the geographic distribution of I. scapularis on a farm near Del Rio, Texas, USA. Babesia odocoilei infections in various deer hosts have been reported in other regions within the I. scapularis geographic range, including eastern Texas and the Texas post oak savannah, Oklahoma, Minnesota, Wisconsin, Indiana, Virginia, and Massachusetts (Emerson, 1969; Waldrup et al., 1989a, b; Perry et al., 1985; Goethert and Telford, 2003). In addition, B. odocoilei has been identified molecularly in I. scapularis ticks in Maine, Massachusetts, and Wisconsin (Armstrong et al., 1998).
Gene sequence analysis of the small subunit ribosomal RNA (SSU rRNA) gene was used to definitively characterize parasites as B. odocoilei from infections in animals in New Hampshire, New York, Pennsylvania, and California. Furthermore, this report identifies two members of the family Bovidae, desert bighorn sheep (Ovis canadensis nelsoni) and musk oxen (Ovibos moschatus), as additional vertebrate hosts naturally infected by this parasite.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The desert bighorn sheep isolate (CA BH), which was obtained from free-ranging, clinically normal animals in the San Bernardino Mountains, California, has been described previously (Goff et al., 1993). A bull elk (NH Elk) in New Hampshire (Farm 1: 42°42'N, 71°10'W) became ill in October 2002 with clinical babesiosis. Three cases of fatal babesiosis in reindeer occurred during 2003 on farms in New York (42°42'N, 73°36'W; designated as NY R1 and NY R2, respectively) and Pennsylvania (39°42'N, 76°19'W; designated as PA R). Two musk oxen (MN MO1 and MN MO2) housed at the Minnesota Zoological Garden, Apple Valley, Minnesota, USA, developed acute cases of fatal babesiosis in September 2003 and November 2003, respectively. Subsequently, two additional musk oxen at the zoo (MN MO3 and MN MO4) tested positive for B. odocoilei by specific polymerase chain reaction (PCR) testing but did not show clinical signs.
Parasite DNA was obtained from an original cryopreserved blood stabilate of the CA BH isolate (Goff et al., 1993), from blood samples from the reindeer (kindly provided by C. Cripps, Battenkill Veterinary Bovine PC, Greenwich, New York, USA, and J. A. Cohagan, Muddy Creek Veterinary Service, Fawn Ridge, Pennsylvania, USA) and musk oxen, and from cultured parasites from the NH Elk (Table 1
). The parasite culture was established as described previously, and DNA was extracted from the cultures when the parasitemia was 10% (Holman et al., 2003).
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Giemsa-stained blood smears (NY reindeer, PA reindeer, MN musk ox, and NH elk) or Giemsa-stained erythrocyte smears from culture (CA bighorn sheep parasite) were examined under oil at 1,000x magnification. Parasites at the periphery of the erythrocyte (accolé position), small paired piriforms, and erythrocytes containing four or more parasites are characteristic of B. odocoilei (Holman et al., 1988; Gray et al., 1991).
Indirect fluorescent antibody test
Elk serum samples collected from 15 cohort elk on Farm 1, 17 elk on Farm 2 (43°8'N, 70°57'W), and 26 elk on Farm 3 (43°37'N, 72°19'W) in New Hampshire were tested for B. odocoilei antibody activity by the indirect fluorescent antibody test using standard protocols (Goff et al., 1993). Fluorescein-labeled protein G (Sigma) served as the conjugate (Goff et al., 1993). Antigen slides were prepared previously from cultures of B. odocoilei in white-tailed deer erythrocytes and stored frozen at –20 C until use. All serum samples and appropriate positive- and negative-control samples were tested at a 1:80 dilution in phosphate-buffered saline (pH 7.2). All tests were observed under oil immersion at 1,000x magnification on a Labophot-2 model microscope (Nikon, Tokyo, Japan) and scored as positive or negative reactions.
SSU rRNA gene sequence analysis
Genomic DNA was purified from each sample using a standard phenol-chloroform extraction method facilitated by the use of Phase Lock Gel tubes (Phase Lock Gel System, Eppendorf AG, Hamburg, Germany). The SSU rRNA genes were amplified from approximately 50–100 ng of template genomic DNA using 1 pmol each of primers A and B (Fig. 1 and Table 1
) (Sogin, 1990) in a 25-236l reaction volume (Advantage 2 PCR Enzyme System, BD Biosciences, Palo Alto, California) as described previously (Holman et al., 2003). PCR products were separated by electrophoresis through a 1% (w/v) agarose gel alongside a 100 BP marker (Invitrogen Corp., Carlsbad, California, USA) stained with ethidium bromide and the bands visualized by ultraviolet transillumination. In the case of the MN MO3 and MN MO4 isolates, a nested PCR was performed as described above but using 1 µl of the primary PCR product as template. The nested primer set AN and BN was designed just internal to primers A and B, with primer AN located approximately 30 bases downstream of the 5' end and BN located approximately 25 bases upstream of the 3' end (Fig. 1 and Table 1). The DNA isolation, PCR master mix preparation, template addition to the PCR master mix aliquots, and cloning procedures were conducted in separate locations in hoods with dedicated equipment and materials for each step to prevent cross-contamination of samples. Distilled water template controls were included in each PCR experiment to ensure that no carryover contamination occurred. For nested PCR, two water controls were included, one using the primary water control as template and a second with new water as template. Plasmid DNA containing the B. odocoilei SSU rRNA gene insert served as a positive control. Each isolate was handled at a different time frame to prevent cross-contamination.
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. Gene-specific primer sequences are shown in Figure 1. All sequencing reactions and automated sequencing were performed by services either at the Gene Technologies Laboratory (Department of Biology) or the DNA Technologies Laboratory (Department of Veterinary Pathobiology) of Texas A&M University, College Station, Texas, USA. The obtained sequences were analyzed using Sequencher 3.11 software (Gene Codes Corporation, Inc., Ann Arbor, Michigan, USA). A consensus sequence for each cloned isolate was determined. BLAST similarity searches (Altschul et al., 1990) were performed for all SSU rRNA gene sequences obtained (GenBank database, National Center for Biotechnology Information, National Institutes of Health, Bethesda, Maryland, USA). An alignment of the obtained direct and consensus sequences was created using the ClustalW 1.8 Program (http://searchlauncher.bcm.tmc.edu/multi-align/multi-align.html).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). All are small piroplasms (<2.5 µm in length for paired piroplasms) (Levine, 1985). They frequently are seen as tetrads, and they often are located peripherally in the host erythrocyte, as described previously for B. odocoilei (Holman et al., 1988, Gray et al., 1991). Voucher specimens from each host species were deposited at the National Parasite Collection, Beltsville, Maryland, USA, as follows: MN MO1, USNCP 95152 B. odocoilei ex O. moschatus; NH Elk, USNCP 95153 B. odocoilei ex C. e. canadensis; NY R1, USNCP 95154 B. odocoilei ex R. t. tarandus; PA R, USNCP 95155 B. odocoilei ex R. t. tarandus; and CA BH, USNCP 95156 B. odocoilei ex O. c. nelsoni.
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). Sequence details for each isolate are given in Table 1. A consensus sequence from a minimum of three cloned sequences per isolate was deposited into the GenBank database (accession nos. as follows: NY R1, AY661504; NY R2, AY661505; PA R, AY661506; MN MO2, AY661508; MN MO3, AY661509; MN MO 4, AY661510) (Table 1). The sequences obtained directly for CA BH, NH Elk, and MN MO1 were deposited in the GenBank database under accession numbers AY661502, AY661503, and AY661507, respectively (Table 1).
The resulting SSU rRNA sequences for all nine isolates were aligned with the 1,723-bp SSU rRNA sequence for B. odocoilei in the GenBank database (Engeling Texas white-tailed deer isolate, GenBank accession no. U16369) (Holman et al., 2000) (graphic alignment not shown for present study). The consensus SSU rRNA gene sequences from the three reindeer, the New Hampshire elk, and the four musk oxen isolates all showed 100% identity to the B. odocoilei U16369 sequence (Fig. 1). The CA BH isolate showed 99.9% identity to U16369, with a thymidine substituted for a cytosine at position 1290, as indicated in Figure 1.
A serosurvey of elk on three farms in New Hampshire revealed prevalence rates of 100% on Farm 1 (15/15 positive), 100% on Farm 2 (17/17 positive), and 12% on Farm 3 (3/26 positive) for indirect fluorescent antibody activity to B. odocoilei.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The absence of carriers among the caribou raises the question of whether caribou are more prone to severe clinical disease after infection. Because no further cases in caribou have been reported, this question cannot be answered unequivocally. All cases in reindeer, a sister subspecies to caribou (R. t. tarandus and R. t. caribou, respectively), reported to date (including the three current cases and a previous case described by Holman et al., 2003) presented as acute babesiosis, however, and were rapidly fatal. A previous case involved a 7-mo-old reindeer, which strongly suggests that the phenomenon of age-related immunity does not play a role in B. odocoilei infections in reindeer. With some Babesia spp., most notably the agents of bovine and equine babesiosis, young animals are susceptible to infection without clinical manifestations but mount an immune response directed against the infecting parasite (Levine, 1985). Thus, a carrier state is established without clinical disease. As long as the animal maintains a carrier infection, it is refractory to disease unless stressed or otherwise immune compromised. This state may exist in elk, because subclinical carriers have been identified (Gallatin et al., 2003).
With the discovery of B. odocoilei–infected reindeer in New York and Pennsylvania and the infected elk in New Hampshire, the known range of the parasite in mammalian hosts is extended to New England states beyond Massachusetts (Goethert and Telford, 2003). Serosurveys of elk herds at different localities in New Hampshire showed a prevalence rate of 100% at two and a low prevalence of exposure (12%) at the third. These data suggest that B. odocoilei is endemic in New Hampshire. Moreover, molecular epidemiologic studies demonstrated B. odocoilei DNA in I. scapularis ticks as far north as Maine (Armstrong et al., 1998). The known range of B. odocoilei will, in time, likely correlate with the leading territorial edge of the vector tick distribution.
Babesia spp. parasites of clinically normal, free-ranging desert bighorn sheep (O. c. nelsoni) in the San Bernardino Mountains near Los Angeles, California, USA, were reported in 1993 (Goff et al., 1993; Thomford et al., 1993). The B. odocoilei–like parasites described in a Rocky Mountain bighorn lamb (Ovis canadensis canadensis) subinoculated with pooled blood from the free-ranging animals (Goff et al., 1993) were confirmed in the present study by molecular analysis to be B. odocoilei. Three additional morphologically distinct small piroplasm isolates, designated as BH1, BH2, and BH3, were obtained by cultivation of infected blood from desert bighorn sheep (Thomford et al., 1993). Both BH1 and BH2 were similar in size to B. odocoilei, but they were not found in the accolé position characteristic of B. odocoilei. Unlike B. odocoilei, BH3 is a large piroplasm (piriform average length, 2.6 µm) (Thomford et al., 1993). Subsequent SSU rRNA gene sequencing of BH1 and BH3 showed them to be divergent from B. odocoilei (Kjemtrup et al., 2000); the SSU rRNA gene sequence for BH2 has not been reported.
The definitive identification of the desert bighorn sheep Babesia sp. isolated by Goff et al. (1993) as B. odocoilei is significant, because this is the first recognition of a vertebrate host for this parasite outside the family Cervidae. Furthermore, this delineation expands the known geographic range of the hemoparasite to California, which is outside the geographic distribution of the known vector tick, I. scapularis.
The geographic range of I. scapularis includes the state of Florida in the southeastern United States north to the Canadian provinces of Nova Scotia and Prince Edward Island, west to North Dakota and South Dakota, and south to the Mexican state of Coahuila (Keirans et al., 1996), excluding the western coastal United States. In fact, using a predictive model, Brownstein et al. (2003) conclude that the California habitat is unsuitable for supporting I. scapularis. Therefore, an alternate vector tick species for B. odocoilei must be active in California. Ixodes sp. ticks infested bighorn sheep in the geographic region from which the CA BH isolate was obtained (Goff et al., 1993). This observation suggests that Ixodes pacificus may vector B. odocoilei in California. Clinically affected elk infected with B. odocoilei in Texas were infested with D. albipictus ticks, a species that is widely distributed in California but concentrated in the central coastal and Sierra foothill areas of California (Holman et al., 1994; Furman and Loomis, 1984). The roles of these two tick species in the biology of B. odocoilei remain to be confirmed.
The addition of musk ox (O. moschatus) as another member of the family Bovidae that is susceptible to B. odocoilei infection is noteworthy. Babesia odocoilei is not transmissible to domestic sheep, goats, or splenectomized calves (Emerson and Wright, 1968). The fact that two members of the Bovidae family have now emerged as competent, naturally infected hosts suggests that other as-yet-unidentified host species may exist and indicates the potential for babesiosis outbreaks in previously unknown hosts.
Molecular tools may aid in predicting the future geographic distribution of B. odocoilei infections. Armstrong et al. (1998) showed molecular evidence of B. odocoilei in salivary glands of I. scapularis ticks in Wisconsin, Massachusetts, and Maine. In Wisconsin, B. odocoilei infections in elk and reindeer are documented, and B. odocoilei has been confirmed molecularly in white-tailed deer in Massachusetts (Goethert and Telford, 2003; Holman et al., 2003). To date there have been no reports of infected animals in Maine. The parasite is likely to emerge in additional animal hosts and geographic areas as it is introduced via translocation of infected animals into new environments or as naïve animals are introduced into endemic environments. Effective tick-control measures and judicious animal management practices should be implemented to prevent transmission of this tick-borne pathogen to susceptible animals.
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ACKNOWLEDGMENTS |
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Received for publication 27 September 2004.
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, 
). The New Hampshire (NH) elk isolate sequence begins at position 55 and ends at position 1,662 (>, <). The California (CA) bighorn sheep and MN musk ox 1 isolate sequences begin at position 56 and end at position 1,660 (
, 
). All sequences were identical to that of U16369 except for a single base substitution in the CA bighorn sheep isolate sequence (thymidine for a cytosine at position 1,290; bold type, underlined). The positions of primers A and B are indicated by broken underlining, of primers AN and BN by dotted underlining, and of 528F by bold underlining. Primer orientation is indicated by arrows.