A Fatal Case of Babesiosis in Missouri

Identification of Another Piroplasm That Infects Humans
Annals of Internal Medicine 1 April 1996. 124:643-650. 

Barbara L. Herwaldt, MD, MPH; David H. Persing, MD, PhD; Eric A. Précigout, PhD; W. L. Goff, PhD; Dane A. Mathiesen, BS; Philip W. Taylor, MD; M. L. Eberhard, PhD; and André F. Gorenflot, PhD 

Objective: To characterize the etiologic agent (MO1) of the first reported case of babesiosis acquired in Missouri. 

Design: Case report, serologic testing, animal inoculations, and molecular studies. 

Setting: Southeastern Missouri. 

Patient: A 73-year-old man who had had a splenectomy and had a fatal case of babesiosis. 

Measurements: Serum specimens from the patient were assayed by indirect immunofluorescent antibody testing and immunoprecipitation for reactivity with antigens from various Babesia species. Whole blood obtained from the patient before treatment was inoculated into hamsters and jirds and into calves and bighorn sheep that had had splenectomy and were immunosuppressed with dexamethasone. Piroplasm-specific nuclear small-subunit ribosomal DNA was recovered from the patient's blood by using broad-range amplification with the polymerase chain reaction; a 144 base-pair region of the amplification product was sequenced; and phylogenetic analysis was done to compare MO1 with various Babesia species. 

Results: Indirect immunofluorescent antibody testing showed that the patient's serum had strong reactivity with Babesia divergens, which causes babesiosis in cattle and humans in Europe, but that it had minimal reactivity with B. microti and WA1, which are the piroplasms previously known to cause zoonotic babesiosis in the United States. Immunoprecipitations showed that MO1 is more closely related to B. divergens than to B. canis (a canine parasite). None of the experimentally inoculated animals became demonstrably parasitemic. Phylogenetic analyses, after DNA sequencing, showed that MO1 is most closely related to B. divergens (100% similarity). 

Conclusions: Although MO1 is probably distinct from B. divergens, the two share morphologic, antigenic, and genetic characteristics; MO1 probably represents a Babesia species not previously recognized to have infected humans. Medical personnel should be aware that patients in the United States can have life-threatening babesiosis even though they are seronegative to B. microti and WA1 antigen. 

Ann Intern Med. 1996;124:643-650. Annals of Internal Medicine 

From the Centers for Disease Control and Prevention, Atlanta, Georgia; Mayo Clinic and Foundation, Rochester, Minnesota; Université Montpellier, Montpellier, France; U.S. Department of Agriculture, Pullman, Washington; and Cape Girardeau Physician Associates, Cape Girardeau, Missouri.   

For current author addresses, see end of text.

Human cases of the tick-borne disease babesiosis are caused by the bovine parasite Babesia divergens in Europe (1, 2), by the rodent parasite B. microti in the northeastern and upper midwestern United States (2, 3), and by WA1-type piroplasms in Washington and California (4-6). We describe the first reported zoonotic case of babesiosis acquired in Missouri and provide evidence to show that this fatal case was caused by an intraerythrocytic piroplasm (MO1) that is probably distinct from but shares morphologic, antigenic, and molecular characteristics with B. divergens. 

Case Report  

A 73-year-old man was hospitalized on 1 July 1992 because of fever, a rigor, and thrombocytopenia. He had developed a dry cough, mild headache, sore throat, and joint pain 4 days before admission and had had a temperature of 38.9 °C and a platelet count of 70 × 109/L (baseline count, 100 × 109/L to 150 × 109/L) 2 days before admission. He began receiving erythromycin therapy on an outpatient basis but did not improve. 

His medical history included systemic lupus erythematosus, which had been diagnosed in 1979 and for which he was taking prednisone (10 mg/d). He had had a splenectomy in 1979 because of hemolytic anemia and thrombocytopenia and had had an intracerebral hemorrhage in 1989 because of thrombocytopenia. Except for proteinuria due to membranous glomerulonephritis, his systemic lupus erythematosus had been quiescent since that time. His medical history was also notable for a myocardial infarction in 1986 and recurrent supraventricular tachycardia, for which he was taking digoxin. 

The patient lived with his wife on 1 acre of land (all mowed or gardened) in a rural area of southeastern Missouri (Cape Girardeau County). He primarily stayed indoors, but he mowed the lawn with a riding mower and did some gardening. He had not traveled outside Missouri in the previous 3 to 4 years, had not traveled outside the midwestern United States in the previous 10 years, and had never been in a country other than the United States. He had no pets or known tick exposures. He had intermittently been employed to feed dairy cattle, which neighbors kept about 1 mile from his home, until 8 years before his hospitalization on 1 July 1992. 

On admission to the hospital, the patient was febrile (Table 1), had a small effusion in one knee joint, and had slight pain on shoulder rotation. He was thrombocytopenic (Table 1), his total bilirubin and lactase dehydrogenase levels were elevated, a 24-hour urine specimen contained 11.4 g of protein, and his creatinine clearance was 0.95 mL/s (57 mL/min). Complement levels were normal, no anti-DNA antibody was detectable, and his antinuclear antibody titer was 80 (speckled pattern), suggesting that his systemic lupus erythematosus was quiescent. The patient tested negative for antibody to the human immunodeficiency virus, and blood and urine cultures obtained on 1 July and periodically thereafter also tested negative. He was treated with aztreonam, cefazolin, and an increased dosage of prednisone (80 mg/d). 

On 2 July, babesiosis was diagnosed after intraerythrocytic ring forms were noted on the patient's blood smear (Table 1, Figure 1). The antibiotic regimen was changed to oral quinine sulfate, 650 mg three times daily, and intravenous clindamycin, 600 mg three times daily. The patient became afebrile on 3 July, but his parasitemia level continued to increase. His lactate dehydrogenase, total bilirubin, and creatinine levels also increased. Hemodialysis was instituted on 6 July and was provided periodically thereafter. By 11 July, the prednisone dosage had been reduced to 10 mg/day. By 13 July, the 12th day of therapy for babesiosis, the patient's parasitemia level had markedly decreased. 

On 15 July, the patient had a cardiopulmonary arrest that was attributed to hypoxemia; he was intubated and resuscitated. Diffuse pulmonary infiltrates were noted and were thought to be at least partly due to volume overload. After his arrest, the patient had a generalized seizure and never fully regained his baseline mental status. Intravenous methylprednisolone therapy (10 mg three times daily) was started. The quinine dosage was decreased to 650 mg twice daily because of high quinine levels (as high as 7.1 µg/mL). On 16 July, the patient again became febrile, but no parasites were noted on his blood smear; ceftazidime therapy was started because of Enterobacter cloacae pneumonia. On 17 July, the patient developed ventricular tachycardia and was cardioverted. On 20 July, the 20th day of hospitalization, supportive therapy was discontinued, and the patient died. No autopsy was done. 

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Methods:  

Serologic Assays 

At the Centers for Disease Control and Prevention, indirect immunofluorescent antibody testing was used to assay serum specimens, in serial fourfold dilutions, for reactivity to B. microti and WA1 antigens (4, 7). At the Laboratoire de Biologie Cellulaire, serum specimens were tested for antibody to B. divergens and B. canis (a canine piroplasm). Indirect immunofluorescent antibody testing and immunoprecipitation assays were done as previously described (8, 9); however, the immunoprecipitation assays were done on long-term cultures of B. divergens (human isolate Rouen 1987) (9) but on short-term cultures of B. canis (isolate Gignac 1994) (10). Babesia divergens had been obtained from a naturally infected human and maintained in jirds (Mongolian gerbils [Meriones unguiculatus]) by syringe passage twice weekly (11) or in long-term in vitro culture (9). Babesia canis had been obtained from a naturally infected dog. 

At the U.S. Department of Agriculture, serum specimens were tested for antibody to bovine isolates of B. divergens (German isolate) and B. bovis (Mexican isolate); a Babesia species from desert bighorn sheep (Ovis canadensis nelsoni; California isolate) that is morphologically similar to B. divergens and serologically crossreacts with it to some degree (12); and B. odocoilei (Texas isolate), a parasite of white-tailed deer (Odocoileus virginianus) (13, 14). The techniques for obtaining in vitro-derived antigens from these isolates have been described previously (12, 15-18). Indirect immunofluorescent antibody testing was done as previously described (19), except that fluorescein-conjugated recombinant protein G was used to detect specific IgG (20). When human serum specimens were tested, fluorescein-conjugated goat antihuman IgG (Kirkegaard and Perry Laboratories, Gaithersburg, Maryland) was used. 

Animal Inoculations 

Whole blood from the patient was inoculated into hamsters (Mesocricetus auratus), jirds (some of which were immunosuppressed with dexamethasone), and calves and bighorn sheep that had had splenectomy and were immunosuppressed (Table 2). Hamsters and jirds are suitable animal hosts for both B. microti and WA1 (4); jirds and calves are suitable hosts for B. divergens (2, 11); and bighorn sheep (or the bighorn sheep culture system) are suitable hosts for various Babesia species that infect wild ruminants (12, 23). Sheep BHR-32 (Table 2) was challenged intravenously on day 63 after inoculation with a stabilate of the bighorn Babesia species (2 × 108 merozoites) that had been cryopreserved in polyvinylpyrollidone-40 (Sigma, St. Louis, Missouri). On day 27, calf C-03 was challenged intravenously with a cryopreserved stabilate of B. bovis (2 × 108 merozoites), and sheep BHR-34 was challenged intravenously with a cryopreserved stabilate of the bighorn Babesia species (2 × 108 merozoites). 

In Vitro Culturing 

At the Laboratoire de Biologie Cellulaire, 0.5-mL aliquots of whole blood obtained from the patient before treatment on 2 July and cryopreserved in 10% dimethyl sulfoxide were used for each of two in vitro culture systems: B. divergens (9) and B. canis (10). Human erythrocytes were used for the former; canine erythrocytes were used for the latter. At the U.S. Department of Agriculture, in vitro culturing of blood from bighorn sheep (before and after inoculation with the patient's blood; Table 2) was attempted as previously described (16, 17), except that bighorn sheep erythrocytes and medium supplemented with bighorn sheep serum were used. The sheep blood was cultured fresh, with the exception of the specimen taken before inoculation from the sheep inoculated in May (Table 2); this specimen had been cryopreserved in polyvinylpyrollidone-40. The cultures were monitored for 30 days. 

Molecular Studies 

At the Mayo Clinic, MO1 DNA was isolated (24) from whole blood that had been obtained from the patient on 2 July and cryopreserved in 10% dimethyl sulfoxide. Broad-range amplification with the polymerase chain reaction, to recover piroplasm-specific nuclear small-subunit ribosomal DNA, and DNA sequence analysis of a 144 base-pair region of the amplification product were done as previously described (5, 6). Phylogenetic analysis was done by maximum parsimony analysis in PAUP (phylogenetic analysis using parsimony) version 3.1.1 (25); the analysis included 119 alignable nucleotides and 28 phylogenetically informative positions. The sequences for the other pathogens included in the analysis were previously known (5, 6, 26); the GenBank accession number for the nuclear small-subunit ribosomal RNA gene for B. odocoilei is U16369 (26). 

Results:  
Morphologic Analysis 

Most of the intraerythrocytic parasites noted on the patient's blood smear (Figure 1) were in a subcentral position; those in a subperipheral position did not protrude from the erythrocytes. Most erythrocytes were multiply infected. The parasites were polymorphic. Punctiform (<1 µm in diameter), annular (1 mm to 2.5 mm in diameter), piriform (1 µm to 2.5 mm in length), and tetrad ("Maltese cross") forms were noted. These morphologic features are consistent with but not diagnostic for human infections with B. divergens (1, 2, 9). 

Serologic Assays 

Indirect immunofluorescent antibody testing was done to determine whether MO1 could be classified by species through differential reactivity with antigen from various Babesia species. The patient's serum specimens (one obtained on 2 July, the other on 16 July) had minimal reactivity to B. microti (titer, 16), WA1 (titers, 16 on 2 July and 64 on 16 July), and B. odocoilei (titers, negative on 2 July and <40 on 16 July); modest reactivity to B. canis (titer, 160) and B. bovis (titers, <80 on 2 July and 160 on 16 July); moderate reactivity to the bighorn Babesia species (titers, 80 on 2 July and 1280 on 16 July); and strong reactivity to B. divergens (titers, 1024 on 2 July and 4096 on 16 July at the Laboratoire de Biologie Cellulaire and 1280 on 2 July and 5120 on 16 July at the U.S. Department of Agriculture). Serum specimens obtained on 17 July from the patient's daughter and wife did not react to B. microti or WA1 and had minimal reactivity to B. divergens (titers of 16 [daughter] and 32 [wife] compared with 4 [human negative control]) that may have been the result of nonspecific fluorescence. 

Immunoprecipitation assays done with a serum specimen from the patient showed that MO1 is more closely related to B. divergens than to B. canis (Figure 2). In contrast, B. microti did not show crossreactivity with B. divergens or B. canis, except for a 70-kd antigen that is a heat shock protein common to Babesia and Plasmodia species (27). 

Animal Inoculations  

To determine the host specificity of MO1, various animals were inoculated experimentally with the patient's blood (Table 2). None of the animals, even those that were immunosuppressed, became demonstrably parasitemic. However, one calf (C-674BL) died 8 days after inoculation from ulcerative colitis attributed to corticosteroid treatment, and another (C-204) died on day 16 after inoculation because of medical problems unrelated to babesiosis. The only animals that developed clinical or laboratory signs suggestive of infection (but of unknown cause) were two sheep (BHR-32 and BHR-33) whose hematocrits decreased and temperatures increased (Table 2). Cultures of sheep blood (before inoculation and on day 20 after inoculation) were negative for piroplasms, and the inoculated sheep did not develop demonstrable antibody to bighorn Babesia species antigen. 

For the animals experimentally inoculated in September 1994, cultures of sheep blood before and after inoculation tested negative for piroplasms. The sheep and calves did not develop detectable reactivity (by indirect immunofluorescent antibody testing) to bighorn Babesia species or B. bovis antigen. Testing of sheep and calf serum specimens obtained before and after inoculation (May and September inoculations) at the Laboratoire de Biologie Cellulaire did not show specific immunoprecipitation of B. divergens antigen. 

The sheep (BHR-32 and BHR-34) that were subsequently challenged with the bighorn Babesia species and the calf (C-03) that was subsequently challenged with B. bovis became clinically ill, anemic, and parasitemic (for example, BHR-32 developed a parasitemia level of >80%, a hematocrit of 0.09, and severe hemoglobinuria and died on day 5). 

In Vitro Culturing  

Rare live parasites resembling B. divergens were noted 24 hours after initiation of the B. divergens (but not the B. canis) culture system. None were seen 12 hours later. 

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Molecular Studies 

Sequence analysis (Figure 3) of a region of nuclear small-subunit ribosomal DNA previously shown to be useful for phylogenetic analysis for piroplasms (5, 6) showed that MO1 and B. divergens had identical sequences (100% similar). The percentages of similarity between MO1 and B. odocoilei, B. canis, B. microti, WA1, and Toxoplasma gondii were 97.6%, 93.4%, 86.6%, 85.9%, and 75.8%, respectively. Phylogenetic analysis confirmed that MO1 is most closely related to B. divergens and lies in a phylogenetic cluster that includes B. odocoilei (Figure 4). The other known zoonotic pathogens (WA1, CA1, and B. microti) lie in more remote clusters. 

Discussion  

We have described the first reported human case of babesiosis acquired in Missouri and have presented evidence to show that the etiologic agent of this case (MO1) is distinct from B. microti and WA1-type parasites, the piroplasms previously known to cause zoonotic babesiosis in the United States. In our attempts to classify MO1 by species, we considered morphologic, host-specificity, antigenic, and molecular criteria. We have concluded that MO1 probably is distinct from but shares features with the bovine parasite B. divergens, which causes babesiosis in cattle and humans in Europe and reportedly is not present in the United States. 

Traditionally, morphologic and host-specificity criteria have been used to classify Babesia organisms, but these criteria are inadequate for definitive species identification (2, 4). The morphologic features of MO1 by light microscopy are consistent with those of B. divergens in humans. However, piroplasms of various species can have similar morphologic features, and piroplasms of a given species can look dissimilar in different hosts. 

Our attempts to infect hamsters, jirds, calves, and bighorn sheep with MO1 were unsuccessful. Even severely immunosuppressed animals were refractory to infection. We were particularly interested in whether jirds and calves would become parasitemic, because these animals are good hosts for B. divergens (2, 11), at least for the B. divergens isolates from human patients in Europe. Our efforts at experimental infection may have been hindered because the immunosuppressed animals were inoculated with cryopreserved, rather than fresh, blood and because we had only limited supplies of the patient's blood to use for the inoculations. 

On the basis of serologic criteria, we conclude that MO1 is much more closely related to B. divergens and somewhat more closely related to the bighorn Babesia species than to B. microti, WA1-type parasites, B. odocoilei, B. canis, B. bovis, or another bovine parasite, B. bigemina (the relatedness of MO1 to B. bigemina was determined by indirect immunofluorescent antibody testing. Carson A, Bailey C. Personal communication). Various Babesia species not known to be zoonotic crossreact with B. divergens (12, 23). Because we did not obtain an isolate of MO1 and thus did not generate homologous antigen for serologic testing, we could not compare the reactivity of our patient's serum with B. divergens and with MO1 antigen. In addition, because our knowledge about the antigenic diversity among B. divergens strains is limited (28, 29), we do not know whether strain (as opposed to species) differences could account for the different results obtained with immunoprecipitation assays for MO1 serum and for serum from a European patient infected with B. divergens (Table 2, top). However, in Europe, serum specimens from patients infected with B. divergens who have been tested to date have reacted with the same B. divergens (isolate Rouen 1987) antigens (Figure 2, top), as have serum specimens from cattle infected with various isolates of B. divergens (28, 29). 

Our molecular data are also consistent with the conclusions that MO1 is closely related to B. divergens and is very different from the other known zoonotic pathogens, B. microti and WA1-type parasites; a well-pedigreed strain of B. divergens (30) was used for the genetic analyses. Because the DNA of relatively few Babesia species has been sequenced, we do not know whether any other species are indistinguishable from B. divergens in the DNA region that we sequenced. After B. divergens, MO1 is most closely related to, but clearly distinct from, B. odocoilei. The bighorn Babesia species, which is more closely related than B. odocoilei to MO1 by serologic criteria, has not yet been sequenced. Additional molecular data (from DNA hybridization studies) distinguish MO1 from B. bovis and B. bigemina (Carson A, Bailey C. Personal communication). 

The clinical implications of zoonotic infection with MO1 (for example, whether infection would typically cause severe disease in humans) remain to be elucidated. Our patient was elderly, had had his spleen removed, and was receiving prednisone therapy for systemic lupus erythematosus, factors that may have placed him at increased risk for a severe case of babesiosis. However, because no autopsy was done, we do not know the extent to which his systemic lupus erythematosus (which apparently was quiescent at the time he was hospitalized) and medical problems other than babesiosis contributed to his acute renal failure and death. In addition, we do not know whether more aggressive therapy for babesiosis, such as an exchange transfusion, would have changed his clinical course and outcome. 

In conclusion, MO1 appears to be B. divergens-like but distinct from B. divergens, and it seems to represent a species not previously recognized to be zoonotic. Although, to our knowledge, no other cases of MO1 infection have been diagnosed, this case highlights the need for vigilance for vector- borne infections not formerly reported in the United States. Medical personnel should be aware that patients can have babesiosis even if they have not been in areas known to be endemic for babesiosis, do not recall exposure to ticks, and are not seroreactive to B. microti or WA1 antigen. 

Acknowledgments: The authors thank the patient and his family and Patricia A. Conrad, DVM, PhD, Jennifer W. Dickerson, BA, K. Friedhoff, DMV (for provision of B. divergens in vitro antigen), Theodore J. Grieshop, MD, W. Carl Johnson, MS, Donald O. Miles, PhD, Stanley D. Sides, MD, John W. Thomford, PhD, Essie M. Walker, Doris A. Ware, and Marianna Wilson, MS, for their contributions. 

Grant Support: Dr. Persing is supported by Public Health Service grants AI32403, AR41497, and AI30548. 

Requests for Reprints: Barbara L. Herwaldt, MD, MPH, Centers for Disease Control and Prevention, Division of Parasitic Diseases, Mailstop F-22, 4770 Buford Highway NE, Atlanta, GA 30341-3724. 

Current Author Addresses: Drs. Herwaldt and Eberhard: Centers for Disease Control and Prevention, Division of Parasitic Diseases, Mailstop F-22, 4770 Buford Highway NE, Atlanta, GA 30341-3724. 
Dr. Persing and Ms. Mathiesen: Division of Experimental Pathology, Department of Laboratory Medicine and Pathology, and Division of Infectious Diseases, Department of Medicine, Mayo Clinic and Foundation, Rochester, MN 55905. 
Drs. Précigout and Gorenflot: Université Montpellier, Laboratoire de Biologie Cellulaire, Faculté de Pharmacie, 15, Avenue Charles Flahault, 34060 Montpellier Cedex, France. 
Dr. Goff: Animal Disease Research Unit, Agricultural Research Service, United States Department of Agriculture, Room 337, Bustad Hall, Washington State University, Pullman, WA 99164-7030. 
Dr. Taylor: Cape Girardeau Physician Associates, 14 Doctors' Park, Cape Girardeau, MO 63703-4993. 

References

1. Brasseur P, Gorenflot A. Human babesiosis in Europe. Mem Inst Oswaldo Cruz. 1992;87(Suppl 3):131-2. 
2. Telford SR 3d, Gorenflot A, Brasseur P, Spielman A. Babesial infections in humans and wildlife. Parasitic Protozoa. 1993;5:1-47. 
3. Herwaldt BL, Springs FE, Roberts PP, Eberhard ML, Case K, Persing DH, et al. Babesiosis in Wisconsin: a potentially fatal disease. Am J Trop Med Hyg. 1995;53:146-51. 
4. Quick RE, Herwaldt BL, Thomford JW, Garnett ME, Eberhard ML, Wilson M, et al. Babesiosis in Washington State: a new species of Babesia? Ann Intern Med. 1993;119:284-90. 
5. Thomford JW, Conrad PA, Telford SR 3d, Mathiesen D, Bowman BH, Spielman A, et al. Cultivation and phylogenetic characterization of a newly recognized human pathogenic protozoan. J Infect Dis. 1994;169:1050-6. 
6. Persing DH, Herwaldt BL, Glaser C, Lane RS, Thomford JW, Mathiesen D, et al. Infection with a babesia-like organism in northern California. N Engl J Med. 1995;332:298-303. 
7. Chisholm ES, Ruebush TK 2d, Sulzer AJ, Healy GR. Babesia microti infection in man: evaluation of an indirect immunofluorescent antibody test. Am J Trop Med Hyg. 1978;27(1 Pt 1):14-9. 
8. Gorenflot A, Précigout E, Bissuel G, Lecointre O, Brasseur P, Vidor E, et al. Identification of major Babesia divergens polypeptides that induce protection against homologous challenge in gerbils. Infect Immun. 1990;58:4076-82. 
9. Gorenflot A, Brasseur P, Précigout E, L'Hostis M, Marchand A, Schrevel J. Cytological and immunological responses to Babesia divergens in different hosts: ox, gerbil, man. Parasitol Res. 1991;77:3-12. 
10. Schetters TP, Kleuskens J, Scholtes N, Bos HJ. Vaccination of dogs against Babesia canis infection using parasite antigens from in vitro culture. Parasite Immunol. 1992;14:295-305. 
11. Lewis D, Williams H. Infection of the Mongolian gerbil with the cattle piroplasm Babesia divergens. Nature. 1979;278:170-1. 
12. Goff WL, Jessup DA, Waldrup KA, Thomford JW, Conrad PA, Boyce WM, et al. The isolation and partial characterization of a Babesia sp. from desert bighorn sheep (Ovis canadensis nelsoni). J Eukaryot Microbiol. 1993;40:237-43. 
13. Waldrup KA, Kocan AA, Qureshi T, Davis DS, Baggett D, Wagner GG. Serological prevalence and isolation of Babesia odocoilei among white-tailed deer (Odocoileus virginianus) in Texas and Oklahoma. J Wildl Dis. 1989;25:194-201. 
14. Kocan AA, Kocan KM. Tick-transmitted protozoan diseases of wildlife in North America. Bulletin of the Society of Vector Ecology. 1991;16:94-108. 
15. Winger CM, Canning EU, Culverhouse JD. A monoclonal antibody to Babesia divergens which inhibits merozoite invasion. Parasitology. 1987;94:17-27. 
16. Goff WL, Yunker CE. Babesia bovis: increased percentage parasitized erythrocytes in cultures and assessment of growth by incorporation of [3H]hypoxanthine. Exp Parasitol. 1986;62:202-10. 
17. Goff WL, Yunker CE. Effects of pH, buffers and medium-storage on the growth of Babesia bovis in vitro. Int J Parasitol. 1988;18:775-8. 
18. Holman PJ, Waldrup KA, Wagner GG. In vitro cultivation of a Babesia isolated from a white-tailed deer (Odocoileus virginianus). J Parasitol. 1988;74:111-5. 
19. Goff WL, Wagner GG, Craig TM, Long RF. The bovine immune response to tick-derived Babesia bovis infection: serological studies of isolated immunoglobulins. Vet Parasitol. 1982;11:109-20. 
20. Tibbitts T, Goff W, Foreyt W, Stiller D. Susceptibility of two Rocky Mountain bighorn sheep to experimental infection with Anaplasma ovis. J Wildl Dis. 1992;28:125-9. 
21. Goff WL, Davis WC, Palmer GH, McElwain TF, Johnson WC, Bailey JF, et al. Identification of Babesia bovis merozoite surface antigens by using immune bovine sera and monoclonal antibodies. Infect Immun. 1988;56:2363-8. 
22. Kock MD, Jessup DA, Clark RK, Franti CE. Effects of capture on biological parameters in free-ranging bighorn sheep (Ovis canadensis): evaluation of drop-net, drive-net, chemical immobilization and the net-gun. J Wildl Dis. 1987;23:641-51. 
23. Goff WL, Alperin D, Holman PJ, Wagner GG. Comparison of a Babesia sp isolated from desert bighorn sheep with other wild ruminant Babesia spp and Babesia odocoilei [Abstract]. In: Proceedings of the Ninth International Veterinary Hemoparasite Disease Conference; October 6-9, 1993; Merida, Yucatan, Mexico. 
24. Pancholi P, Kolbert CP, Mitchell PD, Reed KD, Dumler JS, Bakken JS, et al. Ixodes dammini as a potential vector of human granulocytic ehrlichiosis. J Infect Dis. 1995;172:1007-12. 
25. Swofford DL. PAUP: phylogenetic analysis using parsimony. Version 3.1.1. Champaign, IL: Illinois Natural History Survey; 1991. 
26. Holman PJ. Veterinary Pathobiology. Thesis. College Station, TX: Texas A&M University; 1994. 
27. Carcy B, Précigout E, Valentin A, Gorenflot A, Reese RT, Schrével J. Heat shock response of Babesia divergens and identification of the hsp70 as an immunodominant early antigen during ox, gerbil and human babesiosis. Biol Cell. 1991;72:93-102. 
28. Phillips RS, Reid GM, McLean SA, Pearson CD. Antigenic diversity in Babesia divergens: preliminary results with three monoclonal antibodies to the rat-adapted strain. Res Vet Sci. 1987;42:96-100. 
29. Précigout E, Gorenflot A, Valentin A, Bissuel G, Carcy B, Brasseur P, et al. Analysis of the immune responses of different hosts to Babesia divergens isolates from different geographic areas and capacity of culture-derived exoantigens to induce efficient cross-protection. Infect Immun. 1991;59:2799-805. 
30. Purnell RE, Brocklesby DW, Hendry DJ, Young ER. Separation and recombination of Babesia divergens and Ehrlichia phagocytophila from a field case of redwater from Eire. Vet Rec. 1976;99:415-7. 

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