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Texas Babesia

  1. Parasit Vectors. 2010 Apr 8;3(1):36. [Epub ahead of print]

    One Health Approach to Identify Research Needs in Bovine and Human Babesioses: Workshop Report.

    Perez de Leon AA, Strickman DA, Knowles DP, Fish D, Thacker E, de la Fuente J, Krause PJ, Wikel SK, Miller RS, Wagner GG, Almazan C, Hillman R, Messenger MT, Ugstad PO, Duhaime RA, Teel PD, Ortega-Santos A, Hewitt DG, Bowers EJ, Bent SJ, Cochran MH, McElwain TF, Scoles GA, Suarez CE, Davey R, Howell Freeman JM, Lohmeyer K, Li AY, Guerrero FD, Kammlah DM, Phillips P, Pound JM, And Development In The U S GF.

    ABSTRACT: BACKGROUND: Babesia are emerging health threats to humans and animals in the United States. A collaborative effort of multiple disciplines to attain optimal health for people, animals and our environment, otherwise known as the One Health concept, was taken during a research workshop held in April 2009 to identify gaps in scientific knowledge regarding babesioses. The impetus for this analysis was the increased risk for outbreaks of bovine babesiosis, also known as Texas cattle fever, associated with the re-infestation of the U.S. by cattle fever ticks. RESULTS: The involvement of wildlife in the ecology of cattle fever ticks jeopardizes the ability of state and federal agencies to keep the national herd free of Texas cattle fever. Similarly, there has been a progressive increase in the number of cases of human babesiosis over the past 25 years due to an increase in the white-tailed deer population. Human babesiosis due to cattle-associated Babesia divergens and Babesia divergens-like organisms have begun to appear in residents of the United States. Research needs for human and bovine babesioses were identified and are presented herein. CONCLUSIONS: The translation of this research is expected to provide veterinary and public health systems with the tools to mitigate the impact of bovine and human babesioses. However, economic, political, and social commitments are urgently required, including increased national funding for animal and human Babesia research, to prevent the re-establishment of cattle fever ticks and the increasing problem of human babesiosis in the United States.

    PMID: 20377902 [PubMed - as supplied by publisher]

  2. Vet Clin North Am Small Anim Pract. 2009 Nov;39(6):1035-53, v.

    Canine hepatozoonosis and babesiosis, and feline cytauxzoonosis.

    Holman PJ, Snowden KF.

    Department of Veterinary Pathobiology, College of Veterinary Medicine, Mailstop 4467, Texas A&M University, College Station, TX 77843-4467, USA.

    The apicomplexan protozoans of the genera Hepatozoon, Babesia, and Cytauxzoon are emerging parasite pathogens that are increasingly diagnosed in the pet population. These tick-transmitted apicomplexan parasites are becoming more widely recognized as serious canine or feline pathogens. This article reviews the epidemiology, diagnosis, treatment, and control of canine hepatozoonosis and babesiosis, and feline cytauxzoonosis.

    PMID: 19932361 [PubMed - indexed for MEDLINE]

  3. Vet Parasitol. 2010 Feb 10;167(2-4):216-26. Epub 2009 Sep 20.

    In silico predicted conserved B-cell epitopes in the merozoite surface antigen-2 family of B. bovis are neutralization sensitive.

    Dominguez M, Echaide I, Echaide ST, Mosqueda J, Cetrá B, Suarez CE, Florin-Christensen M.

    Institute of Pathobiology, Center of Agriculture and Veterinary Research, National Institute of Agriculture Technology, Castelar, Argentina.

    The merozoite surface antigens MSA-2 of Babesia bovis constitute a family of polymorphic GPI-anchored glycoproteins located at the parasite cell surface, that contain neutralization-sensitive B-cell epitopes. These are therefore putative vaccine candidates for bovine babesiosis. It was previously shown that (i) the MSA-2 antigens of the biologically cloned Mo7 strain are encoded by four tandemly organized genes: msa-2a(1), a(2), b and c, and (ii) at least one allele of each of these genes is present in the Argentine R1A strain with a moderate degree of polymorphism. The present work was aimed at defining neutralization-sensitive B-cell epitopes in the MSA-2 family, that are conserved among different B. bovis geographical isolates. To this end, msa-2a, b and c alleles from different isolates from Argentina, USA and Mexico were amplified by PCR, cloned and sequenced. Bioinformatic analysis by ClustalW alignments and B-cell epitope prediction algorithms performed on these sequences allowed the identification of several regions containing putative conserved B-cell epitopes. Four peptides representing these regions: (KDYKTMVKFCN from msa-2a(1); YYKKHIS, from msa-2b; and THDALKAVKQLIKT and ELLKLLIEA from msa-2c) were chemically synthesized, conjugated to keyhole limpet hemocyanin and used to inoculate mice to obtain immune sera. Anti-peptide antibodies recognized B. bovis merozoite extracts in all cases in ELISA tests. In addition, these sera reacted with the surface of merozoites of an Argentine and a Mexican B. bovis strains in immunofluorescence assays, and sera against two of the selected peptides inhibited invasion of erythrocytes by in vitro cultured merozoites. Taken together, the results show that the peptide sequences selected by bioinformatic analysis represent expressed and geographically conserved B. bovis B-cell epitopes that might be strong candidates for development of subunit vaccines.

    PMID: 19850413 [PubMed - indexed for MEDLINE]

  4. Vet Parasitol. 2010 Feb 10;167(2-4):196-204. Epub 2009 Sep 19.

    Development of a tandem repeat-based multilocus typing system distinguishing Babesia bovis geographic isolates.

    Perez-Llaneza A, Caballero M, Baravalle E, Mesplet M, Mosqueda J, Suarez CE, Echaide I, Katzer F, Pacheco GM, Florin-Christensen M, Schnittger L.

    Institute of Pathobiology, CICVyA, INTA-Castelar, Buenos Aires, Argentina.

    Mini- and microsatellite sequences have proven to be excellent tools for the differentiation of strains and populations in several protozoan parasites due to their high variability. In the present work we have searched the genome of the tick-transmitted bovine hemoprotozoon Babesia bovis for tandem repeats (TRs) that could be useful for a multilocus typing system. Hundred and nineteen sequences were shortlisted and tested in five common B. bovis reference isolates originating from distinct geographic locations of North and South America: Texas, USA (T2Bo), Mexico (RAD and Mo7), and Santa Fe and Salta, Argentina (R1A and S2P, respectively). Satellite sequences were PCR-amplified using specific primers, separated by polyacrylamide gel electrophoresis, visualized by silver staining and sized. Fourteen TR sequences could be reliably amplified in all isolates and displayed length polymorphism. All primers used were specific for B. bovis and did not amplify genomic DNA from the bovine host or from Babesia bigemina, the principal co-infecting bovine parasite in the Americas, allowing their future use in field surveys. The 14 satellite markers identified are distributed throughout the four chromosomes of B. bovis as follows: chromosome 1 (n=3), chromosome 2 (n=2), chromosome 3 (n=5), and chromosome 4 (n=4). Within the five B. bovis isolates we identified nine satellite marker loci with two alleles, three with three alleles, one with four and another with five alleles. In comparison to Theileria parva, a bovine hemoprotozoan that pertains to the same piroplasmida order and own a genome of similar size, the number of polymorphic TRs and the average number of alleles per TR locus seem to be significantly reduced in the B. bovis genome. Furthermore, the ratio of micro- to minisatellites in both B. bovis and T. parva is considerably lower than in other eukaryotes, as confirmed by bioinformatic analysis. The multilocus genotype of the five B. bovis isolates was assessed and the genetic distance between each other determined followed by cluster analysis based on neighbor joining. The resulting phenogram showed that B. bovis isolates segregated into three clusters according to their geographic origin. The presented marker system is suitable to explore various parameters of B. bovis populations such as genetic diversity, infection dynamics and their structure under different epidemiological situations, which are of crucial importance for improved control strategies.

    PMID: 19833439 [PubMed - indexed for MEDLINE]

  5. J Am Vet Med Assoc. 2009 Oct 1;235(7):851-4.

    Detection of a large unnamed Babesia piroplasm originally identified in dogs in North Carolina in a dog with no history of travel to that state.

    Holman PJ, Backlund BB, Wilcox AL, Stone R, Stricklin AL, Bardin KE.

    Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843, USA. pholman@cvm.tamu.edu

    CASE DESCRIPTION: A 12-year-old 46-kg (101.2-lb) sexually intact male Labrador Retriever was evaluated because of lymphadenomegaly. The dog resided in Texas, and its travel history included many southeastern and eastern shore states but not North Carolina. CLINICAL FINDINGS: Following evaluation of the dog, a diagnosis of stage IVa intermediate- to large-cell lymphoma was made. A cyclophosphamide-hydroxydaunorubicin (doxorubicin)-vincristine-prednisone chemotherapy protocol was initiated. One week after the first chemotherapeutic treatment, a routine blood smear evaluation revealed single and paired intraerythrocytic large piroplasms that resembled Babesia canis. Via molecular testing, the organism was identified as a Babesia sp that had been detected previously in dogs in North Carolina. TREATMENT AND OUTCOME: The dog was administered imidocarb diproprionate (7 mg/kg [3.2 mg/lb], IM) on 2 occasions (3-week interval). At 1, 4, 15, and 50 weeks after the second treatment, blood samples were analyzed specifically for the North Carolina Babesia sp via PCR assay; the result of each assay was positive. CLINICAL RELEVANCE: Because of the morphologic similarity of the large piroplasm detected in dogs in North Carolina to B canis, molecular testing of large piroplasms detected in dogs is needed to definitively identify the infective Babesia sp. In the dog of this report, the infection was not eliminated following treatment with imidocarb diproprionate, which may have been a result of the immunocompromised state of the dog or the drug's ineffectiveness against this parasite. If imidocarb diproprionate is ineffective against the North Carolina Babesia sp, treated dogs may act as reservoirs of infection.

    PMID: 19793016 [PubMed - indexed for MEDLINE]

  6. Vox Sang. 2008 Nov;95(4):331-4.

    Transmission of Babesia microti by blood transfusion in Texas.

    Cangelosi JJ, Sarvat B, Sarria JC, Herwaldt BL, Indrikovs AJ.

    Department of Pathology, University of Texas Medical Branch, Galveston, TX 77555-0435, USA.

    In the USA, seasonal tickborne transmission of Babesia microti occurs in the Northeast and upper Midwest. A resident of Texas became infected through a red blood cell transfusion from an asymptomatic local donor who had summered in Massachusetts. The patient's infection was diagnosed by blood smear examination in January, 7 weeks post-transfusion. He died 1 week later from variceal haemorrhage complicated by haemolysis. Premortem patient specimens and archived blood from the donor unit tested positive for B. microti antibodies and DNA. Babesiosis should be included in the differential diagnosis of post-transfusion haemolytic anaemia or thrombocytopenia, regardless of the geographical region or season.

    PMID: 19138264 [PubMed - indexed for MEDLINE]

  7. Vet Parasitol. 2008 Feb 14;151(2-4):150-7. Epub 2007 Nov 12.

    In vitro cultivation of a newly recognized Babesia sp. in dogs in North Carolina.

    Lehtinen LE, Birkenheuer AJ, Droleskey RE, Holman PJ.

    Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843-4467, United States.

    A novel large Babesia sp. from an infected dog was cultivated in vitro by microaerophilous stationary phase culture methodology. A primary culture initiated in enriched RPMI-1640 medium supplemented with 40% canine serum and incubated in a 2% oxygen environment supported parasite growth in vitro. Subsequent subcultures into enriched HL-1 medium with 20% fetal bovine serum also supported parasite propagation. Cultures were successfully introduced to 5% carbon dioxide in air atmosphere at passage 4. To date, the parasites have been continuously cultured through 35 passages, although the parasitemias are low, ranging from 0.2 to 0.3%. Parasites cultured in RPMI with canine serum were cryopreserved and successfully recovered from liquid nitrogen storage. The small subunit ribosomal rRNA gene sequence was identical in blood-derived and culture-derived parasites, differing in a single base position from the previously reported sequence for this Babesia sp. The ultrastructure of the parasite was consistent with that of other large Babesia spp., except that the spherical body contained numerous round particles unlike the inclusions previously described in Babesia spp.

    PMID: 18083310 [PubMed - indexed for MEDLINE]

  8. J Wildl Dis. 2007 Jul;43(3):504-7.

    Immunologic and molecular identification of Babesia bovis and Babesia bigemina in free-ranging white-tailed deer in northern Mexico.

    Cantu A, Ortega-S JA, Mosqueda J, Garcia-Vazquez Z, Henke SE, George JE.

    Texas A&M University-Kinsgsville, Caesar Kleberg Wildlife Research Institute, MSC 218, 700 University Blvd., Kingsville, Texas 78363, USA.

    The suitability of white-tailed deer (Odocoileus virginianus) as hosts for the cattle ticks Rhipicephalus (Boophilus) microplus and Rhipicephalus (Boophilus) annulatus, has been well documented. These ticks have a wide host range, and both transmit Babesia bovis and Babesia bigemina, the agents responsible for bovine babesiosis. Although this disease and its vectors have been eradicated from the United States and some states in northern Mexico, it still is a problem in other Mexican states. It is not known if wild cervids like white-tailed deer can act as reservoirs for bovine babesiosis. The purpose of this study was to determine if B. bovis and B. bigemina or antibodies against them occur in white-tailed deer in the states of Nuevo Leon and Tamaulipas, Mexico. Twenty blood samples from white-tailed deer from two ranches were collected and tested with a nested polymerase chain reaction (nested PCR) and indirect immunofluorescence antibody test (IFAT) for B. bovis and B. bigemina. Eleven samples were positive for B. bigemina and four for B. bovis by nested PCR; amplicon sequences were identical to those reported in GenBank for B. bovis (Rap 1) and B. bigemina. Results of the IFA test showed the presence of specific antibodies in serum samples. This is the first report of the presence of B. bovis and B. bigemina in white-tailed deer using these techniques and underscores the importance of cervids as possible reservoirs for bovine babesiosis.

    PMID: 17699089 [PubMed - indexed for MEDLINE]

  9. Vet Parasitol. 2007 Apr 10;145(1-2):156-63. Epub 2006 Dec 18.

    Detection of Babesia bigemina infection in strains of Rhipicephalus (Boophilus) microplus collected from outbreaks in south Texas.

    Guerrero FD, Bendele KG, Davey RB, George JE.

    USDA-ARS Knipling-Bushland, US Livestock Insects Research Laboratory, Kerrville, TX 78028, USA. felix.guerrero@ars.usda.gov

    The sudden death of several cattle infested experimentally with Rhipicephalus (Boophilus) microplus led to a clinical investigation into the reasons for the unexpected mortality. Microscopic evidence for Babesia bigemina infection was found in blood smears from the affected animals and a PCR assay was designed to detect the presence of B. bigemina and Babesia bovis in all R. microplus strains received and propagated at the laboratory. The assay utilizes a nested PCR approach with the first PCR amplifying a well-conserved segment from the Babesia 18S ribosomal RNA gene followed by a nested PCR with Babesia species-specific primers and annealing temperatures enabling amplification of the 18S ribosomal RNA gene fragment specific to either B. bigemina or B. bovis. DNA from groups of 50 larvae was extracted using a rapid DNA preparation protocol, which consisted of grinding the frozen tick larvae in PCR buffer and boiling the mixture for 5min. The assay sensitivity allowed for the detection of the equivalent of a single infected tick larva. R. microplus eggs were also analyzed, but yolk protein viscosity created inconsistent results with the crush and boil DNA isolation protocol, necessitating the use of a more extensive proteinase K digestion-based DNA purification method. We detected the presence of B. bigemina in all strains of R. microplus currently reared at the laboratory and 4 of 26 strains collected from infestation outbreaks in Texas by the U.S. Cattle Fever Tick Eradication Program.

    PMID: 17178440 [PubMed - indexed for MEDLINE]

  10. Ann N Y Acad Sci. 2006 Oct;1081:518-25.

    Phylogenetic and biologic evidence that Babesia divergens is not endemic in the United States.

    Holman PJ.

    Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843, USA. pholman@cvm.tamu.edu

    The causative agent of human babesiosis in a Kentucky case, which was first identified as Babesia divergens, is identical to a parasite of eastern cottontail rabbits on Nantucket Island, Massachusetts based on piroplasm size, morphology, and ribosomal RNA sequence analysis. Studies showing differential infectivity for cattle, host erythrocyte specificity in vitro, parasite size and morphology in vitro, and ribosomal RNA sequences clearly demonstrate that the parasite from the rabbit (conspecific with the human Kentucky agent) is not the same organism as B. divergens.

    PMID: 17135561 [PubMed - indexed for MEDLINE]

  11. J Wildl Dis. 2006 Apr;42(2):366-74.

    Molecular detection and characterization of Cytauxzoon felis and a Babesia species in cougars from Florida.

    Yabsley MJ, Murphy SM, Cunningham MW.

    Southeastern Cooperative Wildlife Disease Study, Department of Population Health, College of Veterinary Medicine, University of Georgia, Athens, Georgia 30602, USA. myabsley@uga.edu

    Piroplasms, morphologically indistinguishable from Cytauxzoon felis, previously were detected in 36% of cougars in Florida. We utilized a nested 18S rRNA assay, which amplifies DNA from all piroplasms, to screen blood samples collected from 41 cougars from Florida (39 native Florida panthers [Puma concolor coryi] and two translocated Texas cougars [P. c. stanleyana]) from 1989-2005. Thirty-nine of the 41 cougars (95%) were positive for piroplasms; however, sequence analysis and restriction enzyme digestion revealed that only five were positive for C. felis. Samples from 32 cougars were positive for a Babesia sp. Two cougars were co-infected with both C. felis and the Babesia sp. Phylogenetic analysis of 18S rRNA gene sequence indicated that the Florida panther Babesia sp. was most closely related to a Babesia sp. reported from Ixodes ovatus from Japan, Babesia divergens, and Babesia odocoilei. This study indicates that Florida panthers harbor two distinct piroplasms, C. felis and a Babesia sp., and that some individuals are infected with both organisms. The infectivity and pathogenicity of this Babesia sp. for domestic cats is unknown. This represents the first report of a feline Babesia sp. in North America.

    PMID: 16870859 [PubMed - indexed for MEDLINE]

  12. Biochem Biophys Res Commun. 2006 Aug 25;347(2):527-33. Epub 2006 Jun 30.

    Small heat shock proteins differentially affect Abeta aggregation and toxicity.

    Lee S, Carson K, Rice-Ficht A, Good T.

    Department of Chemical Engineering, Texas A&M University, College Station, TX 77843-3122, USA.

    beta-Amyloid (Abeta) is the primary protein component of senile plaques in Alzheimer's disease (AD) and has been implicated in neurotoxicity associated with the disease. Abeta aggregates readily in vitro and in vivo, and its toxicity has been linked to its aggregation state. Prevention of Abeta aggregation has been investigated as a means to prevent Abeta toxicity associated with AD. Recently we found that Hsp20 from Babesia bovis prevented both Abeta aggregation and toxicity [S. Lee, K. Carson, A. Rice-Ficht, T. Good, Hsp20, a novel alpha-crystallin, prevents Abeta fibril formation and toxicity, Protein Sci. 14 (2005) 593-601.]. In this work, we examined the mechanism of Hsp20 interaction with Abeta1-40 and compared its activity to that of other small heat shock proteins, carrot Hsp17.7 and human Hsp27. While all three small heat shock proteins were able to prevent Abeta aggregation, only Hsp20 was able to attenuate Abeta toxicity in cultured SH-SY5Y cells. Understanding the mechanism of the Hsp20-Abeta interaction may provide insights into the design of the next generation of Abeta aggregation and toxicity inhibitors.

    PMID: 16828710 [PubMed - indexed for MEDLINE]

  13. J Parasitol. 2006 Apr;92(2):333-40.

    In vitro host erythrocyte specificity and differential morphology of Babesia divergens and a zoonotic Babesia sp. from eastern cottontail rabbits (Sylvilagus floridanus).

    Spencer AM, Goethert HK, Telford SR 3rd, Holman PJ.

    Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, Texas 77843, USA.

    A Babesia sp. isolated from eastern cottontail rabbits (Sylvilagus floridanus) is morphologically similar and genetically identical, based on SSU rRNA gene comparisons, to 2 agents responsible for human babesiosis in the United States. This zoonotic agent is closely related to the European parasite, Babesia divergens. The 2 organisms were characterized by in vitro comparisons. In vitro growth of the rabbit Babesia sp. was supported in human and cottontail rabbit erythrocytes, but not in bovine cells. Babesia divergens was supported in vitro in bovine and human erythrocytes, but not in cottontail rabbit cells. Morphometric analysis classifies B. divergens as a small babesia in bovine erythrocytes, but the parasite exceeds this size in human erythrocytes. The rabbit Babesia sp. is large, the same size in both human or rabbit erythrocytes, and is significantly larger than B. divergens. Eight or more rabbit Babesia sp. parasites may occur within a single erythrocyte, sometimes in a floret array, unlike B. divergens. The erythrocyte specificity and morphological differences reported in this study agree with previous in vivo results and validate the use of in vitro methods for characterization of Babesia species.

    PMID: 16729690 [PubMed - indexed for MEDLINE]

  14. Vector Borne Zoonotic Dis. 2006 Spring;6(1):7-13.

    Detection of Babesia and Anaplasma species in rabbits from Texas and Georgia, USA.

    Yabsley MJ, Romines J, Nettles VF.

    Southeastern Cooperative Wildlife Disease Study and Department of Population Health, College of Veterinary Medicine, University of Georgia, Athens, Georgia 30602, USA. myabsley@uga.edu

    Rabbits have been shown to harbor a suite of zoonotic organisms, including a Babesia species, Borrelia burgdorferi, and Anaplasma phagocytophilum. In this study, we conducted a molecular survey for various tick-borne pathogens in three species of rabbits from Texas and Georgia. Of 18 black-tailed jackrabbits (Lepus californicus) tested from Texas, six (28%) were polymerase chain reaction (PCR) positive for Babesia, and nucleotide sequencing revealed two distinct species or strains. Two jackrabbits were infected with a Babesia species or strain (Babesia sp. A) that was nearly identical (99.9%) to a piroplasm previously detected in humans from Washington state, and the remaining four jackrabbits were infected with a Babesia species (Babesia sp. B) that was most similar (99.7%) to a Babesia species detected in cottontail rabbits from Massachusetts and humans from Kentucky and Missouri. Eleven (61%) black-tailed jackrabbits were positive for A. bovis, and one was positive for A. phagocytophilum. Two of four desert cottontails (Sylvilagus audubonii) from Texas were positive for the Babesia sp. B, and one desert cottontail each was positive for A. bovis and A. phagocytophilum. One of these desert cottontails was coinfected with the Babesia sp. B and A. phagocytophilum, and five jackrabbits were coinfected with Babesia species and A. bovis. Of 19 eastern cottontails (S. floridanus) from Georgia, only one (5.3%) was positive for A. phagocytophilum, and three (15.8%) were positive for A. bovis. No rabbits from Texas or Georgia were positive for Borrelia species. The only tick species detected on the Texas and Georgia rabbits was the rabbit tick, Haemaphysalis leporispalustris. These data extend the geographic and host range of these pathogens, and because both the Babesia species and A. phagocytophilum are potential zoonotic pathogens, it is important to be aware that these organisms are enzootic in parts of the southern United States.

    PMID: 16584322 [PubMed - indexed for MEDLINE]

  15. J Wildl Dis. 2005 Oct;41(4):683-90.

    New ruminant hosts and wider geographic range identified for Babesia odocoilei (Emerson and Wright 1970).

    Schoelkopf L, Hutchinson CE, Bendele KG, Goff WL, Willette M, Rasmussen JM, Holman PJ.

    Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University, College Station, Texas 77843, USA.

    Babesia odocoilei was found to infect two previously unknown host species, desert bighorn sheep (Ovis canadensis nelsoni) and musk oxen (Ovibos moschatus), both of which are members of the family Bovidae. Previously, B. odocoilei has been reported in only Cervidae hosts. New geographic regions where B. odocoilei infections have not been reported previously include Pennsylvania and New York, where fatal babesiosis has occurred in reindeer (Rangifer tarandus tarandus); New Hampshire, where elk (Cervus elaphus canadensis) have been affected; and California, home of the infected desert bighorn sheep. Infection with B. odocoilei in these hosts was confirmed by parasite small subunit ribosomal RNA gene sequence analysis. A serosurvey for B. odocoilei antibody activity in New Hampshire showed prevalence rates of 100% at two elk farms and 12% at another farm. Control of potential vector ticks, Ixodes scapularis, especially when translocating livestock, is imperative to prevent outbreaks of babesiosis in managed herds of potential host species.

    PMID: 16456156 [PubMed - indexed for MEDLINE]

  16. Am J Trop Med Hyg. 2005 Nov;73(5):865-70.

    Comparative infectivity of Babesia divergens and a zoonotic Babesia divergens-like parasite in cattle.

    Holman PJ, Spencer AM, Telford SR 3rd, Goethert HK, Allen AJ, Knowles DP, Goff WL.

    Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A and M University, College Station, Texas, USA.

    Babesia divergens-like parasites identified in human babesiosis cases in Missouri and Kentucky and in eastern cottontail rabbits (Sylvilagus floridanus) on Nantucket Island, Massachusetts, share identical small subunit ribosomal RNA gene sequences. This sequence is 99.8% identical to that of Babesia divergens, suggesting that the U.S. parasite may be B. divergens, a causative agent of human and bovine babesiosis in Europe. Holstein-Friesian calves were inoculated with cultured Nantucket Island Babesia sp. (NR831) and B. divergens parasites and monitored by clinical signs, Giemsa-stained blood films, PCR, and culture. The NR831 recipients did not exhibit clinical signs of infection and remained negative for all assays. The B. divergens recipients developed clinical infections and became positive by all assays. NR831 recipients were fully susceptible upon challenge inoculation with B. divergens. This study confirms that the Nantucket Island Babesia sp. is not conspecific with B. divergens based on host specificity for cattle.

    PMID: 16282295 [PubMed - indexed for MEDLINE]

  17. J Clin Microbiol. 2005 Aug;43(8):3995-4001.

    In vitro cultivation of a zoonotic Babesia sp. isolated from eastern cottontail rabbits (Sylvilagus floridanus) on Nantucket Island, Massachusetts.

    Holman PJ, Spencer AM, Droleskey RE, Goethert HK, Telford SR 3rd.

    Department of Veterinary Pathobiology, Texas A&M University, College Station, TX 77843-4467, USA. holman@cvm.tamu.edu

    A Babesia sp. found in eastern cottontail rabbits (Sylvilagus floridanus) on Nantucket Island, Massachusetts, is the same organism that caused human babesiosis in Missouri and Kentucky, on the basis of morphology and identical small-subunit rRNA (SSU rRNA) gene sequences. Continuous cultures of the rabbit parasite were established from infected blood samples collected from two cottontail rabbits livetrapped on Nantucket Island. HL-1 medium or minimal essential medium alpha medium supplemented with 20% human serum best supported in vitro propagation of the parasite in human or cottontail erythrocytes, respectively. Parasite growth was not sustained in domestic-rabbit erythrocytes or in medium supplemented with domestic-rabbit serum. The cultured parasites were morphologically indistinguishable from the Kentucky human isolate. Transmission electron microscopy revealed similar fine structures of the parasite regardless of the host erythrocyte utilized in the cultures. Two continuous lines of the zoonotic Babesia sp. were established and confirmed to share identical SSU rRNA gene sequences with each other and with the Missouri and Kentucky human Babesia isolates.

    PMCID: PMC1233898 PMID: 16081941 [PubMed - indexed for MEDLINE]

  18. Protein Sci. 2005 Mar;14(3):593-601.

    Hsp20, a novel alpha-crystallin, prevents Abeta fibril formation and toxicity.

    Lee S, Carson K, Rice-Ficht A, Good T.

    Department of Chemical Engineering, Texas A&M University, College Station, Texas 77843-3122, USA.

    Beta-amyloid (Abeta) is a major protein component of senile plaques in Alzheimer's disease, and is neurotoxic when aggregated. The size of aggregated Abeta responsible for the observed neurotoxicity and the mechanism of aggregation are still under investigation; however, prevention of Abeta aggregation still holds promise as a means to reduce Abeta neurotoxicity. In research presented here, we show that Hsp20, a novel alpha-crystallin isolated from the bovine erythrocyte parasite Babesia bovis, was able to prevent aggregation of denatured alcohol dehydrogenase when the two proteins are present at near equimolar levels. We then examined the ability of Hsp20 produced as two different fusion proteins to prevent Abeta amyloid formation as indicated by Congo Red binding; we found that not only was Hsp20 able to dramatically reduce Congo Red binding, but it was able to do so at molar ratios of Hsp20 to Abeta of 1 to 1000. Electron microscopy confirmed that Hsp20 does prevent Abeta fibril formation. Hsp20 was also able to significantly reduce Abeta toxicity to both SH-SY5Y and PC12 neuronal cells at similar molar ratios. At high concentrations of Hsp20, the protein no longer displays its aggregation inhibition and toxicity attenuation properties. Size exclusion chromatography indicated that Hsp20 was active at low concentrations in which dimer was present. Loss of activity at high concentrations was associated with the presence of higher oligomers of Hsp20. This work could contribute to the development of a novel aggregation inhibitor for prevention of Abeta toxicity.

    PMCID: PMC2279291 PMID: 15722443 [PubMed - indexed for MEDLINE]

  19. Parasitol Res. 2003 Nov;91(5):378-83. Epub 2003 Sep 18.

    Ribosomal RNA analysis of Babesia odocoilei isolates from farmed reindeer (Rangifer tarandus tarandus) and elk (Cervus elaphus canadensis) in Wisconsin.

    Holman PJ, Bendele KG, Schoelkopf L, Jones-Witthuhn RL, Jones SO.

    Department of Veterinary Pathobiology, Texas Veterinary Medical Center, Texas A&M University, College Station, TX 77843-4467, USA. pholman@cvm.tamu.edu

    Piroplasms isolated from a farmed reindeer and elk in Wisconsin were determined to be Babesia odocoilei, based on morphology and ribosomal RNA (rRNA) analysis. Different clinical manifestations were observed in the two host species. The reindeer was parasitemic and exhibited acute babesiosis resulting in death, while the elk showed no parasites in blood smears and no overt clinical signs of babesiosis. B. odocoilei was, however, readily cultured from elk erythrocytes. Small subunit rRNA gene sequences from the two isolates were identical to that previously reported for B. odocoilei. Internal transcribed spacers 1 and 2 and 5.8S rRNA sequence analysis showed an overall identity range of 94.3-98.1% to corresponding sequences from three previously reported B. odocoilei isolates, but the Wisconsin reindeer B. odocoilei shared only 87.3% identity with a previously reported Babesia sp. isolated from a reindeer in California (RD61).

    PMID: 14505046 [PubMed - indexed for MEDLINE]

  20. Wien Klin Wochenschr. 2002 Jul 31;114(13-14):479-81.

    Theobald Smith--the discoverer of ticks as vectors of disease.

    Assadian O, Stanek G.

    Clinical Institute of Hygiene and Medical Microbiology, University of Vienna, Vienna, Austria. ojan.assadian@akh-wien-ac.at

    The cause of Texas fever in cattle, which is characterised by lysis of erythrocytes leading to anaemia, icterus, haemoglobinuria, and death, remained unsolved for many decades and assorted theories were proposed as an explanation for a disease being transmitted by apparently healthy animals. From 1889 to 1893, Theobald Smith and Frederick L. Kilbourne could demonstrate in elegantly conducted experiments how the disease was spread from cattle to cattle by ticks serving as the vector of transmission. Furthermore, they were able to identify the pathogen of Texas fever, an intra-erythrocytic protozoan which Smith named Pyrosoma bigeminum. Today it is recognised that either of two species of the now renamed genus Babesia, Babesia bigemina and Babesia bovis, may be involved in Texas fever and that babesiosis is generally transmitted by ticks. In animals, genera like Boophilus spp., Dermacentor spp. and Rhipicephalus spp. are possible vectors. The first case of tick-transmitted babesiosis in a human was reported by Skrabalo and Deanovic in 1957 and occurred near Ljubliana in the small town of Strmec, Croatia. In humans, the vectors of most reported cases are ticks of the genus Ixodes, which are among the most predominant ticks in Austria. However, cases of human babesiosis in Austria remain to be studied. Smith and Kilbourne's work was the first demonstration that ticks transmit disease of any kind. Furthermore, by proving that ticks carry Babesia microti--which causes babesiosis in animals and humans--this is the first account of a zoonotic disease and the foundation of all later work on the animal host and the arthropod vector.

    PMID: 12422586 [PubMed - indexed for MEDLINE]

  21. Parasitol Res. 2002 May;88(5):405-11.

    Genotypically unique Babesia spp. isolated from reindeer (Rangifer tarandus tarandus) in the United States.

    Holman PJ, Swift PK, Frey RE, Bennett J, Cruz D, Wagner GG.

    Department of Veterinary Pathobiology, The Texas Veterinary Medical Center, Texas A&M University, College Station 77843-4467, USA. pholman@cvm.tamu.edu

    Two morphologically dissimilar Babesia spp. were cultured from reindeer (Rangifer tarandus tarandus) in Placer County, Calif. The smaller isolate, designated RD61, was morphologically similar to Babesia odocoilei. Serum from RD61-infected reindeer reacted equally strongly to B. odocoilei and RD61 parasites in the indirect fluorescent antibody (IFA) test. Small subunit ribosomal RNA (SSU rRNA) gene-sequence analysis showed 99.0% identity to that of B. odocoilei. The larger piroplasm, designated RD63, resembled larger babesia organisms, such as Babesia caballi and Babesia bigemina. Serum from RD63-infected reindeer also reacted with both B. odocoilei and RD61 parasites in the indirect fluorescent antibody test. The SSU rRNA gene showed 94.2% identity to that of B. bigemina. Further studies are needed to determine whether these parasites are the same as the Babesia spp. previously documented in Siberian reindeer.

    PMID: 12049456 [PubMed - indexed for MEDLINE]

  22. Mol Biochem Parasitol. 2002 Feb;119(2):295-300.

    A cathepsin L-like cysteine protease is conserved among Babesia equi isolates.

    Holman PJ, Hsieh MM, Nix JL, Bendele KG, Wagner GG, Ball JM.

    Department of Veterinary Pathobiology, The Texas Veterinary Medical Center, Texas A&M University, College Station, TX 77843-4467, USA. pholman@cvm.tamu.edu

    PMID: 11814583 [PubMed - indexed for MEDLINE]

  23. J Wildl Dis. 2000 Jul;36(3):518-30.

    Antigenic, phenotypic and molecular characterization confirms Babesia odocoilei isolated from three cervids.

    Holman PJ, Madeley J, Craig TM, Allsopp BA, Allsopp MT, Petrini KR, Waghela SD, Wagner GG.

    Department of Veterinary Pathobiology, Texas Veterinary Medical Center, Texas A&M University, College Station 77843-4467, USA. pholman@cvm.tamu.edu

    Babesia isolates from an elk (Cervus elaphus canadensis) and a caribou (Rangifer tarandus caribou) with fatal infections were compared to Babesia odocoilei (Engeling isolate) from white-tailed deer (Odocoileus virginianus) by experimental infection, serologic, and small subunit ribosomal RNA (SSU rRNA) gene sequence analysis studies. Both the indirect fluorescent antibody test and immunoprecipitation assays demonstrated antigenic variation among the isolates. Experimental infection studies showed no clinical differences among the isolates. Nucleotide sequence analysis showed that the elk and caribou Babesia sp. isolates possessed SSU rRNA genes with identical sequences to that of B. odocoilei. A phylogenetic tree constructed from SSU rRNA gene sequences shows that B. odocoilei is most closely related to Babesia divergens, both of which branch together in the true babesia clade.

    PMID: 10941738 [PubMed - indexed for MEDLINE]

  24. Mol Biochem Parasitol. 2000 Jun;109(1):67-72.

    Phylogenetic analysis with newly characterized Babesia bovis hsp70 and hsp90 provides strong support for paraphyly within the piroplasms.

    Ruef BJ, Ward TJ, Oxner CR, Conley PG, Brown WC, Rice-Ficht AC.

    Department oe Medical Biochemistry and Genetics, Texas A&M University, College Station 77843-1114, USA. bjruef@tamu.edu

    PMID: 10924758 [PubMed - indexed for MEDLINE]

  25. Int J Parasitol. 2000 Jan;30(1):59-64.

    A common high molecular weight antigen of Babesia bovis isolates from Mexico.

    Sahagun Ruiz A, Waghela SD, Romany MM, Holman PJ, Melendy D, Cruz D, Wagner GG.

    Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University, College Station, TX 77843-4467, USA.

    Cattle from an area of Mexico endemic with Babesia bovis infections have a dominant antibody response to a 152kDa antigen of the Tamaulipas strain of B. bovis. A mAb termed PB/5, showing a specific reactivity to this 152kDa antigen in Western blots, was identified. The mAb which reacted with the blunt end of B. bovis in an indirect fluorescent antibody test also reacted to a 152kDa antigen in two other isolates (Nuevo Leon and Yucatan), and a 175kDa antigen in the Huasteca B. bovis isolate from Mexico. Polyclonal monospecific sera from a calf inoculated with mAb-affinity purified 152kDa antigen (Tamaulipas strain) identified B. bovis by the indirect fluorescent antibody test and two antigens of B. bovis (65kDa and 152kDa) in Western blot. Since the epitope reacting to the mAb PB/5 is conserved, this antigen provides a basis for developing a diagnostic test or an immunogen.

    PMID: 10675745 [PubMed - indexed for MEDLINE]

  26. Mol Biochem Parasitol. 2000 Jan 5;105(1):1-12.

    A unique Babesia bovis spherical body protein is conserved among geographic isolates and localizes to the infected erythrocyte membrane.

    Ruef BJ, Dowling SC, Conley PG, Perryman LE, Brown WC, Jasmer DP, Rice-Ficht AC.

    Department of Medical Biochemistry and Genetics, Texas A&M University, System Health Science Center, College Station 77843-1114, USA.

    Using monoclonal antibody (mAb) 70/52.9, generated from a Babesia bovis fraction enriched for spherical body organelles, we have identified a 135-kDa protein containing an epitope conserved in B. bovis strains from Texas, Mexico, and Australia. The protein was localized to the spherical bodies of the babesial apical complex and was designated spherical body protein 3 (SBP3), according to the established nomenclature. Immunofluorescence studies showed binding of the 70/52.9 mAb to the infected-erythrocyte membrane region but not to their uninfected counterparts, demonstrating a host-cell association shared with the previously isolated B. bovis spherical body proteins, SBP1 and SBP2. Using mAb 70/52.9, the full-length cDNA encoding SBP3 was isolated from an expression library, sequenced, and oligonucleotide primers synthesized to amplify the genomic copy by polymerase chain reaction. The genomic copy contained no introns and was identical to the cDNA sequence with each containing a single, large open reading frame encoding a protein of 1089 residues. Analysis of the SBP3 amino acid sequence revealed no significant amino acid identity to SBP1 and SBP2 and a lack of repeated epitopes, a notable feature of the other two spherical body proteins. Labeled probes derived from the coding region of SBP3 hybridized to single fragments on Southern blots containing B. bovis genomic DNA indicating a single copy gene. With the identification of this third spherical body protein, which associates with the cytoplasmic face of the infected-erythrocyte membrane, a complement of distinct B. bovis proteins have been identified that are likely to contribute to intracellular survival, growth, and development for this parasite. The encoded protein should be valuable for functional investigations and evaluation of potential targets for host immunity.

    PMID: 10613694 [PubMed - indexed for MEDLINE]

  27. J Clin Microbiol. 1999 Aug;37(8):2598-601.

    Isolation of a new subspecies, Bartonella vinsonii subsp. arupensis, from a cattle rancher: identity with isolates found in conjunction with Borrelia burgdorferi and Babesia microti among naturally infected mice.

    Welch DF, Carroll KC, Hofmeister EK, Persing DH, Robison DA, Steigerwalt AG, Brenner DJ.

    Laboratory Corporation of America, Dallas, Texas 75230, USA. da_welchd@rodeo.biomed.com

    Bacteremia with fever due to a novel subspecies of Bartonella vinsonii was found in a cattle rancher. The subspecies shared major characteristics of the genus Bartonella in terms of most biochemical features and cellular fatty acid profile, but it was distinguishable from other subspecies of B. vinsonii by good growth on heart infusion agar supplemented with X factor and by its pattern of enzymatic hydrolysis of peptide substrates. DNA relatedness studies verified that the isolate belonged to the genus Bartonella and that it was genotypically related to B. vinsonii. The highest level of relatedness was observed with recently characterized strains from naturally infected mice that were coinfected with Borrelia burgdorferi and Babesia microti. We propose the name Bartonella vinsonii subsp. arupensis subsp. nov. as the new subspecies to accommodate these human and murine isolates.

    PMCID: PMC85292 PMID: 10405408 [PubMed - indexed for MEDLINE]

  28. J Parasitol. 1998 Aug;84(4):696-9.

    Babesia equi field isolates cultured from horse blood using a microcentrifuge method.

    Holman PJ, Becu T, Bakos E, Polledo G, Cruz D, Wagner GG.

    Department of Veterinary Pathobiology, Texas A&M University, College Station 77843-4467, USA.

    Babesia equi, a causative agent of equine piroplasmosis, was isolated from horses in the Chaco Province of Argentina, a known piroplasmosis endemic region. Fifteen B. equi field isolates were acquired by culture from 23 actively working horses from 2 ranches. The horses appeared healthy with no clinical signs or histories indicative of equine piroplasmosis. All 23 horses had B. equi-specific antibody activity by the indirect fluorescent antibody test and 18 were also complement fixation test positive for B. equi. Equine erythrocytes were prepared for parasite culture using a microcentrifuge tube method. This method greatly reduces the time involved in cell handling and parasite exposure to ambient conditions. By this method, B. equi cultures can be initiated from very small quantities of blood.

    PMID: 9714196 [PubMed - indexed for MEDLINE]

  29. Vet Parasitol. 1997 Dec 15;73(1-2):53-63.

    Biotin-labeled DNA probe in a PCR-based assay increases detection sensitivity for the equine hemoparasite Babesia caballi.

    Sahagun-Ruiz A, Waghela SD, Holman PJ, Chieves LP, Wagner GG.

    Department of Veterinary Pathobiology, Texas A&M University, College Station 77843, USA.

    A DNA probe from Babesia caballi (Bc1) was selected by antibody screening of a genomic library. The Bc1 probe hybridized specifically to B. caballi genomic DNA. A polymerase-chain-reaction-based assay for B. caballi DNA was developed from primers deduced from the probe nucleotide sequence. An amplified product of 1.6 kb was detected from as little as 500 fg B. caballi template DNA. Sensitivity increased 1000-fold when the biotin-labeled Bc1 probe was hybridized to the amplicons in a Southern blot.

    PMID: 9477492 [PubMed - indexed for MEDLINE]

  30. J Clin Microbiol. 1997 Feb;35(2):474-6.

    Case report: field-acquired subclinical Babesia equi infection confirmed by in vitro culture.

    Holman PJ, Hietala SK, Kayashima LR, Olson D, Waghela SD, Wagner GG.

    Department of Veterinary Pathobiology, Texas Agricultural Experiment Station, Texas A&M University, College Station, USA.

    A horse with no prior clinical history of equine piroplasmosis tested negative for Babesia caballi and Babesia equi in the complement fixation test before importation into the United States from France. After 5 years in residence in the United States, the animal tested serologically positive for B. equi by the complement fixation test, the immunofluorescent antibody test, and Western blot analysis. The carrier status of the horse was confirmed by culture of B. equi parasites. In vitro culture offers an efficient and comparatively inexpensive method to determine the carrier status of horses suspected of harboring B. equi.

    PMCID: PMC229603 PMID: 9003619 [PubMed - indexed for MEDLINE]

  31. J Interferon Cytokine Res. 1997 Jan;17(1):45-54.

    Immunization with Babesia bigemina rhoptry-associated protein 1 induces a type 1 cytokine response.

    Ruef BJ, Tuo W, Rodriguez SD, Roussel AJ, Chitko-McKown CG, Palmer GH, McElwain TF, Canals A, Zarlenga DS, Gasbarre LC, Brown WC.

    Department of Veterinary Pathobiology, Texas A & M University, College Station 77843, USA.

    Rhoptry-associated protein-1 (RAP-1) homologues of Babesia bigemina and Babesia bovis are promising candidates for inclusion in subunit vaccines against these hemoprotozoan parasites. Partial protection against challenge infection has been achieved with native forms of these antigens, but the mechanism of immunity has not been thoroughly defined. We previously demonstrated that a panel of antigen-specific T helper cell clones derived from B. bigemina RAP-1-immunized cattle expressed relatively high levels of interferon-gamma (IFN-gamma) protein and transcript and low levels of interleukin-4 (IL-4), indicative of a type 1 immune response. In the current study we present evidence that subcutaneous immunization with native B. bigemina RAP-1 protein in RIBI adjuvant induces a predominant type 1 immune response in vivo, characterized by relatively high levels of IFN-gamma and IL-2 and low levels of IL-4 and IL-10 mRNA in the draining prescapular lymph node. Ex vivo restimulation of draining lymph node lymphocytes with specific antigen resulted in proliferation and enhanced expression of IL-2 and IFN-gamma, whereas IL-4 and IL-10 transcript levels remained relatively low. These findings show that our previously described cytokine profiles of antigen-specific cloned T cell lines are representative of autologous in vivo responses and confirm that type 1 recall responses to B. bigemina RAP-1 can be evoked in immunized animals by native parasite antigen.

    PMID: 9041471 [PubMed - indexed for MEDLINE]

  32. Infect Immun. 1996 Aug;64(8):3341-50.

    Babesia bovis rhoptry-associated protein 1 is immunodominant for T helper cells of immune cattle and contains T-cell epitopes conserved among geographically distant B. bovis strains.

    Brown WC, McElwain TF, Ruef BJ, Suarez CE, Shkap V, Chitko-McKown CG, Tuo W, Rice-Ficht AC, Palmer GH.

    Department of Veterinary Pathiobiology, Texas A & M University, College Station 77843, USA.

    The ability of rhoptry-associated protein 1 (RAP-1) of Babesia bovis and Babesia bigemina to confer partial protective immunity in cattle has stimulated interest in characterizing both B-cell and T-cell epitopes of these proteins. It was previously shown that B. bovis RAP-1 associates with the merozoite surface as well as rhoptries and expresses B-cell epitopes conserved among otherwise antigenically different B. bovis strains. An amino-terminal 307-amino-acid domain of the molecule that is highly conserved in the B. bigemina RAP-1 homolog did not contain cross-reactive B-cell epitopes. The studies reported here demonstrate that B. bovis RAP-1 is strongly immunogenic for T helper (Th) cells from B. bovis-immune cattle and that like B-cell epitopes, Th-cell epitopes are conserved in different B. bovis strains but not in B. bigemina RAP-1. Lymphocytes from cattle immune to challenge with the Mexico strain of B. bovis proliferated against recombinant B. bovis RAP-1 protein derived from the Mexico strain. T-cell lines established by stimulating lymphocytes with recombinant RAP-1 protein responded against B. bovis, but not B. bigemina, merozoites. T-cell lines established by repeated stimulation of lymphocytes with B. bovis membrane antigen proliferated strongly against RAP-1, demonstrating the immunodominant nature of this protein. RAP-1-specific CD4+ T cell clones recognized Mexico, Texas, Australia, and Israel strains of B. bovis but neither B. bigemina merozoites nor recombinant B. bigemina RAP- 1. Analysis of cytokine mRNA in RAP-1-specific Th cell clones revealed strong expression of gamma interferon but little or no expression of interleukin-2 (IL-2), IL-4, or IL-10. Gamma interferon production was confirmed by enzyme-linked imunosorbent assay. These results indicate the potential to use selected B. bovis RAP-1 peptides as immunogens to prime for strong, anamnestic, strain-cross-reactive type 1 immune responses upon exposure to B. bovis.

    PMCID: PMC174227 PMID: 8757873 [PubMed - indexed for MEDLINE]

  33. Ann N Y Acad Sci. 1996 Jul 23;791:128-35.

    Characterization of helper T cell responses against rhoptry-associated protein 1 (RAP-1) of babesial parasites.

    Brown WC, Rodriguez SD, Hotzel I, Ruef BJ, Chitko-McKown CG, McElwain TF, Palmer GH.

    Department of Veterinary Pathobiology, Texas A & M University College Station 77843-4467, USA.

    PMID: 8784494 [PubMed - indexed for MEDLINE]

  34. Infect Immun. 1996 Jun;64(6):2079-87.

    CD4+ T-helper lymphocyte responses against Babesia bigemina rhoptry-associated protein I.

    Rodríguez SD, Palmer GH, McElwain TF, McGuire TC, Ruef BJ, Chitko-McKown MG, Brown WC.

    Department of Veterinary Pathobiology, Texas A&M University, College Station 77843, USA.

    A multigene family of 58- to 60-kDa proteins, which are designated rhoptry-associated protein 1 (RAP-1) and which come from the parasites Babesia bigemina and Babesia bovis, is a target for vaccine development. The presence of multiple gene copies and conserved sequences and epitopes of RAP-1 implies that these proteins are functionally important for the survival of these parasites. Furthermore, it was previously shown that B. bigemina RAP-1 induced partial protection against challenge infection. However, the lack of correlation between protective immunity to B. bigemina infection and antibody titers against a merozoite surface-exposed, neutralization-sensitive epitope of B. bigemina RAP-1 indicated the potential importance of RAP-1-specific T helper (Th) cells in the observed protection. To begin to understand the mechanism of RAP-1-induced protective immunity, RAP-1-specific T-cell responses were characterized in cattle. Vigorous and sustained proliferative responses of peripheral blood mononuclear cells from native RAP-1-immunized cattle were observed. The anamnestic response in immunized cattle was specific for B. bigemina RAP-1 and predominantly comprised CD4+ T cells, which upon cloning expressed type 1 cytokine mRNA profiles and high levels of gamma interferon protein. The T cells responded to both native and recombinant forms of RAP-1, indicating the potential to use recombinant protein or epitopes derived therefrom as a vaccine that could evoke specific recall responses after exposure to natural infection. The differential responses of peripheral blood mononuclear cells and seven Th-cell clones derived from RAP-1-immunized cattle to different Central American strains of B. bigemina indicated the presence of at least one conserved and one variable Th-cell epitope. The lack of response to B. bovis RAP-1 indicated that a strictly conserved 14-amino-acid peptide shared by the two babesial species was not immunogenic for Th cells in these experiments. However, the Th-cell epitope conserved among strains of B. bigemina may be a useful component of a RAP-1 subunit vaccine.

    PMCID: PMC174039 PMID: 8675310 [PubMed - indexed for MEDLINE]

  35. J Interferon Cytokine Res. 1995 Oct;15(10):915-22.

    Interleukin-10 downregulates proliferation and expression of interleukin-2 receptor p55 chain and interferon-gamma, but not interleukin-2 or interleukin-4, by parasite-specific helper T cell clones obtained from cattle chronically infected with Babesia bovis or Fasciola hepatica.

    Chitko-McKown CG, Ruef BJ, Rice-Ficht AC, Brown WC.

    Department of Veterinary Pathobiology, Texas A&M University, College Station 77843, USA.

    Human recombinant interleukin-10 (IL-10) was previously shown to inhibit accessory cell (AC)-dependent proliferation of bovine parasite-specific T helper 1 (Th1), Th2, and Th0 cells in an IL-2-reversible manner (Brown, W.C., Woods, V.M., Chitko-McKown, C.G., Hash, S.M., and Rice-Ficht, A.C., 1994. Infect. Immun. 62, 4697-4708). The present study was therefore designed to determine whether the effect of IL-10 on T cell proliferation corresponded with downregulated expression of cytokines, or their receptors, important for T cell growth. The effects of IL-10 on cellular proliferation and expression of IL-2, IL-4, IL-2 receptor (IL-2R; p55), and IFN-gamma by Babesia bovis- or Fasciola hepatica-specific Th cell clones were simultaneously evaluated. As shown previously, IL-10 strongly inhibited proliferation of all types of Th cell clones, although this did not correspond with reduced expression of IL-2 or IL-4 mRNA or their products. In contrast, expression of IL-2R mRNA was consistently reduced in the IL-10-treated clones. These results indicate that IL-10 does not inhibit AC-dependent proliferation of bovine Th cells by downregulating T cell cytokines; rather, IL-10 may act by downregulating IL-2R p55 expression and subsequent signal transduction leading to decreased cellular proliferation. IFN-gamma production was also consistently downregulated in the presence of IL-10.

    PMID: 8564714 [PubMed - indexed for MEDLINE]

  36. Infect Immun. 1995 Aug;63(8):3106-16.

    Identification of Babesia bovis merozoite antigens separated by continuous-flow electrophoresis that stimulate proliferation of helper T-cell clones derived from B. bovis-immune cattle.

    Brown WC, Logan KS, Zhao S, Bergman DK, Rice-Ficht AC.

    Department of Veterinary Pathobiology, Texas A & M University, College Station 77843, USA.

    To characterize Babesia bovis merozoite antigens that stimulate anamnestic T helper (Th)-cell responses from B. bovis-immune cattle, B. bovis-specific Th-cell lines and clones, previously assigned to different antigenic groups (W. C. Brown, S. Zhao, A. C. Rice-Ficht, K. S. Logan, and V. M. Woods, Infect. Immun. 60:4364-4372, 1992), were tested in proliferation assays against fractionated merozoite antigens. The antigenic groups were determined by the patterns of response of Th clones to different parasite isolates and soluble or membrane forms of merozoite antigen. Soluble antigen fractionated by anion-exchange chromatography or gel filtration by using fast-performance liquid chromatography resolved two or three antigenic peaks, respectively. To enable fractionation of membrane-associated proteins and to resolve more precisely the proteins present in homogenized merozoites, a novel technique of continuous-flow electrophoresis was employed. Merozoite membranes or whole merozoites were homogenized and solubilized in sodium dodecyl sulfate-sample buffer, electrophoresed under reducing conditions on 15% or 10% acrylamide gels, eluted, and collected as fractions. Individual fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and tested for the ability to stimulate Babesia-specific CD4+ T-cell lines and clones. CD4+ Th-cell lines from two cattle displayed differential patterns of reactivity and detected numerous peaks of antigenic activity, ranging from < 14 to 76 kDa. Th-cell clones previously categorized into different antigenic groups detected antigenic peaks unique for clones representative of a given group. Antigens of 29, 51 to 52, and 85 to 95 kDa (group I), 40 kDa (group III), 20 kDa (group IV), 58 to 60 kDa (group VI), and 38, 45, and 83 kDa (group VII) were identified in the stimulatory fractions. Immunization of rabbits with selected fractions produced a panel of antisera that reacted specifically on Western blots (immunoblots) with merozoite antigens of similar sizes, leading to the tentative identification of candidate antigens of B. bovis merozoites with molecular masses of 20, 40, 44, 51 to 52 or 95, and 58 to 60 kDa that stimulate proliferation of Th clones representative of five different antigenic groups. These antisera may be useful for isolating recombinant proteins that are immunogenic for Th cells of immune cattle and therefore potentially useful for vaccine development.

    PMCID: PMC173424 PMID: 7622238 [PubMed - indexed for MEDLINE]

  37. Infect Immun. 1994 Nov;62(11):4697-708.

    Interleukin-10 is expressed by bovine type 1 helper, type 2 helper, and unrestricted parasite-specific T-cell clones and inhibits proliferation of all three subsets in an accessory-cell-dependent manner.

    Brown WC, Woods VM, Chitko-McKown CG, Hash SM, Rice-Ficht AC.

    Department of Veterinary Pathobiology, Texas A&M University, College Station 77843.

    Murine interleukin-10 (IL-10) is produced by type 2 helper (Th2) cells and selectively inhibits cytokine synthesis by type 1 helper (Th1) cells, whereas human IL-10 is produced by and inhibits proliferation and cytokine synthesis by both Th1 and Th2 subsets. This study reports that bovine IL-10 mRNA is expressed by Th0, Th1, and Th2 clones of bovine T cells specific for either Babesia bovis or Fasciola hepatica but not by two CD8+ T-cell clones. The antigen-induced proliferative responses of all three subsets of CD4+ cells were inhibited by human IL-10, and low levels (10 U/ml) of exogenous human IL-2 restored the suppressed response. However, proliferation of one Th1 clone was never inhibited but was enhanced by IL-10. Human IL-10 also inhibited the expression of gamma interferon and IL-4 mRNA in Th0 clones. In the absence of accessory cells (AC), the responses of Th clones to concanavalin A or IL-2 were not inhibited by IL-10, whereas antigen-specific responses of Th1 and Th2 cells were reduced when IL-10-pretreated macrophages were used as AC. Together, our results with bovine T cells support the concept that IL-10 primarily affects AC function and does not directly inhibit CD4+ T cells and demonstrate that the immunoregulatory effects of IL-10 are not selectively directed at Th1 populations, as they are in mice.

    PMCID: PMC303176 PMID: 7927745 [PubMed - indexed for MEDLINE]

  38. J Wildl Dis. 1994 Jul;30(3):460-5.

    Culture isolation and partial characterization of a Babesia sp. from a North American elk (Cervus elaphus).

    Holman PJ, Craig TM, Crider DL, Petrini KR, Rhyan J, Wagner GG.

    Department of Veterinary Pathobiology, Texas A&M University, College Station 77843.

    Three North American yearling elk (Cervus elaphus) died with clinical symptoms suggestive of babesiosis. Babesia sp. organisms similar in morphology to B. odocoilei of white-tailed deer (Odocoileus virginianus) were observed in Giemsa-stained blood films from one of the elk. Continuous cultures of the parasite were established. Antiserum raised against the elk Babesia sp. isolate was compared to B. odocoilei specific antiserum in an immunofluorescent antibody assay; we found evidence of differences in reactivity to several Babesia spp. isolated from wildlife and domestic ruminants. Cultured parasites from the elk were not infective to either intact or splenectomized Bos taurus steers.

    PMID: 7933298 [PubMed - indexed for MEDLINE]

  39. J Parasitol. 1994 Apr;80(2):232-6.

    Babesia equi erythrocytic stage continuously cultured in an enriched medium.

    Holman PJ, Chieves L, Frerichs WM, Olson D, Wagner GG.

    Department of Veterinary Pathobiology, Texas A&M University, College Station 77843-4467.

    Babesia equi was continuously cultured through 90 passages in an enriched chemically defined basal medium (HL-1) supplemented with 20% fetal bovine serum and serum replacement factors, including lipid-rich bovine serum albumin, bovine insulin, and human transferrin. Cryopreservation and subsequent recovery of B. equi were easily achieved. Inoculation of a splenectomized and an intact horse with cultured infected erythrocytes resulted in parasitemias and B. equi in vitro reisolation from both animals. In vitro forms of the parasite resembled in vivo forms. After establishment, parasitemias of 10-15% were commonly observed.

    PMID: 8158466 [PubMed - indexed for MEDLINE]

  40. J Wildl Dis. 1994 Apr;30(2):195-200.

    In vitro isolation and cultivation of a Babesia from an American woodland caribou (Rangifer tarandus caribou).

    Holman PJ, Petrini K, Rhyan J, Wagner GG.

    Department of Veterinary Pathobiology, Texas A&M University, College Station 77843.

    A Babesia species isolated from a captive caribou (Rangifer tarandus caribou) with clinical signs of babesiosis and a circulating parasitemia was cultured in vitro. Normal adult caribou erythrocytes supported the growth of the Babesia sp., as did erythrocytes from white-tailed deer (Odocoileus virginianus). Two basal media (M-199 and RPMI-1640) and a defined medium (HL-1) each supplemented with adult bovine serum were compared. The most favorable growth of the parasite occurred in HL-1 medium with 20% adult bovine serum. The morphology of this Babesia sp. isolate shared some characteristics with B. odocoilei and B. divergens.

    PMID: 7913142 [PubMed - indexed for MEDLINE]

  41. Infect Immun. 1994 Mar;62(3):818-27.

    CD4+ T-cell clones obtained from cattle chronically infected with Fasciola hepatica and specific for adult worm antigen express both unrestricted and Th2 cytokine profiles.

    Brown WC, Davis WC, Dobbelaere DA, Rice-Ficht AC.

    Department of Veterinary Pathobiology, Texas A&M University, College Station 77843.

    The well-established importance of helper T (Th)-cell subsets in immunity and immunoregulation of many experimental helminth infections prompted a detailed study of the cellular immune response against Fasciola hepatica in the natural bovine host. T-cell lines established from two cattle infected with F. hepatica were characterized for the expression of T-cell surface markers and proliferative responses against F. hepatica adult worm antigen. Parasite-specific T-cell lines contained a mixture of CD4+, CD8+, and gamma/delta T-cell-receptor-bearing T cells. However, cell lines containing either fewer than 10% CD8+ T cells or depleted of gamma/delta T cells proliferated vigorously against F. hepatica antigen, indicating that these T-cell subsets are not required for proliferative responses in vitro. Seventeen F. hepatica-specific CD4+ Th-cell clones were examined for cytokine expression following concanavalin A stimulation. Biological assays to measure interleukin-2 (IL-2) or IL-4, gamma interferon (IFN-gamma), and tumor necrosis factor and Northern (RNA) blot analysis to verify the expression of IL-2, IL-4, and IFN-gamma revealed that the Th-cell clones expressed a spectrum of cytokine profiles. Several Th-cell clones were identified as Th2 cells by the strong expression of IL-4 but little or no IL-2 or IFN-gamma mRNA. The majority of Th-cell clones were classified as Th0 cells by the expression of either all three cytokines or combinations of IL-2 and IL-4 or IL-4 and IFN-gamma. No Th1-cell clones were obtained. All of the Th-cell clones expressed a typical memory cell surface phenotype, characterized as CD45Rlow, and all expressed the lymph node homing receptor (L selectin). These results are the first to describe cytokine responses of F. hepatica-specific T cells obtained from infected cattle and extend our previous analysis of Th0 and Th1 cells from cattle immune to Babesia bovis (W. C. Brown, V. M. Woods, D. A. E. Dobbelaere, and K. S. Logan, Infect. Immun. 61:3273-3281, 1993) to include F. hepatica-specific Th2 cells.

    PMCID: PMC186188 PMID: 7509319 [PubMed - indexed for MEDLINE]

  42. Parasitol Today. 1994 Apr;10(4):145-9.

    Use of helper T cells to identify potential vaccine antigens of Babesia bovis.

    Brown WC, Rice-Ficht AC.

    Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A & M University, College Station, TX 77843, USA.

    Babesia bovis is an economically important hemoprotozoon parasite o f cattle that is prevalent in many, tropical and subtropical regions o f the world. Effective vaccines against this tick-transmitted parasite consist o f live organisms attenuated by passage through splenectomized calves. However, the nature o f acquired resistance to challenge infection with heterologous isolates of this parasite has not been clearly defined. Unsuccessful attempts to select protective antigens have relied upon the use of antibodies to identify immunodominant proteins. In this review, Wendy Brown and Allison Rice-Ficht discuss the limitations of this approach and the rationale behind using helper T cells to select potential vaccine antigens.

    PMID: 15275482 [PubMed]

  43. Cell Immunol. 1994 Jan;153(1):9-27.

    Functional and phenotypic characterization of WC1+ gamma/delta T cells isolated from Babesia bovis-stimulated T cell lines.

    Brown WC, Davis WC, Choi SH, Dobbelaere DA, Splitter GA.

    Department of Veterinary Pathobiology, Texas A & M University, College Station 77843.

    Functional studies with WC1+ gamma/delta T cell lines were performed to clarify the role of this subpopulation of gamma/delta T cells in the in vitro immune response to Babesia bovis. As CD4+ T cells decreased and gamma/delta T cells increased in B. bovis-stimulated T cell lines, antigen-specific proliferation declined to background levels. One irradiated gamma/delta T cell line inhibited proliferation of autologous Th1 cells, although unirradiated gamma/delta T cells either synergized with or had no effect on Th cell proliferation. gamma/delta T cells were not cytolytic for bovine alpha/beta T cells, but expressed natural killer (NK)-like cytotoxicity when assayed on xenogeneic NK-sensitive target cells. The gamma/delta T cells were IL-2 dependent and expressed IFN-gamma and TNF-alpha, but not TNF-beta, IL-2, or IL-4 mRNA. Together, these results raise the possibility that WC1+ gamma/delta T cells respond in vitro to autoantigens present on CD4+ T cells or to cytokines secreted by activated CD4+ T cells, resulting in modulation of the CD4+ T cell response and outgrowth of the gamma/delta T cells in parasite-stimulated lines.

    PMID: 7507005 [PubMed - indexed for MEDLINE]

  44. Exp Parasitol. 1993 Aug;77(1):97-110.

    Babesia bovis: characterization of the T helper cell response against the 42-kDa merozoite surface antigen (MSA-1) in cattle.

    Brown WC, Palmer GH, McElwain TF, Hines SA, Dobbelaere DA.

    Department of Veterinary Pathobiology, Texas A&M University, College Station 77843.

    The Babesia bovis major merozoite surface antigen (MSA-1) is a 42-kDa integral membrane glycoprotein previously shown to induce immunodominant antibody responses in cattle protectively immune to B. bovis and to induce neutralizing antibody. Recent studies have also shown that MSA-1 B cell epitopes common to New World strains of B. bovis are not present in either Israel or Australia strains. To understand the potential role of this protein in protective immunity, T helper cell responses specific for MSA-1 were characterized in Babesia-immune cattle. Peripheral blood mononuclear cells from immune cattle proliferated against affinity-purified recombinant MSA-1 protein expressed in Escherichia coli. MSA-1 preferentially stimulated the growth of CD4+ T cells in cell lines cultured with antigen for 4 weeks. MSA-1-reactive cell lines responded to a membrane fraction of B. bovis merozoites, suggesting recognition of the native protein. However, B. bovis-reactive T cell lines and T helper clones established by stimulation with crude parasite membrane antigen failed to respond to recombinant MSA-1, indicating that this antigen is not immunodominant for T cells. The majority of MSA-1-specific T helper clones reacted to unfractionated merozoite membrane antigen from New World B. bovis strains, but none of the clones responded to Australia B. bovis or to a Mexico strain of Babesia bigemina. Several T helper clones produced low levels of cytokines when stimulated with concanavalin A and interleukin-2. Northern blot analysis revealed the expression of interleukin-2, interleukin-4, interferon-gamma, and tumor necrosis factor-alpha messenger RNA in mitogen-stimulated T helper clones, showing that the clones examined expressed an unrestricted T helper phenotype. We conclude that the MSA-1 protein, although serologically immunodominant and capable of inducing neutralizing antibodies as well as a T helper cell response, is not an immunodominant T cell antigen. Furthermore, the parasite strain specificity of the Th clones supports previous findings of extensive polymorphism in the MSA-1 glycoprotein and suggests that like B cell epitopes, T cell epitopes reside in a nonconserved portion of the protein.

    PMID: 8344411 [PubMed - indexed for MEDLINE]

  45. Infect Immun. 1993 Aug;61(8):3273-81.

    Heterogeneity in cytokine profiles of Babesia bovis-specific bovine CD4+ T cells clones activated in vitro.

    Brown WC, Woods VM, Dobbelaere DA, Logan KS.

    Department of Veterinary Pathobiology, Texas A & M University, College Station 77843.

    The central role of T cells in the immune response against hemoprotozoan parasites, both as helper cells for T cell-dependent antibody production and as effector cells acting on intracellular parasites through the elaboration of cytokines, has prompted an investigation of the bovine cellular immune response against Babesia bovis antigens. CD4+ T helper (Th) cell clones generated from four B. bovis-immune cattle by in vitro stimulation with a soluble or membrane-associated merozoite antigen were characterized for reactivity against various forms of antigen and against different geographical isolates of B. bovis and B. bigemina and analyzed for cytokine production following mitogenic stimulation with concanavalin A. Biological assays to measure interleukin-2 (IL-2), IL-4, gamma interferon (IFN-gamma), and tumor necrosis factor alpha or tumor necrosis factor beta and Northern (RNA) blot analysis to verify the expression of IL-2, IL-4, IFN-gamma, and tumor necrosis factor alpha revealed differential production of cytokines by the Th cell clones. The majority of clones expressed the Th0 pattern of cytokines: IFN-gamma, IL-4, and IL-2. One clone expressed the Th1 profile (IFN-gamma and IL-2 but not IL-4), whereas none of the clones expressed the Th2 profile. All of the Th cell clones examined expressed the low-molecular-weight isoform of the leukocyte common antigen associated with a memory cell phenotype (CD45RO), and all expressed the lymph node homing receptor (L-selectin). These results extend our previous finding of differential cytokine expression by B. bovis-specific Th cell clones and confirm the identity of the specific cytokines produced, showing that a Th0 response is preferentially induced in a panel of 20 CD4+ T cell clones obtained from immune cattle.

    PMCID: PMC280999 PMID: 8335361 [PubMed - indexed for MEDLINE]

  46. J Parasitol. 1993 Jun;79(3):424-34.

    Ultrastructural characteristics of Babesia odocoilei in vitro.

    Droleskey RE, Holman PJ, Waldrup KA, Corrier DE, Wagner GG.

    USDA/ARS, Food Animal Protection Research Laboratory, College Station, Texas 77845.

    Babesia odocoilei continuously cultured in white-tailed deer (Odocoileus virginianus) erythrocytes was examined by transmission and scanning electron microscopy. Merozoites, trophozoites, intermediate-stage forms, and dividing forms were observed. Merozoites possessed a single nucleus, inner membrane complex, rhoptries, free ribosomes, rough endoplasmic reticulum, and single membrane-bound vesicles. Trophozoites lacked an inner membrane complex and rhoptries. Intermediate stages were characterized by distinct segments of inner membrane complex. Dividing forms ranged from cells with an elongated nucleus to mature daughter cells joined by a ringlike structure. Babesia odocoilei was characterized by its close proximity to the erythrocyte membrane, membranous structures resembling feeding organelles, and reproduction via a method resembling budding sensu stricto.

    PMID: 8501601 [PubMed - indexed for MEDLINE]

  47. J Parasitol. 1993 Apr;79(2):233-7.

    In vitro growth of Babesia bovis in white-tailed deer (Odocoileus virginianus) erythrocytes.

    Holman PJ, Waldrup KA, Droleskey RE, Corrier DE, Wagner GG.

    Texas Agricultural Experiment Station, Department of Veterinary Pathobiology, Texas A&M University, College Station 77843.

    Babesia bovis cultured in bovine erythrocytes was passaged into white-tailed deer (Odocoileus virginianus) erythrocytes and medium containing either white-tailed deer serum or bovine serum. Deer erythrocytes supported the growth of the parasite only in the presence of bovine serum. Cryopreserved cultures were recovered successfully in white-tailed deer erythrocytes. By light and electron microscopy, B. bovis structure appeared similar in host cells of either species.

    PMID: 8459334 [PubMed - indexed for MEDLINE]

  48. J Clin Microbiol. 1993 Mar;31(3):698-701.

    Culture confirmation of the carrier status of Babesia caballi-infected horses.

    Holman PJ, Frerichs WM, Chieves L, Wagner GG.

    Department of Veterinary Pathobiology, Texas A&M University, College Station 77843.

    Culture of horse blood for Babesia caballi identified four carrier horses among nine previously infected horses. Three of the carriers had no detectable parasitemias on stained blood smears, and sera from two carrier horses were complement fixation test negative. Three cultures were continuously cultivated. Cryopreserved fourth-passage B. caballi was successfully reestablished in vitro. Blood from a 10th horse previously subinoculated with blood from a suspected carrier was cultured, with negative results.

    PMCID: PMC262846 PMID: 8458966 [PubMed - indexed for MEDLINE]

  49. Rev Elev Med Vet Pays Trop. 1993;46(1-2):65-9.

    Babesia bovis-specific CD4+ T cell clones from immune cattle express either the Th0 or Th1 profile of cytokines.

    Brown WC, Zhao S, Woods VM, Dobbelaere DA, Rice-Ficht AC.

    Department of Veterinary Pathobiology, Texas A & M University, College Station.

    The central role of T cells in the immune response against hemoprotozoan parasites, both as helper cells for T-dependent antibody production, and as effector cells acting directly or indirectly on intracellular parasites through the elaboration of cytokines, has prompted us to investigate the bovine cellular immune response against B. bovis antigens. T cell clones generated from four B. bovis-immune cattle by in vitro stimulation with soluble or membrane associated merozoite antigen were characterized for reactivity against various forms of antigen and different geographical isolates of B. bovis and B. bigemina. The clones were categorized into seven different groups based on differential patterns of reactivity. This panel of T cell clones and additional clones specific for either the 77 kDa merozoite apical complex associated protein (Bb-1) or the 42 kDa major merozoite protein (MSA-1) were analyzed for cytokines. Biological assays to measure IL-2/IL-4, IFN-gamma and TNF-alpha/TNF-beta and Northern blot analysis to detect mRNA encoding bovine IL2, IL-4, IFN-gamma, TNF-beta and TNF-alpha revealed the differential production of cytokines by clones with different antigen specificities. Two Bb-1-specific T cell clones produced the Th1 pattern of cytokines: IL-2, IFN-gamma, TNF-beta and TNF-alpha, but not IL-4. Clones specific for the 42 kDa protein produced undetectable levels of all cytokines, but expressed an unrestricted or Th0 pattern of cytokine mRNA: IL-2, IL-4, IFN-gamma and TNF-alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

    PMID: 7907805 [PubMed - indexed for MEDLINE]

  50. Infect Immun. 1993 Jan;61(1):236-44.

    Identification of two Th1 cell epitopes on the Babesia bovis-encoded 77-kilodalton merozoite protein (Bb-1) by use of truncated recombinant fusion proteins.

    Brown WC, Zhao S, Woods VM, Tripp CA, Tetzlaff CL, Heussler VT, Dobbelaere DA, Rice-Ficht AC.

    Department of Veterinary Pathobiology, Texas A&M University, College Station 77843.

    Previous studies have demonstrated the serologic and T-cell immunogenicity for cattle of a recombinant form of the apical complex-associated 77-kDa merozite protein of Babesia bovis, designated Bb-1. The present study characterizes the immunogenic epitopes of the Bb-1 protein. A series of recombinant truncated fusion proteins spanning the majority of the Bb-1 protein were expressed in Escherichia coli, and their reactivities with bovine peripheral blood mononuclear cells and T-cell clones derived from B. bovis-immune cattle and with rabbit antibodies were determined. Lymphocytes from two immune cattle were preferentially stimulated by the N-terminal half of the Bb-1 protein (amino acids 23 to 266, termed Bb-1A), localizing the T-cell epitopes to the Bb-1A portion of the molecule. CD4+ T-cell clones derived by stimulation with the intact Bb-1 fusion protein were used to identify two T-cell epitopes in the Bb-1A protein, consisting of amino acids SVVLLSAFSGN VWANEAEVSQVVK and FSDVDKTKSTEKT (residues 23 to 46 and 82 to 94). In contrast, rabbit antiserum raised against the intact fusion protein reacted only with the C-terminal half of the protein (amino acids 267 to 499, termed Bb-1B), which contained 28 tandem repeats of the tetrapeptide PAEK or PAET. Biological assays and Northern (RNA) blot analyses for cytokines revealed that following activation with concanavalin A, T-cell clones reactive against the two Bb-1A epitopes produced interleukin-2, gamma interferon, and tumor necrosis factors beta and alpha, but not interleukin-4, suggesting that the Bb-1 antigen preferentially stimulates the Th1 subset of CD4+ T cells in cattle. The studies described here report for the first time the characterization, by cytokine production, of the Th1 subset of bovine T cells and show that, as in mice, protozoal antigens can induce Th1 cells in ruminants. This first demonstration of B. bovis-encoded Th1 cell epitopes provides a rationale for incorporation of all or part of the Bb-1 protein into a recombinant vaccine.

    PMCID: PMC302710 PMID: 7678098 [PubMed - indexed for MEDLINE]

  51. Infect Immun. 1992 Oct;60(10):4364-72.

    Bovine helper T cell clones recognize five distinct epitopes on Babesia bovis merozoite antigens.

    Brown WC, Zhao S, Rice-Ficht AC, Logan KS, Woods VM.

    Department of Veterinary Pathobiology, Texas A&M University, College Station 77843.

    Helper T cell clones from two Babesia bovis-immune cattle were characterized for use in identification of potentially protective immunogens of B. bovis merozoites. Proliferation assays with 11 CD4+ clones revealed a differential pattern of response to soluble cytosolic antigen, membrane-enriched antigen, detergent extracts of the membrane-enriched antigen, soluble culture supernatant exoantigen, and different geographical isolates of B. bovis as well as Babesia bigemina parasites. When the data were combined, the clones could be grouped according to five different patterns of response. One group recognized only the membrane-enriched fraction of New World and Australian parasites. Four remaining groups recognized antigens found in the cytosolic as well as the membrane-enriched fraction, and clones representative of each group were used to identify cytosolic antigens fractionated by anion-exchange chromatography with the use of fast-performance liquid chromatography. One clone (C97.3C3), which responded to all B. bovis isolates and to B. bigemina, recognized a single peak of activity that eluted with 0.25 M NaCl and contained protein bands of 70 and 75 kDa. The remaining clones were stimulated by a second antigenic peak that eluted between 0.35 and 0.45 M NaCl and contained protein bands of 42, 47, 56, and 84 kDa. The majority of the clones produced interferon, whereas tumor necrosis factor alpha/tumor necrosis factor beta production was less frequent. These studies provide the basis for using helper T cell clones to identify potentially protective immunogens of B. bovis and delineate a minimum of five helper T cell epitopes recognized by two immune cattle.

    PMCID: PMC257473 PMID: 1383149 [PubMed - indexed for MEDLINE]

  52. Mol Biochem Parasitol. 1992 Oct;55(1-2):85-94.

    Neutralization-sensitive merozoite surface antigens of Babesia bovis encoded by members of a polymorphic gene family.

    Hines SA, Palmer GH, Jasmer DP, McGuire TC, McElwain TF.

    Department of Infectious Diseases, University of Florida, Gainesville.

    Monospecific antibodies against native and recombinant versions of the major merozoite surface antigen (MSA-1) of Babesia bovis neutralize the infectivity of merozoites from Texas and Mexico strains in vitro. Sequence analysis shows that MSA-1 and a related, co-expressed 44 kDa merozoite surface protein (MSA-2) are encoded by members of a multigene family previously designated BabR. BabR genes, originally described in Australia strains of B. bovis, are notable because their marked polymorphism is apparently mediated by chromosomal rearrangements, but protein products of BabR genes have not previously been identified. The 3' terminal 173 nucleotides of the MSA-1 gene, including 60 nucleotides of untranslated sequence, are highly similar to the 3' terminal sequences of BabR 0.8 (84% identity) and MSA-2 (94% identity). Alignment of the predicted protein sequences demonstrates significant overall homology between MSA-1 and MSA-2, and between both proteins and the amino terminal BabR sequence. MSA-1 nucleic acid probes also hybridize weakly to genomic DNA from the Australia 'L' strain, even though this strain does not express merozoite surface epitopes cross-reactive with MSA-1 or MSA-2. Hybridization of these same probes to genomic DNA from the cloned Mexico strain reveals a pattern of bands compatible with two copies each of MSA-1 and MSA-2. Proteins encoded by this B. bovis gene family have been designated variable merozoite surface antigens (VMSA). The extent and mechanism of VMSA polymorphism among strains will be important when evaluating the role these surface proteins have in the host-parasite interaction, including immunity to blood stages.

    PMID: 1279421 [PubMed - indexed for MEDLINE]

  53. J Wildl Dis. 1992 Jul;28(3):457-9.

    Monthly incidence of Theileria cervi and seroconversion to Babesia odocoilei in white-tailed deer (Odocoileus virginianus) in Texas.

    Waldrup KA, Moritz J, Baggett D, Magyar S, Wagner GG.

    Department of Veterinary Pathobiology, Texas A&M University, College Station 77843.

    Monthly monitoring of fawns collected from an area in Texas endemic for Theileria cervi and Babesia odocoilei showed that transmission of T. cervi occurred during July and August, a time period consistent with the occurrence of Amblyomma americanum. Seroconversion to B. odocoilei occurred during October to December and possibly continued through January and February. The time of seroconversion was more suggestive of transmission of B. odocoilei by Ixodes scapularis than by Amblyomma americanum.

    PMID: 1512881 [PubMed - indexed for MEDLINE]

  54. J Clin Microbiol. 1992 Jun;30(6):1374-9.

    Detection of Babesia bovis carrier cattle by using polymerase chain reaction amplification of parasite DNA.

    Fahrimal Y, Goff WL, Jasmer DP.

    Department of Veterinary Microbiology and Pathology, Washington State University, Pullman.

    Carrier cattle infected with Babesia bovis are difficult to detect because of the low numbers of parasites that occur in peripheral blood. However, diagnosis of low-level infections with the parasite is important for evaluating the efficacies of vaccines and in transmission and epidemiological studies. We used the polymerase chain reaction (PCR) to amplify a portion of the apocytochrome b gene from the parasite and tested the ability of this method to detect carrier cattle. The target sequence is associated with a 7.4-kb DNA element in undigested B. bovis genomic DNA (as shown previously), and the amplified product was detected by Southern and dot blot hybridization. The assay was specific for B. bovis, since no amplification was detected with Babesia bigemina, Trypanosoma brucei, Anaplasma marginale, or leukocyte DNA. The target sequence was amplified in DNA from B. bovis Mexico, Texas, and Australia S and L strains, demonstrating the applicability of the method to strains from different geographic regions. The sensitivity of the method ranged from 1 to 10 infected erythrocytes extracted from 0.5 ml of blood. This sensitivity was about 1,000 times greater than that from the use of unamplified parasite DNA. By the PCR method, six B. bovis carrier cattle were detected 86% of the time (range, 66 to 100%) when they were tested 11 times, while with microscopic examination of thick blood smears, the same carrier cattle were detected only 36% of the time (range, 17 to 66%). The method provides a useful diagnostic tool for detecting B. bovis carrier cattle, and the sensitivity is significantly improved over that of current methods. The results also suggest that characteristics of the apocytchrome b gene may make this a valuable target DNA for PCR-based detection of other hemoparasites.

    PMCID: PMC265295 PMID: 1624551 [PubMed - indexed for MEDLINE]

  55. Exp Parasitol. 1992 Mar;74(2):188-99.

    Babesia bovis: bovine helper T cell lines reactive with soluble and membrane antigens of merozoites.

    Brown WC, Logan KS.

    Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University, College Station 77843-4467.

    Babesia bovis-specific T cell lines were established from cattle infected with either tick-derived or cultured parasites by stimulation of peripheral blood mononuclear cells with a crude parasite membrane fraction. Induction and enrichment of CD4+ T cells occurred over time. All cell lines responded vigorously and in a dose-dependent, MHC-restricted manner to intact merozoites, and to soluble and membrane fractions derived from merozoites by homogenization and high-speed centrifugation. Solubilization of the membrane fraction with nondenaturing zwitterionic or nonionic detergents yielded antigenic extracts which also stimulated the T cells. However, a differential response was observed, in that cell lines from one animal proliferated vigorously to the detergent extracts of the membrane fraction, whereas cell lines from a second animal proliferated only weakly to these extracts. SDS-PAGE analysis revealed common protein bands of 90 and 22 kDa in the various immunogenic fractions. Cell lines from the animal infected with cultured parasites also responded to parasite culture supernatant "exoantigens" and to the related parasite, Babesia bigemina. We conclude that antigens present in merozoite membranes and soluble parasite extracts preferentially stimulate CD4+ T cells from cattle immune to Babesia bovis. The differential pattern of response of T cell lines from different cattle suggests that more than one protein or epitope is immunodominant for T cells.

    PMID: 1371257 [PubMed - indexed for MEDLINE]

  56. Infect Immun. 1992 Feb;60(2):644-52.

    Induction of proliferative responses of T cells from Babesia bovis-immune cattle with a recombinant 77-kilodalton merozoite protein (Bb-1).

    Tetzlaff CL, Rice-Ficht AC, Woods VM, Brown WC.

    Department of Veterinary Pathobiology, Texas A & M University, College Station 77843.

    A major portion of a Babesia bovis-specific gene encoding a 77-kDa merozoite protein (Bb-1) produced during natural infection in cattle and in microaerophilous culture was subcloned into the pGEX1N expression vector. Recombinant Bb-1 protein fused to glutathione S-transferase (Bb-1-GST) was used to examine cellular immune responses in B. bovis-immune cattle. Sera from rabbits immunized with Bb-1-GST reacted with fusion protein and with the native antigen present in crude B. bovis but not with B. bigemina merozoites. Bb-1-GST but not GST induced strong proliferation of T lymphocytes from these immune cattle, and Bb-1-reactive T-cell lines which consisted of a mixed population of either CD4+ and CD8+ cells or CD4+, CD8+, and "null" (gamma delta T) cells were established by in vitro stimulation of peripheral blood mononuclear cells with the recombinant fusion protein. Three CD4+ CD8- and three CD4- CD8+ Bb-1-specific T-cell clones were identified after limiting-dilution cloning of the cell lines. The studies described here demonstrate that the 77-kDa protein of B. bovis contains T-cell epitopes capable of eliciting proliferation of two types of T cells in immune cattle, an important consideration for the design of a recombinant subunit vaccine.

    PMCID: PMC257678 PMID: 1730498 [PubMed - indexed for MEDLINE]

  57. Mem Inst Oswaldo Cruz. 1992;87 Suppl 3:193-9.

    Non-immunologic methods of diagnosis of babesiosis.

    Wagner G, Cruz D, Holman P, Waghela S, Perrone J, Shompole S, Rurangirwa F.

    Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University, College Station 77843-4467.

    The diagnosis of tick-borne diseases such as babesiosis still depends on observing the parasite in the infected erythrocyte. Microscopic observation is tedious and often problematic in both early and carrier infections. Better diagnostic methods are needed to prevent clinical disease, especially when susceptible cattle are being moved into disease enzootic areas. This study evaluates two techniques for early diagnosis of Babesia bovis infections in cattle, DNA probes specific for the organism and fluorescent probes specific for nucleic acid. The radioisotopically labeled DNA probes are used in slot blot hybridizations with lysed blood samples, not purified DNA. Thusfar, the probe is specific for B. bovis and can detect as few as 1000 B. bovis parasites in 10 microliters of blood. The specificity of the fluorescent probe depends on the characteristic morphology of the babesia in whole blood samples, as determined microscopically. The fluorescent probe detects as few as 10,000 B. bovis parasites in 10 microliters os blood. The application of each method for laboratory and field use is discussed.

    PMID: 1343690 [PubMed - indexed for MEDLINE]

  58. Infect Immun. 1991 Jul;59(7):2418-26.

    Cell-mediated immune responses to Babesia bovis merozoite antigens in cattle following infection with tick-derived or cultured parasites.

    Brown WC, Logan KS, Wagner GG, Tetzlaff CL.

    Department of Veterinary Pathobiology, Texas A&M University, College Station 77843.

    Peripheral blood mononuclear cells from cattle experimentally infected with Babesia bovis were examined for parasite-specific cell-mediated immune responses. Unfractionated merozoites and soluble and membrane fractions derived from merozoites were all antigenic for immune cattle, although the membrane fraction was the most stimulatory. Cattle responded to different antigenic fractions in a differential manner, and only that animal immunized with autologous cultured parasites responded to parasitized erythrocyte culture supernatants. Plastic-adherent cells (presumably monocytes/macrophages) were required for a proliferative response to babesial antigens but not to the T-cell mitogen concanavalin A, suggesting that babesial proteins are not simply mitogenic for T cells. Lymphocyte responses directed against a different hemoparasite from Mexico, Babesia bigemina, indicate that this parasite shares cross-reactive T-cell epitopes with B. bovis. These studies define a system whereby T lymphocytes from babesia-immune cattle can be used in proliferation assays to identify babesial merozoite antigens which are immunogenic for T cells. Because identification of helper T-cell epitopes is important for the design of a babesial subunit vaccine which will evoke anamnestic responses, the studies described here provide a basis for such experiments.

    PMCID: PMC258027 PMID: 2050406 [PubMed - indexed for MEDLINE]

  59. Mol Biochem Parasitol. 1991 May;46(1):45-52.

    Characterization of the gene encoding a 60-kilodalton Babesia bovis merozoite protein with conserved and surface exposed epitopes.

    Suarez CE, Palmer GH, Jasmer DP, Hines SA, Perryman LE, McElwain TF.

    Department of Veterinary Microbiology and Pathology, Washington State University, Pullman.

    A clone expressing a surface exposed, conserved epitope of a 60-kDa merozoite polypeptide was identified in a cDNA library constructed from a cloned Mexico strain of Babesia bovis. Sequencing of the 1.9-kb insert (pBv60) revealed an open reading frame encoding a 65-kDa polypeptide with a signal peptide and a tandemly repeated region. Monoclonal antibody 23/56.156, which binds a surface exposed epitope on the native polypeptide, specifically immunoprecipitated [35S]methionine-labeled polypeptides ranging from 60-30 kDa from pBv60 directed transcription and translation. Antibodies raised in rabbits against recombinant polypeptide reacted with the live merozoite surface in a polar immunofluorescence pattern, immunoprecipitated the native 60-kDa polypeptide, and were used to deplete the polypeptide by adsorption from a preparation of native [35S]methionine-labeled merozoite antigen. Restriction enzyme analysis indicated a single gene copy and the absence of introns. Hybridization demonstrated the presence of the gene in Mexico, Australia 'L', and Texas strains of B. bovis, but not in Babesia bigemina. A slightly different hybridization pattern was present in uncloned Australia 'L' B. bovis, indicating sequence diversity in the Bv60 gene among isolates. Cloning and structural analysis of pBv60 provides a source of defined antigen for determining the role of conserved merozoite surface epitopes in protective immunity against babesiosis.

    PMID: 1712911 [PubMed - indexed for MEDLINE]

  60. J Wildl Dis. 1991 Jan;27(1):86-91.

    Comparative studies of Babesia spp. from white-tailed and sika deer.

    Gray JS, Murphy TM, Waldrup KA, Wagner GG, Blewett DA, Harrington R.

    Department of Environmental Resource Management, University College, Belfield, Dublin, Ireland.

    Babesia odocoilei from white-tailed deer (Odocoileus virginianus) in Texas (USA) and B. capreoli isolated from sika deer (Cervus nippon) in Ireland were compared morphologically and antigenically. Babesia odocoilei and B. capreoli paired pyriforms resembled each other closely when in sika deer, but B. odocoilei pyriforms in white-tailed deer were slightly different. Babesia odocoilei in white-tailed deer also differed from B. odocoilei and B. capreoli in sika deer in the frequency of its developmental forms. Indirect immunofluorescence antibody test titres showed that there was some antigen cross-reactivity, but not as much as between B. capreoli and the bovine parasite, B. divergens. The Babesia spp. from deer that we studied appear to be distinct but related species. The low infectivity of B. odocoilei for a splenectomised sika deer suggests that sika deer in North America are probably not very susceptible to this parasite in the wild.

    PMID: 2023332 [PubMed - indexed for MEDLINE]

  61. J Med Entomol. 1990 Nov;27(6):1067-70.

    Microwave fixation: in situ tick (Acari: Ixodidae) histoanatomy, thin sectioning of tick tissues, and antigen preservation in mouse spleen.

    Carranza GA, Cruz D, Wagner GG.

    Department of Veterinary Microbiology and Parasitology, College of Veterinary Medicine, Texas A&M University, College Station 77843.

    Microwave irradiation was used for the fixation of eggs, nymphs, and adult Boophilus spp. ticks. Although optimal temperatures for fixation of the different tick stages varied, heating to 58 degrees C of adult ticks submerged in either PBS or fixative was found to be sufficient. After microwave fixation, whole adult ticks, hand held, were sectioned with a sharp razor blade. The resulting sections revealed the in situ histoanatomy of the tick. Thin sections of ticks were obtained after either paraffin or polyester wax embedding. Microwave fixation combined with polyester wax embedding made serial thin sections of the different stages of Boophilus ticks possible. The technique preserved antigens as demonstrated by the immunostaining of lymphocytes and erythrocytes infected with Babesia microti in mouse tissues subjected to the same treatment as the ticks. With the microwave fixation-polyester wax technique, the specimen preparation time from fixation to the section on the glass slide was reduced to less than 8 h.

    PMID: 2280393 [PubMed - indexed for MEDLINE]

  62. J Wildl Dis. 1990 Jul;26(3):390-1.

    Transmission of Babesia odocoilei in white-tailed deer (Odocoileus virginianus) by Ixodes scapularis (Acari: Ixodidae).

    Waldrup KA, Kocan AA, Barker RW, Wagner GG.

    Department of Veterinary Microbiology and Parasitology, College of Veterinary Medicine, Texas A&M University, College Station 77843.

    Laboratory reared Ixodes scapularis proved to be an efficient vector of Babesia odocoilei Emerson and Wright between white-tailed deer (Odocoileus virginianus). Transtadial survival of the babesia occurred between nymph and adult stages of the tick, and the adult stage transmitted the babesia.

    PMID: 2388362 [PubMed - indexed for MEDLINE]

  63. Mol Biochem Parasitol. 1990 May;40(2):183-92.

    Isolation and characterization of a gene associated with a virulent strain of Babesia microti.

    Tetzlaff CL, McMurray DN, Rice-Ficht AC.

    Department of Medical Microbiology and Immunology, Texas A&M University, College Station 77843.

    Babesia microti genomic DNA was purified from parasitized murine erythrocytes, digested with mung bean nuclease and used to construct an expression library in lambda gt11. Polyspecific antisera from mice infected with virulent B. microti organisms (ATCC30221) were used to screen the genomic library for genes encoding major immunogens. High titer antisera selected a recombinant phage, Bm13, containing 3.3 kb of B. microti DNA. Hybridization analysis confirmed the parasite origin of the clone; affinity-purified antibody revealed a native molecular weight of 54,000 for the B. microti protein encoded by the recombinant. Only genomic DNA isolated from the virulent strain of B. microti contained sequences which hybridized to Bm13. Genomic DNA prepared from the Peabody attenuated strain of B. microti or from Babesia bovis DNA did not contain any complementary sequences. These data suggest a possible role for the gene in the virulence of the organism.

    PMID: 2362602 [PubMed - indexed for MEDLINE]

  64. Exp Parasitol. 1989 Oct;69(3):211-25.

    Babesia bovis: gene isolation and characterization using a mung bean nuclease-derived expression library.

    Tripp CA, Wagner GG, Rice-Ficht AC.

    Department of Veterinary Microbiology and Parasitology, Texas A&M University, College Station 77843.

    Genomic DNA prepared from erythrocyte cultures of Babesia bovis merozoites was digested with mung bean nuclease and used to construct a lambda gt11 expression library of B. bovis recombinants. Immunoscreening with two polyclonal antibody probes detected multiple recombinants from which two, designated Bb-1 and Bb-3, were chosen for further analysis. Monospecific immunoglobulins isolated from the screening sera using nitrocellulose-bound fusion proteins were employed to determine the native molecular weight and the intracellular location of the babesial proteins encoded by the recombinants. Clone Bb-1 encodes an antigen of 77,000 Da located at the apical end of the intraerythrocytic parasite. A protein of 75,000 Da encoded by clone Bb-3 is associated with the infected red blood cell cytoplasm and/or membrane but not with the merozoite.

    PMID: 2676576 [PubMed - indexed for MEDLINE]

  65. J Wildl Dis. 1989 Apr;25(2):194-201.

    Serological prevalence and isolation of Babesia odocoilei among white-tailed deer (Odocoileus virginianus) in Texas and Oklahoma.

    Waldrup KA, Kocan AA, Qureshi T, Davis DS, Baggett D, Wagner GG.

    Department of Veterinary Microbiology and Parasitology, College of Veterinary Medicine, Texas A&M University, College Station 77843.

    Serum samples collected from 581 white-tailed deer (Odocoileus virginianus) from Texas and from 124 white-tailed deer from Oklahoma were tested by the indirect fluorescent antibody technique against Babesia odocoilei. Prevalence of seropositive reactors varied from site to site in both states. Prevalence rates were statistically ranked as high, intermediate or low. Deer less than 12-mo-old had a significantly lower prevalence than all other age classes.

    PMID: 2654422 [PubMed - indexed for MEDLINE]

  66. Res Vet Sci. 1988 Sep;45(2):262-3.

    Effect of carrier erythrocytes containing inositol hexaphosphate on Babesia microti infection.

    Deloach JR, Corrier DE, Wagner GG.

    USDA, Agriculture Research Service, College Station, Texas 77841.

    Erythrocytes containing inositol hexaphosphate (IHP) were administered to mice. Mice were then challenged with Babesia microti. Mice receiving IHP carrier erythrocytes had significantly lower percentages of parasitaemias on days 3, 5 and 7 after infection. Carrier erythrocytes containing IHP have altered P50 oxygen values. Thus, carrier erythrocytes containing IHP may be useful in treating naive animals before transporting into areas endemic for babesiosis.

    PMID: 3194600 [PubMed - indexed for MEDLINE]

  67. Antimicrob Agents Chemother. 1988 Mar;32(3):391-4.

    Reduced parasitemia observed with erythrocytes containing inositol hexaphosphate.

    Mintzer CL, Deloron P, Rice-Ficht A, Durica D, Struck DK, Roessner CA, Nicolau C, Ihler GM.

    Department of Medical Biochemistry and Genetics, Texas A & M College of Medicine, College Station.

    Chemicals entrapped in erythrocytes by hypotonic hemolysis can be assessed for possible antiparasitic activity both in vivo and in vitro, regardless of whether they are able to diffuse into erythrocytes readily. Inositol hexaphosphate, a highly charged compound, produced a dramatic lowering of the percentage of cells infected by Babesia microti in vivo and both B. microti and Plasmodium falciparum in vitro. Several possible mechanisms for this observation are discussed.

    PMCID: PMC172182 PMID: 3364957 [PubMed - indexed for MEDLINE]

  68. J Parasitol. 1988 Feb;74(1):111-5.

    In vitro cultivation of a Babesia isolated from a white-tailed deer (Odocoileus virginianus).

    Holman PJ, Waldrup KA, Wagner GG.

    Department of Veterinary Microbiology and Parasitology, Texas A&M University, College Station 77843.

    Pyriforms and ring forms of Babesia odocoilei were detected in thin blood smears obtained from a white-tailed deer killed by a hunter in Anderson County, Texas. Erythrocytes from the deer were cultured and the parasites maintained through 8 serial subcultures during 1 mo. The parasite was successfully established in culture using Medium 199 supplemented with either 20% deer serum or 40% normal adult bovine serum. The highest parasitemia observed was 30% and more than 4 parasites per erythrocyte were often observed. Cultured B. odocoilei remained infective for a susceptible white-tailed deer.

    PMID: 3357095 [PubMed - indexed for MEDLINE]

  69. Med Microbiol Immunol. 1988;177(6):305-15.

    Reduced dietary protein content suppresses infection with Babesia microti.

    Tetzlaff CL, Carlomagno MA, McMurray DN.

    Department of Medical Microbiology and Immunology, College of Medicine, Texas A&M University, College Station 77843.

    The influence of acute dietary protein restriction on the development of Babesia microti infection in the mouse model was investigated. Female mice consuming a diet either devoid of protein or adequate with respect to protein were infected with B. microti-parasitized erythrocytes and sacrificed 7 days later. Absence of dietary protein resulted in a delay in the onset of infection and a significantly reduced peak parasitemia. Non-specific antibody responses to heterologous erythrocytes and specific anti-babesial antibody titers were impaired in mice consuming the protein-free diets, suggesting that the enhanced resistance to experimental babesiosis observed in protein-malnourished mice is not an antibody-mediated phenomenon. In addition, protein-malnourished mice did not demonstrate significantly lower concentrations of the serum complement component, C3, which has been implicated as a participant in the invasion process of host erythrocytes by parasites. Serum C3 levels were significantly reduced in infected mice consuming both diets. The mechanism by which acute protein deprivation protects mice against lethal babesiosis remains to be determined.

    PMID: 3216813 [PubMed - indexed for MEDLINE]

  70. Infect Immun. 1984 Sep;45(3):697-700.

    Common and isolate-restricted antigens of Anaplasma marginale detected with monoclonal antibodies.

    McGuire TC, Palmer GH, Goff WL, Johnson MI, Davis WC.

    Anaplasma marginale-infected erythrocytes were examined for the presence of maturation, isolate-restricted, and isolate-common antigens by indirect immunofluorescence with monoclonal antibodies. A panel of 18 monoclonal antibodies was used; none of the antibodies reacted with Anaplasma ovis, Babesia bigemina, babesia bovis, Trypanosoma brucei, Trypanosoma congolense, or uninfected bovine erythrocytes. Antigens common to all six A. marginale isolates were detected by nine antibodies. Single isolates from Florida, Southern Idaho, Northern Texas, and Virginia and two isolates from Washington state had four patterns of reactivity with a second panel of nine antibodies. Antigenically distinct stages were not detected, as sequential smears taken daily during acute infection had the same pattern of reactivity. The results demonstrate antigenic heterogeneity among isolates of A. marginale and the presence of common antigens. This information allows grouping of different isolates and, more importantly, provides a method for the identification and isolation of common antigens for diagnostic tests.

    PMCID: PMC263352 PMID: 6205996 [PubMed - indexed for MEDLINE]

  71. J Am Vet Med Assoc. 1983 May 1;182(9):978-82.

    Babesiosis in the Greyhound.

    Breitschwerdt EB, Malone JB, MacWilliams P, Levy MG, Qualls CW Jr, Prudich MJ.

    Babesiosis was diagnosed in five 11- to 18-day old Greyhound pups. In 3 pups, Babesia canis organisms were identified by examination of a Wright's-Giemsa-stained smear of blood. In 2 pups, the diagnosis was established by examination of a splenic impression smear obtained at necropsy. Findings in the 3 clinical cases included depression, weakness, anorexia, pallor, anemia, and thrombocytopenia. Subcutaneous administration of diminazene aceturate resulted in rapid clinical recovery in these cases. In the 2 pups that were necropsied, variable numbers of erythrocytes containing Babesia organisms were observed in the microvasculature of tissues. Subinoculation of blood into an intact dog and a splenectomized dog resulted in parasitemia and B canis serum titers, as determined by indirect fluorescent antibody testing. A site visit to the kennel from which the pups had originated led to identification of numerous Rhipicephalus sanguineus in small buildings and pens. Of 107 dogs from this kennel that were tested, 63 had an indirect fluorescent antibody titer for B canis. A limited serologic survey of Greyhound kennels in West Virginia, Oklahoma, Texas, Mississippi, and Florida identified a large number of dogs with indirect fluorescent antibody titers for B canis.

    PMID: 6853321 [PubMed - indexed for MEDLINE]

  72. Am J Vet Res. 1977 Feb;38(2):153-6.

    Comparisons of the complement-fixation and indirect fluorescent antibody reactions in the detection of bovine babesiasis.

    Kuttler KL, Adams LG, Todorovic RA.

    Complement-fixation (CF) and indirect fluorescent antibody (IFA) antigens were prepared from Babesia bigemina isolates obtained in Texas. These serologic procedures were evaluated on 130 serum samples sequentially collected from 5 B bigemina-infected mature cattle, beginning on the day of exposure and continuing for 175 day thereafter. Both tests were effective in detecting specific antibodies for the first 84 days of infection, with 57 of 60 (95%) serums tested being positive on the CF test and 57 of 57 (100%) tests being positive to the IFA test. During the interval from 98 to 175 days, 24 of 60 (40%) of the serums tested were positive with the CF test, and 53 of 56 (95%) were positive with the IFA test. During the first 84 days, a similar linear regression occurred in both CF and IFA serum titers, but after 98 days the IFA regression flattened out, whereas the CF titers decreased below the sensitivity threshold in 60% of the serums tested.

    PMID: 320922 [PubMed - indexed for MEDLINE]

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