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Florida Babesia

  1. Mol Biochem Parasitol. 2010 Jun;171(2):81-8. Epub 2010 Mar 10.

    The Babesia bovis VESA1 virulence factor subunit 1b is encoded by the 1beta branch of the ves multigene family.

    Xiao YP, Al-Khedery B, Allred DR.

    University of Florida, Department of Infectious Diseases and Pathology, Gainesville, FL, USA.

    Babesia bovis, an intraerythrocytic parasite of cattle, establishes persistent infections of extreme duration. This is accomplished, at least in part, through rapid antigenic variation of a heterodimeric virulence factor, the variant erythrocyte surface antigen-1 (VESA1) protein. Previously, the VESA1a subunit was demonstrated to be encoded by a 1alpha member of the ves multigene family. Since its discovery the 1beta branch of this multigene family has been hypothesized to encode the VESA1b polypeptide, but formal evidence for this connection has been lacking. Here, we provide evidence that products of ves1beta genes are rapidly variant in antigenicity and size-polymorphic, matching known VESA1b polypeptides. Importantly, the ves1beta-encoded antigens are co-precipitated with VESA1a during immunoprecipitation with anti-VESA1a monoclonal antibodies, and antisera to ves1beta polypeptide co-precipitate VESA1a. Further, the ves1beta-encoded antigens significantly co-localize with VESA1a on the infected-erythrocyte membrane surface of live cells. These characteristics all match known properties of VESA1b, allowing us to conclude that the ves1beta gene divergently apposing the ves1beta gene within the locus of active ves transcription (LAT) encodes the 1b subunit of the VESA1 cytoadhesion ligand. However, the extent and stoichiometry of VESA1a and 1b co-localization on the surface of individual cells is quite variable, implicating competing effects on transcription, translation, or trafficking of the two subunits. These results provide essential information facilitating further investigation into this parasite virulence factor. Copyright 2010 Elsevier B.V. All rights reserved.

    PMCID: PMC2856709 [Available on 2011/6/1] PMID: 20226217 [PubMed - in process]

  2. Onderstepoort J Vet Res. 2009 Mar;76(1):53-8.

    Persistence mechanisms in tick-borne diseases.

    Barbet AF.

    Department of Infectious Diseases & Pathology, College of Veterinary Medicine, University of Florida, Gainesville, Florida, USA.

    The use of new, highly sensitive diagnostic methods has revealed persistent infections to be a common feature of different tick-borne diseases, such as babesiosis, anaplasmosis and heartwater. Antigenic variation can contribute to disease persistence through the continual elaboration of new surface structures, and we know in several instances how this is achieved. Known or suspected mechanisms of persistence in babesial parasites include cytoadhesion and rapid variation of the adhesive ligand in Babesia bovis and genetic diversity in several merozoite stage proteins of different Babesia spp. In Anaplasma, extensive variation in the pfam01617 gene family accompanies cycling of organism levels in chronic infection. One result from the pioneering research at Onderstepoort is the definition of a related polymorphic gene family that is likely involved in immunity against heartwater disease. We are beginning to understand the sizes of the antigenic repertoires and full definition is close, with the possibility of applying simultaneous high-throughput sequencing to the order of 1000 small genomes. We also, for the first time, can consider modifying these genomes and looking at effects on persistence and virulence. However, important biological questions remain unanswered; for example, why we are seeing a new emerging Anaplasma infection of humans and is infection of endothelial cells by Anaplasma significant to persistence in vivo.

    PMID: 19967928 [PubMed - indexed for MEDLINE]

  3. J Wildl Dis. 2009 Oct;45(4):907-20.

    Blood parasites in birds from Madagascar.

    Savage AF, Robert V, Goodman SM, Raharimanga V, Raherilalao MJ, Andrianarimisa A, Ariey F, Greiner EC.

    Department of Infectious Diseases and Pathology, College of Veterinary Medicine, University of Florida, Box 110880, Gainesville, Florida 32611, USA. haemosporida@yahoo.com

    Madagascar has long been recognized for its unique and diverse biota. In particular, significant effort has been made to establish baseline population data to better conserve the endemic avifauna. During field expeditions between 1993 and 2004, birds were mist-netted at 11 different sites, at elevations from 60 m to 2,050 m above sea level. Data on endemic status, forest type, and habitat preference were recorded. Thin blood films from 947 birds, belonging to 26 families and 64 species, were examined by light microscopy to determine the prevalence of blood parasites. Of these 947 birds, 30.7% were infected by at least one species of blood parasite, 26.8% of which were infected by more than one species. Species of Haemoproteus were the most prevalent (17.4%), followed by microfilariae (11.0%), Leucocytozoon spp. (9.4%), Plasmodium spp. (1.9%), Trypanosoma spp. (0.9%), and Babesia spp. (0.2%). Species level identifications confirmed the presence of 47 species of hemosporidians and trypanosomes, which is notably high and mirrors the diversity of their avian hosts. Eleven (23.4%) of these parasite species were new to science and thought to be endemic to the island. Significant differences in prevalence were observed by sample site, forest type (humid vs. dry), and habitat preference. Birds from all elevational zones sampled were infected, although not all parasite genera were present in each zone. Four of the six endemic avian families or subfamilies (Bernieridae, Brachypteraciidae, Philepittinae [Eurylaimidae], and Vangidae) were sampled and found to be parasitized. Of the families with the largest sample sizes, the Zosteropidae and Ploceidae had the highest prevalence of infection (65.6% and 49.3%, respectively). The vectors of hematozoan parasites in Madagascar are currently unknown. These results add to the current knowledge of avian parasitism in Madagascar and are of particular interest for the conservation of endemic species, as well as threatened or endangered populations.

    PMID: 19901367 [PubMed - indexed for MEDLINE]

  4. Mol Biochem Parasitol. 2009 Jul;166(1):47-53. Epub 2009 Feb 25.

    Universal primers suitable to assess population dynamics reveal apparent mutually exclusive transcription of the Babesia bovis ves1alpha gene.

    Zupańska AK, Drummond PB, Swetnam DM, Al-Khedery B, Allred DR.

    Department of Infectious Diseases and Pathology, University of Florida, Gainesville, FL 32611-0880, USA.

    Babesia bovis is an intraerythrocytic hemoparasite of widespread distribution, which adversely affects livestock production in many regions of the world. This parasite establishes persistent infections of long duration, at least in part through rapid antigenic variation of the VESA1 protein on the infected-erythrocyte surface. To understand the dynamics of in vivo antigenic variation among the parasite population it is necessary to have sensitive and broadly applicable tools enabling monitoring of variation events in parasite antigen genes. To address this need for B. bovis, "universal" primers for the polymerase chain reaction have been designed for the ves1alpha gene, spanning from exon 2 to near the 3' end of cysteine-lysine-rich domain (CKRD) sequences in exon 3. These primers robustly amplified this segment, with minimal bias, from essentially the entire repertoire of full-length ves1alpha sequences in the B. bovis Mexico isolate genome, and are equivalently present in other isolates. On purified genomic DNA, this primer set can achieve a sensitivity of 10 genome equivalents or less. When applied to the amplification of cDNA derived from the B. bovis C9.1 clonal line evidence consistent with mutually exclusive transcription of the ves1alpha gene was obtained, concomitant with detection of numerous mutational events among members of the parasite population. These characteristics of the primers will facilitate the application of polymerase chain reaction-based methodologies to the study of B. bovis population and antigenic switching dynamics.

    PMCID: PMC2681252 [Available on 2010/7/1] PMID: 19428672 [PubMed - indexed for MEDLINE]

  5. Trop Anim Health Prod. 2009 Oct;41(7):1465-73. Epub 2009 Apr 3.

    Seroprevalence estimation and management factors associated with high herd seropositivity for Babesia bovis in commercial dairy farms of Puerto Rico.

    Urdaz-Rodríguez JH, Fosgate GT, Waghela SD, Alleman AR, Rae DO, Donovan GA, Melendez P.

    Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL 32610, USA. tuatero@yahoo.com

    A cross-sectional study was conducted to determine individual cow seroprevalence of Babesia bovis in adult lactating dairy cattle of Puerto Rico (PR), to assess the associations of farm management factors on herd seroprevalence, and to document the species of ticks infesting cattle within these farms. Antibody activity against B. bovis was determined using an indirect fluorescent antibody test (IFAT). Serum samples were obtained from 2,414 adult lactating dairy cattle from 76 randomly selected commercial dairy farms. Herd seroprevalence ranged from 0 to 51% with an overall individual cow seroprevalence for B. bovis of 26%. Ticks were collected from animals on 7 (9%) of the 76 participating commercial dairy farms. All collected ticks (n = 87) were Rhipicephalus (Boophilus) microplus. Factors associated with high herd seropositivity were dairy farms with calf but not heifer raising facilities (OR = 16, 95% CI = 3.0-86), having more than 4 neighbors with cattle (OR = 17, 95% CI = 1.6-178), same producer owning more than one farm (OR = 7.2, 95% CI = 1.6-32), and use of government services to apply amitraz on cattle (OR = 5.5, 95% CI = 1.5-20).

    PMID: 19343529 [PubMed - indexed for MEDLINE]

  6. J Zoo Wildl Med. 2007 Jun;38(2):285-91.

    Laboratory findings in acute Cytauxzoon felis infection in cougars (Puma concolor couguar) in Florida.

    Harvey JW, Dunbar MR, Norton TM, Yabsley MJ.

    Department of Physiological Sciences, Box 100144, College of Veterinary Medicine, University of Florida, Gainesville, Florida 32610, USA. harveyj@mail.vetmed.ufl.edu

    Intraerythrocytic piroplasms, morphologically indistinguishable from Cytauxzoon felis, were identified in stained blood films from more than one third of free-ranging cougars (Puma concolor couguar) in southern Florida in a study that failed to demonstrate negative effects of piroplasm infection on measured hematologic parameters. However, a recent study with a nested 18s rRNA polymerase chain reaction (PCR) assay identified only 9% of the free-ranging cougars in southern Florida as infected with C. felis but found 83% of these animals were infected with an unnamed small Babesia sp. In this study, hematology and clinical chemistry parameters were determined during the initial appearance of piroplasms in stained blood films of three western cougars housed in northern Florida. One animal became ill, but the remaining two animals did not exhibit clinical signs of disease. The hematocrit decreased in all three cougars concomitant with the first recognized parasitemia. A regenerative response to anemia (increased polychromasia, increased mean cell volume, and increased red cell distribution width) was recognized in two cougars that were examined twice during the following 2 weeks. Thrombocytopenia and probable leukopenia occurred in one animal. The most consistent clinical chemistry findings were increased serum bilirubin concentrations and increased alanine aminotransferase and aspartate aminotransferase activities at the time of initial recognition of parasitemia. Serum protein findings were not consistent in these cougars. The use of PCR and determination of 18S rRNA gene sequences in the blood from these three animals revealed infection with C. felis, but not with the Babesia sp. In this report, we demonstrate that mild hemolytic anemia, and probably liver injury, occurs concomitant with the initial discovery of C. felis piroplasms in stained blood films.

    PMID: 17679513 [PubMed - indexed for MEDLINE]

  7. Vet Parasitol. 2007 May 15;146(1-2):170-4. Epub 2007 Mar 13.

    Dynamics of anemia progression and recovery in Babesia bigemina infection is unrelated to initiating parasite burden.

    Allred DR.

    University of Florida, Department of Infectious Diseases and Pathology, P.O. Box 110880, Gainesville, FL 32611-0880, USA. allredd@mail.vetmed.ufl.edu

    To be informative, immunization-and-challenge experiments in support of vaccine development rely on host responses that enable distinctions to be made in the responses of immunized and non-immunized animals to infectious challenge. It is therefore important that animals be challenged with standardized infectious doses that allow such distinctions to be made. We report here the results of a challenge titration experiment in which cattle were challenged with Babesia bigemina, at dosages ranging over six orders of magnitude. No significant dose-dependent differences were observed in the maximum fever attained, duration of fever, minimum hematocrit reached, kinetics with which anemia developed or was resolved, or animal weight gain. Significant differences were noted only in the length of time post-infection required to initiate fever, reach maximum fever, and attain maximum reduction in hematocrit. These results suggest that, in the absence of further supporting evidence, it is not possible to conclude any direct anti-parasite effects from reductions in maximum fever or hematocrit drop during B. bigemina immunization-and-challenge experiments. However, lengthening of the time to reduction in hematocrit may be a useful indicator of overt suppression of the challenge inoculum.

    PMID: 17353098 [PubMed - indexed for MEDLINE]

  8. 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]

  9. Antigenic variation as an exploitable weakness of babesial parasites.

    Allred DR, Al-Khedery B.

    Department of Pathobiology, University of Florida, Gainesville, FL 32611-0880, USA. allredd@ufl.edu

    Babesia bovis and its bovine host interact in many ways, resulting in a range of disease and infection phenotypes. Host responses to the parasite elicit or select for a variety of responses on the part of the parasite, the full range of which is not yet known. One well-established phenomenon, thought to aid parasite survival by evasion of host adaptive immune responses, is the sequential expansion of antigenically variant populations during an infection, a phenomenon referred to as "antigenic variation". Antigenic variation in B. bovis, like that in the human malarial parasite, Plasmodium falciparum, is intimately linked to a second survival mechanism, cytoadhesion. In cytoadhesion, mature parasite-containing erythrocytes bind to the capillary and post-capillary venous endothelium through parasite-derived ligands. The reliance of these parasites on both functions, and on their linkage, may provide opportunities to develop anti-babesial and, perhaps, anti-malarial protection strategies. The development of inhibitors of DNA metabolism in B. bovis may be used to abrogate the process of antigenic variation, whereas small molecular mimics may provide the means to vaccinate against a wide range of variants or to prevent the surface export of variant antigen ligands. In this article, aspects of antigenic variation and cytoadhesion in bovine babesiosis are explored, with a discussion of opportunities for prophylactic or therapeutic intervention in these intertwined processes.

    PMID: 16517078 [PubMed - indexed for MEDLINE]

  10. Mol Microbiol. 2006 Jan;59(2):402-14.

    Antigenic variation in Babesia bovis occurs through segmental gene conversion of the ves multigene family, within a bidirectional locus of active transcription.

    Al-Khedery B, Allred DR.

    Department of Pathobiology, University of Florida, Gainesville, 32611, USA. Al-KhederyB@mail.vetmed.ufl.edu

    Antigenic variation in Babesia bovis is one aspect of a multifunctional virulence/survival mechanism mediated by the heterodimeric variant erythrocyte surface antigen 1 (VESA1) protein that also involves endothelial cytoadhesion with sequestration of mature parasitized erythrocytes. The ves1alpha gene encoding the VESA1a subunit was previously identified. In this study, we present the unique organization of the genomic locus from which ves1alpha is transcribed, and identify a novel branch of the ves multigene family, ves1beta. These genes are found together, closely juxtaposed and divergently oriented, at the locus of active transcription. We provide compelling evidence that variation of both transcriptionally active genes occurs through a mechanism of segmental gene conversion involving sequence donor genes of similar organization. These results also suggest the possibility of epigenetic regulation through in situ switching among gene loci, further expanding the potential repertoire of variant proteins.

    PMID: 16390438 [PubMed - indexed for MEDLINE]

  11. Mol Biochem Parasitol. 2004 Mar;134(1):27-35.

    Antigenic variation and cytoadhesion in Babesia bovis and Plasmodium falciparum: different logics achieve the same goal.

    Allred DR, Al-Khedery B.

    Department of Pathobiology, College of Veterinary Medicine, University of Florida, Gainesville, FL 32611-0880, USA. allredd@mail.vetmed.ufl.edu

    Babesia bovis is a protozoal hemoparasite of cattle which behaves in certain crucial respects like Plasmodium falciparum, despite being phylogenetically distant and having many differences in its life cycle. The shared behavioral attributes of rapid antigenic variation and cytoadhesion/sequestration are thought to contribute significantly to immune evasion, establishment of persistent infections, and disease pathology. Although differing in their genetic and biochemical strategies for achieving these behaviors, information from studies of each parasite may further our understanding of the overall host-parasite interaction. In this review we contrast the molecular basis and 'genetic logic' for these critical behaviors in the two parasites, with emphasis on the biology of B. bovis.

    PMID: 14747140 [PubMed - indexed for MEDLINE]

  12. Trends Parasitol. 2003 Feb;19(2):51-5.

    Babesiosis: persistence in the face of adversity.

    Allred DR.

    Dept of Pathobiology, College of Veterinary Medicine, University of Florida, Gainesville, FL 32611-0880, USA. allredd@mail.vetmed.ufl.edu

    Many babesial parasites establish infections of long duration in immune hosts. Among different species, at least four mechanisms are known that could facilitate evasion of the host immune response, although no one species is (yet) known to use them all. This update strives to illustrate the ramifications of these mechanisms and the interplay between them.

    PMID: 12586467 [PubMed - indexed for MEDLINE]

  13. Clin Diagn Lab Immunol. 2002 Nov;9(6):1301-6.

    Conformational dependence and conservation of an immunodominant epitope within the babesia equi erythrocyte-stage surface protein equi merozoite antigen 1.

    Cunha CW, Kappmeyer LS, McGuire TC, Dellagostin OA, Knowles DP.

    Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, Washington 99164-7040, USA.

    Equi merozoite antigen 1 (EMA-1) is an immunodominant Babesia equi erythrocyte-stage surface protein. A competitive enzyme-linked immunosorbent assay (ELISA), based on inhibition of monoclonal antibody (MAb) 36/133.97 binding to recombinant EMA-1 by equine anti-B. equi antibodies, detects horses infected with strains present throughout the world. The objectives of this study were to define the epitope bound by MAb 36/133.97 and quantify the amino acid conservation of EMA-1, including the region containing the epitope bound by MAb 36/133.97. The alignment of the deduced amino acid sequence of full-length EMA-1 (Florida isolate) with 15 EMA-1 sequences from geographically distinct isolates showed 82.8 to 99.6% identities (median, 98.5%) and 90.5 to 99.6% similarities (median, 98.9%) between sequences. Full-length and truncated recombinant EMA-1 proteins were expressed and tested for their reactivities with MAb 36/133.97. Binding required the presence of amino acids on both N- and C-terminal regions of a truncated peptide (EMA-1.2) containing amino acids 1 to 98 of EMA-1. This result indicated that the epitope defined by MAb 36/133.97 is dependent on conformation. Sera from persistently infected horses inhibited the binding of MAb 36/133.97 to EMA-1.2 in a competitive ELISA, indicating that equine antibodies which inhibit binding of MAb 36/133.97 also recognize epitopes in the same region (the first 98 residues). Within this region, the deduced amino acid sequences had 85.7 to 100% identities (median, 99.0%), with similarities of 94.9 to 100% (median, 100%). Therefore, the region which binds to both MAb 36/133.97 and inhibiting equine antibodies has a median amino acid identity of 99.0% and a similarity of 100%. These data provide a molecular basis for the use of both EMA-1 and MAb 36/133.97 for the detection of antibodies against B. equi.

    PMCID: PMC130086 PMID: 12414764 [PubMed - indexed for MEDLINE]

  14. Vet Parasitol. 2001 Nov 22;101(3-4):261-74.

    Molecular technology and antigenic variation among intraerythrocytic hemoparasites: do we see reality?

    Allred DR.

    Department of Pathobiology, University of Florida, P.O. Box 110880, Gainesville, FL 32611-0880, USA. allredd@ufl.edu

    Antigenic variation is one mechanism of immune evasion utilized by many microorganisms--encompassing such broad evolutionary groups as viruses, bacteria, and protozoa--to survive the onslaught of a specifically activated host immune system. Because of its importance to the survival of many infectious agents there is considerable interest in understanding this phenomenon. With knowledge of the molecular mechanisms by which these microbes deliberately manipulate their genomes, it may be possible to disrupt the molecular machinery of the responsible genetic mechanisms. Among intraerythrocytic parasites, genetic mechanisms that have been observed or postulated to control antigenic variation include segmental gene conversion, epigenetically controlled in situ transcriptional switching, alterations of chromosomal structure associated with transcriptional control, and recombination during sexual reproduction. Likely, more than one type of mechanism is used by all organisms that undergo antigenic variation. In this paper, both the observed mechanisms and some of the molecular technology used to detect these mechanisms are discussed. While often seemingly straightforward from a technical standpoint, sometimes subtle differences in the methods used to study this process may affect what is observed. Some examples of this phenomenon are discussed in the context of a small selection of intraerythrocytic parasites.

    PMID: 11707301 [PubMed - indexed for MEDLINE]

  15. Vet Parasitol. 2001 Oct 31;101(1):9-21.

    Detection of Babesia equi (Laveran, 1901) by nested polymerase chain reaction.

    Nicolaiewsky TB, Richter MF, Lunge VR, Cunha CW, Delagostin O, Ikuta N, Fonseca AS, da Silva SS, Ozaki LS.

    Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, 91501-970 Porto Alegre, RS, Brazil.

    We describe a nested polymerase chain reaction (PCR) for the detection of Babesia equi in equine infected erythrocytes using oligonucleotides designed on the published sequence of a B. equi merozoite antigen gene (ema-1). A 102bp DNA fragment is specifically amplified from B. equi but not from Babesia caballi, Babesia bovis or Babesia bigemina DNA. In a mock infection we were able to detect down to six infected cells in 10(8) equine erythrocytes or to detect the parasite in blood with an equivalent parasitemia of 0.000006%. Furthermore, gene polymorphism was found by performing a PCR-RFLP (PCR combined with restriction fragment length polymorphism) on both the 102bp and the entire ema-1 gene DNA amplified from two B. equi isolates, Florida (USA) and Pelotas (Southern Brazil) isolates. The polymorphism was confirmed by sequencing the entire ema-1 gene from the B. equi isolate Pelotas. Our results demonstrate that the ema-1 based nested PCR is a valuable technique for routine detection of B. equi in chronically infected horses. It may be used for epidemiological and phylogenetic studies of the parasite as well as monitoring B. equi infected horses in chemotherapeutic trials.

    PMID: 11587829 [PubMed - indexed for MEDLINE]

  16. Microbes Infect. 2001 May;3(6):481-91.

    Antigenic variation in babesiosis: is there more than one 'why'?

    Allred DR.

    Department of Pathobiology, University of Florida, Gainesville, FL 32611-0880, USA. allredd@mail.vetmed.ufl.edu

    Many intraerythrocytic hemoparasites survive the host immune system through rapid antigenic variation. Among babesial parasites antigenic variation has been demonstrated convincingly only for Babesia bovis and Babesia rodhaini. The molecular basis for antigenic variation in babesial parasites and its possible connection with cytoadherence and sequestration are discussed.

    PMID: 11377210 [PubMed - indexed for MEDLINE]

  17. Mol Cell. 2000 Jan;5(1):153-62.

    The ves multigene family of B. bovis encodes components of rapid antigenic variation at the infected erythrocyte surface.

    Allred DR, Carlton JM, Satcher RL, Long JA, Brown WC, Patterson PE, O'Connor RM, Stroup SE.

    Department of Pathobiology, University of Florida, Gainesville 32611, USA. allredd@mail.vetmed.ufl.edu

    B. bovis, an intraerythrocytic protozoal parasite, establishes chronic infections in cattle in part through rapid variation of the polymorphic, heterodimeric VESA1 protein on the infected erythrocyte surface and sequestration of mature parasites. We describe the characterization of the ves1 alpha gene encoding the VESA1a subunit, thus providing a description of a gene whose product is involved in rapid antigenic variation in a babesial parasite. This three-exon gene, a member of a multigene family (ves), encodes a polypeptide with no cleavable signal sequence, a single predicted transmembrane segment, and a cysteine/lysine-rich domain. Variation appears to involve creation and modification or loss of a novel, transcribed copy of the gene.

    PMID: 10678177 [PubMed - indexed for MEDLINE]

  18. J Immunol. 2000 Feb 15;164(4):2037-45.

    Selection of Babesia bovis-infected erythrocytes for adhesion to endothelial cells coselects for altered variant erythrocyte surface antigen isoforms.

    O'Connor RM, Allred DR.

    Department of Pathobiology, College of Veterinary Medicine, University of Florida, Gainesville, FL 32611, USA.

    Sequestration of Babesia bovis-infected erythrocytes (IRBCs) in the host microvasculature is thought to constitute an important mechanism of immune evasion. Since Ig is considered to be important for protection from disease, an in vitro assay of B. bovis sequestration was used to explore the ability of anti-B. bovis Ig to interfere with IRBC cytoadhesion, and to identify IRBC surface Ags acting as endothelial cell receptors. Bovine infection sera reactive with the IRBC surface inhibited and even reversed the binding of IRBCs to bovine brain capillary endothelial cells (BBECs). This activity is at least partially attributable to serum IgG. IgG isolated from inhibitory serum captured the variant erythrocyte surface ag 1 (VESA1) in surface-specific immunoprecipitations of B. bovis-IRBCs. Selection for the cytoadhesive phenotype concurrently selected for antigenic and structural changes in the VESA1 Ag. In addition, the anti-VESA1 mAb, 4D9.1G1, proved capable of effectively inhibiting and reversing binding of adhesive, mAb-reactive parasites to BBECs, and by immunoelectron microscopy localized VESA1 to the external tips of the IRBC membrane knobs. These data are consistent with a link between antigenic variation and cytoadherence in B. bovis and suggest that the VESA1 Ag acts as an endothelial cell ligand on the B. bovis-IRBC.

    PMID: 10657656 [PubMed - indexed for MEDLINE]

  19. Infect Immun. 1999 Aug;67(8):3921-8.

    Cytoadherence of Babesia bovis-infected erythrocytes to bovine brain capillary endothelial cells provides an in vitro model for sequestration.

    O'Connor RM, Long JA, Allred DR.

    Department of Pathobiology, College of Veterinary Medicine, University of Florida, Gainesville, Florida, USA.

    Babesia bovis, an intraerythrocytic parasite of cattle, is sequestered in the host microvasculature, a behavior associated with cerebral and vascular complications of this disease. Despite the importance of this behavior to disease etiology, the underlying mechanisms have not yet been investigated. To study the components involved in sequestration, B. bovis parasites that induce adhesion of the infected erythrocytes (IRBCs) to bovine brain capillary endothelial cells (BBEC) in vitro were isolated. Two clonal lines, CD7(A+I+) and CE11(A+I-), were derived from a cytoadherent, monoclonal antibody 4D9.1G1-reactive parasite population. This antibody recognizes a variant, surface-exposed epitope of the variant erythrocyte surface antigen 1 (VESA1) of B. bovis IRBCs. Both clonal lines were cytoadhesive to BBEC and two other bovine endothelial cell lines but not to COS7 cells, FBK-4 cells, C32 melanoma cells, or bovine brain pericytes. By transmission electron microscopy, IRBCs were observed to bind to BBEC via the knobby protrusions on the IRBC surface, indicating involvement of components associated with these structures. Inhibition of protein export in intact, trypsinized IRBCs ablated both erythrocyte surface reexpression of parasite protein and cytoadhesion. IRBCs allowed to recover surface antigen expression regained the ability to bind endothelial cells, demonstrating that parasite protein export is required for cytoadhesion. We propose the use of this assay as an in vitro model to study the components involved in B. bovis cytoadherence and sequestration.

    PMCID: PMC96673 PMID: 10417157 [PubMed - indexed for MEDLINE]

  20. Mol Biochem Parasitol. 1999 May 15;100(1):125-9.

    Selection and recovery of minor parasite populations expressing unique infected-erythrocyte phenotypes.

    O'Connor RM, Long JA, Allred DR.

    Department of Pathobiology, University of Florida, College of Veterinary Medicine, Gainesville 32611-0880, USA.

    PMID: 10377000 [PubMed - indexed for MEDLINE]

  21. Mol Cell Probes. 1999 Apr;13(2):107-13.

    Development and evaluation of an extra chromosomal DNA-based PCR test for diagnosing bovine babesiosis.

    Salem GH, Liu X, Johnsrude JD, Dame JB, Roman Reddy G.

    College of Veterinary Medicine, University of Florida, Gainesville, FL 32610, USA.

    Subclinical infections of bovine babesiosis, caused primarily by Babesia bigemina or Babesia bovis are a challenge to current diagnostic methods. In this study, the development and evaluation of a PCR test for sensitive and specific detection of B. bigemina or B. bovis is described. The target selected for amplification is part of the apocytochrome b gene, conserved in both Babesia spp. and located on the linear approximately 6.0 kb extra chromosomal DNA. The test was evaluated to detect the parasites over a period of 5 (B. bigemina) and 10 months (B. bovis) post infection in experimentally infected cattle. Analysis of DNA extracted from blood samples drawn from the experimental cattle in a blind study revealed an overall sensitivity of 85 and 64% for B. bovis and B. bigemina respectively, while the specificity was 97% for B. bovis and 91% for B. bigemina. The test results were compared with the recently developed ribosomal DNA-based polymerase chain reaction (PCR) test and to the complement fixation test for both Babesia spp. The extra chromosomal DNA-based test was 20% more sensitive than that of ribosomal DNA-based tests. This test may be a more desirable alternative to the currently used, complement fixation test. Copyright 1999 Academic Press.

    PMID: 10208801 [PubMed - indexed for MEDLINE]

  22. Ann Trop Med Parasitol. 1998 Jun;92(4):461-72.

    Antigenic variation in Babesia bovis: how similar is it to that in Plasmodium falciparum?

    Allred DR.

    Department of Pathobiology, College of Veterinary Medicine, University of Florida, Gainesville 32611-0880, USA. AllredD@mail.vetmed.ufl.edu

    Despite significant differences in some aspects of their life-cycles, the Apicomplexan parasites Babesia bovis and Plasmodium falciparum share many parallels. Significant among these are participation in rapid, clonal antigenic variation, and cyto-adherence and sequestration in the deep vasculature. Antigenic variation has long been thought to be primarily a mechanism of escape from antibody-mediated mechanisms of the host's immune system. In each species, the components demonstrated to participate in antigenic variation are parasite-derived proteins expressed on the infected erythrocyte's surface. Recently, the malarial component PfEMP1 has been found to be a multifunctional protein that is not only subject to antigenic variation, but also participates in cyto-adherence and rosetting (adhesion to uninfected erythrocytes). In the present review, the antigens elaborated on the surface of an erythrocyte infected with B. bovis, for immune evasion via antigenic variation, are described, and compared and contrasted with those from P. falciparum. The significance of the similarities between B. bovis and P. falciparum, and the potential for contributions to be made to our understanding of malaria through the study of babesiosis are discussed.

    PMID: 9683897 [PubMed - indexed for MEDLINE]

  23. Methods. 1997 Oct;13(2):177-89.

    Immunochemical methods for identification of Babesia bovis antigens expressed on the erythrocyte surface.

    Allred DR.

    Department of Pathobiology, College of Veterinary Medicine, University of Florida, Gainesville, Florida, 32611-0880, USA. allredD@mail.vetmed.ufl.edu

    Intraerythrocytic parasites, such as Babesia bovis, modify the erythrocyte plasma membrane structurally, antigenically, and functionally. For such parasites the infected erythrocyte surface also is thought to be a primary site for interaction with the host immune system. These properties demand characterization of the various alterations to understand the overall host-parasite interaction, immunity to disease or infection, and bases for parasite persistence. A paucity of adequate methods exists for characterization of parasite-derived components of the parasitized erythrocyte surface. To facilitate such studies we developed or modified several techniques to detect, identify, and localize parasite-induced alterations on the B. bovis-infected erythrocyte surface. These methods, which we present here, should be adaptable to a variety of intraerythrocytic parasite-host combinations. Copyright 1997 Academic Press.

    PMID: 9405201 [PubMed - indexed for MEDLINE]

  24. Mol Biochem Parasitol. 1997 Nov;89(2):259-70.

    Characterization of a variant erythrocyte surface antigen (VESA1) expressed by Babesia bovis during antigenic variation.

    O'Connor RM, Lane TJ, Stroup SE, Allred DR.

    Department of Pathobiology, University of Florida College of Veterinary Medicine, Gainesville 32611-0880, USA.

    Babesia bovis, an intraerythrocytic, protozoal parasite of cattle, undergoes clonal antigenic variation (Allred DR, Cinque RM, Lane TJ, Ahrens KP. Infect Immun 1994;62:91-98). This ability could provide a mechanism by which the parasite escapes host immune defenses to establish chronic infection. Previous work identified two parasite-derived antigens of Mr 128,000 and 113,000 that were present on the surface of the infected erythrocyte and appeared to be associated with clonal antigenic variation (Allred DR, Cinque RM, Lane TJ, Ahrens KP. Infect Immun 1994;62:91 98). Two monoclonal antibodies (mAbs), 3F7.1H11 and 4D9.1G1, which recognize the variant erythrocyte surface antigen (VESA1) have been identified. These mAbs react only with the surface of erythrocytes infected with the B. bovis C9.1 clone in live-cell immunofluorescence assays. In both conventional and surface immunoprecipitations, the mAbs precipitate a variant antigen doublet that matches in mass the infected red blood cell (IRBC) surface antigens precipitated with bovine serum. In contrast, Western blot analysis revealed that only the Mr 128,000 polypeptide is recognized by the mAbs. Neither mAb recognizes antigenically variant progenitor or progeny parasite clones in any of the immunoassays, confirming the involvement of this antigen in rapid clonal antigenic variation. Failure to label this antigen with [9,10(n)-3H]myristic acid, [9,10(n)-3H]palmitic acid or D-[6-3H]glucosamine indicates that these polypeptides are neither N-glycosylated nor fatty acylated. Identity of the variant antigen recognized by the mAbs with that putatively identified with immune serum was confirmed by comparison of partial proteolytic digestion products. Unambiguous identification of the VESA1 antigen as a component of antigenic variation will facilitate characterization of the events leading to antigenic variation on the B. bovis-infected erythrocyte surface and its significance to parasite survival during chronic infection.

    PMID: 9364970 [PubMed - indexed for MEDLINE]

  25. J Clin Microbiol. 1996 Nov;34(11):2748-55.

    Monitoring Babesia bovis infections in cattle by using PCR-based tests.

    Calder JA, Reddy GR, Chieves L, Courtney CH, Littell R, Livengood JR, Norval RA, Smith C, Dame JB.

    Department of Pathobiology, College of Veterinary Medicine, University of Florida, Gainesville 32611, USA. Cald103w@wonder.em.cdc.gov

    The sensitivity and specificity of PCR tests based on the small-subunit rRNA gene sequence of Babesia bovis were compared in a blind study of experimentally infected cattle with the corresponding parameters of the complement fixation (CF) test currently used in the United States to screen for bovine babesiosis. Cattle were experimentally infected with a single inoculum of a cloned laboratory strain of B. bovis. Blood samples were collected and tested over a period covering from the day of infection to 10 months postinfection. The level of parasitemia (percent infected erythrocytes) present in each sample was estimated from test results and was plotted as a function of time postinfection. These data are the first describing the course of infection by methods capable of detecting parasitemias in the range of 10(-7)%, which frequently occur in the carrier state. Parasitemias in the samples tested strongly influenced the sensitivity and negative predictive value of the PCR-based tests which varied with time postinfection. The average sensitivities of the three PCR-based tests for B. bovis ranged from 58 to 70% for a single determination, while the sensitivity of the CF test was only 6%. Both PCR-based and CF tests for B. bovis had high specificity values ranging from 96 to 100%.

    PMCID: PMC229398 PMID: 8897177 [PubMed - indexed for MEDLINE]

  26. Parasitol Today. 1996 May;12(5):195-8.

    Control of Babesia equi parasitemia.

    Knowles DP Jr.

    Animal Disease Research Unit, Agricultural Research Service, US Department of Agriculture, Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA 99164-7030, USA.

    Infection of horses with the hemoprotozoan Babesia equi has been reported in southern Florida, US Virgin Islands, part of Asia, Russia, India, the Middle East, Europe, Africa, Australia, South America, Central America, Mexico, Philippine Islands and some Caribbean islands. The restrictions placed on the international movement of infected horses has refocused attention on potential methods to control or eliminate infection. Don Knowles here discusses the primary chemotherapeutic compounds that have been used; the current knowledge concerning immune responses that potentially contribute to control of the parasite, and the development of infection of severe combined immuno-deficient foals as a model to dissect potential mechanisms of immunological control.

    PMID: 15275213 [PubMed]

  27. J Med Entomol. 1996 May;33(3):297-318.

    Ixodes (Ixodes) scapularis (Acari:Ixodidae): redescription of all active stages, distribution, hosts, geographical variation, and medical and veterinary importance.

    Keirans JE, Hutcheson HJ, Durden LA, Klompen JS.

    Institute of Arthropodology and Parasitology, Georgia Southern University, Statesboro 30460-8056, USA.

    The blacklegged tick, Ixodes (Ixodes) scapularis Say, 1821, is redescribed, based on laboratory reared specimens originating in Bulloch County, Georgia. Information on distribution, host associations, morphological variation, and medical/veterinary importance is also presented. A great deal of recent work has focused on this species because it is the principal vector of the agent of Lyme disease (Borrelia burgdorferi Johnson, Schmidt, Hyde, Steigerwaldt & Brenner) in eastern North America. Its distribution appears to be expanding, and includes the state of Florida in the southeastern United States north to the provinces of Nova Scotia and Prince Edward Island, Canada, west to North and South Dakota, United States, and south to the state of Coahuila, Mexico. Although I. scapularis feeds on at least 125 species of North American vertebrates (54 mammalian, 57 avian, and 14 lizard species), analysis of the U.S. National Tick Collection holdings show that white-tailed deer, Odocoileus virginianus (Zimmermann), cattle, Bos taurus L., dogs, Canis lupus L., and other medium-to-large sized mammals are important hosts for adults as are native mice and other small mammals, certain ground-frequenting birds, skinks, and glass lizards for nymphs and larvae. This tick is a polytypic species exhibiting north-south and east-west morphological clines. Analysis of variance and Student-Newman-Keuls multiple comparisons revealed significant interpopulational variation that is expressed most significantly in the nymphal stage. Nymphs from northern (Minnesota, Massachusetts, Maryland) populations had relatively larger basis capituli with shorter cornua (except Maryland) than southern (North Carolina, Georgia) populations. Midwestern populations (Minnesota, Missouri) differed from eastern populations (Massachusetts, Maryland, North Carolina, Georgia) in idiosomal characters (broader scuta, larger coxae III, and IV). In addition to Lyme disease, this tick is also a primary vector of the agent of human and rodent babesiosis, Babesia microti Franca. Under laboratory conditions it has transmitted the agents of deer babesiosis, Babesia odocoilei Emerson & Wright, tularemia, Francisella tularensis McCoy & Chapin, and anaplasmosis, Anaplasma marginale Theiler. Moreover, I. scapularis can reach pest proportions on livestock, and females can cause tick paralysis in dogs.

    PMID: 8667375 [PubMed - indexed for MEDLINE]

  28. Trop Anim Health Prod. 1995 Aug;27(3):129-44.

    Effects of tick infestation and tick-borne disease infections (heartwater, anaplasmosis and babesiosis) on the lactation and weight gain of Mashona cattle in south-eastern Zimbabwe.

    Meltzer MI, Norval RA, Donachie PL.

    Department of Infectious Diseases, College of Veterinary Medicine, University of Florida, Gainesville 32611-0880, USA.

    The effects of ticks and tick-borne disease infections on the lactation and weight gain of Mashona cattle were studied at Mbizi Quarantine Station in the south-eastern lowveld of Zimbabwe. Twenty-nine Mashona cows were allocated to 2 balanced groups and kept in separate paddocks at a stocking rate of one animal per 8 ha. One group received regular acaricide treatment to control bont (Amblyomma hebraeum) and other ticks. The other group was left untreated. The cows were artificially inseminated. The acaricide-treated cows and calves were essentially tick free throughout the experiment, while the untreated cows and calves were continuously tick infested. There was a drought-related decline in tick infestations in the second year of the experiment. Antibodies to Cowdria ruminantium, Babesia bigemina and Anaplasma marginale were detected in cows and calves from both groups, though the untreated group had significantly higher titres to C. ruminantium (P < 0.001). The total, measured amount of milk suckled by untreated calves was significantly more than treated calves (273 kg vs. 241 kg, P < or = 0.05). By interpolating between the twice weekly measurements, it was calculated that over the entire lactation untreated calves suckled an average of 935 kg/hd vs. 837 kg/hd for the treated group. There were no statistical differences in the weights of the 2 groups of calves at birth, weaning, 180 and 210 days post partum (P < 0.05). For cows, there were no statistically significant differences in gestation periods (288 vs. 279 days), reconception rates or weight patterns over time (P < 0.05). The results show that intensive acaricide treatment in areas of Zimbabwe where heartwater is enzootically stable is uneconomical. The maintenance of enzootic stability for tick-borne diseases through minimal tick control is clearly a more economic and practical control option.

    PMID: 7502343 [PubMed - indexed for MEDLINE]

  29. Parasitol Today. 1995 Mar;11(3):100-5.

    Immune evasion by Babesia bovis and Plasmodium falciparum: cliff-dwellers of the parasite world.

    Allred DR.

    Department of Infectious Diseases, College of Veterinary Medicine, University of Florida, Gainesville 32611-0880, USA. allred.vetmed3@mail.health.ufl.edu

    Erythrocyte-dwelling parasites, such as Babesia bovis and Plasmodium falciparum, are not accessible to the host immune system during most of their asexual reproductive cycle because they are intracellular. While intracellular, the host immune response must be directed toward the surface of the infected erythrocyte. Immune individuals mount protective antibody and cell-mediated responses which eliminate most of the parasites, yet some survive to establish chronic infections. In this review, David Allred discusses some of the mechanisms used by these parasites to evade individual immune mechanisms targeting the infected erythrocyte to survive in the hostile environment of an effective immune response.

    PMID: 15275361 [PubMed]

  30. Exp Parasitol. 1995 Feb;80(1):76-84.

    Babesia bigemina: host factors affecting the invasion of erythrocytes.

    Kania SA, Allred DR, Barbet AF.

    Department of Infectious Diseases, University of Florida, Gainesville 32611.

    Babesia bigemina merozoites enter their host's erythrocytes by an unknown mechanism that likely involves parasite surface components. Identification of the parasite ligands involved in invasion is hampered by a lack of basic information about the invasion characteristics of Babesia bigemina. Therefore, restrictions on the species of red blood cells (RBC) that are susceptible to invasion were examined as well as the roles of erythrocyte ligands. An invasion assay and a proliferation assay were developed for this study. Unlike some other species of Babesia that infect cattle, B. bigemina failed to enter RBC from most animals that are not natural hosts, suggesting that a species restricted receptor mechanism mediates invasion. Two carbohydrates which are prominent on the surface of bovine erythrocytes, N-acetylglucosamine and N-acetylgalactosamine, when added to cultures, reduced the ability of B. bigemina merozoites to invade erythrocytes. Neuraminidase or trypsin treatment of bovine erythrocytes significantly decreased their susceptibility to invasion whereas chymotrypsin had little effect. These data imply that proteinaceous erythrocyte ligands and carbohydrate residues may be involved in the invasion process. Identification of a species-specific pattern of invasion and RBC treatments that render cells refractory to invasion may provide the basis for the characterization of B. bigemina erythrocyte binding molecules based on their differential binding to invasion competent and refractory cells.

    PMID: 7821413 [PubMed - indexed for MEDLINE]

  31. Infect Immun. 1994 Jan;62(1):91-8.

    Antigenic variation of parasite-derived antigens on the surface of Babesia bovis-infected erythrocytes.

    Allred DR, Cinque RM, Lane TJ, Ahrens KP.

    Department of Infectious Diseases, University of Florida, Gainesville 32611-0880.

    The hemoparasite Babesia bovis antigenically alters the bovine erythrocyte membrane surface by expression of isolate-specific, parasite-derived polypeptides. To determine whether antigenic variation also occurred on the infected erythrocyte surface, a calf was infected once with parasitized erythrocytes carrying the C9.1 clonal line of B. bovis. In vitro cultures then were established periodically from the peripheral blood and analyzed with sequentially collected sera from the same animal. The surface reactivity of infected erythrocytes cultured from the infected animal varied over time, on the basis of reactivity in live cell immunofluorescence, surface immunoprecipitation, and panning assays. Subclones C8 and H10, established from day 41 cultures, were analyzed immunochemically. A loss of immunoreactivity was observed in antigens corresponding to the 113- and 128-kDa parasite-derived antigens of clone C9.1, demonstrating epitopic variation in these antigens; the immunochemical recognition of these antigens paralleled the results of live cell immunofluorescence and panning assays. Concomitant size polymorphism suggested polypeptide structural variation of these antigens as well. Calves infected by inoculation of infected blood or by injection of cloned parasites from in vitro cultures rapidly developed antibodies which cross-reacted among the clonal variant lines, suggesting the presence of common as well as unique epitopes. These results demonstrate that antigenic variation occurs on the surface of B. bovis-infected erythrocytes and that the parasite-derived antigens of 113 and 128 kDa compose at least a part of the antigens undergoing variation.

    PMCID: PMC186072 PMID: 8262654 [PubMed - indexed for MEDLINE]

  32. Mol Biochem Parasitol. 1993 Jul;60(1):121-32.

    Isolate-specific parasite antigens of the Babesia bovis-infected erythrocyte surface.

    Allred DR, Hines SA, Ahrens KP.

    Department of Infectious Diseases, University of Florida, Gainesville 32611-0880.

    Bovine erythrocytes taken from in vitro cultures of Babesia bovis parasites from Mexico and the United States were assayed for the presence of new epitopes on the erythrocyte surface. New surface-exposed epitopes were detected by means of a whole-cell antigen capture assay. These epitopes were subsequently demonstrated only on infected erythrocytes by immunofluorescence staining of intact, living cells. Parasite-synthesized antigens were identified on each isolate using a surface-specific immunoprecipitation technique to analyze metabolically-labeled infected erythrocytes. In the Mexico isolate these antigens were 120 kDa and 107 kDa, whereas in the United States isolate polypeptides of 135, 120 and 107 kDa were detected. In each of these assays, reaction of immune sera with the infected erythrocyte surface was found to be isolate-specific.

    PMID: 8366886 [PubMed - indexed for MEDLINE]

  33. J Parasitol. 1993 Apr;79(2):274-7.

    A nonsubjective assay for antigenic modifications of the Babesia bovis-parasitized erythrocyte surface.

    Allred DR, Ahrens KP.

    Department of Infectious Diseases, College of Veterinary Medicine, University of Florida, Gainesville 32611-0880.

    Intracellular protozoan parasites induce numerous alterations in the invaded host cell, including antigenic modifications of the host cell plasma membrane. We have developed a quantifiable, non-subjective assay for the detection of novel antigenic reactivities on the host cell surface using as a model system bovine erythrocytes infected with Babesia bovis. Infected erythrocytes, metabolically labeled with L-[35S]methionine, were sensitized by incubation with bovine immune serum, then were captured in microtiter plates coated with rabbit anti-bovine IgG antibody. This technique enabled specific capture of B. bovis-infected cells with immune infection sera raised against B. bovis but not with similar sera raised against Babesia bigemina. This assay should be easily applicable to the study of other parasitic diseases.

    PMID: 8459338 [PubMed - indexed for MEDLINE]

  34. 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]

  35. Vet Immunol Immunopathol. 1992 Sep;33(4):339-52.

    Development and persistence of Cowdria ruminantium specific antibodies following experimental infection of cattle, as detected by the indirect fluorescent antibody test.

    Semu SM, Mahan SM, Yunker CE, Burridge MJ.

    University of Florida/USAID/SADCC Heartwater Research Project, c/o Veterinary Research Laboratory, Causeway, Zimbabwe.

    Different breeds of cattle were experimentally infected with Palm River, a Zimbabwean isolate, or Ball-3, a South African isolate of Cowdria ruminantium, derived from tissue culture or tick or blood stabilates. C. ruminantium specific antibody responses were detected by an indirect fluorescent antibody test (IFAT) using C. ruminantium-infected bovine aortic endothelial (BAE) cell cultures as antigen. The first detection of antibodies to C. ruminantium generally coincided with the peak of the febrile reaction and the antibodies remained detectable for a period of 8-30 weeks in the Palm River infected group and 18-30 weeks in the Ball-3 infected group. Peak reciprocal antibody titres in both groups ranged from 64 to 2048 between 3 and 6 weeks post-infection. No apparent serological differences were observed among the various C. ruminantium isolates when tested in homologous and heterologous IFATs. Post-infection sera to Anaplasma marginale, Theileria parva parva, Babesia bigemina and Rickettsia conorii did not exhibit reactivity with the C. ruminantium antigen. These results indicate the possible use of C. ruminantium-infected cultures as antigen in IFATs to detect similar C. ruminantium-specific antibody responses in the field in clinically sick, recovered and carrier animals.

    PMID: 1441219 [PubMed - indexed for MEDLINE]

  36. J Clin Microbiol. 1992 Jul;30(7):1811-4.

    rRNA-based method for sensitive detection of Babesia bigemina in bovine blood.

    Reddy GR, Dame JB.

    Department of Infectious Diseases, College of Veterinary Medicine, University of Florida, Gainesville 32611.

    Three synthetic oligonucleotide probes complementary to unique regions of Babesia bigemina small-subunit rRNA were developed for detecting the parasite in bovine blood. These probes specifically detected a parasitemia of 2 x 10(-5)% by autoradiography in less than 24 h by using a 200-microliters sample of bovine blood. These probes did not bind to total RNA or genomic DNA isolated from another closely related species, Babesia bovis, or to bovine leukocyte RNA. This method detected B. bigemina infections in calves inoculated with as few as 1,000 infected erythrocytes from the second day onward for 16 days.

    PMCID: PMC265386 PMID: 1629339 [PubMed - indexed for MEDLINE]

  37. Mol Biochem Parasitol. 1992 Jul;53(1-2):149-58.

    Isolation, sequence and differential expression of the p58 gene family of Babesia bigemina.

    Mishra VS, McElwain TF, Dame JB, Stephens EB.

    Department of Infectious Diseases, College of Veterinary Medicine, University of Florida, Gainesville 32610.

    Four copies of the gene encoding the merozoite surface protein p58 from the protozoan hemoparasite Babesia bigemina were amplified from genomic DNA by polymerase chain reaction (PCR) techniques, molecularly cloned and subjected to DNA sequence analysis. The amplified DNA (Bbg7, Bbg9, Bbg13, Bbg14) could be placed into 2 classes with respect to its size and the length of the open reading frame (ORF). With the exception of a single base substitution, the sequence of Bbg13 is identical to the cDNA sequence published earlier [1]. The Bbg7 and Bbg14 copies of p58 diverged from Bbg13 sequence at regions towards the 3' and 5' ends, respectively. In contrast, Bbg9 has incorporated both regions of divergence within its sequence. Using a cloned strain of B. bigemina, RNA-PCR and Northern blot analyses demonstrate the in vivo transcription of 3 of the 4 copies, although one of the 3 expressed copies is present in very low abundance. The relative abundance and size of the two p58 mRNA species detected are consistent with the 58- and 55-kDa proteins detected by in vitro translation of B. bigemina poly(A)+ mRNA by immunoprecipitation with an anti-p58 monospecific antibodies. These results indicate that the gene encoding p58 exists as a multigene family that appears to be differentially expressed in the blood stage of the parasite's life cycle.

    PMID: 1501634 [PubMed - indexed for MEDLINE]

  38. J Clin Microbiol. 1992 Apr;30(4):981-6.

    A cloned DNA probe for Cowdria ruminantium hybridizes with eight heartwater strains and detects infected sheep.

    Mahan SM, Waghela SD, McGuire TC, Rurangirwa FR, Wassink LA, Barbet AF.

    Department of Infectious Diseases, University of Florida, Gainesville 32611.

    The DNA probe pCS20, which was cloned from the DNA of the Crystal Springs heartwater strain from Zimbabwe, cross-reacted with DNAs of heartwater strains from all endemic areas, including four heartwater strains from Zimbabwe, two strains from South Africa, one strain from Nigeria, and the Gardel strain from the Caribbean island of Guadeloupe. By nucleic acid hybridization, the pCS20 DNA probe detected Cowdria ruminantium DNA in all DNA preparations made from plasma samples from infected sheep before and during the febrile reaction. Synthetic oligonucleotides were prepared for amplification of specific C. ruminantium DNA sequences by the polymerase chain reaction (PCR). Amplification of two DNA products (181 and 279 bp) from pCS20 DNA and C. ruminantium genomic DNA of heartwater strains was demonstrated. In contrast, amplification of these products or any other products was not possible from genomic DNAs of Anaplasma marginale, Babesia bigemina, Trypanosoma brucei brucei, Escherichia coli, and bovine endothelial cells. The cross-reactivities of the 32P-labeled PCR products with genomic DNAs from several heartwater strains were similar to those with the pCS20 DNA probe. A nucleic acid-based test that uses hybridization assays and PCR provides a sensitive method for the detection of heartwater in both animals and ticks and has applications in epidemiological studies for the disease, which may allow for improved disease control.

    PMCID: PMC265197 PMID: 1572987 [PubMed - indexed for MEDLINE]

  39. J Med Entomol. 1992 Mar;29(2):246-58.

    Computer simulation of Babesia bovis (Babes) and B. bigemina (Smith & Kilborne) transmission by Boophilus cattle ticks (Acari: Ixodidae).

    Haile DG, Mount GA, Cooksey LM.

    Medical and Veterinary Entomology Research Laboratory, USDA-ARS, Gainesville, Florida 32604.

    A computer model was developed to simulate the processes involved in transmission of the cattle fever parasites Babesia bovis (Babes) and Babesia bigemina (Smith & Kilborne) between cattle and Boophilus ticks. The model of Babesia transmission was combined with a dynamic life history model for population dynamics of the tick vectors, Boophilus microplus (Canestrini) and B. annulatus (Say). Epidemiological parameters and relationships in the model include the reduction in fecundity of infected ticks, rate of transovarial transmission, effect of cattle type and inoculation rate on infectivity of cattle, variation of infected cattle recovery rate with age of infection, inoculation rate, and species of parasite. Some parameters in the model were fitted by iterative simulations to produce realistic rates of Babesia infection in larval ticks. Comparisons of simulated and reported epidemiological data from one location in Australia indicated a reasonable level of validity for the model. Theoretical tick density thresholds for maintenance of Babesia in cattle and for inoculation of greater than or equal to 99.5% calves were determined by iterative simulations at 10 locations with B. microplus and six locations with B. annulatus. The model and transmission thresholds can serve as the basis for further simulation studies on strategies for control or eradication of babesiosis.

    PMID: 1495038 [PubMed - indexed for MEDLINE]

  40. J Am Vet Med Assoc. 1992 Jan 1;200(1):47-50.

    Seroprevalence of babesiosis in Greyhounds in Florida.

    Taboada J, Harvey JW, Levy MG, Breitschwerdt EB.

    Department of Veterinary Clinical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge 70803-8410.

    An indirect fluorescent antibody test was used to serologically survey Greyhounds from 10 kennels that are part of the racing Greyhound industry in Florida. Age of dogs ranged from 11 months to 11 years. Additionally, 50 adult non-Greyhound pet dogs were consecutively surveyed. Of 393 Greyhounds tested, 181 (46%) were seropositive for babesiosis; pet dogs were seronegative. Slightly higher percentage of seropositive males than females was observed, but this difference was only significant (P less than 0.01) in the 2- to 5-year age class. Male dogs less than 2 years old had significantly (P less than 0.01) lower seroprevalence than did male dogs greater than 2 years old. All 46 Greyhounds that were actively racing at the time of sample collection were seronegative. Dogs were classified into 2 groups on the basis of whether the kennel owner had sought veterinary attention for anemic pups. The 5 kennel owners that had sought veterinary attention (group A) had significantly (P less than 0.01) higher seroprevalence (78.5%), compared with the 5 that had not sought veterinary attention (group B; 23.0%). Seroprevalence of babesiosis in Greyhounds in Florida was comparable to that reported in a limited survey of other southeastern states. It appears to be higher than that in the pet population. Breeding kennels in Florida and other southeastern states from which anemic pups originate should be screened for babesiosis.

    PMID: 1537689 [PubMed - indexed for MEDLINE]

  41. Mem Inst Oswaldo Cruz. 1992;87 Suppl 3:21-6.

    Polypeptides reactive with antibodies eluted from the surface of Babesia bovis-infected erythrocytes.

    Ahrens KP, Allred DR.

    Department of Infectious Diseases, University of Florida, Gainesville 32611-0880.

    A technique was sought that would enable identification of surface-exposed parasite antigens on Babesia bovis-infected erythrocytes (BbIE) that are not detectable by surface-specific immunoprecipitations. Antibodies which bind to the surface of BbIE were recovered from intact cells using a low pH wash procedure. The eluted antibodies were then used in conventional immunoprecipitation assays to identify parasite-synthesized polypeptides carrying epitopes that are exposed on the surface or are cross-reactive with such epitopes. The results of these experiments support our previous data, obtained using a surface-specific immunoprecipitation technique, in the identification of a repertoire of parasite-derived antigens on the surface of infected erythrocytes (Allred et al., 1991). In addition, two polypeptides of M(r) 68,000 and 185,000 were identified which react strongly with the eluted antibodies but which are not detected by surface-immunoprecipitation. These data illustrate the potential of this approach for identification of parasite polypeptides which carry epitopes exposed on, or cross-reactive with exposed epitopes of the infected erythrocyte surface.

    PMID: 1343693 [PubMed - indexed for MEDLINE]

  42. Mol Biochem Parasitol. 1991 Aug;47(2):207-12.

    Immunogenicity and sequence analysis of recombinant p58: a neutralization-sensitive, antigenically conserved Babesia bigemina merozoite surface protein.

    Mishra VS, Stephens EB, Dame JB, Perryman LE, McGuire TC, McElwain TF.

    Department of Infectious Diseases, College of Veterinary Medicine, University of Florida, Gainesville.

    The gene encoding the conserved, neutralization-sensitive surface protein p58 of Babesia bigemina was cloned and sequenced. An open reading frame of 1440 bases was found to encode a protein with a predicted size of 54 kDa. A transmembrane hydrophobic domain and signal peptide were present at the amino-terminus. The polypeptide encoded by a nearly full length cDNA was expressed in bacteria and contained epitope(s) reactive with anti-p58 polyclonal and monoclonal antibodies. Serum antibodies from rabbits immunized with a lysate of recombinant bacteria specifically immunoprecipitated native p58 from [35S]methionine-labeled B. bigemina antigens. In addition, the sera contained antibodies that bound to the surface of live merozoites from 4 geographically different Latin American isolates, confirming the presence and immunogenicity of conserved, surface-exposed epitopes on the recombinant polypeptide. This molecular clone will now enable immunization trials in cattle designed to better evaluate the ability of p58 to induce immune protection by vaccinating with constructs containing only conserved, neutralization-sensitive epitopes.

    PMID: 1944417 [PubMed - indexed for MEDLINE]

  43. J Wildl Dis. 1991 Jul;27(3):486-90.

    Hemoparasites of raccoons (Procyon lotor) in Florida.

    Telford SR Jr, Forrester DJ.

    Department of Infectious Diseases, College of Veterinary Medicine, University of Florida, Gainesville 32610.

    Four hemoparasite species (Babesia lotori, Trypanosoma cruzi, Dirofilaria tenuis and Mansonella llewellyni) were found in raccoons (Procyon lotor) collected from 1972 to 1974 in Duval (n = 14) and Collier (n = 170) counties, Florida (USA). Trypanosoma cruzi was found in thin blood smears from one raccoon at each locality. The prevalence of B. lotori was 79% and 80% in samples taken in December 1973 in Collier and Duval counties, respectively. No patent infections by B. lotori were detected in raccoons collected in Collier County in December 1972, but 42% of the raccoons examined in September 1973 were infected. In Collier County there were no significant differences in the prevalence of B. lotori by host sex or age. In Duval County, overall D. tenuis prevalence was 7%, whereas that of M. llewellyni was 14%; the latter species was not found in Collier County. Adult raccoons had a significantly greater prevalence of D. tenuis (32%) than did subadults and juveniles (7%), and male raccoons showed a significantly greater prevalence (51%) than did females (8%).

    PMID: 1920671 [PubMed - indexed for MEDLINE]

  44. Infect Immun. 1991 Jul;59(7):2412-7.

    A monoclonal antibody defines a geographically conserved surface protein epitope of Babesia equi merozoites.

    Knowles DP Jr, Perryman LE, Goff WL, Miller CD, Harrington RD, Gorham JR.

    Animal Disease Research Unit, U.S. Department of Agriculture, Pullman, Washington 99164-7030.

    Babesiosis is a tick-borne hemoparasitic disease affecting horses worldwide. To investigate mechanisms of immunity to this parasite, the antibody response of infected horses to Babesia equi merozoite proteins was evaluated. Immunoprecipitation of B. equi merozoite antigens with sera from infected horses revealed 11 major proteins of 210, 144, 108, 88, 70, 56, 44, 36, 34, 28, and 25 kDa. Monoclonal antibody (MAb) 36/133.97, which binds to live merozoites, immunoprecipitated proteins of 44, 36, 34, and 28 kDa. When immunoprecipitations were performed with in vitro translation products of merozoite mRNA, MAb 36/133.97 immunoprecipitated proteins of 38, 28, 26, and 23 kDa which comigrated with proteins immunoprecipitated by sera from infected horses at 10(-3) to 10(-4) dilutions. In Western blot analysis, MAb 36/133.97 recognized proteins of 44, 36, 34, and 28 kDa, and a 28-kDa protein was identified by sera from infected horses at a dilution of 10(-4). MAb 36/133.97 bound to B. equi isolates from Florida and Europe. Furthermore, the binding of MAb 36/133.97 to merozoite proteins was inhibited by sera of infected horses from 19 countries. Collectively, these data indicate MAb 36/133.97 binds to a geographically conserved peptide epitope on multiple B. equi merozoite proteins, including a merozoite surface protein, and MAb 36/133.97 reacts with a B. equi protein immunodominant in infected horses.

    PMCID: PMC258026 PMID: 1711016 [PubMed - indexed for MEDLINE]

  45. Nucleic Acids Res. 1991 Jul 11;19(13):3641-5.

    Sequence microheterogeneity of the three small subunit ribosomal RNA genes of Babesia bigemina: expression in erythrocyte culture.

    Reddy GR, Chakrabarti D, Yowell CA, Dame JB.

    Department of Infectious Diseases, College of Veterinary Medicine, University of Florida, Gainesville 32611.

    Three distinct ribosomal RNA (rRNA) transcription units (rDNA units), designated A, B and C, were identified in the intraerythrocytic protozoan parasite, Babesia bigemina. These rDNA units were cloned, and restriction maps were constructed showing the approximate location of the small and large rRNA coding regions. The arrangement of the genes in the genome and copy number analysis suggests the presence of a single copy of each rDNA unit per haploid genome. The complete nucleotide sequence of the small subunit rRNA coding region (1693 bp) and parts of the 5' and 3' flanking regions were determined for all three units. Units A and B have identical sequences, but unit C differs from units A and B at ten nucleotide positions, two in the small subunit rRNA coding region and four each in the adjacent 5' and 3' flanking regions. The differences in the coding region are confirmed in genomic DNA and RNA from two different isolates of B.bigemina. The RNA of both sequence types is transcribed in parasites from erythrocyte culture, however, the products of gene units A + B accumulate at a ratio of approximately 4:1 compared with the product of unit C.

    PMCID: PMC328392 PMID: 1840661 [PubMed - indexed for MEDLINE]

  46. J Med Entomol. 1991 Mar;28(2):223-40.

    Computer simulation of Boophilus cattle tick (Acari: Ixodidae) population dynamics.

    Mount GA, Haile DG, Davey RB, Cooksey LM.

    Insects Affecting Man and Animals Research Laboratory, USDA-ARS, Gainesville, Florida 32604.

    A comprehensive computer model was developed for simulation of the population dynamics of the cattle ticks, Boophilus microplus (Canestrini) and B. annulatus (Say). The model is deterministic and based on a dynamic life table with weekly time steps. The model simulates the effects of major environmental variables, such as ambient temperature, saturation deficit, precipitation, type of pasture, type of cattle, and cattle density on Boophilus cattle tick population dynamics. General validity of the model is established by comparing simulated and observed yearly densities of standard female ticks/host/day. B. microplus population comparisons were made for a series of years using weekly weather data from two locations in Queensland, Australia. The model also produced acceptable values for initial population growth rate, generation time, and 3-yr population density when historical weather at 7 locations in Australia and 23 locations in the Americas were used. This model provides a framework for the study of Babesia transmission by Boophilus ticks, and can be used to study the effects of control technologies and to develop more efficient and environmentally acceptable eradication strategies for Boophilus ticks.

    PMID: 2056504 [PubMed - indexed for MEDLINE]

  47. Mol Biochem Parasitol. 1989 Nov;37(1):1-9.

    Molecular characterization of Babesia bovis merozoite surface proteins bearing epitopes immunodominant in protected cattle.

    Hines SA, McElwain TF, Buening GM, Palmer GH.

    Department of Infectious Diseases, University of Florida, Gainesville.

    Eight surface-radioiodinated merozoite proteins from a cloned, pathogenic isolate of Babesia bovis can be immunoprecipitated by antibody from cattle that are completely protected against clinical babesiosis. Among these eight surface proteins, the 55- and 42-kDa molecules are biosynthetically labeled with [3H]glucosamine. The 42-kDa glycoprotein can also be labeled with [3H]myristic acid and partitions exclusively into the detergent phase in Triton X-114 extracts, indicating that it is an integral membrane protein and suggesting that it is anchored by a glycosylphosphatidylinositol moiety. Antibody-mediated protection against B. bovis merozoites most probably requires a high level of circulating antibody to ensure antibody-merozoite binding during the parasite's brief extra-erythrocytic phase. Antibodies in diluted sera selectively recognize the 120-, 85-, 55- and 42-kDa surface proteins. Only the 42-kDa integral membrane protein is reactive with serum antibodies diluted greater than or equal to 1:16,000. Thus, we hypothesize that these immunodominant proteins, especially the transmembrane 42-kDa glycoprotein, are important to the induction of the protective immune response and are candidates for an improved vaccine against babesiosis.

    PMID: 2482443 [PubMed - indexed for MEDLINE]

  48. Adv Vet Sci Comp Med. 1989;33:345-75.

    Vaccines for parasitic infections.

    Barbet AF.

    Department of Infectious Diseases, College of Veterinary Medicine, University of Florida, Gainesville.

    PMID: 2648776 [PubMed - indexed for MEDLINE]

  49. J Am Vet Med Assoc. 1988 Jun 15;192(12):1751-2.

    Babesiosis in a litter of pups.

    Harvey JW, Taboada J, Lewis JC.

    Department of Physiological Sciences, College of Veterinary Medicine, University of Florida, Gainesville 32610.

    Babesia canis infection was diagnosed in a litter of seven 3-week-old Mastiff pups kept in a north Florida kennel. The pups were evaluated because of poor weight gain; the smallest pup also was markedly lethargic. Six of the pups were anemic and thrombocytopenic. A positive linear correlation between PCV and absolute reticulocyte count suggested that the variation in PCV may have been related more to the ability of a pup to increase erythrocyte production than to a difference in magnitude of erythrocyte destruction. All pups recovered from clinical signs and hematologic abnormalities attributable to babesiosis within 2.5 weeks after treatment with diminazene aceturate. Transient neurologic signs observed in 1 pup 3 days after treatment were believed to represent an adverse drug reaction. The dam of the litter had a serum titer of 1:640 for B canis, but appeared healthy, as did approximately 30 other adult dog in the kennel. The strain of B canis infecting dogs in the kennel caused severe illness and death in some pups, but clinically inapparent disease in adult dogs.

    PMID: 3410795 [PubMed - indexed for MEDLINE]

  50. 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]

  51. 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]

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