Degradation of bovine complement C3 by trichomonad extracellular proteinase.

Bovine trichomoniasis is a local infection of the reproductive tract making interaction with mucosal host defenses crucial. Since the parasite is susceptible to killing by bovine complement, we investigated the role of the third component of complement (C3) in host parasite interactions. Bovine C3 was purified by anionic and cationic exchange chromatography. The purified protein was characterized by immunoreactivity, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and peptide sequencing of the amino terminus of the beta chain. When purified bovine C3 was incubated for varying time periods with trichomonad extracellular proteinases, SDS-PAGE gels revealed digestion of the alpha chain to small fragments. Such degradation in vivo would prevent formation of C3b and completion of the complement cascade, resulting in evasion of killing. To evaluate the relevance of this data, we determined whether C3 was present in bovine genital secretions. With a quantitative ELISA assay, C3 could be demonstrated in both uterine and vaginal washes. To our knowledge, this is the first demonstration of bovine C3 in genital secretions. The C3 concentration increased significantly in vaginal secretions by 8 and 10 weeks in heifers infected with Tritrichomonas foetus. An increase was also seen in uterine secretions of infected heifers, but sample numbers were insufficient for statistical analysis. Transcription of the major extracellular cysteine proteinase (TFCP8) was demonstrated in T. foetus cells from uterine secretions of infected heifers by RT-PCR and Southern blotting. The results indicate that C3 may be important in genital defense and that trichomonad extracellular proteinases may play a role in evasion of complement-mediated killing.

Phylogenetic relationships of human and wildlife piroplasm isolates in the western United States inferred from the 18S nuclear small subunit RNA gene.

The 18S nuclear small subunit ribosomal RNA gene of piroplasms from wildlife and human cases of babesiosis in the western USA were isolated by PCR and sequenced. Phylogenetic analyses of these sequences and comparisons with sequences from other Babesia and Theileria species revealed that piroplasm isolates from the human cases were indistinguishable from some of the isolates from the western wildlife species, most notably the isolates from mule deer (Odocoileus hemionus). These results suggest that large ungulates may serve as reservoirs for human piroplasm infection. The western piroplasm isolates from humans and wildlife formed a distinct clade, separate from other piroplasms found worldwide.

Tritrichomonas foetus extracellular cysteine proteinase cleavage of bovine IgG2 allotypes.

Bovine trichomoniasis is a sexually transmitted disease associated with reproductive failure. Systemic immunization results in protective IgG antibodies in uterine and vaginal secretions. Because bovine IgG2 is a better opsonin than IgG1, it is potentially important in defense. Yet, Tritrichomonas foetus extracellular cysteine proteinase (TFECP) cleaves bovine IgG2, evading protective IgG2 responses. Variations in resistance of the 2 IgG2 allotypes to digestion may explain inherited differences in protection. To address this hypothesis, TFECP was incubated with both IgG2 allotypes at different concentrations and times. The digestion products were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, stained, and quantitated by image analysis. IgG2a was digested faster by TFECP than IgG2b. Differences in the sizes and numbers of digestion products were observed, but the presence of bands the size of Fc and Fd fragments indicated that both allotypes were cleaved at the hinge. Cysteine in the digestion mixture reduced the antibody molecules and increased the rate of digestion, but IgG2a was still more susceptible to cleavage than IgG2b in the absence of cysteine. Thus, not only reduced H chains can be cleaved by cysteine proteinase secreted by T. foetus but also intact functional antibody molecules. Because parasites may evade protective antibody responses by cleaving IgG2, animals with the more resistant IgG2b allotype may be better protected by immunization than animals with the more readily digested IgG2a allotype.

Clinical, anatomic, and immunopathologic characterization of Babesia gibsoni infection in the domestic dog (Canis familiaris).

The pathology associated with acute, chronic, and recrudescent Babesia gibsoni infections was characterized in a group of 6 naturally or experimentally infected, spleen-intact and splenectomized dogs. All experimentally infected dogs became acutely parasitemic, lethargic, anemic, thrombocytopenic, and hemoglobinuric. Anatomic lesions associated, with the disease included diffuse nonsuppurative periportal and centrilobular hepatitis, multifocal necrotizing arteritis, membranoproliferative glomerulonephritis, reactive lymphadenopathy, diffuse erythrophagocytosis, and extramedullary hematopoiesis. The density of CD3+ lymphocytes within the liver sinusoids was markedly increased. Aggregates of large mononuclear cells with immunohistochemical features of activated macrophages were demonstrated in the central veins of the liver. Kupffer cells throughout the hepatic sinusoids appeared hypertrophic and prominent. The density of sinusoidal T lymphocytes, macrophages in central veins, and the degree of Kupffer cell hypertrophy were greatest in the splenectomized dogs. Multifocal deposits of IgM antibody were immunohistochemically demonstrated within the walls of inflamed arteries and renal glomeruli. The results of this study suggest that intense immunostimulation resulting in activation and expansion of T and B lymphocyte populations, macrophage recruitment and activation, vasculitis, glomerulonephritis and anemia contribute to the pathology associated with B. gibsoni infections.

Characterization of extracellular proteinases of Tritrichomonas foetus.

Proteinases released from Tritrichomonas foetus into a reducing buffer were characterized because we previously showed that they digested host proteins important in defense of the reproductive tract. These proteinases were shown to be extracellular because cell numbers did not decrease and trichomonads remained motile during their 3.5-hr incubation. Detectable proteinase activity was attributable to enzymes of the cysteine class by spectrophotometric and fluorometric automated assays for peptide substrate specificity. Proteinases from the trichomonad-conditioned buffer were partially purified by bacitracin affinity chromatography. Separation of the eluate on nondenaturing SDS-PAGE gels copolymerized with gelatin, revealed primarily low molecular weight proteinases. Bacitracin-purified T. foetus extracellular cysteine proteinase (TFECP) was separated in a denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel, electroblotted, and a 31-kDa band cut out for amino acid sequencing. Four internal fragments were sequenced, 1 of which contained the highly conserved asparagine region of the cysteine proteinase active site. The combined sequences of these enzyme fragments were 66% and 55% identical to and the corresponding deduced amino acid sequences of 2 T. foetus cysteine proteinase genes (TFCP1 and TFCP2, respectively), which we previously cloned. These results indicate that this enzyme (TFECP) is a distinct cysteine proteinase. The extracellular release of TFECP from T. foetus suggests that it is a potential virulence factor in bovine trichomoniasis.

Seroprevalence of two Babesia spp. isolates in selected bighorn sheep (Ovis canadensis) and mule deer (Odocoileus hemionus) populations in California.

Sera from 111 bighorn sheep (Ovis canadensis) and 95 mule deer (Odocoileus hemionus) were tested using an indirect immunofluorescence assay for antibodies to two isolates of Babesia spp. recently obtained from these hosts in California (USA). The study populations were from six locations: three areas of real or potential sympatry of bighorn sheep and deer, one area with deer only, and two areas with bighorn sheep only. Antibody titers from seroreactive individuals were similar with both babesial isolate antigens (P < 0.05), and seroprevalence was highest in the areas of host sympatry. A moderate to high seroprevalence (> or = 30%) in some of the study populations was evidence that babesial parasites may be common in bighorn sheep and mule deer in some areas of California.

Infection with a babesia-like organism in northern California.

BACKGROUND: Human babesiosis is a tick-transmitted zoonosis associated with two protozoa of the family Piroplasmorida: Babesia microti (in the United States) and B. divergens (in Europe). Recently, infection with an unusual babesia-like piroplasm (designated WA1) was described in a patient from Washington State. We studied four patients in California who were identified as being infected with a similar protozoal parasite. All four patients had undergone splenectomy, three because of trauma and one because of Hodgkin's disease. Two of the patients had complicated courses, and one died. METHODS: Piroplasm-specific nuclear small-subunit ribosomal DNA was recovered from the blood of the four patients by amplification with the polymerase chain reaction. The genetic sequences were compared with those of other known piroplasm species. Indirect immunofluorescent-antibody testing of serum from the four patients and from other potentially exposed persons was performed with WA1 and babesia antigens. RESULTS: Genetic sequence analysis showed that the organisms from all four patients were nearly identical. Phylogenic analysis showed that this strain is more closely related to a known canine pathogen (B. gibsoni) and to theileria species than to some members of the genus babesia. Serum from three of the patients was reactive to WA1 but not to B. microti antigen. Serologic testing showed WA1-antibody seroprevalence rates of 16 percent (8 of 51 persons at risk) and 3.5 percent (4 of 115) in two geographically distinct areas of northern California. CONCLUSIONS: A newly identified babesia-like organism causes infections in humans in the western United States. The clinical spectrum associated with infection with this protozoan ranges from asymptomatic infection or influenza-like illness to fulminant, fatal disease.

Cultivation and phylogenetic characterization of a newly recognized human pathogenic protozoan.

An intraerythrocytic protozoan (WA1) recently isolated from a patient in Washington State was shown to be morphologically identical to Babesia microti but biologically and genetically distinct. Continuous growth of WA1 was established in stationary erythrocyte cultures. Hybridization of a chemiluminescent Babesia-specific DNA probe to Southern blots of restriction enzyme-digested genomic DNA showed that WA1 could be distinguished from other Babesia species that were antigenically cross-reactive (Babesia gibsoni and babesial parasites from desert bighorn sheep, Ovis canadensis nelsoni) or known to infect humans (B. microti, Babesia divergens, and Babesia equi), or both. A 1436-bp portion of the nuclear small subunit rRNA gene of WA1 was sequenced and analyzed. Genetic distance analysis showed that WA1 is most closely related to the canine pathogen B. gibsoni and lies within a phylogenetic cluster with Theileria species and B. equi. The methodology described will be useful for improved diagnosis and identification of human protozoal pathogens.

Diagnosis of babesiosis: evaluation of a serologic test for the detection of Babesia microti antibody.

To assess the possibility of standardization of a commonly used indirect immunofluorescent antibody (IFA) test for detection of Babesia microti antibody in human sera, the results from four reference laboratories were compared. Patients with babesiosis from southern New England (n = 25) and subjects with no history of babesiosis from southern New England (n = 55) and Iceland (n = 50) were enrolled in the study. Anti-Babesia antibody titers were determined in a blinded fashion by IFA test. The range of test results in the four laboratories was 88%-96% sensitivity, 90%-100% specificity, 69%-100% positive predictive value, and 96%-99% negative predictive value. Interlaboratory and intralaboratory concordance ranged from 84% to 85% and 94% to 100%, respectively. This B. microti IFA procedure is a sensitive, specific, and reproducible method for diagnosing babesiosis and is suitable for use as a standard in laboratories testing human sera for B. microti antibody.

Evaluation of the indirect fluorescent antibody test for diagnosis of Babesia gibsoni infections in dogs.

We determined the extent of the serologic cross-reactivity in the indirect fluorescent antibody (IFA) test for Babesia gibsoni, and the optimal cut-off titer for seropositivity in the test. The lowest titer to B gibsoni detected in a dog with naturally acquired clinical babesiosis was 1,280, and 7 of 12 dogs had titer between 10,240 and 20,480. Two experimentally infected normosplenic dogs developed high titer (40,960 to 81,920) to B gibsoni, and the same sera reacted in IFA tests for B canis (titer < or = 640), Toxoplasma gondii (titer < or = 2,560), and Neospora caninum (titer < or = 10,240). Dogs that were experimentally infected with B canis and T gondii had titer < or = 160 to B gibsoni. Dogs that were experimentally infected with N caninum had titer (80 to 10,240) to N caninum, but failed to have serologic reactivity to B gibsoni. Serologic titer of healthy dogs from Australia, a country where B gibsoni is not known to exist, was < or = 160 to B gibsoni. On the basis of these findings, a cut-off titer of 320 was considered to be appropriate for serodiagnosis of B gibsoni in dogs with clinical signs of babesiosis. A more conservative titer of 1,280 was established as the cut-off titer for seroepidemiologic studies based on relative costs and benefits of false-positive results and failure to isolate B gibsoni parasites after splenectomy and immunosuppression from a clinically normal dog with B gibsoni titer of 640.(ABSTRACT TRUNCATED AT 250 WORDS)

Babesiosis in Washington State: a new species of Babesia?

OBJECTIVE: To characterize the etiologic agent (WA1) of the first reported case of babesiosis acquired in Washington State. DESIGN: Case report, and serologic, molecular, and epizootiologic studies. SETTING: South-central Washington State. PATIENT: A 41-year-old immunocompetent man with an intact spleen who developed a moderately severe case of babesiosis. MEASUREMENTS: Serum specimens from the patient were assayed by indirect immunofluorescent antibody (IFA) testing for reactivity with seven Babesia species and with WA1, which was propagated in hamsters inoculated with his blood. A Babesia-specific, ribosomal-DNA (rDNA) probe was hybridized to Southern blots of restriction-endonuclease-digested preparations of DNA from WA1, Babesia microti, and Babesia gibsoni. Serum specimens from 83 family members and neighbors were assayed for IFA reactivity with WA1 and B. microti. Small mammals and ticks were examined for Babesia infection. RESULTS: The patient's serum had very strong IFA reactivity with WA1, strong reactivity with B. gibsoni (which infects dogs), but only weak reactivity with B. microti. DNA hybridization patterns with the rDNA probe clearly differentiated WA1 from B. gibsoni and B. microti. Four of the patient's neighbors had IFA titers to WA1 of 256. The tick vector and animal reservoir of WA1 have not yet been identified, despite trapping 83 mammals and collecting 235 ticks. CONCLUSIONS: WA1 is morphologically indistinguishable but antigenically and genotypically distinct from B. microti. Some patients elsewhere who were assumed to have been infected with B. microti may have been infected with WA1. Improved serodiagnostic and molecular techniques are needed for characterizing Babesia species and elucidating the epidemiology of babesiosis, an emergent zoonosis.

The isolation and partial characterization of a Babesia sp. from desert bighorn sheep (Ovis canadensis nelsoni).

A novel Babesia parasite of desert bighorn sheep was isolated. Its taxonomic description, host range, pathogenicity and antigenic relatedness were investigated. The parasite was infective for black-tailed and white-tailed deer, but with host-specific differences compared to that of bighorn sheep. A splenectomized calf and domestic sheep were refractory to infection. A comparative immunofluorescence assay detected antigens cross-reactive with Babesia odocoilei, B. divergens, B. equi and B. caballi, but not with B. bovis or B. bigemina. Babesia odocoilei was also infective for bighorn sheep, allowing comparison by a cross-challenge experiment, the results of which supported the conclusion that this parasite was not B. odocoilei. However, the bighorn sheep Babesia cannot currently be distinguished from B. capreoli described from roe deer in northern Germany. Data indicate that, while this parasite may not present a problem for domestic animals, it may cause disease in bighorn sheep and deer populations.

Experimental Lyme disease in dogs produces arthritis and persistent infection.

Lyme disease was reproduced in specific pathogen-free beagle dogs by exposure to Borrelia burgdorferi-infected ticks (Ixodes dammini). Seroconversion and disease frequency were higher after exposure to infected adult ticks than to infected nymphs. Young pups developed clinical disease more readily than older dogs. The incubation period lasted 2-5 months. Acute recurrent lameness with fibrinopurulent arthritis was the dominant clinical sign. Dogs recovered but developed persistent mild polyarthritis. B. burgdorferi persisted in recovered dogs for at least 1 year. Isolation of B. burgdorferi and detection by polymerase chain reaction was most successful from skin biopsies at the site of the tick bite. Antibody to B. burgdorferi antigens was first detected by ELISA and Western blots by 4-6 weeks after exposure. High serum levels persisted during 17 months of observation. In contrast to infection from ticks, inoculation of dogs with cultured B. burgdorferi resulted in seroconversion with a shorter duration of antibody persistence and no clinical disease.

Isolation and in vitro cultivation of Babesia parasites from free-ranging desert bighorn sheep (Ovis canadensis nelsoni) and mule deer (Odocoileus hemionus) in California.

Protozoal parasites of the genus Babesia were isolated for the first time from free-ranging desert bighorn sheep (Ovis canadensis nelsoni) and mule deer (Odocoileus hemionus) populations in California by in vitro culture of host blood. These naturally infected animals did not have microscopically detectable parasitemia at the time blood was collected for parasite cultivation. Three isolates of small Babesia parasites were cultured from different sample groups of bighorn sheep, and 2 isolates of large Babesia parasites were cultured from a group of bighorn sheep and a group of mule deer, respectively. The size and structure of the various forms of piroplasms from each isolate remained consistent throughout the period of cultivation. Statistical comparison of the sizes of the piroplasms among the isolates indicated that there were at least 2 distinct morphotypes. Four of the 5 isolates were maintained with continuous growth in cultures containing erythrocytes from uninfected donor bighorn sheep, mule deer, and domestic sheep. Cryopreservation or storage of cultures at 4 C for 7 days did not affect viability of the isolates. These results demonstrate the potential for use of in vitro cultivation methods for the isolation of Babesia parasites from free-ranging artiodactylids.

Detection of Babesia microti by polymerase chain reaction.

Human babesiosis, which is caused by infection with the intraerythrocytic malarialike protozoan Babesia microti, has recently been diagnosed with increasing frequency in residents of New England. Diagnosis is difficult because of the small size of the parasite and the sparse parasitemia that is characteristic of most infections with this pathogen. We generated B. microti-specific DNA sequence information by universal primer amplification of a portion of the eukaryotic 16S-like gene; this was followed by direct DNA sequence analysis. Specific primers were synthesized on the basis of this sequence information for use in the polymerase chain reaction (PCR). The PCR-based system demonstrates a strong bias for detection of B. microti as opposed to Babesia gibsoni and does not amplify vertebrate DNA. The analytical sensitivity of the system is approximately three merozoites. Blood specimens from 12 patients with clinically diagnosed and parasitologically confirmed babesiosis from Nantucket Island, Mass., were PCR positive in a blinded test of this procedure. Thus, DNA amplification may provide an adjunct to conventional methods for the diagnosis of human babesiosis and may provide a new means of monitoring therapy or enhancing epidemiological surveillance for this emerging pathogen.

Ribosomal DNA probe for differentiation of Babesia microti and B. gibsoni isolates.

The objective of this study was to determine whether different isolates of Babesia microti could be distinguished from morphologically similar isolates of B. gibsoni by using a ribosomal DNA (rDNA) probe. A Babesia-specific rDNA probe was obtained by polymerase chain reaction amplification of sequences from B. microti DNA using universal primers directed against highly conserved portions of the eukaryotic 16S-like rRNA gene. The chemiluminescent rDNA probe hybridized to Southern blots of restriction endonuclease-digested DNA preparations of different isolates of B. gibsoni from infected dogs and B. microti from infected humans and white-footed mice. Restriction fragment length polymorphisms served to differentiate these species. Although the hybridization patterns seen with DNAs from six B. microti isolates did not vary, those of the five B. gibsoni isolates did indicate genotypic variation. We concluded that isolates of B. microti and B. gibsoni can be differentiated on the basis of restriction fragment length polymorphism detected with a chemiluminescent rDNA probe.

Hemolytic anemia caused by Babesia gibsoni infection in dogs.

Babesia gibsoni caused severe hemolytic anemia in 11 dogs from southern California. The most common clinical signs of B gibsoni infection were lethargy, anorexia, anemia, and thrombocytopenia. Acute infection with B gibsoni may be misdiagnosed as autoimmune hemolytic anemia. Diagnosis was most reliably determined by identification of the intraerythrocytic parasites on Giemsa-stained blood smears. The pathogenicity of B gibsoni, difficulties in diagnosis, the parasite's resistance to treatment with available drugs, and frequent interstate movement of dogs indicate that this disease may be a serious threat to dogs throughout the United States.

Developmental changes in Hymenolepis citelli and Hymenolepis diminuta during patency.

Stem cell frequency, wet weight, proglottid number, and egg production were measured in Hymenolepis citelli at specific intervals between 20 and 120 days postinfection in an effort to correlate changes in stem cell frequency to other developmental parameters. Considerable variability was seen in wet weight and proglottid number, but differences did not seem to reflect any relation between these parameters and stem cell frequency. Significant differences were observed in egg production at specific postinfection periods. These appeared to correspond to changes seen in stem cell frequency during patency. Similar changes in egg production which also correspond to measured changes in stem cell frequency were recorded for Hymenolepis diminuta. Differences were also seen in number of eggs contained within gravid proglottids at various times postinfection for both species.