Amino acid sequences of proteins from Leptospira serovar pomona.

This report describes a partial amino acid sequences from three putative outer envelope proteins from Leptospira serovar pomona. In order to obtain internal fragments for protein sequencing, enzymatic and chemical digestion was performed. The enzyme clostripain was used to digest the proteins 32 and 45 kDa. In situ digestion of 40 kDa molecular weight protein was accomplished using cyanogen bromide. The 32 kDa protein generated two fragments, one of 21 kDa and another of 10 kDa that yielded five residues. A fragment of 24 kDa that yielded nineteen residues of amino acids was obtained from 45 kDa protein. A fragment with a molecular weight of 20 kDa, yielding a twenty amino acids sequence from the 40 kDa protein.

Detection of Leptospira spp. in the aqueous humor of horses with naturally acquired recurrent uveitis.

Leptospiral organisms have long been presumed to be associated with the presence of equine recurrent uveitis. This project was undertaken to determine the presence of Leptospira spp. in the aqueous humor of horses with uveitis to determine if there was an association with inflammation. Thirty horses were determined to have recurrent uveitis based on clinical evaluation or history. Sixteen horses were judged clinically and historically to be free of uveitis and were used as controls. Aqueous humor samples were cultured and evaluated by PCR for the presence of Leptospira DNA. Serum was collected and evaluated for the presence of antibodies against five serovars in a leptospirosis panel. Twenty-one of 30 horses with recurrent uveitis and one of 16 uveitis-free horses were positive by PCR for the presence of Leptospira DNA. Six of these 21 horses with uveitis were culture positive for leptospires from the aqueous humor. Serologic results did not correlate well with the presence of Leptospira DNA or organisms in the aqueous humor. Leptospira spp. are present in a high percentage of horses with naturally occurring recurrent uveitis.

DNA sequence similarity between California isolates of Cryptosporidium parvum.

We evaluated whether nucleic acid amplification with primers specific for Cryptosporidium parvum followed by automated DNA sequence analysis of the PCR amplicons could differentiate between California isolates of C. parvum obtained from livestock, humans, and feral pigs. Almost complete sequence identity existed among the livestock isolates and between the livestock and human isolates. DNA sequences from feral pig isolates differed from those from livestock and humans by 1.0 to 1.2%. The reference sequence obtained by Laxer et al. (M. A. Laxer, B. K. Timblin, and R. J. Patel, Am. J. Trop. Med. Hyg. 45:688-694, 1991.) differed from California isolates of C. parvum by 1.8 to 3.2%. These data suggest that DNA sequence analysis of the amplicon of Laxer et al. does not allow for differentiation between various strains of C. parvum or that our collection of isolates obtained from various hosts from across California was limited to one strain of C. parvum.

Immunization with outer surface protein (Osp) A, but not OspC, provides cross-protection of mice challenged with North American isolates of Borrelia burgdorferi.

The identification of antigens with the capacity to induce a broad spectrum of protective immunity is an important consideration in the design of a Lyme disease vaccine. In this study, the range of protection provided by outer surface protein (Osp) A or OspC vaccination was compared. Mice actively immunized with OspA or OspC were challenged with 3 North American isolates of Borrelia burgdorferi. OspA-immunized mice were fully protected from infection with each of the isolates, whereas mice immunized with OspC were protected from infection with the homologous isolate but not with 2 heterologous isolates. Sequence analysis revealed that the ospA genes from these 3 isolates were >99% homologous, whereas the ospC genes shared only 81%-85% homology. Western blot analysis suggested antigenic heterogeneity associated with OspC but not OspA. These results indicate that genetic and antigenic heterogeneity may limit the usefulness of OspC as a vaccine constituent.

Antibodies to OspB prevent infection of C3H mice challenged with Borrelia burgdorferi isolates expressing truncated OspB antigens.

Truncation of outer surface protein B (OspB) of the Lyme disease agent, Borrelia burgdorferi, may allow the organism to escape immunological destruction and render an OspB-based vaccine ineffective. To investigate this possibility, we have identified two isolates, 297 and CA4, which predominantly express a truncated form of the OspB antigen. In each case, nucleic acid sequencing revealed that truncation of the OspB antigen resulted from a nonsense mutation within the 3', end of the ospB gene. Growth inhibition and protection studies demonstrated that both isolates were neutralized by an anti-OspB serum. Our results indicate that truncated forms of the OspB antigen possess epitopes that may represent important targets for neutralizing antibodies and thus, support the inclusion of OspB as a component of a subunit vaccine.

Identification and characterization of a surface-exposed, 66-kilodalton protein from Borrelia burgdorferi.

The surface-exposed antigens of Borrelia burgdorferi represent important targets for the development of a protective immune response. We have identified a proteinase K-accessible, 66-kDa protein from B. burgdorferi and have demonstrated that at least a portion of this protein is surface exposed. The 66-kDa protein was purified by sequential extraction of spirochetes with butanol and Triton X-114 followed by preparative gel electrophoresis. Polyclonal antibodies developed against the purified 66-kDa protein were Borrelia spp. specific, whereas a monoclonal antibody, Route 66, displayed a genospecies-specific pattern of recognition for the 66-kDa protein. N-terminal amino acid sequence was obtained from an internal fragment, a truncated version, and the full-length form of the 66-kDa protein. A search of protein and gene databases for homologous sequences yielded a match with the predicted amino acid sequence from a segment of B. burgdorferi chromosomal DNA (P. A. Rosa, D. Hogan, and T. G. Schwan, J. Clin. Microbiol. 29:524-532, 1991). The construction of primers based on this DNA sequence and the N-terminal amino acid sequence allowed the amplification and cloning of the 66-kDa-protein gene. The identity of the cloned gene was verified by the recognition of the expressed gene product by Route 66. Pulsed-field gel electrophoresis and Southern blot analysis were performed to confirm the chromosomal location of the 66-kDa-protein gene. This study describes the identification and cloning of the first chromosomally encoded, surface-exposed protein from B. burgdoferi.

Polymerase chain reaction for detection of Borrelia coriaceae, putative agent of epizootic bovine abortion.

The nucleotide sequence of a chromosomally encoded antigen-expressing gene of Borrelia coriaceae was determined and used as a target for the polymerase chain reaction (PCR). Two primer sets were designed specifying the amplification of 269- and 701-bp DNA fragments. Primer set I, producing the short amplicon, was tenfold more sensitive than primer set II. As little as 10 fg of purified B coriaceae DNA could consistently be detected. The PCR assays, containing controlled numbers of whole spirochetes, allowed detectable amplification of 2 to 10 organisms. An internal, nonradioactively labeled gene-specific probe verified specificity of the PCR amplicons. Neither primer set cross-reacted with other related spirochetes. This PCR assay was adapted and found suitable for identification of B coriaceae in biological samples, such as blood and thymus. Evidence for presence of B coriaceae in biological samples was not found in tissue samples obtained from experimentally infected cows and their fetuses. These data failed to establish a definite association between B coriaceae and epizootic bovine abortion.

Protection of C3H/HeN mice from challenge with Borrelia burgdorferi through active immunization with OspA, OspB, or OspC, but not with OspD or the 83-kilodalton antigen.

Recent advances in the development of animal models for Lyme borreliosis have provided means of identifying potential targets for the design of a subunit vaccine to prevent this disease. The C3H/HeN mouse model was used to study several Borrelia burgdorferi antigens from a single isolate for their ability to elicit borreliacidal and protective antibodies. The ospA, ospB, ospC, ospD, and 83-kDa genes from a California isolate, SON 188, were cloned and expressed in Escherichia coli as proteins fused to the C-terminal end of maltose-binding protein. Active immunization of mice with these fusion proteins elicited high titers of antibodies that recognized the homologous SON 188 antigens upon immunoblotting. Antibodies generated to the OspA and OspB fusion proteins, but not to the OspC, OspD, and the 83-kDa fusion proteins, demonstrated in vitro borreliacidal activity. Challenge of all actively immunized mice with 10(7) SON 188 spirochetes resulted in infection in all mice receiving the OspD or 83-kDa immunogens but not in any mice receiving the OspA, OspB, or OspC fusion proteins. These results demonstrate the potential of OspA, OspB, and OspC as components of a subunit vaccine for the prevention of Lyme borreliosis.

Detection of bovine trichomoniasis with a specific DNA probe and PCR amplification system.

Trichomoniasis is a widespread, economically important venereal disease of cattle which causes infertility and abortion. Effective control of trichomoniasis has been impeded by the insensitivity of traditional diagnostic procedures, which require the isolation and cultivation of the parasite, Tritrichomonas foetus, from infected cattle. We developed a 0.85-kb T. foetus DNA probe by identifying conserved sequences in DNAs from T. foetus that were isolated from cattle in California, Idaho, Nevada, and Costa Rica. The probe hybridized specifically to DNAs of T. foetus isolates from different geographic areas but not to DNA preparations of Trichomonas vaginalis, bovine cells, or a variety of bacteria from cattle. The probe detected DNA from a minimum of 10(5) T. foetus organisms. To improve sensitivity, a partial sequence of the probe was used to identify oligonucleotide primers (TF1 and TF2) which could be used to amplify a 162-bp product from T. foetus DNAs by PCR. A chemiluminescent internal T. foetus sequence probe was hybridized to Southern blots of the amplification product. This system detected as few as one T. foetus organism in culture media or 10 parasites in samples containing bovine preputial smegma. Analysis of 52 clinical samples showed that 47 (90.4%) of the 52 samples were correctly identified, with no false-positive reactions. In comparison, the traditional cultivation method detected 44 (84.6%) of the 52 samples from T. foetus-infected and uninfected bulls. These results indicate that the PCR-based amplification system could be a useful alternative method for the diagnosis of bovine trichomoniasis.

Genetic diversity among Borrelia burgdorferi isolates from wood rats and kangaroo rats in California.

Twenty-nine Borrelia burgdorferi isolates, obtained from dusky-footed wood rats (Neotoma fuscipes) and California kangaroo rats (Dipodomys californicus) in California, were analyzed genetically. Chromosomal DNA was examined by restriction endonuclease analysis (REA) and gene probe restriction fragment length polymorphism. Pulsed-field gel electrophoresis was used to analyze the plasmid profiles of the isolates. REA, the method with the greatest discrimination, disclosed 24 distinct restriction patterns among the 29 isolates. These restriction patterns were sorted into four restriction fragment length polymorphism groups on the basis of their gene hybridization patterns. Results of the REA and plasmid profile analysis supported this grouping. The degree of genetic diversity among Californian isolates demonstrated by our findings is greater than that previously reported among other groups of North American isolates and is similar or greater than the diversity reported among European isolates.

Comparative analysis of genetic variability among Borrelia burgdorferi isolates from Europe and the United States by restriction enzyme analysis, gene restriction fragment length polymorphism, and pulsed-field gel electrophoresis.

The genomes of 62 North American and European Borrelia burgdorferi isolates were examined by restriction endonuclease analysis (REA), gene probe restriction fragment length polymorphism, and pulsed-field gel electrophoresis (PFGE). Hybridization of restriction fragments with the immunologically relevant 83-kDa antigen gene revealed polymorphisms and divided the isolates into three major groups. Group I included all but two of the American isolates and some of the European isolates. One of two Californian isolates (DN 127) and one Ixodes dammini isolate from New York (strain 25015), previously described as atypical, were distinct from the isolates in the three groups. Plasmid profile analysis and REA, the method with the highest level of discrimination, revealed extensive heterogeneity among isolates of the same major group. Our study demonstrates the usefulness of the 83-kDa antigen gene probe for dividing the isolates into major genogroups, whereas REA and plasmid profile analysis allow for a distinction of individual strains within these groups.

Characterization of a chromosomal gene and the antigen it expresses from the Lyme disease agent, Borrelia burgdorferi.

The sequence and characterization of a chromosomal gene from the Lyme disease agent Borrelia burgdorferi and the antigen it encodes are described. The gene was cloned and expressed in transformed Escherichia coli cells. The gene is composed of 597 bases and expresses a predicted protein of 199 amino acids. Antibodies specific for the recombinant antigen reacted with a single B. burgdorferi protein with a molecular mass of approximately 22 kDa. The protein was not susceptible to proteinase digestion but was extracted by n-butanol phase partitioning, suggesting a periplasmic location of the antigen. Sera from humans and canines seropositive for B. burgdorferi reacted with the recombinant antigen. The antigen characterized in this report appears to be immunologically significant in naturally infected hosts.

First isolation of Borrelia burgdorferi in southern California.

The Lyme disease spirochete, Borrelia burgdorferi Johnson, Schmid, Hyde, Steigerwalt & Brenner, was isolated from the blood of a dusky-footed wood rat, Neotoma fuscipes Baird, in the San Bernardino Mountains of southern California. Antigenic, protein, and molecular analyses demonstrated that the isolate varied slightly from most isolates of B. burgdorferi from northern California and was clearly distinct from other species of Borrelia that are endemic to the state. This is the first reported isolate of B. burgdorferi from southern California and demonstrates that the Lyme disease spirochete is enzootic in mountains near the major human population center of the state.

Further characterization of a potent immunogen and the chromosomal gene encoding it in the Lyme disease agent, Borrelia burgdorferi.

Further characterization of a previously reported 83-kDa antigen of Borrelia burgdorferi and the gene encoding it is reported. The DNA sequence of the gene and the amino acid sequence of the protein were determined. On the basis of the amino acid content, the actual size of the antigen was determined to be 79.8 kDa, rather than 83 kDa as previously reported. The expression of the antigen in several representative North American and European B. burgdorferi isolates was demonstrated. The conservation of the gene within the species was demonstrated by DNA hybridization of a labeled gene probe to several North American and European B. burgdorferi isolates. Of other Borrelia spp. assayed (B. hermsii, B. coriaceae, B. duttonii, and B. anserina), only B. anserina, a poultry pathogen, hybridized to the gene probe and expressed the 79.8-kDa antigen.

Protein and antigen profiles of prevalent serovars of Leptospira interrogans.

Whole-cell and detergent-soluble proteins, enriched for outer membrane antigens, of the Leptospira interrogans serovars present in commercially available pentavalent vaccines (hardjo, pomona, icterohaemorrhagiae, grippotyphosa, and canicola) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting (immunoblotting). Protein and antigenic profiles of these serovars, representing several serogroups, were compared with similar profiles of the most common North American bovine pathogen, serovar hardjo type hardjo bovis. The reference strain of serovar balcanica and a hardjoprajitno bovine field isolate (serovar hardjo) were also assayed. Coomassie blue-stained gels revealed extensive protein similarities, and Western blots demonstrated antigenic relatedness throughout the low- and high-molecular-weight regions. Possible quantitative differences in protein expression among the strains were noted, as were similarities in the protein profiles of type hardjo bovis and the balcanica reference strain. A cocktail composed of these homologous antigens may serve as an efficacious subunit vaccine for leptospirosis.

The 83-kilodalton antigen of Borrelia burgdorferi which stimulates immunoglobulin M (IgM) and IgG responses in infected hosts is expressed by a chromosomal gene.

We report the cloning and characterization of a chromosomal gene from Borrelia burgdorferi expressing an 83-kilodalton protein antigen in Escherichia coli. The antigen reacted strongly with antisera from two human Lyme disease patients. The chromosomal gene was expressed in a 6.5-kilobase-pair ClaI fragment cloned into a variable-reading-frame plasmid vector.

Expression of antigens from chromosomal and linear plasmid DNA of Borrelia coriaceae.

Three recombinant plasmids containing DNA from Borrelia coriaceae, the putative agent of epizootic bovine abortion, expressed antigens in Escherichia coli that reacted with antibodies specific for B. coriaceae. Two of the recombinants each expressed a single high-molecular-weight antigen. The third recombinant expressed three smaller antigens. The DNA inserts were sized and mapped. Hybridization of the cloned inserts to pulsed-field electrophoresis samples of B. coriaceae whole-cell DNA revealed the origin of two of the inserts to be located in linear plasmids. One of these, expressing three antigens, was located in a 210-kilobase linear plasmid. A second recombinant expressed a single antigen but hybridized to at least three distinct linear plasmids. The third clone also expressed a single antigen but was demonstrated to be chromosomal in origin.

Unique ribosome structure of Leptospira interrogans is composed of four rRNA components.

All known ribosomes of procaryotic organisms are made up of three rRNA components that are 23, 16, and 5S in size. We now report that in some Leptospira interrogans strains, the classical 23S rRNA is further processed to generate 14 and 17S rRNAs. This processing step was previously known to occur only in some eucaryotes and in a small group of procaryotes. The implications of this finding are discussed.

DNA and protein analyses of tick-derived isolates of Borrelia burgdorferi from California.

Nine isolates of Borrelia burgdorferi from ixodid ticks collected in northern California were characterized. Restriction endonuclease analysis, pulsed-field gel electrophoresis, and Western blot (immunoblot) analysis were used in this study. Four isolates were very similar to each other. The others shared some similarities but were classified as having unique genotypes. A strain from an Ixodes neotomae tick displayed the greatest genetic and antigenic diversity when compared to the isolates collected from Ixodes pacificus ticks. A computerized library based on DNA banding patterns of the isolates by restriction enzyme analysis is also reported. This library was created by using a scanning laser densitometer.

Characterization of Borrelia burgdorferi isolates by restriction endonuclease analysis and DNA hybridization.

Genomes of several Borrelia burgdorferi isolates from North America and Europe were characterized by restriction endonuclease analysis and DNA hybridization using labeled B. burgdorferi whole-cell DNA (strain ATCC 35210). Several different restriction and homology patterns were observed among these isolates, indicating genotypic heterogeneity within this genus and species. It was concluded from this study that restriction endonuclease analysis of B. burgdorferi whole-cell DNA may be a reliable and accurate method for identifying strains or genotypes of the Lyme disease agent.