Immunohistochemical analysis of Lyme disease in the skin of naive and infection-immune rabbits following challenge.

In this study, skin histopathology from naive and infection-derived immune rabbits was compared following intradermal challenge using Borrelia burgdorferi B31 strain. The presence or absence of spirochetes in relationship to host cellular immune responses was determined from the time of intradermal inoculation to the time of erythema migrans (EM) development (approximately 7 days in naive rabbits) and through development of challenge immunity (approximately 5 months in naive rabbits). Skin biopsies were obtained and analyzed for the presence of spirochetes, B cells, T cells, polymorphonuclear cells (PMNs), and macrophages by immunohistochemical techniques. In infected naive animals, morphologically identifiable spirochetes were detected at 2 h and up to 3 weeks postinfection. At 12 and 24 h postinfection there was a marked PMN response that decreased by 36 to 48 h; by 72 h the PMNs were replaced by a few infiltrating macrophages. At the time of EM development and 14 days postinfection, the PMNs and macrophages were replaced by a lymphocytic infiltrate. There was a greater number of spirochetes at 14 days, a time when EM had resolved, than at 7 days postinfection. By 3 weeks postinfection there were few organisms and lymphocytes detectable. In contrast to infected naive rabbits, intact spirochetes were never visualized in skin biopsies from infection-immune rabbits; only spirochetal antigen was detected at 2, 12, and 24 h in the presence of a numerous PMN infiltrate. By 36 h postchallenge, spirochetal antigen could not be detected and the PMN response was replaced by a few infiltrating macrophages. By 72 h postchallenge, PMNs and macrophages were absent from the skin; B and T cells were never detected at any time point in skin from infection-immune rabbits. The destruction of spirochetes in immune animals in the presence of PMNs and in the absence of a lymphocytic infiltrate suggests that infection-derived immunity is antibody mediated.

Homologous and heterologous Borrelia burgdorferi challenge of infection-derived immune rabbits using host-adapted organisms.

We have recently found that strain B31 infection-immune rabbits are completely protected against homologous challenge with large numbers (>10(6)) of host-adapted Borrelia burgdorferi (HAB) (E. S. Shang, C. I. Champion, X. Wu, J. T. Skare, D. B. Blanco, J. N. Miller, and M. A. Lovett, Infect. Immun. 68:4189-4199, 2000). In this study, we have extended these findings to determine whether B31 strain infection-immune rabbits are also protected against heterologous HAB challenge. Infection-immune rabbits challenged with large numbers (>10(6)) of homologous HAB strain B31 were completely protected from erythema migrans (EM) and skin and disseminated infection. In contrast, infection-immune rabbits challenged with heterologous HAB strains N40 and Sh-2-82 were completely susceptible to EM and skin and disseminated infection; challenge with strain 297 also resulted in EM and infection of the skin and viscera, but clearance of infection occurred 3 weeks postchallenge. These findings confirm that immunity elicited in rabbits by B31 strain infection confers complete protection against large-dose homologous HAB challenge but not against a heterologous strain.

Comparison of protection in rabbits against host-adapted and cultivated Borrelia burgdorferi following infection-derived immunity or immunization with outer membrane vesicles or outer surface protein A.

In this study, infection-derived immunity in the rabbit model of Lyme disease was compared to immunity following immunization with purified outer membrane vesicles (OMV) isolated from Borrelia burgdorferi and recombinant outer surface protein A (OspA). Immunization of rabbits with OMV isolated from virulent strain B31 and its avirulent derivative B313 (lacking OspA and DbpA) conferred highly significant protection against intradermal injection with 6 x 10(4) in vitro-cultivated virulent B. burgdorferi. This is the first demonstration of protective immunogenicity induced by OMV. While immunization with OspA and avirulent B31 OMV provided far less protection against this challenge, rabbits with infection-derived immunity were completely protected. Protection against host-adapted B. burgdorferi was assessed by implantation of skin biopsies taken from rabbit erythema migrans (a uniquely rich source of B. burgdorferi in vertebrate tissue) containing up to 10(8) spirochetes. While all of the OMV- and OspA-immunized rabbits were fully susceptible to skin and disseminated infection, rabbits with infection-derived immunity were completely protected. Analysis of the antibody responses to outer membrane proteins, including DbpA, OspA, and OspC, suggests that the remarkable protection exhibited by the infection-immune rabbits is due to antibodies directed at antigens unique to or markedly up-regulated in host-adapted B. burgdorferi.

Renaturation of recombinant Treponema pallidum rare outer membrane protein 1 into a trimeric, hydrophobic, and porin-active conformation.

We have previously observed that while native Treponema pallidum rare outer membrane protein 1 (Tromp1) is hydrophobic and has porin activity, recombinant forms of Tromp1 do not possess these properties. In this study we show that these properties are determined by conformation and can be replicated by proper renaturation of recombinant Tromp1. Native Tromp1, but not the 47-kDa lipoprotein, extracted from whole organisms by using Triton X-114, was found to lose hydrophobicity after treatment in 8 M urea, indicating that Tromp1's hydrophobicity is conformation dependent. Native Tromp1 was purified from 0.1% Triton X-100 extracts of whole organisms by fast-performance liquid chromatography (FPLC) and shown to have porin activity in planar lipid bilayers. Cross-linking studies of purified native Tromp1 with an 11 A cross-linking agent showed oligomeric forms consistent with dimers and trimers. For renaturation studies of recombinant Tromp1 (rTromp1), a 31,109-Da signal-less construct was expressed in Escherichia coli and purified by FPLC. FPLC-purified rTromp1 was denatured in 8 M urea and then renatured in the presence of 0.5% Zwittergent 3,14 during dialysis to remove the urea. Renatured rTromp1 was passed through a Sephacryl S-300 gel exclusion column previously calibrated with known molecular weight standards. While all nonrenatured rTromp1 eluted from the column at approximately the position of the carbonic anhydrase protein standard (29 kDa), all renatured rTromp1 eluted at the position of the phosphorylase b protein standard (97 kDa), suggesting a trimeric conformation. Trimerization was confirmed by using an 11 A cross-linking agent which showed both dimers and trimers similar to that of native Tromp1. Triton X-114 phase separations showed that all of renatured rTromp1, but none of nonrenatured rTromp1, phase separated exclusively into the hydrophobic detergent phase, similar to native Tromp1. Circular dichroism of nonrenatured and renatured rTromp1 showed a marked loss in alpha-helical secondary structure of renatured rTromp1 compared to the nonrenatured form. Finally, renatured rTromp1, but not the nonrenatured form, showed porin activity in planar liquid bilayers. These results demonstrate that proper folding of rTromp1 results in a trimeric, hydrophobic, and porin-active conformation similar to that of the native protein.

Immunization with Treponema pallidum outer membrane vesicles induces high-titer complement-dependent treponemicidal activity and aggregation of T. pallidum rare outer membrane proteins (TROMPs).

The purpose of this study was to determine whether immunization with purified outer membrane vesicles (OMV) from Treponema pallidum (T.p. ) could elicit Abs capable of killing this organism. It is well established that the immunization of rabbits or mice with killed T.p. or with recombinant T.p. Ags has failed to generate serum killing activity comparable with that of infection-derived immunity. Because of the small amount of T.p. OMV obtainable, a single mouse was immunized with purified OMV. The mouse anti-OMV serum and infection-derived immune rabbit serum (IRS) were compared by reactivities on two-dimensional T.p. immunoblots and by the T.p. immobilization test, a complement-dependent killing assay. Whereas IRS detected >40 Ags, the anti-OMV serum identified only 6 Ags corresponding to proteins identified previously in the outer membrane. T.p. immobilization testing showed that IRS had a 100% killing titer of 1:44 and a 50% killing titer of 1:662. By comparison, the mouse anti-OMV serum had a significantly greater 100% killing titer of 1:1,408 and a 50% killing titer of 1:16,896. Absorption of the anti-OMV serum to remove Ab against outer membrane-associated lipoproteins did not change the 100% killing titer. Freeze-fracture analysis of T.p. incubated in IRS or anti-OMV serum showed that T.p. rare membrane-spanning outer membrane proteins were aggregated. This is the first demonstration of high-titer killing Abs resulting from immunization with defined T.p. molecules; our study indicates that the targets for these Abs are T. p. rare outer membrane proteins.

Characterization of leptospiral outer membrane lipoprotein LipL36: downregulation associated with late-log-phase growth and mammalian infection.

We report the cloning of the gene encoding a 36-kDa leptospiral outer membrane lipoprotein, designated LipL36. We obtained the N-terminal amino acid sequence of a staphylococcal V8 proteolytic-digest fragment in order to design an oligonucleotide probe. A Lambda-Zap II library containing EcoRI fragments of Leptospira kirschneri DNA was screened, and a 2.3-kb DNA fragment which contained the entire structural lipL36 gene was identified. Several lines of evidence indicate that LipL36 is lipid modified in a manner similar to that of LipL41, a leptospiral outer membrane lipoprotein we described in a previous study (E. S. Shang, T. A. Summers, and D. A. Haake, Infect. Immun. 64:2322-2330, 1996). The deduced amino acid sequence of LipL36 would constitute a 364-amino-acid polypeptide with a 20-amino-acid signal peptide, followed by an L-X-Y-C lipoprotein signal peptidase cleavage site. LipL36 is solubilized by Triton X-114 extraction of L. kirschneri; phase separation results in partitioning of LipL36 exclusively into the hydrophobic, detergent phase. LipL36 is intrinsically labeled during incubation of L. kirschneri in media containing [3H]palmitate. Processing of LipL36 is inhibited by globomycin, a selective inhibitor of lipoprotein signal peptidase. After processing, LipL36 is exported to the outer membrane along with LipL41 and lipopolysaccharide. Unlike LipL41, there appears to be differential expression of LipL36. In early-log-phase cultures, LipL36 is one of the most abundant L. kirschneri proteins. However, LipL36 levels drop considerably beginning in mid-log phase. LipL36 expression in vivo was evaluated by examining the humoral immune response to leptospiral antigens in the hamster model of leptospirosis. Hamsters surviving challenge with culture-adapted virulent L. kirschneri generate a strong antibody response to LipL36. In contrast, sera from hamsters surviving challenge with host-adapted L. kirschneri do not recognize LipL36. These findings suggest that LipL36 expression is downregulated during mammalian infection, providing a marker for studying the mechanisms by which pathogenic Leptospira species adapt to the host environment.

Isolation and characterization of the outer membrane of Borrelia hermsii.

The outer membrane of Borrelia hermsii has been shown by freeze-fracture analysis to contain a low density of membrane-spanning outer membrane proteins which have not yet been isolated or identified. In this study, we report the purification of outer membrane vesicles (OMV) from B. hermsii HS-1 and the subsequent identification of their constituent outer membrane proteins. The B. hermsii outer membranes were released by vigorous vortexing of whole organisms in low-pH, hypotonic citrate buffer and isolated by isopycnic sucrose gradient centrifugation. The isolated OMV exhibited porin activities ranging from 0.2 to 7.2 nS, consistent with their outer membrane origin. Purified OMV were shown to be relatively free of inner membrane contamination by the absence of measurable beta-NADH oxidase activity and the absence of protoplasmic cylinder-associated proteins observed by Coomassie blue staining. Approximately 60 protein spots (some of which are putative isoelectric isomers) with 25 distinct molecular weights were identified as constituents of the OMV enrichment. The majority of these proteins were also shown to be antigenic with sera from B. hermsii-infected mice. Seven of these antigenic proteins were labeled with [3H]palmitate, including the surface-exposed glycerophosphodiester phosphodiesterase, the variable major proteins 7 and 33, and proteins of 15, 17, 38, 42, and 67 kDa, indicating that they are lipoprotein constituents of the outer membrane. In addition, immunoblot analysis of the OMV probed with antiserum to the Borrelia garinii surface-exposed p66/Oms66 porin protein demonstrated the presence of a p66 (Oms66) outer membrane homolog. Treatment of intact B. hermsii with proteinase K resulted in the partial proteolysis of the Oms66/p66 homolog, indicating that it is surface exposed. This identification and characterization of the OMV proteins should aid in further studies of pathogenesis and immunity of tick-borne relapsing fever.

The Oms66 (p66) protein is a Borrelia burgdorferi porin.

In this study we report the purification and characterization of a 66-kDa protein, designated Oms66, for outer membrane-spanning 66-kDa protein, that functions as a porin in the outer membrane (OM) of Borrelia burgdorferi. Oms66 was purified by fast-performance liquid chromatography and exhibited an average single-channel conductance of 9.62 +/- 0.37 nS in 1 M KCl, as evidenced by 581 individual insertional events in planar lipid bilayers. Electrophysiological characterization indicated that Oms66 was virtually nonselective between cations and anions and exhibited voltage-dependent closure with multiple substates. The amino acid sequence of tryptic peptides derived from purified Oms66 was identical to the deduced amino acid sequence of p66, a previously described surface-exposed protein of B. burgdorferi. Purified Oms66 was recognized by antiserum specific for p66 and serum from rabbits immune to challenge with virulent B. burgdorferi, indicating that p66 and Oms66 were identical proteins and that Oms66/p66 is an immunogenic protein in infected rabbits. In a methodology that reduces liposomal trapping and nonspecific interactions, native Oms66 was incorporated into liposomes, confirming that Oms66 is an outer membrane-spanning protein. Proteoliposomes containing Oms66 exhibited porin activity nearly identical to that of native, purified Oms66, indicating that reconstituted Oms66 retained native conformation. The use of proteoliposomes reconstituted with Oms66 and other Oms proteins provides an experimental system for determinating the relationship between conformation, protection, and biological function of these molecules.

Sequence analysis and characterization of a 40-kilodalton Borrelia hermsii glycerophosphodiester phosphodiesterase homolog.

We report the purification, molecular cloning, and characterization of a 40-kDa glycerophosphodiester phosphodiesterase homolog from Borrelia hermsii. The 40-kDa protein was solubilized from whole organisms with 0.1% Triton X-100, phase partitioned into the Triton X-114 detergent phase, and purified by fast-performance liquid chromatography (FPLC). The gene encoding the 40-kDa protein was cloned from a B. hermsii chromosomal DNA lambda EXlox expression library and identified by using affinity antibodies generated against the purified native protein. The deduced amino acid sequence included a 20-amino-acid signal peptide encoding a putative leader peptidase II cleavage site, indicating that the 40-kDa protein was a lipoprotein. Based on significant homology (31 to 52% identity) of the 40-kDa protein to glycerophosphodiester phosphodiesterases of Escherichia coli (GlpQ), Bacillus subtilis (GlpQ), and Haemophilus influenzae (Hpd; protein D), we have designated this B. hermsii 40-kDa lipoprotein a glycerophosphodiester phosphodiesterase (Gpd) homolog, the first B. hermsii lipoprotein to have a putative functional assignment. A nonlipidated form of the Gpd homolog was overproduced as a fusion protein in E. coli BL21(DE3)(pLysE) and was used to immunize rabbits to generate specific antiserum. Immunoblot analysis with anti-Gpd serum recognized recombinant H. influenzae protein D, and conversely, antiserum to H. influenzae protein D recognized recombinant B. hermsii Gpd (rGpd), indicating antigenic conservation between these proteins. Antiserum to rGpd also identified native Gpd as a constituent of purified outer membrane vesicles prepared from B. hermsii. Screening of other pathogenic spirochetes with anti-rGpd serum revealed the presence of antigenically related proteins in Borrelia burgdorferi, Treponema pallidum, and Leptospira kirschneri. Further sequence analysis both upstream and downstream of the Gpd homolog showed additional homologs of glycerol metabolism, including a glycerol-3-phosphate transporter (GlpT), a glycerol-3-phosphate dehydrogenase (GlpD), and a thioredoxin reductase (TrxB).

Recombinant Treponema pallidum rare outer membrane protein 1 (Tromp1) expressed in Escherichia coli has porin activity and surface antigenic exposure.

We recently reported the cloning and sequencing of the gene encoding a 31-kDa Treponema pallidum subsp. pallidum rare outer membrane porin protein, designated Tromp1 (D. R. Blanco, C. I. Champion, M. M. Exner, H. Erdjument-Bromage, R. E. W. Hancock, P. Tempst, J. N. Miller, and M. A. Lovett, J. Bacteriol. 177:3556-3562, 1995). Here, we report the stable expression of recombinant Tromp1 (rTromp1) in Escherichia coli. rTromp1 expressed without its signal peptide and containing a 22-residue N-terminal fusion resulted in high-level accumulation of a nonexported soluble protein that was purified to homogeneity by fast protein liquid chromatography (FPLC). Specific antiserum generated to the FPLC-purified rTromp1 fusion identified on immunoblots of T. pallidum the native 31-kDa Tromp1 protein and two higher-molecular-mass oligomeric forms of Tromp1 at 55 and 80 kDa. rTromp1 was also expressed with its native signal peptide by using an inducible T7 promoter. Under these conditions, rTromp1 fractionated predominantly with the E. coli soluble and outer membrane fractions, but not with the inner membrane fraction. rTromp1 isolated from the E. coli outer membrane and reconstituted into planar lipid bilayers showed porin activity based on average single-channel conductances of 0.4 and 0.8 nS in 1 M KCl. Whole-mount immunoelectron microscopy using infection-derived immune serum against T. pallidum indicated that rTromp1 was surface exposed when expressed in E. coli. These findings demonstrate that rTromp1 can be targeted to the E. coli outer membrane, where it has both porin activity and surface antigenic exposure.

Porin activity of the native and recombinant outer membrane protein Oms28 of Borrelia burgdorferi.

The outer membrane-spanning (Oms) proteins of Borrelia burgdorferi have been visualized by freeze-fracture analysis but, until recently, not further characterized. We developed a method for the isolation of B. burgdorferi outer membrane vesicles and described porin activities with single-channel conductances of 0.6 and 12.6 nS in 1 M KCI. By using both nondenaturing isoelectric focusing gel electrophoresis and fast-performance liquid chromatography separation after detergent solubilization, we found that the 0.6-nS porin activity resided in a 28-kDa protein, designated Oms28. The oms28 gene was cloned, and its nucleotide sequence was determined. The deduced amino acid sequence of Oms28 predicted a 257-amino-acid precursor protein with a putative 24-amino-acid leader peptidase I signal sequence. Processed Oms28 yielded a mature protein with a predicted molecular mass of 25,363 Da. When overproduced in Escherichia coli, the Oms28 porin fractionated in part to the outer membrane. Sodium dodecyl sulfate-polyacrylamide gel-purified recombinant Oms28 from E. coli retained functional activity as demonstrated by an average single-channel conductance of 1.1 nS in the planar lipid bilayer assay. These findings confirmed that Oms28 is a B. burgdorferi porin, the first to be described. As such, it is potential relevance to the pathogenesis of Lyme borreliosis and to the physiology of the spirochete.

Molecular cloning and sequence analysis of the gene encoding LipL41, a surface-exposed lipoprotein of pathogenic Leptospira species.

We report the cloning of the gene encoding a surface-exposed leptospiral lipoprotein, designated LipL41. In a previous study, a 41-kDa protein antigen was identified on the surface of Leptospira kirschneri (D. A. Haake, E. M. Walker, D. R. Blanco, C. A. Bolin, J. N. Miller, and M. A. Lovett, Infect. Immun. 59:1131-1140, 1991). We obtained the N-terminal amino acid sequence of a staphylococcal V8 proteolytic-digest fragment in order to design an oligonucleotide probe.A Lambda ZAP II library containing EcoRI fragments of L. kirschneri DNA was screened, and a 2.3-kb DNA fragment which contained the entire structural lipL41 gene was identified. The deduced amino acid sequence of LipL41 would encode a 355-amino-acid polypeptide with a 19-amino-acid signal peptide, followed by an L-X-Y-C lipoprotein signal peptidase cleavage site. A recombinant His6-LipL41 fusion protein was expressed in Escherichia coli in order to generate specific rabbit antiserum. LipL41 is solubilized by Triton X-114 extraction of L. kirschneri; phase separation results in partitioning of LipL41 exclusively into the detergent phase. At least eight proteins, including LipL41 and the other major Triton X-114 detergent phase proteins, are intrinsically labeled during incubation of L. kirschneri in media containing [3H] palmitate. Processing of LipL41 is inhibited by globomycin, a selective inhibitor of lipoprotein signal peptidase. Triton X-100 extracts of L. kirschneri contain immunoprecipitable OmpL1 (porin), LipL41, and another lipoprotein, LipL36. However, in contrast to LipL36, only LipL41 and OmpL1 were exposed on the surface of intact organisms. Immunoblot analysis of a panel of Leptospira species reveals that LipL41 expression is highly conserved among leptospiral pathogens.

Virulent strain associated outer membrane proteins of Borrelia burgdorferi.

We have isolated and purified outer membrane vesicles (OMV) from Borrelia burgdorferi strain B31 based on methods developed for isolation of Treponema pallidum OMV. Purified OMV exhibited distinct porin activities with conductances of 0.6 and 12.6 nano-Siemen and had no detectable beta-NADH oxidase activity indicating their outer membrane origin and their lack of inner membrane contamination, respectively. Hydrophobic proteins were identified by phase partitioning with Triton X-114. Most of these hydrophobic membrane proteins were not acylated, suggesting that they are outer membrane-spanning proteins. Identification of palmitate-labeled lipoproteins revealed that several were enriched in the OMV, several were enriched in the protoplasmic cylinder inner membrane fraction, and others were found exclusively associated with the inner membrane. The protein composition of OMV changed significantly with successive in vitro cultivation of strain B31. Using antiserum with specificity for virulent strain B31, we identified OMV antigens on the surface of the spirochete and identified proteins whose presence in OMV could be correlated with virulence and protective immunity in the rabbit Lyme disease model. These virulent strain associated outer membrane-spanning proteins may provide new insight into the pathogenesis of Lyme disease.

The rare outer membrane protein, OmpL1, of pathogenic Leptospira species is a heat-modifiable porin.

The outer membranes of invasive spirochetes contain unusually small amounts of transmembrane proteins. Pathogenic Leptospira species produce a rare 31-kDa surface protein, OmpL1, which has a deduced amino acid sequence predictive of multiple transmembrane beta-strands. Studies were conducted to characterize the structure and function of this protein. Alkali, high-salt, and urea fractionation of leptospiral membranes demonstrated that OmpL1 is an integral membrane protein. The electrophoretic mobility of monomeric OmpL1 was modifiable by heat and reduction; complete denaturation of OmpL1 required prolonged boiling in sodium dodecyl sulfate (SDS), 8 M urea, and 2-mercaptoethanol. When solubilized in SDS at low temperature, a small proportion of OmpL1 exhibited an apparent molecular mass of approximately 90 kDa, indicating the existence of an SDS-unstable oligomer. OmpL1 dimers and trimers were demonstrated by nearest neighbor chemical cross-linking. In order to generate purified protein for functional studies, the ompL1 gene was ligated into the pMMB66 expression plasmid under control of the tac promoter. Although expression in Escherichia coli was toxic, most of the OmpL1 produced was found in the outer membrane, as determined by subcellular fractionation. Purified recombinant OmpL1 was reconstituted into planar lipid bilayers, demonstrating an average single channel conductance of 1.1 nS, similar to the major porin activity of native leptospiral membranes. These findings indicate that OmpL1 spans the leptospiral outer membrane and functions as a porin.

Molecular cloning and sequence analysis of the gene encoding OmpL1, a transmembrane outer membrane protein of pathogenic Leptospira spp.

Pathogenic Leptospira spp. are spirochetes that have a low transmembrane outer membrane protein content relative to that of enteric gram-negative bacteria. In a previous study we identified a 31-kDa surface protein that was present in strains of Leptospira alstoni in amounts which correlated with the outer membrane particle density observed by freeze fracture electron microscopy (D. A. Haake, E. M. Walker, D. R. Blanco, C. A. Bolin, J. N. Miller, and M. A. Lovett, Infect. Immun. 59:1131-1140, 1991). The N-terminal amino acid sequence was used to design a pair of oligonucleotides which were utilized to screen a lambda ZAP II library containing EcoRI fragments of L. alstoni DNA. A 2.5-kb DNA fragment which contained the entire structural ompL1 gene was identified. The structural gene deduced from the sequence of this DNA fragment would encode a 320-amino-acid polypeptide with a 24-amino-acid leader peptide and a leader peptidase I cleavage site. Processing of OmpL1 results in a mature protein with a predicted molecular mass of 31,113 Da. Secondary-structure prediction identified repeated stretches of amphipathic beta-sheets typical of outer membrane protein membrane-spanning sequences. A topological model of OmpL1 containing 10 transmembrane segments is suggested. A recombinant OmpL1 fusion protein was expressed in Escherichia coli in order to immunize rabbits with the purified protein. Upon Triton X-114 extraction of L. alstoni and phase separation, anti-OmpL1 antiserum recognized a single band on immunoblots of the hydrophobic detergent fraction which was not present in the hydrophilic aqueous fraction. Immunoelectron microscopy with anti-OmpL1 antiserum demonstrates binding to the surface of intact L. alstoni. DNA hybridization studies indicate that the ompL1 gene is present in a single copy in all pathogenic Leptospira species that have been tested and is absent in nonpathogenic Leptospira species. OmpL1 may be the first spirochetal transmembrane outer membrane protein for which the structural gene has been cloned and sequenced.

Evaluation of the Hawaiian reef fishes with the solid phase immunobead assay.

This study presents data on the evaluation of a laboratory ciguatera kit based on the solid phase immunobead assay (SPIA) for the detection of ciguatoxin in Hawaiian reef fish. The SPIA was performed on fish catches by volunteer fishermen throughout the State of Hawaii. A total of 1,067 fish of various species were tested for ciguatoxin (CTX) using the SPIA kit. Of the total 1,067 fish tested, 510 were from Oahu, 402 from Hawaii, and 75 from Maui. The number of fish from Molokai, Kauai, and Lanai were 23, 20, and 7 respectively. Twenty percent of the total fish tested were positive, 41% borderline, and 39% negative for ciguatoxin. The highest percentage of SPIA- positive fish were from Hawaii (27%) followed by Oahu (19%) and Kauai (15%). These results correlate with the reported incidents from the Department of Health (DOH) of actual ciguatera poisoning in the State of Hawaii. Fish in all three categories of the SPIA test values were eaten. No false negatives were noted with individuals eating SPIA negative fish. Of the 232 SPIA borderline values eaten, 3 species of fish caused ciguatera poisoning. These fish included 2 papio, 1 mullet, and 1 po'ou. Of the 17 SPIA positive fish eaten, 5 caused ciguatera poisoning: 2 papio, a kole, an uhu, and a weke. The SPIA ciguatera test did protect the public when only SPIA-negative fish were eaten. The borderline and positive SPIA fish were generally unsafe, especially the positive fish. The data indicated that the probability of getting ciguatera with a SPIA positive fish was 1 out of 3.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of the stick enzyme immunoassay in Caranx sp. and Seriola dumerili associated with ciguatera.

The stick enzyme immunoassay (S-EIA) using monoclonal antibody to ciguatoxin (MAb-CTX) was used to examine clinically implicated fish and to pre-screen two species of fish, Caranx sp. (ulua or jack) and Seriola dumerili (kahala or amberjack), supplied by sports fishermen. All of the clinically implicated fish from the Department of Health gave S-EIA values greater than or equal to 1.3. The Caranx sp. and Seriola dumerili considered safe (less than or equal to 1.2 value) and consumed after testing gave no false-negative results. The S-EIA procedure using MAb-CTX proved to be specific, sensitive, and simple to use in the laboratory. It also proved to be useful in screening two large carnivorous fish for ciguatoxin and related polyethers prior to consumption.