Species-specific mAbs Research Articles

Anti‐nerve growth factor monoclonal antibodies for the control of pain in dogs and cats

Nerve growth factor (NGF) is essential for the survival of sensory and sympathetic neurons during development. However, in the adult, NGF and its interaction with tropomyosin receptor kinase A receptor (TrkA) has been found to play a critical role in nociception and nervous system plasticity in pain conditions. Thus, various monoclonal antibody (mAb) therapies targeting this pathway have been investigated in the development of new pharmacotherapies for chronic pain. Although none of the mAbs against NGF are yet approved for use in humans, they look very promising for the effective control of pain. Recently, species-specific anti-NGF mAbs for the management of osteoarthritis (OA)-associated pain in dogs and cats has been developed, and early clinical trials have been conducted. Anti-NGF therapy looks to be both very effective and very promising as a novel therapy against chronic pain in dogs and cats. This review outlines the mechanism of action of NGF, the role of NGF in osteoarthritis, research in rodent OA models and the current status of the development of anti-NGF mAbs in humans. Furthermore, we describe and discuss the recent development of species-specific anti-NGF mAbs for the treatment of OA-associated pain in veterinary medicine.

Single human B cell-derived monoclonal anti-Candida antibodies enhance phagocytosis and protect against disseminated candidiasis

The high global burden of over one million annual lethal fungal infections reflects a lack of protective vaccines, late diagnosis and inadequate chemotherapy. Here, we have generated a unique set of fully human anti-Candida monoclonal antibodies (mAbs) with diagnostic and therapeutic potential by expressing recombinant antibodies from genes cloned from the B cells of patients suffering from candidiasis. Single class switched memory B cells isolated from donors serum-positive for anti-Candida IgG were differentiated in vitro and screened against recombinant Candida albicans Hyr1 cell wall protein and whole fungal cell wall preparations. Antibody genes from Candida-reactive B cell cultures were cloned and expressed in Expi293F human embryonic kidney cells to generate a panel of human recombinant anti-Candida mAbs that demonstrate morphology-specific, high avidity binding to the cell wall. The species-specific and pan-Candida mAbs generated through this technology display favourable properties for diagnostics, strong opsono-phagocytic activity of macrophages in vitro, and protection in a murine model of disseminated candidiasis.

Concentration levels of TSH evaluation with species-specific MABs due to the dog reproduction disorders assessment in the course of hypothyroidism

Concentration levels of TSH evaluation with species-specific MABs due to the dog reproduction disorders assessment in the course of hypothyroidism

Mouse Monoclonal Antibodies Against Human Urinary Protein Sm-UP2IP ofSchistosoma mansoni

There are innumerable clinical and pathological problems associated with schistosomiasis that have necessitated various control programs. Successful control would naturally depend on effective rapid diagnosis in the field. However, the overlapping distribution of urinary and intestinal schistosomiasis in hyperendemic areas calls for differential diagnosis. This study was aimed at producing anti-Schistosoma mansoni monoclonal antibodies (MAbs) for possible utilization in assays to detect antigens in the urine of infected persons. In order to raise antibodies to less immunogenic urinary parasite antigens, BALB/c mice were immunized with Schistosoma mansoni soluble worm antigens (Sm-SWA) while urinary proteins (Sm-UP(2)IP), isolated from infected human urine samples, was used as a final booster before cell fusion. Hybridoma cells were obtained by the fusion of mouse myeloma and spleen cells from the immunized mice, which were screened by microplate ELISA and then studied further to obtain anti-S. mansoni specific MAbs. The MAbs analyzed presented IgM isotypes. The reactivity of anti-S. mansoni MAbs with Sm-UP(2)IP, 13/43 (30.2%), MAbs showed stronger reactivity. It was observed that one of the MAbs cross-reacted with antigen associated with S. haematobium urinary antigen (Sh-UP(2)IP). Nine (9/13, 69.2%) MAbs recognized glycoprotein antigenic epitopes of Sm-UP(2)IP and Sm-SWA. On the other hand, 4/13 (30.8%) MAbs recognized carbohydrate antigenic epitopes. Band size of 8.9 kDa associated with Sm-UP(2)IP was detected by the 13 MAbs. With Sm-SWA, all the MAbs detected band sizes of 177.8 and 158.5 kDa. In addition, three MAbs recognized a 38.9 kDa band. The generation of anti-S. mansoni species-specific MAbs offers opportunities to develop a specific MAb-based diagnostic tool for use in the field to detect Schistosoma mansoni infection in Ghana.

The application of immune complex immunization for the production of monoclonal antibodies against spores of Penicillium brevicompactum

Rationale Most monoclonal antibodies (mAbs) to fungi are often found to cross-react with related and non-related fungi. In this study we attempted to shift the immune response towards species-specific antibodies by immunizing mice with preformed immune complexes rather than spores alone. Methods Balb/c mice were repeatedly immunized with immune complexes composed of the particulate fraction of homogenized spores of P. brevicompactum and mAbs which were previously produced against spores of Penicillium chrysogenum and shown to be highly cross-reactive with P. brevicompactum. The specificities of the resulting mAbs were determined by ELISA against spores of 51 fungal species commonly found in indoor environments. Results None of the 12 mAbs produced was found to be speciesspecific for P. brevicompactum and based on their cross-reactivity profiles the 12 mAbs could be allocated to one of 5 different groups. Although immune complex immunization when compared to conventional immunizations with spores alone resulted in a decrease of mAb reactivity to related species of Penicillium and Aspergillus, the reactivity of mAbs to unrelated species such as Cladosporium, Memnoniella, Stachybotrys or Ulocladium spp. was unaffected and very common. Conclusions Immune complex immunization did not result in species-specific mAbs against P. brevicompactum. The cross-reactivity patterns demonstrate that phylogenetically distant fungi share common epitopes and that these epitopes are expressed at different amounts on a per spore basis by different fungi.

Heat-stable serogroup-specific proteins of Yersinia pseudotuberculosis.

A library of mAbs to the species- and serogroup-specific epitopes of Yersinia pseudotuberculosis serogroups I-VI was developed. These mAbs recognized linear sequential protein epitopes, as shown by ELISA and immunoblotting. Using the mAbs, Y. pseudotuberculosis was found to produce serogroup-specific proteins, whose synthesis was dependent on cultivation temperature. These proteins appeared to be parts of heat-stable O-antigens prepared by heating Y. pseudotuberculosis serogroups I-VI at 100 degrees C for 2 h, and are responsible for the protein serotype specificity of these bacteria. The high specificity of serogroup- or species-specific mAbs obtained in ELISA suggests that they may be effective for serotyping of Y. pseudotuberculosis strains or differentiation from other pathogenic yersiniae.

Induction of unresponsiveness to islet xenograft by MMC treatment of graft and blockage of LFA-1/ICAM-1 pathway.

Induction of unresponsiveness to graft is one of major interest in xenotransplantation. Two different modalities [direct graft treatment by mitomycin C (MMC) and blockage of the lymphocyte function-associated antigen-1/intracellular adhesion molecule-1 (LFA-1/ICAM-1) pathway in recipients by species-specific mAbs] were tested for their ability to produce unresponsiveness to secondary islet xenografts. Collagenase-digested WS (RT1k) rat islets, purified by Ficoll density gradient, were incubated for 30 min with MMC 10 microg/ml, cultured for 20 hr, and transplanted into the renal subcapsular space of streptozotocin-induced diabetic C57BL/6 (H-2b) mice. Recipient mice were divided into experimental groups according to anti-rat ICAM-1 and/or anti-mouse LFA-1 mAb treatment and transplantation of MMC-treated or nontreated islets. MMC pretreatment alone prolonged graft survival, with a mean survival time (MST) of 23.0+/-7.4 days, compared with that of cultured islets (12.4+/-2.7 days; P<0.01). MMC treatment of islets significantly augmented graft survival, compared with that of crude islet grafts under treatment with anti-donor ICAM-1 mAb (MST: >41.3+/-30 vs. 16.6+/-5.4 days, P<0.01), anti-recipient LFA-mAb (MST: >70.3+/-28.9 vs. 30.4+/-10.4 days, P<0.001), or both mAbs (MST: >88.1+/-24.1 vs. 23+/-7.4 days, P<0.0001). One of six, four of nine, and six of eight animals accepted MMC-treated islet xenografts over 100 days after treatment with anti-rat ICAM-1, anti-mouse LFA-1, or both mAbs treatments, respectively, whereas none of the animals accepted nontreated islets under the same treatment. When the mice bearing long-term functioning xenografts were challenged with the secondary graft from the original donor strain, the animals previously treated with anti-recipient LFA-1 and anti-donor ICAM-1 mAbs were more prone to accept it than animals given anti-recipient LFA-1 mAb alone (MST: 55.8+/-25.7 vs. 15+/-2.4 respectively; P<0.001), although they rejected the third-party xenograft and allograft acutely. In the xenogeneic islet transplantation model, MMC graft pretreatment and blockage of the ICAM-1/LFA-1 pathway constitute a potent protocol for inducing unresponsiveness to islet xenografts.

Detection of microsporidia spore-specific antigens by monoclonal antibodies.

Microsporidia (phylum Microspora) are unicellular parasites commonly found in invertebrates, fish, and laboratory animals; however, microsporidiosis is an emerging problem in patients with the acquired immunodeficiency syndrome (AIDS). The infective stage of these parasites is the spore, which possesses a rigid cell wall that protects the parasite outside its host. Little is known about their antigenic composition. Sensitive, reliable, and easily performed methods for identification and speciation are generally not available. Here, we report the production of 21 MAbs specific to spore antigens of several species of Microsporidia. MAbs were generated to purified spores of Encephalitozoon intestinalis and Encephalitozoon hellem, and their reactivities were tested against spores and intracellular developing forms of E. intestinalis, E. hellem, Encephalitozoon cuniculi, and Vittaforma corneae. Both species-specific and broad-reactivity MAbs were produced. Five MAbs reacted against the spores of all four species tested: 7 with 3 species, 6 with 2 species, 1 with E. intestinalis, and 4 with the polar tube of all species. Immunoelectron microscopy confirmed the reactivity of specific MAbs to the spore wall or the polar tube. These MAbs reacted to a few antigens as determined by Western blot, and none of the epitopes were periodate-sensitive. These MAbs may be useful in the diagnosis and speciation of Microsporidia as well in the purification, cloning, and detection of these antigens.

Sub-specific differentiation of intestinal spirochaete isolates by macrorestriction fragment profiling.

Macrorestriction fragment profile analysis by PFGE was used to distinguish intestinal spirochaetes, some of which were isolated from cases of swine dysentery and intestinal spirochaetosis in humans, pigs, mice, chickens and dogs. Macrorestriction fragment profiles using SmaI and SacII restriction enzymes were produced and used in statistical analysis. This permitted the division of the isolates into two major clusters. One cluster contained isolates which were identified as Serpulina pilosicoli and the second cluster contained isolates identified as Serpulina hyodysenteriae by immunoblotting with species-specific mAbs. Both species contained sub-specific groups, although these rarely correlated with the source of the isolates. We conclude that PFGE is capable of sub-specific differentiation of intestinal spirochaetes, but that the current species contain a large variety of genotypes among which cross-species transmission may be feasible.

Apodemus sp.Rodents, Reservoir Hosts for Borrelia afzelii in an Endemic Area in Switzerland

Borrelia burgdorferi is maintained in nature in transmission cycles alternatively involving ticks and reservoir hosts. Small rodents like Apodemus mice and Clethrionomys voles are the primary reservoir of Lyme disease in Europe. In this study, we analyzed by SDS-PAGE and Western blot 20 borrelial isolates from xenodiagnostic ticks fed on four Apodemus sp. mice captured in the Staatswald forest (Switzerland). All isolates but one showed a homogeneous protein pattern expressing an outer surface protein, (Osp) A of 32 kDa and an OspB of 35 kDa and reacted with monoclonal antibody (mAb) I 17.3 specific for B. afzelii. One isolate expressed an OspA of 32.5 kDa and an OspB of 35 kDa and did not react with species-specific mAbs I 17.3, D6 and H3TS, but was shown to belong to B. afzelii by Southern blot analysis. The possibility exists that non-cultivatable borreliae are present in xenodiagnostic ticks. However, our results clearly show that Apodemus sp. are reservoir hosts for B. afzelii, since this genospecies is transmitted from Apodemus sp. to feeding larval ticks.

Differentiation between culture-derived insect stages of T. brucei, T. vivax, T. congolense and T. simiae using a monoclonal antibody-based dot-ELISA.

A sensitive and specific nitrocellulose (NC) membrane-based dot-ELISA, utilizing a panel of monoclonal antibodies (mAbs), was developed for differentiation between in vitro-derived procyclic forms of Trypanosoma brucei, T. congolense and T. simiae, and epimastigotes of T. vivax. Trypanosomes in suspension were applied onto NC membrane in dots and probed with unlabelled trypanosome species-specific mAbs. Bound mAb was revealed by enzyme labelled anti-mouse IgG and precipitable chromogenic substrate. The assay detected the aforementioned trypanosome species in both single and artificially mixed preparations. Ten T. brucei, 4 T. vivax, 7 T. congolense and 3 T. simiae procyclic stocks and clones from different geographical areas were tested and identified using the specific mAbs in the dot-ELISA which had a specificity of 100%. Some of the T. brucei, T. congolense and Nannomonas-specific mAbs could detect as few as 10 trypanosomes/dot, whilst 1 T. vivax mAb was able to detect a minimum of 100 trypanosomes/dot in monospecies preparations. A concentration of 1 x 10(4) trypanosomes/microliters/dot was eventually determined as ideal for testing in the dot-ELISA. Antigen dots stored at 4 degrees C under desiccated conditions did not show any loss in activity for up to 90 days. However, when stored under similar conditions at room temperature (17-26 degrees C), the T. congolense-specific antigen remained unaffected up to 60 days, and then showed decreased activity when tested on day 90.

Interspecies molecular chimeras of kit help define the binding site of the stem cell factor.

The extracellular portion of the kit-encoded receptor for the stem cell factor (SCF) comprises five immunoglobulin (Ig)-like domains. To localize the ligand recognition site, we exploited the lack of binding of human SCF to the murine receptor by using human-mouse hybrids of Kit and species-specific monoclonal antibodies (MAbs) that inhibit ligand binding. Replacement of the three N-terminal Ig-like domains of the murine Kit with the corresponding portion of the human receptor conferred upon the chimeric receptor high-affinity binding of the human ligand as well as of human-specific ligand-inhibitory MAbs. By constructing five chimeric murine Kit proteins which individually contain each of these three human Ig-like units or pairs of them, we found that the second human domain confers upon the mouse Kit high-affinity binding of the human ligand and also binding of species-specific SCF-competitive MAbs. Nevertheless, the flanking Ig-like domains also affect high-affinity recognition of SCF. Moreover, it appears that the determinants that define ligand specificity of the murine and the human receptors do not structurally coincide. This observation allowed us to identify a chimeric receptor that displayed a dual specificity; namely, it bound with high affinity either the human or the murine SCF molecules and reacted with mouse- as well as human-specific ligand-inhibitory MAbs. Conversely, another chimera, which included all of the five Ig-like domains, bound neither ligand. In conclusion, interdomain packing involving the second Ig-like domain of human Kit and noncontiguous structural motifs of the receptor are involved in SCF recognition.

Interspecies Molecular Chimeras of Kit Help Define the Binding Site of the Stem Cell Factor

The extracellular portion of the kit-encoded receptor for the stem cell factor (SCF) comprises five immunoglobulin (Ig)-like domains. To localize the ligand recognition site, we exploited the lack of binding of human SCF to the murine receptor by using human-mouse hybrids of Kit and species-specific monoclonal antibodies (MAbs) that inhibit ligand binding. Replacement of the three N-terminal Ig-like domains of the murine Kit with the corresponding portion of the human receptor conferred upon the chimeric receptor high-affinity binding of the human ligand as well as of human-specific ligand-inhibitory MAbs. By constructing five chimeric murine Kit proteins which individually contain each of these three human Ig-like units or pairs of them, we found that the second human domain confers upon the mouse Kit high-affinity binding of the human ligand and also binding of species-specific SCF-competitive MAbs. Nevertheless, the flanking Ig-like domains also affect high-affinity recognition of SCF. Moreover, it appears that the determinants that define ligand specificity of the murine and the human receptors do not structurally coincide. This observation allowed us to identify a chimeric receptor that displayed a dual specificity; namely, it bound with high affinity either the human or the murine SCF molecules and reacted with mouse- as well as human-specific ligand-inhibitory MAbs. Conversely, another chimera, which included all of the five Ig-like domains, bound neither ligand. In conclusion, interdomain packing involving the second Ig-like domain of human Kit and noncontiguous structural motifs of the receptor are involved in SCF recognition.

Serodiagnosis of cysticercosis by ELISA-inhibition test using monoclonal antibodies.

Monoclonal antibodies (Mabs) were produced against crude scolex extract of T. solium metacestodes, and applied to ELISA-inhibition test for improving the specificity of serodiagnosis of human cysticercosis. Four hybridomas secreting species-specific anticysticercal Mabs (Cya-1, Cya-7, Cya-28 and Cya-31) were selected. Each Mab reacted on antigenic components of 25.5 kDa (Cya-1), 28 kDa (Cya-7), 87.5 kDa (Cya-28), and 12.5 kDa (Cya-31). IFA showed that Cya-1 was located at the calcium corpuscles, and Cya-7 at the loose connective tissue of T. solium metacestode scolex. Cya-28 and Cya-31 reacted on the tegument of the scolex. By conventional ELISA, 23 out of 28 (82.1%) cysticercosis patients were found serologically positive, but 1 out of 9 (11.1%) sparganosis cases and 6 out of 31 (19.4%) paragonimiasis cases showed false positives. By ELISA-inhibition test using species-specific anti-cysticercal Mab Cya-7, 19 out of 28 (67.9%) cysticercosis cases were found serologically positive, but there were no false positives in other parasitic infections.

Development of monoclonal antibodies that identify Vibrio species commonly isolated from infections of humans, fish, and shellfish

Monoclonal antibodies (MAbs) against Vibrio species that infect humans, fish, and shellfish were developed for application in rapid identifications. The pathogens included Vibrio alginolyticus, V. anguillarum, V. carchariae, V. cholerae, V. damsela, V. furnissii, V. harveyi, V. ordalii, V. parahaemolyticus, and V. vulnificus. Three types of MAbs were selected. The first important group included MAbs that reacted with only a single species. A second group comprised a number of MAbs that reacted with two, taxonomically closely related Vibrio species. For example, of 22 MAbs raised against V. alginolyticus, 6 recognized a 52-kDa flagellar H antigen common to both V. alginolyticus and V. parahaemolyticus; V. anguillarum and V. ordalii also shared antigens. A third group included three genus-specific MAbs that reacted with almost all Vibrio species but did not react with other members of the family Vibrionaceae (e.g., members of the Aeromonas, Photobacterium, and Plesiomonas genera) or a wide range of gram-negative bacteria representing many genera. This last group indicated the possible existence of an antigenic determinant common to Vibrio species. Two of these three genus-specific MAbs reacted with heat-stable antigenic determinants of Vibrio species as well as lipopolysaccharide extracted from Vibrio species. The use of the MAbs in blind tests and diagnosis of clinical isolates indicated that three different types of bacteria, viz., live, formalin-fixed, and sodium azide-killed bacteria, were detected consistently. Overall, it was found that the genus-specific MAbs were very useful for rapidly identifying vibrios in the screening of acute infections, while the species-specific MAbs and others were useful for completing the diagnosis.

Monoclonal antibodies specific to sailfish serum albumin: development of an assay for the identification of fish species in the field.

Balb/c mice were immunized with albumin purified from sailfish (Istiophorus albicans) serum. Hybridomas were produced and screened by ELISA for reactivity with the purified albumins of sailfish, blue marlin (Makaira nigricans) and white marlin (Tetrapturus albidus). Monoclonal antibodies (MAbs) from 16 different clones exhibited activity against sailfish albumin. Thirteen of the MAbs showed cross-reactivity with the marlin species. Three MAbs exhibited distinct specificity for sailfish albumin. One of these species specific MAbs (M2D1) was conjugated to horseradish peroxidase (HRP) in order to construct an ELISA for identification of sailfish from serum. The ELISA for sailfish correctly identified eight sailfish from 26 billfish serum samples. The MAb-peroxidase conjugate was highly specific toward sailfish in that no reaction against heterologous species was detected.

Monoclonal Antibodies That React with Leishmania (Viannia) naiffi

Monoclonal antibodies were raised against Leishmania (Viannia) naiffi, which recently has been characterized as a new species. BALB/c mice were immunized with membrane-enriched fractions of a mixture of L. (V.) naiffi isolates. Subsequent fusion of immunized splenocytes with NS-1 myeloma cells resulted in the production of 5 Mabs (N1-N5). Screening by ELISA and indirect immunofluorescence against an extensive cross-panel of Leishmania strains revealed that N3 was species-specific and could thus be used to identify L. (V.) naiffi. N2 and N5 reacted only with strains of L. (V.) naiffi and Leishmania (Viannia) braziliensis, and therefore could be used in conjunction with N3 to identify L. (V.) braziliensis parasites. These species-specific Mabs will therefore be useful additions to the panel of antibodies already available for the identification of Leishmania species. N1 and N4 were found to recognize a small, glycosylated molecule present on all L. (V.) naiffi and L. (V.) braziliensis isolates that is related to the GIPL family of membrane glycophospholipids previously described for Leishmania (Leishmania) major.

Preparation of murine monoclonal antibodies against the yeast phase of the dimorphic fungus Sporothrix schenckii

Three murine monoclonal antibodies (Mabs) were raised against a cytoplasmic antigen of the yeast phase of the pathogenic fungus Sporothrix schenckii using a modification of standard hybridoma technology incorporating the immunosuppressive drug cyclophosphamide. When tested for species-specificity within the pathogenic dimorphic fungi one of these Mabs (S5) showed little cross-reactivity by enzyme-linked immunosorbent assay and Western blot, though there was some recognition of Paracoccidioides brasiliensis antigen. This Mab recognized a 70–75 kDa molecule on reduced Western blots of S. schenckii antigen. The other two Mabs (S12 and S15) showed cross-reactivity with all dimorphic fungal antigens tested, though they appeared to recognize a molecule of similar molecular weight. This is the first report of any attempt to raise species-specific Mabs against this important causative agent of dermatological disease.

A two-site monoclonal antibody ELISA for the quantification of the major Dermatophagoides spp. allergens, Der p I and Der f I

A two-site monoclonal antibody (Mab) ELISA was developed to measure the Group I allergens from Dermatophagoides spp., Der p I from D. pteronyssinus and Der f I from D. farinae. Species-specific Mabs were used to coat microtiter plates which were then incubated with allergen or house dust extracts. Bound allergen was detected using a biotinylated Mab which recognized a common epitope on both Der p I and Der f I, followed by the addition of streptavidin-peroxidase and ABTS/H 2O 2 substrate. The assay had low non-specific binding (∼ 0.08 absorbance units) and had a sensitivity of 5 ng/nl for aqueous allergen extracts (equivalent to 0.1 μg allergen/g dust). 53 dust samples were assayed using the Mab ELISA and an RIA previously described using 125I-labelled Mab. The results showed a very good quantitative correlation between the assays ( r = 0.96, p < 0.001 for Der p I; r = 0.92, P < 0.001 for Der f I). A further 132 dust samples from a different geographical areas were also assayed by both methods and gave correlation coefficients of 0.90 ( P < 0.001) and ( P < 0.001) for Der p I and Der f I, respectively. The Mab ELISA will be useful in epidemiological studies of allergic asthma, both in the assessment of levels of dust mite allergen present in houses and the efficacy of allergen avoidance regimes.

Coagglutination of Vibrio cholerae, Vibrio mimicus, and Vibrio vulnificus with anti-flagellar monoclonal antibody.

Monoclonal antibodies (MAbs) with serological activity for purified flagellar (H) core protein prepared from Vibrio cholerae were identified by enzyme-linked immunosorbent assay. Four of these MAbs reacted with the flagella of V. cholerae and V. mimicus exclusively, while eight MAbs reacted with at least 1 of 30 heterologous Vibrio species tested by enzyme-linked immunosorbent assay or coagglutination. It appears that V. cholerae and V. mimicus express similar, if not identical, H determinants unique to these two Vibrio species. Staphylococcus aureus cells or latex beads armed with the four species-specific MAbs coagglutinated each of 47 isolates identified bacteriologically as V. cholerae or V. mimicus from among 103 Vibrio isolates tested. One coagglutination reagent armed with anti-V. vulnificus H MAb exhibited species specificity in that only V. vulnificus cells were coagglutinated from among the 31 Vibrio species examined. This reagent coagglutinated 20 isolates identified bacteriologically as V. vulnificus in a serological survey. MAb coagglutination reagents offer a rapid, specific, and economical alternative to the classical bacteriological approach to identify the human pathogens V. cholerae, V. mimicus, and V. vulnificus.