Genetic diversity of clinical and environmental isolates of Vibrio cholerae determined by amplified fragment length polymorphism fingerprinting

Vibrio cholerae, the causative agent of major epidemics of diarrheal disease in Bangladesh, South America, Southeastern Asia, and Africa, was isolated from clinical samples and from aquatic environments during and between epidemics over the past 20 years. To determine the evolutionary relationships and molecular diversity of these strains, in order to understand sources, origin, and epidemiology, a novel DNA fingerprinting technique, amplified fragment length polymorphism (AFLP), was employed. Two sets of restriction enzyme-primer combinations were tested for fingerprinting of V. cholerae serogroup O1, O139, and non-O1, O139 isolates. Amplification of HindIII- and TaqI-digested genomic DNA produced 30 to 50 bands for each strain. However, this combination, although capable of separating environmental isolates of O1 and non-O1 strains, was unable to distinguish between O1 and O139 clinical strains. This result confirmed that clinical O1 and O139 strains are genetically closely related. On the other hand, AFLP analyses of restriction enzyme ApaI- and TaqI-digested genomic DNA yielded 20 to 30 bands for each strain, but were able to separate O1 from O139 strains. Of the 74 strains examined with the latter combination, 26 serogroup O1 strains showed identical banding patterns and were represented by the O1 El Tor strain of the seventh pandemic. A second group, represented by O139 Bengal, included 12 strains of O139 clinical isolates, with 7 from Thailand, 3 from Bangladesh, and 2 from India. Interestingly, an O1 clinical isolate from Africa also grouped with the O139 clinical isolates. Eight clinical O1 isolates from Mexico grouped separately from the O1 El Tor of the seventh pandemic, suggesting an independent origin of these isolates. Identical fingerprints were observed between an O1 environmental isolate from a river in Chile and an O1 clinical strain from Kenya, both isolated more than 10 years apart. Both strains were distinct from the O1 seventh pandemic strain. Two O139 clinical isolates from Africa clustered with environmental non-O1 isolates, independent of other O139 strains included in the study. These results suggest that although a single clone of pathogenic V. cholerae appears responsible for many cases of cholera in Asia, Africa, and Latin America during the seventh pandemic, other cases of clinical cholera were caused by toxigenic V. cholerae strains that appear to have been derived locally from environmental O1 or non-O1 strains.     

Spread of a single clone Acinetobacter baumannii strain in an intensive care unit of a teaching hospital in Turkey

Acinetobacter baumannii is an important nosocomial pathogen, especially in immunocomprimised patients and those hospitalized in intensive care units. After the first isolation of A. baumannii strains from the bronchial aspirates of two patients in the intensive care unit (ICU) of our hospital as a pure culture, screening studies were performed to define possible source(s). A. baumannii strains isolated from bronchial aspirates and blood cultures of the patients in ICU were collected as a possible part of the outbreak. A total of 23 screening samples collected from equipment (7), hands (4) and gloves (2) of the staff, and from ten different body regions of the patients in the ICU were cultured. Antimicrobial susceptibility test of the isolates was performed by the standardized disk-diffusion method. All isolates were subtyped by antibiogram, arbitrarily primed polymerase chain reaction (AP-PCR) and pulsed-field gel electrophoresis (PFGE) typing methods. A total of 26 A. baumannii strains including eight clinical and 18 screening isolates were identified. All isolates were susceptible only to meropenem, tobramycin, and imipenem. There was at least a 96% resistance rate to the other antibiotics tested. Antibiogram typing showed that 24 of the 26 isolates were epidemiologically related, two were unique. AP-PCR yielded two types, one of which had 21 isolates, the other had five. PFGE fingerprinting revealed that all isolates were clonally related, including four closely related and 22 indistinguishable strains. Based on the results of PFGE which has been accepted as a reference method it can be concluded that A. baumannii strains isolated from our intensive care unit originated from a single type of strain.     

Isolation of apparently wild strains of poliovirus type 1 from sewage in the Ottawa area.

In the first 4 months of 1974, 140 gauze pad samples of sewage collected in the Ottawa area were analysed by the BS-C-1 cell system for the presence of viruses pathogenic for humans. Viruses were isolated from 111 (79%) of the samples. Of the 72 (65%) isolates identified by serology and electron microscopic examination, 56 (78%) were reoviruses and 16 (22%), enteroviruses. The enterovirus isolates included one coxsackievirus B4, one vaccine strain of poliovirus type 3, nine vaccine strains of poliovirus type 1 and five strains of poliovirus type 1 that proved by serodifferentiation and temperature marker tests to be different from vaccine strains. The fact that these strains were present in the community sewage in readily detectable concentrations at a time when immunity against polioviruses is declining in such communities is a cause for concern.     

广西不同时期IBV分离株S1基因高变区Ⅰ的遗传变异分析

对广西1985-2007年间分离到的22株传染性支气管炎病毒（IBV）的S1基因高变区I（HVRI）进行序列测定,并与发表的其他IBV参考株及鸽子分离的冠状病毒株的基因序列进行比较和分析。系统进化关系显示毒株可分为5个基因群,其中有16个广西分离株属第1群,它们与鸽子冠状病毒分离株的氨基酸序列同源性较高,与Massachusetts（Mass）型疫苗株的同源性较低。有15个分离株在33-34位和34～35之间分别有4个和3个氨基酸残基的插入,GX-NN6在33～34位和34～35位之间则均有4个氨基酸残基的插入;GX-YL1、GX-NN2与常用的Mass型疫苗株的亲缘关系最近,同属于第Ⅱ群;GX-G、GX-XD与日本同一时期分离的毒株JP Miyazaki 89亲缘关系最近,属于第Ⅲ群;GX-YL6、GX-NN7与欧洲毒株4／91亲缘关系较近,属于第V群。结果表明广西存在着多种类型IBV毒株的流行,毒株S1基因HVRI碱基的突变或插入比较普遍,可导致其氨基酸序列的变化,绝大部分毒株与目前常用的Mass型疫苗株的亲缘关系较低。同一时期的分离株同源性较高,但无明显的地域性差异。     

Genetic diversity and antimicrobial susceptibility of motile aeromonads isolated from rainbow trout (Oncorhynchus mykiss, Walbaum) farms

A total of 22 motile Aeromonas strains were detected in 48 (18.53%) of 259 fish and 6 (10.71%) of 56 water samples obtained from seven commercial rainbow trout ( Oncorhynchus mykiss , Walbaum) farms in the province of Mersin, Turkey. These strains were identified by conventional microbiological techniques and by using an ID32GN system. Of these isolates 20 (91.3%) were identified as Aeromonas hydrophila and 2 (8.7%) as Aeromonas sobria . While 8 of the A. hydrophila strains were isolated from water samples, 12 isolates were from fish samples. Whereas A. hydrophila strains were found in all farms, A. sobria was detected in only two farms. Genetic diversity by arbitrarily primed polymerase chain reaction (AP-PCR) and antimicrobial sensitivity tests were carried out on eight A. hydrophila isolates obtained from water samples, and isolates from seven A. hydrophila and one A. sobria from fish samples. The AP-PCR band patterns of motile aeromonads demonstrated weak similarity to the A. hydrophila reference strain ATCC 7966. Five A. hydrophila strains in the water samples displayed genetic similarity, but three others were different. Aeromonas hydrophila isolates from fish samples possessed slight similarities, and A. sobria was genetically distant to all A. hydrophila strains. An antimicrobial sensitivity test of 16 isolates revealed that 100% were sensitive to gentamicin, 87.5% to sulphamethoxazole–trimethoprim, 62.5% to enrofloxacin, 43.8% to oxytetracycline, 37.5% to neomycin, 18.75% to streptomycin and 6.25% to erythromycin. All isolates were resistant to novobiocin.     

Verotoxic Escherichia coli (STEC) from beef and its products

In the present investigation, out of 27 (24.10%) strains of Escherichia coli isolated from 112 beef samples comprising raw meat (45), kabab (36) and kofta (31), 9 (33.33%) belonging to 7 different serotypes were verotoxic as tested by vero cell cytotoxic assay. Serotype O145 was the predominant STEC in raw meat. Interestingly, one STEC-O157 strain was also detected. All the STEC strains were positive for Stx genes by polymerase chain reaction showing stx2 (77.78%) to be most predominant followed by stx1 (22.22%). Phenotypic enterohaemolysin production on washed sheep blood agar supplemented with CaCl2 revealed 6 (66.67%) STEC strains to be positive. Presence of STEC in cooked beef products, viz., kabab and kofta appeared to be a matter of concern and potential threat to public health.     

Occurrence of selected loci of the cps region for capsule K1 or K2 synthesis in epidemic strains of Klebsiella pneumoniae isolated from infants in Poland

Number of 60 epidemic strains of K. pneumoniae and 15 isolated occasionally from stool samples were tested for presence of 4 and 11 selected loci of the cps cluster of K1 and K2. Following open reading frames (ORFs): 1, 2, 3, 4, 7, 9, 10, 14, 15 (gnd) of cps K2 strain Chedid and genes magA, gmd, wzc, wca of cps K1 strain DTS were searched by PCR in tested and reference strains O1:K1 A5054 and O1:K2 B5055. The ORFs 1 to 3 and ORF15 were detected in both the reference and epidemic strains as well as in the greatest majority of the occasional isolates. Thus, such ORFs were found K. pneumoniae cps common domains, while tested ORFs 4 to 14 were observed in strain B5055 and 11 epidemic isolates from patients of the same hospital ward. Only exception was a single strain O3 occasionally isolated from faeces. The tested genes of cps K1 were detected only in strain A5054 and in two O3 occasional isolates from faeces. Interestingly, these genes as well ORFs 4 to 14 were detected together in the appropriate reference and tested strains with only two exceptions. Therefore, the cps sector occupied by ORFs 4 to 14 was found as group-specific domain. The occurrence ratio of cps K2 group-specific loci among epidemic strains from infants was 18%, while the K1 group-specific loci were absent.     

Intraspecific characterization of Vibrio alginolyticus isolates recovered from cultured fish in Spain

AIMS: Intraspecific differentiation and characterization of Vibrio alginolyticus strains isolated from cultured fish in Spain. MATERIALS AND RESULTS: Thirty-four Vibrio alginolyticus strains isolated from cultured fish were intraspecifically characterized on the basis of biochemical and exoenzymatic patterns, outer membrane protein (OMP) profiles, ribotyping and plasmid analyses. The typing methods used did not allow to group V. alginolyticus isolates on the basis of their sources of collection. A higher homogeneity was observed in OMP profiles. A high percentage of isolates were plasmidless. Ribotyping was the highest discriminatory typing method, as all the isolates tested presented 23 profiles using the HindIII restriction enzyme. On the basis of the ribotyping pattern, a similarity matrix and a dendrogram were constructed. CONCLUSIONS: The results obtained indicate that V. alginolyticus strains isolated from southwestern Spain belong to different clonal lineages. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has shown differences with other similar studies carried out in other areas of Europe with strains of V. alginolyticus with respect to the clonal lineages of the strains isolated in southwestern Spain.     

Biofilm acts as a microenvironment for plankton-associated Vibrio cholerae in the aquatic environment of Bangladesh

The role of biofilm as a microenvironment of plankton-associated Vibrio cholerae was investigated using plexiglass as a bait. A total of 72 biofilm samples were tested using culture, direct fluorescent antibody (DFA) and molecular techniques following standard procedures. Culturable V. cholerae (smooth and rugose variants) were isolated from 33% of the samples. V. cholerae O1 were detected by FA technique throughout the year except April and June. All V. cholerae O1 isolates were positive for tcpA, ctxA and ace genes while V. cholerae non-O1, non-O139 isolates lacked these genes. V. cholerae O1 (both Inaba and Ogawa) strains had identical ribotype pattern (R1), but V. cholerae non-O1, non-O139 had different ribotype patterns. All V. cholerae O1 strains were resistant to vibrio-static compound (O/129). All V. cholerae O1 except one were resistant to trimethoprime-sulphamethoxazole, streptomycin, nalidixic acid and furazolidone but sensitive to ciprofloxacin, and tetracycline. This study indicates that plexiglass can act as a bait to form biofilm, a microenvironment that provides shelter for plankton containing V. cholerae in the aquatic environment of Bangladesh.     

Characterization and distribution of Vibrio alginolyticus and Vibrio parahaemolyticus isolated in Indonesia

Previous studies have shown that Vibrio alginolyticus and Vibrio parahaemolyticus can be isolated from similar types of marine samples. In this report, the results of an examination of 567 V. alginolyticus and V. parahaemolyticus strains, isolated from seawater in Jakarta Bay and from more than 30 types of seafood from markets in Jakarta, Indonesia, are presented. Most isolates were from mackerel, shrimp, or squid. Numerical taxonomic analyses clustered 337 isolates and three V. alginolyticus reference strains at S greater than or equal to 80%. These strains produced acid from sucrose, but only approximately 80% produced acetoin or grew in the presence of 10% NaCl. The frequency of occurrence of V. alginolyticus in seawater samples ranged from 0% (in February and March 1972) to 100% (in September and December 1972) and was highest in seafood samples from August to December 1972. A second cluster of 230 isolates and seven V. parahaemolyticus reference strains was observed at S greater than or equal to 82%. These strains did not produce acetoin or acid from sucrose, and approximately 20% grew in the presence of 10% NaCl. V. parahaemolyticus was detected in seawater samples each month, with the highest frequency of occurrence (83.3%) in May 1972. Twenty-nine K antigen serotypes were demonstrated in V. parahaemolyticus isolates, and another 40% were untypable. The modal antibiotic resistance pattern for each species included five drugs. Only 12% of the V. parahaemolyticus strains were Kanagawa positive, and 10% elicited fluid accumulation in ligated rabbit ileal loops. All of the 7 V. alginolyticus strains and 94 (70%) of the V. parahaemolyticus strains tested killed mice when inoculated intraperitoneally.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization and distribution of Vibrio alginolyticus and Vibrio parahaemolyticus isolated in Indonesia.

Previous studies have shown that Vibrio alginolyticus and Vibrio parahaemolyticus can be isolated from similar types of marine samples. In this report, the results of an examination of 567 V. alginolyticus and V. parahaemolyticus strains, isolated from seawater in Jakarta Bay and from more than 30 types of seafood from markets in Jakarta, Indonesia, are presented. Most isolates were from mackerel, shrimp, or squid. Numerical taxonomic analyses clustered 337 isolates and three V. alginolyticus reference strains at S greater than or equal to 80%. These strains produced acid from sucrose, but only approximately 80% produced acetoin or grew in the presence of 10% NaCl. The frequency of occurrence of V. alginolyticus in seawater samples ranged from 0% (in February and March 1972) to 100% (in September and December 1972) and was highest in seafood samples from August to December 1972. A second cluster of 230 isolates and seven V. parahaemolyticus reference strains was observed at S greater than or equal to 82%. These strains did not produce acetoin or acid from sucrose, and approximately 20% grew in the presence of 10% NaCl. V. parahaemolyticus was detected in seawater samples each month, with the highest frequency of occurrence (83.3%) in May 1972. Twenty-nine K antigen serotypes were demonstrated in V. parahaemolyticus isolates, and another 40% were untypable. The modal antibiotic resistance pattern for each species included five drugs. Only 12% of the V. parahaemolyticus strains were Kanagawa positive, and 10% elicited fluid accumulation in ligated rabbit ileal loops. All of the 7 V. alginolyticus strains and 94 (70%) of the V. parahaemolyticus strains tested killed mice when inoculated intraperitoneally.(ABSTRACT TRUNCATED AT 250 WORDS)     

Herpesvirus saimiri strain variability

Herpesvirus saimiri was isolated from 22 squirrel monkeys by cocultivation of peripheral lymphocytes with permissive owl monkey kidney monolayer cells. Comparison of virion DNA fragments produced from restriction endonuclease digestion was used as a sensitive measure of strain variability. Although all isolates contained similarities and common features, 19 of the 22 were readily distinguished. Three of the isolates, however, were indistinguishable and possibly were related epidemiologically. Distinct subtypes of H. saimiri were not evident by these criteria; Peruvian, Colombian, Guyanan, and Bolivian squirrel monkeys yielded isolates without characteristic features peculiar to the geographic region. Three of three colony-born squirrel monkeys that were tested yielded a strain of virus distinct from that obtained from the mother. In separate experiments, two of three animals chosen at random yielded a strain of virus different from that originally obtained 16 and 22 months previously; only one of the three animals examined yielded the same strain of virus 22 months after the original isolation. The degree of restriction endonuclease fragment variability among H. saimiri strains appeared to be greater than previously observed for other herpesviruses.     

Virulence and molecular typing of Vibrio harveyi strains isolated from cultured dentex,gilthead sea bream and European sea bass

Vibrio harveyi was isolated from internal organs or ulcers of diseased and apparently healthy gilthead sea bream (Sparus aurata) and European sea bass (Dicentrarchus labrax) cultured in several fish farms located on the Spanish Mediterranean coast. The prevalence of the bacterium was significantly higher in European sea bass than in gilthead sea bream, and was closely related to the season in both fish species, occurring almost exclusively on warm months (June to November). After phenotypic characterization, a selection of forty five isolates from gilthead sea bream, sea bass, and several isolates previously obtained from common dentex (Dentex dentex) of the same area, were molecularly typed by automated ribotyping and random amplified polymorphic DNA (RAPD) analysis. Cluster analysis of data established 8 RAPD types and 13 ribotypes among wild isolates, and the combination of both techniques allowed to define fourteen different groups and a clear discrimination of all outbreaks and samplings. Several strains isolated from diseased gilthead sea bream and sea bass and also from asymptomatic sea bream, were tested for virulence in both fish species by intracoelomic injection. All the isolates (11) were pathogenic for sea bass, with nine out of the eleven LD50 values ranging from 1.5 x 10(5) to 1.6 x 10(6) cfu/fish. Gilthead sea bream was unaffected by the seven tested strains, even by those more virulent for sea bass, and only one strain caused a 10% mortality at 4.2 x 10(7) cfu/fish. This is the first report on virulence of V. harveyi for sea bass.     

Genetic Diversity of Clinical and Environmental Isolates of Vibrio cholerae Determined by Amplified Fragment Length Polymorphism Fingerprinting

Vibrio cholerae, the causative agent of major epidemics of diarrheal disease in Bangladesh, South America, Southeastern Asia, and Africa, was isolated from clinical samples and from aquatic environments during and between epidemics over the past 20 years. To determine the evolutionary relationships and molecular diversity of these strains, in order to understand sources, origin, and epidemiology, a novel DNA fingerprinting technique, amplified fragment length polymorphism (AFLP), was employed. Two sets of restriction enzyme-primer combinations were tested for fingerprinting of V. cholerae serogroup O1, O139, and non-O1, O139 isolates. Amplification of HindIII- and TaqI-digested genomic DNA produced 30 to 50 bands for each strain. However, this combination, although capable of separating environmental isolates of O1 and non-O1 strains, was unable to distinguish between O1 and O139 clinical strains. This result confirmed that clinical O1 and O139 strains are genetically closely related. On the other hand, AFLP analyses of restriction enzyme ApaI- and TaqI-digested genomic DNA yielded 20 to 30 bands for each strain, but were able to separate O1 from O139 strains. Of the 74 strains examined with the latter combination, 26 serogroup O1 strains showed identical banding patterns and were represented by the O1 El Tor strain of the seventh pandemic. A second group, represented by O139 Bengal, included 12 strains of O139 clinical isolates, with 7 from Thailand, 3 from Bangladesh, and 2 from India. Interestingly, an O1 clinical isolate from Africa also grouped with the O139 clinical isolates. Eight clinical O1 isolates from Mexico grouped separately from the O1 El Tor of the seventh pandemic, suggesting an independent origin of these isolates. Identical fingerprints were observed between an O1 environmental isolate from a river in Chile and an O1 clinical strain from Kenya, both isolated more than 10 years apart. Both strains were distinct from the O1 seventh pandemic strain. Two O139 clinical isolates from Africa clustered with environmental non-O1 isolates, independent of other O139 strains included in the study. These results suggest that although a single clone of pathogenic V. cholerae appears responsible for many cases of cholera in Asia, Africa, and Latin America during the seventh pandemic, other cases of clinical cholera were caused by toxigenic V. cholerae strains that appear to have been derived locally from environmental O1 or non-O1 strains.     

Isolation of Vibrio cholerae from aquatic birds in Colorado and Utah

Vibrio cholerae was isolated from cloacal swabs and freshly voided feces collected from 20 species of aquatic birds in Colorado and Utah during 1986 and 1987. About 17% (198 of 1,131) fecal specimens collected from July 1986 through August 1987 contained the organism. Both O1 and non-O1 V. cholerae strains were isolated from the fecal specimens. Isolates from eight birds (representing five species) agglutinated in O group 1 antiserum. Supernatants of broth cultures from three isolates which typed as V. cholerae O1 serotype Ogawa gave reactions typical of cholera toxin when tested on Y-1 mouse adrenal cell cultures. Several serovars of non-O1 V. cholerae were isolated from the fecal specimens; serovar 22 was the most prevalent type. All non-O1 isolates were cytotoxic to Y-1 mouse adrenal cells. Only non-O1 V. cholerae was detected in water samples collected from the habitat of the birds. The results of this study suggest that aquatic birds serve as carriers and disseminate V. cholerae over a wide area.     

Isolation of Vibrio cholerae from aquatic birds in Colorado and Utah.

Vibrio cholerae was isolated from cloacal swabs and freshly voided feces collected from 20 species of aquatic birds in Colorado and Utah during 1986 and 1987. About 17% (198 of 1,131) fecal specimens collected from July 1986 through August 1987 contained the organism. Both O1 and non-O1 V. cholerae strains were isolated from the fecal specimens. Isolates from eight birds (representing five species) agglutinated in O group 1 antiserum. Supernatants of broth cultures from three isolates which typed as V. cholerae O1 serotype Ogawa gave reactions typical of cholera toxin when tested on Y-1 mouse adrenal cell cultures. Several serovars of non-O1 V. cholerae were isolated from the fecal specimens; serovar 22 was the most prevalent type. All non-O1 isolates were cytotoxic to Y-1 mouse adrenal cells. Only non-O1 V. cholerae was detected in water samples collected from the habitat of the birds. The results of this study suggest that aquatic birds serve as carriers and disseminate V. cholerae over a wide area.     

Identification and biochemical characterization of Leishmania strains isolated in Peru, Mexico, and Spain

Eight Leishmania promastigotes were isolated from different geographical areas: three (LP1, LP2, and LP3) from the provincial department La Libertad and the fourth (LP4) from the department of Cajamarca (northern Peru); another three (LM1, LM2, and LM3) in the province of Campeche (Mexico); and the last (LS1) from a clinical case of a dog in Madrid (Spain). The isolates were characterized by carbohydrate cell-surface residues using agglutinations with four purified lectins, by isoenzyme analysis using different isoenzymes, by analysis of kinetoplast DNA (kDNA) restriction fragment length polymorphism using four different restriction endonucleases and by the final metabolite patterns after in vitro culture. These isolates were compared with four reference strains and typified as: Leishmania (Leishmania) donovani, two strains of L. (L.) infantum, and one species of L. (Viania) peruviana. According to our results and the statistical study, the Peruvian isolates represent three different strains: one would be L. (V.) peruviana, another the strain isolated in Cajamarca (LP4) and the third would include the three strains from the department of La Libertad (LP1, LP2, and LP3), these latter three isolates being phylogenetically closer to the reference strain L. (L.) donovani. Meanwhile, the three isolates from Mexico form a group with close phylogenetic relationships to each other. The isolate from Spain belongs to the species L. (L.) infantum. Thus, a close correlation was drawn between the identity of each strain and its geographical origin.     

Comparative evaluation of plasmid profiling and ribotyping in the analysis of Lactobacillus plantarum strain heterogeneity in silage

F. DUFFNER AND M. O'CONNELL. 1995. Seventy-two Lactobacillus plantarum isolates were recovered from six uninoculated grass silages for the purposes of firstly evaluating the usefulness of (1) restriction endonuclease digestion of total genomic DNA, (2) plasmid profiling and (3) ribotyping in Lact. plantarum strain differentiation and secondly, examining the strain heterogeneity in well preserved silage.
The three methods for differentiation were applied to 72 of the isolates and allowed at least 32 different strains to be identified. Twenty-five different plasmid profiles were detected (26 if the absence of plasmids is included as a profile). Ribotyping with Eco RI identified only 11 patterns among the silage isolates. A variety of restriction enzymes was screened to increase the sensitivity of ribotyping to detect strain differences and Bam HI was used successfully for this purpose, differentiating all of the strains tested.
Two dominant strains (I and II) were identified in one particular silage, comprising 47% and 17% respectively of the isolates, while strains III and V comprised 37% and 25% of the Lact. plantarum population isolated from another of the silages.     

Phenotypic Characteristics and Virulence of Vibrio anguillarum-Related Organisms

The phenotypic, molecular, and virulence properties of 46 Vibrio anguillarum-related (VAR) strains isolated from diseased fish and shellfish and from the environment were investigated. Twelve reference strains belonging to the 10 serotypes of V. anguillarum and the Vibrio splendidus type strain were included for comparison. Numerical taxonomy studies allowed us to group the isolates into four phena. The main phenotypic traits to differentiate VAR strains from V. anguillarum were fermentation of arabinose and mannitol, indole and Voges-Proskauer reactions, gelatin and casein hydrolysis, hemolytic activity, growth at 37 and 4°C, and resistance to ampicillin. Serological analysis confirmed that phena I and II were composed mainly of strains of V. anguillarum, while phena III and IV included VAR strains. Excluding the reference strains, the typeable isolates belonged to serotypes O3 (15 strains), O4 (3 strains), and O5 (2 strains) of V. anguillarum. The infectivity trials showed that only 9 of a total of 24 strains tested displayed virulence for rainbow trout. Virulent strains (50% lethal dose ranging from 10² to 10⁶ cells) included V. anguillarum strains belonging to serotypes O1 (one strain), O2 (one strain), O3 (three isolates), and O4 (one isolate) and only three strains of the VAR group. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of lipopolysaccharide and outer membrane proteins showed heterogeneity not only among the 10 V. anguillarum serotypes but also within the VAR group. Immunoblot assays demonstrated a close relationship among V. anguillarum strains from the same serotype, while strains from different serotypes were not antigenically related. The VAR strains did not share antigenic components with the serotypes of V. anguillarum tested (serotypes O1 to O5). Plasmids were detected in only 19 of the total of 59 strains. The majority of the strains carrying plasmids were grouped within phenon IV, in which plasmid bands of 27 and 36 MDa were found in all the isolates. No correlation between the plasmid content of VAR microorganisms and their phenotypic or virulence characteristics was observed. From these results it can be concluded that VAR strains associated with disease should be included together with V. anguillarum in the formulation of vaccines against vibriosis.