Identification of Shigella flexneri subserotype 1c in rural Egypt

In a population-based study of diarrhea in rural, northern Egypt, 60 Shigella flexneri strains were identified, of which 10 could not be definitively serotyped. Serological analysis with commercial reagents suggested that they were serotype 1, but the strains failed to react with subserotype 1a- or 1b-specific antibodies. All 10 strains reacted with MASF 1c, a monoclonal antibody specific for a provisional S. flexneri subserotype, 1c, first identified in Bangladesh and not previously detected outside of that region. Our results show that S. flexneri subserotype 1c is not unique to Bangladesh and that the inability to detect it may reflect both the limited use of suitable screening methods and the rarity of this subserotype.     

Comparative study of biological properties and electrophoretic characteristics of lipopolysaccharide from eel-virulent and eel-A virulent Vibrio vulnificus strains

In Vibrio vulnificus, virulence for eels is associated with serovar E strains. In this study, we investigated some biological properties of purified lipopolysaccharides (LPSs) from serovar E and non-serovar E strains. Purified LPSs retained their O-polysaccharidic side chains and did not show any differences that could be related to host specificity, except for serological differences.     

"Black holes" and bacterial pathogenicity: a large genomic deletion that enhances the virulence of Shigella spp. and enteroinvasive Escherichia coli

Plasmids, bacteriophages, and pathogenicity islands are genomic additions that contribute to the evolution of bacterial pathogens. For example, Shigella spp., the causative agents of bacillary dysentery, differ from the closely related commensal Escherichia coli in the presence of a plasmid in Shigella that encodes virulence functions. However, pathogenic bacteria also may lack properties that are characteristic of nonpathogens. Lysine decarboxylase (LDC) activity is present in approximately 90% of E. coli strains but is uniformly absent in Shigella strains. When the gene for LDC, cadA, was introduced into Shigella flexneri 2a, virulence became attenuated, and enterotoxin activity was inhibited greatly. The enterotoxin inhibitor was identified as cadaverine, a product of the reaction catalyzed by LDC. Comparison of the S. flexneri 2a and laboratory E. coli K-12 genomes in the region of cadA revealed a large deletion in Shigella. Representative strains of Shigella spp. and enteroinvasive E. coli displayed similar deletions of cadA. Our results suggest that, as Shigella spp. evolved from E. coli to become pathogens, they not only acquired virulence genes on a plasmid but also shed genes via deletions. The formation of these "black holes," deletions of genes that are detrimental to a pathogenic lifestyle, provides an evolutionary pathway that enables a pathogen to enhance virulence. Furthermore, the demonstration that cadaverine can inhibit enterotoxin activity may lead to more general models about toxin activity or entry into cells and suggests an avenue for antitoxin therapy. Thus, understanding the role of black holes in pathogen evolution may yield clues to new treatments of infectious diseases.     

Evolutionary relationships among pathogenic and nonpathogenic Escherichia coli strains inferred from multilocus enzyme electrophoresis and mdh sequence studies

Within the species Escherichia coli, there are commensal strains and a variety of pathogenic strains, including enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), and urinary tract infection (UTI) strains. The pathogenic strains are identified by serotype and by possession of specific virulence determinants (toxins and adhesions, etc.) encoded by either monocistronic genes, plasmids, or pathogenicity islands. Although there are studies on the relationships between selected pathogenic strains, the relatedness among the majority of the pathogenic forms to each other, to commensal E. coli, and to the genus Shigella (which has often been suggested to be part of E. coli) has not been determined. We used multilocus enzyme electrophoresis (MLEE) at 10 enzyme loci and the sequence of the mdh housekeeping gene to study the genetic relationships of pathogenic E. coli strains (including Shigella clones), namely, 5 EPEC strains (serotypes O111 and O55), 3 EHEC strains (serotype O157), 6 ETEC strains (serotypes O78, O159, and O148), 5 EIEC strains (serotypes O124, O28, and O112), and 13 Shigella strains representing clones Flexneri, Dysenteriae, Boydii, and Sonnei, to commensal E. coli strains. Both the MLEE and mdh sequence trees reveal that EPEC, EHEC, ETEC, EIEC, and UTI strains are distributed among the ECOR set groups, with no overall clustering of EPEC, ETEC, EIEC, or UTI strains. The genus Shigella is shown to comprise a group of closely related pathogenic E. coli strains. Six pathogenic strains, i.e., M502 (EIEC; O112ac:NM), M503 (EPEC; O111:H12), M526 (ETEC; O159:H4), M522 (EPEC; O111ac:H12), M524 (ETEC; O78:H11), and M506 (ETEC; O78:H11), were found to have mdh sequences identical to those of five ECOR group A strains (ECOR5, ECOR10, ECOR14, ECOR6, and K-12). All 11 strains are closely related by MLEE. The results indicate that pathogenic strains of E. coli do not have a single evolutionary origin within E. coli but have arisen many times. The results also suggest the possibility that any E. coli strain acquiring the appropriate virulence factors may give rise to a pathogenic form.     

Phenotypic and genotypic differentiation of several human and avian isolates of Cryptococcus neoformans

The Cryptococcus neoformans strains isolated from two human cases could be diagnosed as Cr. neoformans var. neoformans by differentiation on the basis of their characteristics determined by proline, canavanine and EDTA urease tests. The results of the serovar assignment were: for the isolate from the meningoencephalitis patient with lethal outcome, serovar A; for the strain isolated from the osteomyelitis patient with benign course, serovar D. Also, the PCR fingerprinting using primers (GACA)4, (CAC)5 and FM 1 resulted in a clear and reproducible assignment of the Cr. neoformans strains to the varieties neoformans and gattii, respectively, and, in addition, it confirmed the serovar assignment. No statistically confirmed differences in virulence between the osteomyelitis and the meningoencephalitis strain could be established by i.v. testing in mice, nor did the PCR with several primers provide any clues to a genetically determined higher virulence of the meningoencephalitis strain. The different classification as serovars A and D does not allow any conclusions concerning different virulence. It was not possible to retrospectively establish the sources of infection of the two Cr. neoformans infections, but pigeon faeces may well have played a role as a reservoir for one of the illnesses.     

Identification of enteroinvasive Escherichia coli and Shigella strains in pediatric patients by an IpaC-specific enzyme-linked immunosorbent assay

A new method, a monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) recognizing a secreted, invasion plasmid-coded protein antigen (IpaC), was used to identify enteroinvasive Escherichia coli and Shigella strains among colonies from 859 cultures of fecal samples from children in Kuwait. A total of 33.8% of the samples were diarrheal. By the immunoassay, enteroinvasive E. coli strains were identified from two diarrheal samples but from none of the samples from children without diarrhea. These strains were fully virulent and belonged to serogroup O28ac. In addition, 26 Shigella strains were also recognized by the ELISA, while only 23 were isolated by routine biotyping and serotyping. For two diarrheal patients, Shigella was identified by culture only. The study showed that the IpaC-specific immunoassay is a simple and useful tool for identifying enteroinvasive strains. Furthermore, by reporting the first enteroinvasive E. coli isolates from Kuwait, the study indicates the presence of this group of pathogens as a potential source of diarrhea in the region.     

Shigellae isolated in 1997--plasmid profiles and antibiotic resistance

INTRODUCTION: Shigella spp is one of the most frequently isolated bacteria causing acute diarrhea with us. Genetics of pathogenicity of Shigella spp. includes chromosomal and plasmid genes. Most virulence factors are coded by invasion plasmid antigen genes residing on a 180-230 MDa plasmid. There is a big problem with multiple resistance of Shigella spp. strains, which is mostly plasmid-borne. Genetic analysis of bacterial cells, that is plasmid profile analysis, is important for investigation of sources and ways of spreading of the infection. All isolates originating from the same clone have identical plasmid profiles, i.e. number and size of plasmids. The aim of the investigation was: comparing the type of resistance to antimicrobical agents found in epidemic and nonepidemic. Shigella strains isolated in 1997, analyzing plasmid profiles of these isolates and confirming their epidemic connection. MATERIAL AND METHODS: Susceptibility to antibiotics was examined by a standard disc-diffusion method. Plasmid profiles of 40 strains (20 from the outbreak and 20 from sporadic cases) were tested using a method of alkaline lysis by Birnboim and Doly followed by electrophoresis in agarose gel. RESULTS: Shigella strains were resistant to antimicrobial agents which are most commonly used. Epidemic isolates shared the same resistance type, they were resistant to cephalexin, streptomycin and co-trimoxazole. The dominant type of resistance of nonepidemic strains was to ampicillin, streptomycin and co-trimoxazole. Strains isolated during the outbreak had identical plasmid profiles (2 plasmid bands of 55 and 1.5 MDa). Non-epidemic isolates had different plasmid profiles as well as type of resistance. CONCLUSION: Strains of Shigella spp. isolated during an outbreak had the same type of resistance and the same plasmid profiles, which indicated their origin from the same clone. The plasmid profile analysis is a reliable and precise method for determination of epidemic connection of Shigella isolates.     

H-NS regulates DNA repair in Shigella

We report a new role for H-NS in Shigella spp.: suppression of repair of DNA damage after UV irradiation. H-NS-mediated suppression of virulence gene expression is thermoregulated in Shigella, being functional at 30 degrees C and nonfunctional at 37 to 40 degrees C. We find that H-NS-mediated suppression of DNA repair after UV irradiation is also thermoregulated. Thus, Shigella flexneri M90T, incubated at 37 or 40 degrees C postirradiation, shows up to 30-fold higher survival than when incubated at 30 degrees C postirradiation. The hns mutants BS189 and BS208, both of which lack functional H-NS, show a high rate of survival (no repression) whether incubated at 30 or 40 degrees C postirradiation. Suppression of DNA repair by H-NS is not mediated through genes on the invasion plasmid of S. flexneri M90T, since BS176, cured of plasmid, behaves identically to the parental M90T. Thus, in Shigella the nonfunctionality of H-NS permits enhanced DNA repair at temperatures encountered in the human host. However, pathogenic Escherichia coli strains (enteroinvasive and enterohemorrhagic E. coli) show low survival whether incubated at 30 or 40 degrees C postirradiation. E. coli K-12 shows markedly different behavior; high survival postirradiation at both 30 and 40 degrees C. These K-12 strains were originally selected from E. coli organisms subjected to both UV and X irradiation. Therefore, our data suggest that repair processes, extensively described for laboratory strains of E. coli, require experimental verification in pathogenic strains which were not adapted to irradiation.     

Colicin U, a novel colicin produced by Shigella boydii

A novel colicin, designated colicin U, was found in two Shigella boydii strains of serovars 1 and 8. Colicin U was active against bacterial strains of the genera Escherichia and Shigella. Plasmid pColU (7.3 kb) of the colicinogenic strain S. boydii M592 (serovar 8) was sequenced, and three colicin genes were identified. The colicin U activity gene, cua, encodes a protein of 619 amino acids (Mr, 66,289); the immunity gene, cui, encodes a protein of 174 amino acids (Mr, 20,688); and the lytic protein gene, cul, encodes a polypeptide of 45 amino acids (Mr, 4,672). Colicin U displays sequence similarities to various colicins. The N-terminal sequence of 130 amino acids has 54% identity to the N-terminal sequence of bacteriocin 28b produced by Serratia marcescens. Furthermore, the N-terminal 36 amino acids have striking sequence identity (83%) to colicin A. Although the C-terminal pore-forming sequence of colicin U shows the highest degree of identity (73%) to the pore-forming C-terminal sequence of colicin B, the immunity protein, which interacts with the same region, displays a higher degree of sequence similarity to the immunity protein of colicin A (45%) than to the immunity protein of colicin B (30.5%). Immunity specificity is probably conferred by a short sequence from residues 571 to residue 599 of colicin U; this sequence is not similar to that of colicin B. We showed that binding of colicin U to sensitive cells is mediated by the OmpA protein, the OmpF porin, and core lipopolysaccharide. Uptake of colicin U was dependent on the TolA, -B, -Q, and -R proteins. pColU is homologous to plasmid pSB41 (4.1 kb) except for the colicin genes on pColU. pSB41 and pColU coexist in S. boydii strains and can be cotransformed into Escherichia coli, and both plasmids are homologous to pColE1.     

Emergence of a unique O3:K6 clone of Vibrio parahaemolyticus in Calcutta, India, and isolation of strains from the same clonal group from Southeast Asian travelers arriving in Japan

Active surveillance of Vibrio parahaemolyticus infection among hospitalized patients in Calcutta, India, was initiated in January 1994. The incidence of cases of V. parahaemolyticus infection suddenly increased in February 1996 and has remained high since then. One hundred thirty-four strains of V. parahaemolyticus isolated from January 1994 to August 1996 were examined for serovar, the presence of the thermostable direct hemolysin gene (tdh) and tdh-related hemolysin genes (trh1 and trh2), production of urease, and antibiogram. Strains of the O3:K6 serovar appeared for the first time in February 1996. The O3:K6 serovar strains accounted for 50 to 80% of the strains isolated during the high-incidence period (February to August 1996). All of the serovar O3:K6 strains carried the tdh gene but not the trh genes and did not produce urease. All of the isolates except two were sensitive to all of the antibiotics tested. These and the results of analysis by an arbitrarily primed PCR method indicated that the O3:K6 serovar strains belong to a unique clone. When the O3:K6 serovar strains, isolated from travelers arriving in Japan from Southeast Asian countries, were compared by the arbitrarily primed PCR method, the strains isolated between 1982 and 1993 were distinct from Calcutta O3:K6 while the strains isolated in 1995 and 1996 were indistinguishable from the Calcutta O3:K6 strains. The results suggest that this unique O3:K6 clone may have become prevalent not only in Calcutta but also in Southeast Asian countries very recently. Not only the O3:K6 strains but also the non-O3:K6, tdh-bearing strains isolated in 1996 produced thermostable direct hemolysin at high levels, and thus the level of hemolysin produced does not appear to have influenced the high incidence of serovar O3:K6 strains.     

Differences in the association of Chlamydia trachomatis serovar E and serovar L2 with epithelial cells in vitro may reflect biological differences in vivo

Chlamydia trachomatis serovar E is one of the most common bacterial sexually transmitted pathogens. Since it is an obligate intracellular bacterium, efficient colonization of genital mucosal epithelial cells is crucial to the infectious process. Serovar E elementary bodies (EB) metabolically radiolabeled with 35S-Cys-Met and harvested from microcarrier bead cultures, which significantly improves the infectious EB-to-particle ratio, provided a more accurate picture of the parameters of attachment of EB to human endometrial epithelial cells (HEC-1B) than did less infectious 14C-EB harvested from flask cultures. Binding of serovar E EB was (i) equivalent at 35 and 4 degrees C, (ii) decreased by preexposure of EB to heat or the topical microbicide C31G, (iii) comparable among common eukaryotic cell lines (HeLa, McCoy), and (iv) significantly increased to the apical surfaces of polarized cells versus nonpolarized cells. In parallel experiments with C. trachomatis serovar L2, serovar E attachment was not affected by heparin or heparan sulfate whereas these glucosaminoglycans dramatically reduced serovar L2 attachment. These data were confirmed by competitive inhibition of serovar E binding and infectivity by excess unlabeled live and UV-inactivated serovar E EB but not by excess serovar L2 EB. The noninvasive serovar E strains in the lumen of the genital tract enter and exit the apical domains of target columnar epithelial cells to spread canalicularly in an ascending fashion from the lower to the upper genital tract. In contrast, the invasive serovar L2 strains are primarily submucosal pathogens and likely use the glucosaminoglycans concentrated in the extracellular matrix to colonize the basolateral domains of mucosal epithelia to perpetuate the infectious process.     

Evaluation of the usefulness of tests for production of Beta-D-glucuronidase and propylene glycol utilization for the differentiation of enterobacteriaceae rods

The aim of the study was to inquire about the diagnostic usefulness of determining the activity of glucuronidase and utilisation of propylene glycol in Enterobacteriaceae rods. The study included 1511 strains: 411- E. coli, 278 - Klebsiella, 231 - Salmonella, 159 - Yersinia, 97 - Citrobacter, 75 - Shigella and 260 strains representing 6 other kinds of enteric rods. Determination was performed in a liquid medium containing in 1 ml 25 mcg MUG and 100 mcg ONPG. Propylene glycol (PG) utilisation was observed in peptone water with 2% of the substrate and with the Andrade indicator. In comparative tests Rambach commercial medium and MacConkey agar from the Fluorocult series were used. In the test with MUG a positive result was obtained from 81.8% E. coli, 65% - Shigella and 13% - Salmonella subgenus I. Only exceptionally was this test positive with Providencia, Enterobacter and Yersinia strains (1-5%) but negative with Citrobacter, Klebsiella, Serratia, Hafnia, Proteus and Morganella strains. Glucuronidase production is not sufficiently characteristic of E. coli strains isolated from humans to be the only basis for the preliminary differentiation of these rods from other Enterobacteriaceae. The test with ONPG was positive from 95-100% E. coli, Yersinia, Citrobacter, Klebsiella, Enterobacter and Hafnia strains; 61% - Shigella, 9% - Salmonella and 3% - Providencia, but negative with Serratia, Proteus and Morganella strains. Propylene glycol was decomposed by 74% Salmonella strains of subgenus I, 65-94% - Klebsiella, Yersinia and Citrobacter. Shigella, Enterobacter, Serratia, Proteus, Providencia and Morganella rods did not decompose propylene glycol. Evidence that among strains non-decomposing propylene glycol were all the studied S. typhi, S. paratyphi A, S. paratyphi C, S. choleraesuis, S. virchow and S. gallinarum strains as well as a significant percentage of strains representing 8 other Salmonella serotypes frequently detected allows to believe that the use of activity against propylene glycol even simultaneously with the test for galactosidase as basis for the differentiation of Salmonella rods of subgenus I from other Enterobacteriaceae can lead to errors already at the onset of diagnostic procedure.     

Characterization of leptospiral serovars by randomly amplified polymorphic DNA fingerprinting

Randomly amplified polymorphic DNA (RAPD) fingerprinting of 14 laboratory strains of leptospiral serovars (serovars australis, autumnalis, ballum, bataviae, canicola, grippotyphosa, hardjoprajitno, hebdomadis, icterohaemorrhagiae, javanica, pomona, pyrogenes, panama, and tarassovi) was carried out by using a pair of primers. Each serovar had a unique and distinct fingerprint pattern. DNAs of other bacterial species, including Escherichia coli, Pasteurella multocida, Salmonella spp., Pseudomonas spp., and Klebsiella spp., did not show any amplification. RAPD fingerprinting was found to be a rapid and sensitive method for serovar identification when it was compared to DNA restriction enzyme analysis, which produced a larger number of bands that made it more difficult to compare serovars.     

Analysis of genome-type, serovar and antibiotic susceptibility of Pseudomonas aeruginosa isolated in Beijing Hospital China in 1991 to 1993]

We analyzed the in vitro antibiotic susceptibility pattern and serovar for 192 strains of Pseudomonas aeruginosa isolated at Beijing Hospital(China) from October 1991 to October 1993. The frequency of resistant strains was high, more than 15%, for ceftazidime, cefsulodin, cefoperazone, aztreonam, gentamicin, tobramycin, tosufloxacin, ofloxacin and fosfomycin. The incidence of resistants against piperacillin and imipenem was significantly low. Among the 192 strains, 16 were designated as being multi-drug resistant strains(i.e.; resistant to more than 8 drugs out of 11 drugs), and all of the multi-drug resistant strains were isolated from inpatients. Predominant serovar of 192 strains were 60(31.3%) for G, 28(14.7%) for E, and 24(12.5%) for I. Multi-drug resistant strains have no characteristic serovar. Restriction enzyme SpeI digestion analysis(using pulse field electrophoresis) of P. aeruginosa-genome yielded several common patterns, and was shown to be useful for tracing the route of nosocomial infection. Further, isolates with closely related genome type, in which the size of one digested band was different, showed a different minimum inhibitory concentration of fosfomycin in one genome type or new quinolones in the other genome type. Analysis of genome type and antibiotic susceptibility pattern may exhibit the antibiotic resistant gene.     

Study of the antibiotic sensitivity of Shigella newcastle isolated in Zaporozhe Province

Sensitivity to antibacterial drugs of 510 strains of Shigella Newcastle isolated from both sporadic patients and cases during the disease outbreaks was studied. The studies revealed a rather high percentage of Shigella Newcastle resistant to the antibiotics widely used in clinical practice. Most of the strains, i.e. 90.5 per cent were resistant to tetracycline and a significant number of the isolates, i.e. 55.6--53.3 per cent were resistant to streptomycin and erythromycin. A statistically reliable predominance of the Shigella resistant to levomycetin and streptomycin in the disease foci was noted as compared to the Shigella isolated from the sporadic patients. Most of the isolates were simultaneously resistant to several antibiotics. Thus, resistance to 2,3--4 and 5--6 antibiotics was found in 28, 54.3 and I.I per cent of the cultures respectively. At the same time almost all Shigella Newcastle strains were sensitive to rifampicin, neomycin and monomycin (aminoglycoside antibiotics), enteroseptol (oxychinoline drug) and furazolidone (nitrofuran drug).     

Resistance to antibiotics of Shigella strains isolated in Somalia

The resistance to antibiotics of 240 Shigella strains isolated in Somalia from 1973 to 1976 was studied. Many strains, particularly those of Shigella dysenteriae type 1, were found to be resistant to more than one drug. In view of their resistance to ampicillin, chloramphenicol, streptomycin, tetracycline, and sulfonamides, it is suggested that polymyxin B or M sulfate - which have proved to be effective in vivo - should be used for the treatment of clinically typical cases of bacillary dysentery.     

Hemolysin-positive enteroaggregative and cell-detaching Escherichia coli strains cause oncosis of human monocyte-derived macrophages and apoptosis of murine J774 cells

Infection of human monocyte-derived macrophages (HMDM) and J774 cells (murine macrophage cell line) with several enteroaggregative and cytodetaching Escherichia coli (EAggEC and CDEC, respectively) strains demonstrated that some strains could induce macrophage cell death accompanied by release of lactate dehydrogenase activity and interleukin 1beta (IL-1beta) into culture supernatants. The mode of cell death differed in the two types of macrophages. Damage to macrophage plasma membrane integrity without changes in nuclear morphology resulted in cytolysis of HMDM. This mechanism of cell death has been previously described for virulent Shigella infection of HMDM and is termed oncosis. In contrast, infection of J774 cells by EAggEC and CDEC strains resulted in apoptosis. The presence of alpha-hemolysin (Hly) in EAggEC and CDEC strains appears to be critical for both oncosis in HMDM and apoptosis in J774 cells. Bacteria lacking Hly, including Hly- EAggEC strains as well as enterotoxigenic, enteropathogenic, and enterohemorrhagic E. coli strains, behaved like avirulent Shigella flexneri in that the macrophage monolayers were intact, with no release of lactate dehydrogenase activity or IL-1beta into the culture supernatants.     

Transforamtion of twitching strains of Streptococcus sanguis

Ninety-five strains of S. sanguis, 90 of which were twitching, were screened for competence in transformation with DNA from the "Challis" strain. Seventy-two strains, 68 of sero-group H and 4 of the provisional group 10043, were competent. Fourteen of the competent strains and all strains which appeared to be incompetent were tested with DNA from 3 other strains. The 14 competent strains were transformed by all the 3 DNAs. One of the apparently incompetent strains was transformed by autologous DNA only. Among 8 reference strains (including ATCC 10577, Type II of Washburn et al.) 5 were competent. Three of these did not show spreading or twitching. Among 16 non-spreading strains of alphahaemolytic streptococci which did not possess either the H or the 10043 group antigen, only one showed competence. The results indicate that twitching mobility is not a prerequisite for competence.     

Shigella isolates in stool

A total of 447 Shigella strains were isolated from stool samples during 1989-1991. Of these 270 (60%) were from children. Among the different species and serotypes Sh. flexneri 60 (13.4%) and Sh. sonnei Phage 139 (65%) were the most frequently isolated strains. 154 (34.4%) strains were resistant to three and 179 (40%) to more than three antibiotics. Some strains of Shigella were found to be resistant to furazolidine and neomycin.     

The copper, manganese and zinc content of some edible mushrooms

Salmonella enterica subsp. enterica serovar Typhimurium (Salm. Typhimurium) live vaccine strain Zoosaloral H was characterized by pulsed-field gel electrophoresis (PFGE). Each of the two suitable restriction enzymes, XbaI and SpeI, produced a unique restriction fragment pattern for this live vaccine strain which was not shared by field isolates of the same serovar. The characteristic fragment pattern proved to be stable during a 22 month observation period and was also not altered after animal passage of the vaccine strains. Thus PFGE analysis proved to be a helpful tool in the identification of Salm. Typhimurium live vaccine strain Zoosaloral H and its differentiation from wild-type isolates of the same serovar.