Some safety aspects of Salmonella vaccines for poultry: in vivo study of the genetic stability of three Salmonella typhimurium live vaccines

Live vaccine strains of Salmonella should be avirulent, immunogenic and genetically stable. Some isolates of three commercially available live vaccine strains of Salmonella typhimurium, sampled during a study on their persistence in a vaccinated flock of chickens, were analyzed for genetic stability using macrorestriction analysis of their genome. Two out of the three vaccine strains showed genetic instabilities. Two of the 51 isolates of Zoosaloral vaccine strain and nine of the 32 analyzed isolates of chi(3985), a genetically modified organism, were variants and showed different macrorestriction profiles.     

Serum survival and plasmid possession by strains of Salmonella enteritidis, Salm. typhimurium and Salm. virchow

Strains of Salmonella enteritidis, Salm. typhimurium and Salm. virchow , carrying different numbers of plasmids, were examined for the ability to multiply in sera. Viable counts were performed to monitor the kinetics of growth of bacteria when in human, chicken and turkey sera. The presence of plasmids in Salm. enteritidis, Salm. typhimurium and Salm. virchow reduced considerably the ability of strains of these serotypes to multiply in serum. SDS-PAGE was used to show that growth of Salm. enteritidis in serum did not involve changes in outer membrane proteins or lipopolysaccharide. It was concluded that the carriage of plasmids may be disadvantageous for the survival in serum of certain common salmonella serotypes.     

Investigation of the genetic diversity among isolates of Salmonella enterica serovar Dublin from animals and humans from England,Wales and Ireland

AIMS: To assess the degree of genetic diversity among animal Salmonella Dublin UK isolates, and to compare it with the genetic diversity found among human isolates from the same time period. METHODS AND RESULTS: One hundred isolates (50 human and 50 animal) were typed using plasmid profiling, XbaI-pulsed field gel electrophoresis (PFGE) and PstI-SphI ribotyping. Antimicrobial resistance data to 16 antibiotics was presented, and the presence of class-I integrons was investigated by real-time PCR. Seven different plasmid profiles, 19 ribotypes and 21 PFGE types were detected. A combination of the three methods allowed clear differentiation of 43 clones or strains. Eighteen isolates were resistant to at least one antimicrobial; five of them were multi-resistant and of these, only three presented class I integrons. CONCLUSIONS: Ribotyping data suggest the existence of at least three very different clonal lines; the same distribution in well-defined groups was not evident from the PFGE data. The existence of a variety of clones in both animals and humans has been demonstrated. A few prevalent clones seem to be widely disseminated among different animal species and show a diverse geographical and temporal distribution. The same clones were found in animals and humans, which may infer that both farm and pet animals may act as potential vehicles of infection for humans. Some other clones seem to be less widely distributed. Clustering analysis of genomic fingerprints of Salmonella Dublin and Salm. Enteritidis isolates confirms the existence of a close phylogenetic relationship between both serotypes. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper describes the utility of a multiple genetic typing approach for Salm. Dublin. It gives useful information on clonal diversity among human and animal isolates.     

Survival of Salmonella enteritidis PT4 and Salm. typhimurium Swindon in aerosols

A.S. McDERMID AND M.S. LEVER. 1996. Small particle aerosols of plate-grown Salmonella enteritidis and Salm. typhimurium were generated and maintained within a rotating drum at 75% relative humidity and 24°C for 2 h. Plate-grown organisms were found to be more aerosol-stable than broth-grown organisms. Differences were observed between the two species; plate-grown Salm. typhimurium retained 100% viability after 2 h compared to approximately 70% for plate-grown Salm. enteritidis . A large proportion of cells of both serotypes remained viable in aerosols after 2 h, confirming the potential for airborne transmission for these organisms, e.g. within henhouses and during food     

Use of Polymerase Chain Reaction and Restriction Enzyme Analysis to directly detect and identify Salmonella typhimurium in food

A primer set of oligonucleotides (Salm 3 and Salm 4) from the inv A gene of Salmonellae has been evaluated for the specific detection of Salmonella spp. by the polymerase chain reaction (PCR). This primer set amplified 33 Salmonella serovars but did not amplify 16 non- Salmonella bacteria. Moreover, after PCR amplification, it was possible to identify Salm. typhimurium by restriction enzyme analysis. The PCR-RE method developed could represent a helpful tool for detecting Salmonella spp., and for directly and rapidly identifying Salm. typhimurium in food.     

Salmonella enterica serotype Enteritidis phage types 4, 7, 6, 8, 13a, 29 and 34: a comparative analysis of genomic fingerprints from geographically distant isolates

AIMS: To evaluate genetic heterogeneity in the most common phage types of Salmonella enterica serovar Enteritidis. METHODS AND RESULTS: A total of 233 isolates of Salm. Enteritidis from England, Northern Ireland, Spain, Hong Kong and the USA belonging to phage types (PT) 4 (n=88), PT7 (n=12), PT6 (n=72), PT8 (n=14), PT13a (n=29), PT29 (n=14) and PT34 (n=4) were characterized by PstI-SphI (PS) ribotyping and pulsed-field gel electrophoresis after digestion of DNA with XbaI. PS ribotyping differentiated the isolates into 53 different PS types and PFGE showed 14 different macrorestriction profiles; with the combination of both methods, 73 combined types were identified. Some of these clones appeared to be present within several countries. Movement of foodstuffs, animals or people may have been involved in the spread of these strains. On the other hand, some clones were only found in specific locations. CONCLUSIONS: Several well defined clonal lines seem to co-exist within the different phage types included in this study, and a combined typing approach may constitute a useful tool for epidemiological investigations. Clustering analysis of ribotypes and PFGE types agree with previous studies and suggest that phage types that share receptor binding properties can be distinguished as two families: the PT4 family including PT7 and PT6, and the PT8 family including PT13a. The other phage types are difficult to place in a family unless the geographical site of isolation is known. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper reports on an extensive assessment of the use of molecular tools for the study of the epidemiology of the enteric pathogen Salm. Enteritidis. It also gives new information regarding relationships among some common phage types.     

Analysis of Salmonella enteritidis strains isolated from food-poisoning cases in Taiwan by pulsed field gel electrophoresis, plasmid profile and phage typing

AIMS: To establish the molecular typing data for Salmonella enteritidis due to its increasing role in Salmonella infections in Taiwan. METHODS AND RESULTS: Sixty-three Salm. enteritidis strains isolated from related and unrelated patients suffering from food-borne poisoning during 1991-97 were collected and subjected to pulsed field gel electrophoresis (PFGE), plasmid analysis and phage typing. For PFGE, XbaI, SpeI and NotI restriction enzymes were used for chromosomal DNA digestion. The results showed that, for these 63 Salmonella strains, 10 PFGE pattern combinations were found. Of these, pattern X3 S3 N3 was the major subtype, since 46 strains isolated from different locations at different times during 1991-97 showed this PFGE pattern. Plasmid analysis showed only three plasmid profiles and phage typing showed that most of the Salmonella strains were of the phage type PT4. CONCLUSION: Most of the Salm. enteritidis strains circulating in Taiwan are of very similar genetic types or are highly related and that strains of PFGE pattern X3 S3 N3 are the prevalent and recirculating strains of Salm. enteritidis which caused food-poisoning cases in Taiwan in 1991-97. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides information that in Salmonella infection, certain subtypes of Salm. enteritidis should be scrutinized.     

Use of pulsed field gel electrophoresis as an epidemiological tool for analysis of sporadic associated strains of Salmonella typhi isolated in Taiwan

In order to characterize the subtypes of Salmonella typhi which cause sporadic disease in Taiwan, 55 isolates of Salm. typhi obtained from unrelated patients of sporadic cases during 1992-96 were subjected to chromosomal DNA digestion and pulsed field gel electrophoresis (PFGE). When DNAs of these 55 Salm. typhi strains were digested with XbaI, 41 PFGE patterns were observed. Strains sharing the same XbaI digestion pattern could not be further discriminated by PFGE analysis using SpeI and NotI as digestion enzymes. Thus, considerable genetic diversity exists among the Salm. typhi isolates. Although strains of the same patterns were mainly isolated during the same time, recirculation of certain infectious strains could be possible. When 12 antibiotics, i.e. ampicillin, trimethoprim/sulfamethoxazole, erythromycin, norfloxacin, tetracycline, sulphonamide, streptomycin, neomycin, chloramphenicol, kanamycin, cefoperazone and gentamycin were used to test the antibiotic susceptibility for these Salmonella isolates, only three antibiogram patterns were obtained and 49 of the 55 Salm. typhi isolates were found to belong to one pattern. Phage typing and plasmid profiles were also poor in discriminating these strains. Thus, PFGE alone may be used as a powerful tool for analysis of sporadic associated Salm. typhi strains.     

Distribution of pathogen inhibition in the Lactobacillus isolates of a commercial probiotic consortium

Pure strains of Lactobacillus ssp. isolated from a commercial probiotic consortium were checked in a double layer solid medium for their inhibition activities against selected pathogenic bacteria including serotypes of Listeria monocytogenes, Escherichia coli and Salmonella. The antagonistic properties of the Lactobacillus strains may be related to the production of bacteriocin-like compounds. All the pathogens tested were inhibited by one or a few strains of Lactobacillus, the best inhibition was observed against L. monocytogenes but the inhibition was also satisfactory against E. coli, Salm. typhimurium and Salm. enteritidis.     

Identification of a conjugative plasmid carrying antibiotic resistance and salmonella plasmid virulence (spv) genes in epidemic strains of Salmonella typhimurium phage type 193

A plasmid pDEP34 that codes for resistance to ampicillin, streptomycin, sulphonamides and tetracyclines has been identified in strains of Salmonella typhimurium phage type 193 which have become increasingly common in England and Wales since 1988. pDEP34 is also self-conjugative, carries the genes responsible for the virulence of host strains for BALB/c mice ( spv genes) and is closely related to the Salm. typhimurium 'serotype-specific' plasmid pSLT.     

A method for the enumeration of male-specific bacteriophages in sewage

Male-specific bacteriophages adsorb to F-pili and thus can only infect male host strains. A method was developed for the selective enumeration of these phages, based on the observation that in sewage there are few phages capable of infecting F- -salmonellas--usually less than 10 pfu/ml. Using a male Salmonella strain, constructed by the introduction of the plasmid F'42 lac::Tn5 into Salmonella typhimurium phage type 3, plaque counts in secondary effluent were found to be in the range of 60-8200 pfu/ml. Practically all the phages detected had a host range restricted to male Salmonella or Escherichia coli strains, were resistant to chloroform and their infectivity was inhibited by RNase. Electron microscopy of lysates revealed phage particles that were morphologically identical to the male-specific single-strand RNA phages. Similar results were obtained with a strain of Salm. indiana carrying F'42 lac. A derivative of the Salm. typhimurium LT2 strain carrying an F-plasmid (F'42 lac fin P301) derepressed for fertility inhibition by the resident plasmid pSLT was equally sensitive to male-specific phages, but from sewage samples many other phages infecting F- E. coli but not F- Salmonella were isolated using this host strain.     

Hazard analysis applied to microbial growth in foods: development of mathematical models describing the effect of water activity

Mathematical models have been developed which describe the effect of lowering the water activity on the growth kinetics of Staphylococcus aureus and Salmonella typhimurium. By treating the lag phase and exponential phase kinetics separately predictions can be made on the extent of microbial growth over successive time/temperature cycles. Staph. aureus was far more tolerant than Salm. typhimurium to lowered water activity and under near growth limiting conditions of water activity and temperature was showing lag periods as long as ca 40 d. The maximum lag period observed for Salm. typhimurium was ca 5 d. Under these conditions the predicted generation times for Staph. aureus were 2-3 d and for Salm. typhimurium.     

Detection of low numbers of Salmonella in environmental water, sewage and food samples by a nested polymerase chain reaction assay

A polymerase chain reaction (PCR) assay with two nested pairs of primers selected from conserved sequences within a 2.3 kb randomly cloned DNA fragment from the Salmonella typhimurium chromosome was developed. The nested PCR assay correctly identified 128 of a total of 129 Salmonella strains belonging to subspecies I, II, IIIb and IV. One strain of Salm. arizona (ssp. IIIa) tested negative. No PCR products were obtained from any of the 31 non-Salmonella strains examined. The sensitivity of the assay was 2 cfu, as determined by analysis of proteinase K-treated boiled lysates of Salm. typhimurium. The performance of the assay was evaluated for environmental water, sewage and food samples spiked with Salm. typhimurium. Water and sewage samples were filtered and filters were enriched overnight in a non-selective medium. Prior to PCR, the broth cultures were subjected to a rapid and simple preparation procedure consisting of centrifugation, proteinase K treatment and boiling. This assay enabled detection of 10 cfu 100 ml(-1) water with background levels of up to 8700 heterotrophic organisms ml(-1) and 10000 cfu of coliform organisms 100 ml(-1) water. Spiked food samples were analysed with and without overnight enrichment in a non-selective medium using the same assay as above. Nested PCR performed on enriched broths enabled detection of <10 cfu g(-1) food. Variable results were obtained for food samples examined without prior enrichment and most results were negative. This rapid and simple assay provides a sensitive and specific means of screening drinking water or environmental water samples, as well as food samples, for the presence of Salmonella spp.     

Occurrence and clonal relatedness of sec/tst-gene positive Staphylococcus aureus isolates of quartermilk samples of cows suffering from mastitis

AIMS: To investigate the prevalence of sec/tst-gene positive Staphylococcus aureus in bovine mastitis and to get information about the clonal relatedness of these clinical isolates. METHODS AND RESULTS: A total of 533 Staph. aureus strains isolated from bovine mastitic quartermilk samples at 493 randomized dairy farms in Hessia, Germany, from January 1997 until June 1998 were examined for enterotoxin C (sec) gene and toxic shock syndrome toxin (tst) gene by multiplex polymerase chain reaction. Fifty-three (9.3%) of the strains were sec/tst-gene positive. Phenotypic TSST-1 production was found in all positive strains by reversed passive latex agglutination test. With DNA macrorestriction analysis, sec/tst-gene positive strains were divided into five different macrorestriction types. Type I (10 isolates) and III (40 isolates) were found to be the predominant types in terms of frequency of isolation in the investigated area. These DNA macrorestriction types differed in only two bands in the 500 and 270 bp region. CONCLUSIONS: Closely related Staph. aureus strains seem to be responsible for an unusual large proportion of bovine mastitis cases in geographically widely distinct locations. SIGNIFICANCE AND IMPACT OF THE STUDY: This is one of the first reports about the relatedness of sec/tst-gene positive Staph. aureus clinical isolates from bovine mastitis.     

Hazard analysis applied to microbial growth in foods: Development of mathematical models describing the effect of water activity

Mathematical models have been developed which describe the effect of lowering the water activity on the growth kinetics of Staphylococcus aureus and Salmonella typhimurium. By treating the lag phase and exponential phase kinetics separately predictions can be made on the extent of microbial growth over successive time-temperature cycles. Staph. aureus was far more tolerant than Salm. typhimurium to lowered water activity and under near growth limiting conditions of water activity and temperature was showing lag periods as long as ca 40 d. The maximum lag period observed for Salm. typhimurium was ca 5 d. Under these conditions the predicted generation times for Staph. aureus were 2–3 d and for Salm. typhimurium 1–4 d.     

Antagonistic effects of intestinal Lactobacillus isolates on pathogens of chicken

L.Z. JIN, Y.W. HO, N. ABDULLAH, M.A. ALI AND S. JALALUDIN. 1996. Twelve Lactobacillus strains isolated from chicken intestine, which demonstrated a strong and moderate capacity to adhere to the ileal epithelial cells in vitro , were used to investigate their inhibitory ability against five strains of salmonella, i.e. Salmonella enteritidis 935/79, Salm. pullorum, Salm. typhimurium, Salm. blockley and Salm. enteritidis 94/448, and three serotypes of Escherichia coli , viz. E. coli O1 : K1, O2 : K1 and O78 : K80. The results showed that all the 12 Lactobacillus isolates were able to inhibit the growth of the five strains of salmonella, and the three strains of E. coli in varying degrees. Generally, they were more effective in inhibiting the growth of salmonella than E. coli . Inhibition of the pathogenic bacteria was probably due to the production of organic acids by the Lactobacillus isolates.     

Modulation of Salmonella infection by the lectins of Canavalia ensiformis (Con A) and Galanthus nivalis (GNA) in a rat model in vivo

The plant lectins, Concanavalin A (Con A) and Galanthus nivalis agglutinin (GNA) have been prefed to rats for 3 d pre- and 6 d postinfection with Salmonella typhimurium S986 or Salm. enteritidis 857. Con A significantly increased numbers of Salm. typhimurium S986 in the large intestine and in faeces, and severely impaired growth of the rats, more severely than is the case of infection with Salmonella typhimurium alone. Con A had much less effect on rats infected with Salm. enteritidis 857 only showing a significant increase in numbers in the colon, accompanied by intermittent increases of Salmonella in the faeces during the study. GNA significantly reduced pathogen numbers in the lower part of the small bowel and the large intestine of rats infected with Salm. typhimurium S986 and significantly improved rat growth. GNA had little effect on infection by Salm. enteritidis 857 with slight decreases in Salmonella numbers in the small intestine and large intestine and transient increases in the faeces.     

Sandwich capture ELISA by a murine monoclonal antibody against a genus-specific LPS epitope for the detection of different common serotypes of salmonellas.

A sandwich capture ELISA based on a murine monoclonal antibody against a genus-specific epitope in the outer core region of the Salmonella lipopolysaccharide is described for the detection of different common serotypes of salmonellas. Four h broth cultures of seven standard and 24 wild strains of salmonellas were all detected by the capture ELISA while overnight broth cultures of 21 non-salmonella standard strains were all negative. The capture ELISA detected 1 ng/ml of Ra lipopolysaccharide, 10(6)/ml of a smooth wild strain of Salm. typhimurium, and 1120 cells of Salm. heidelberg after enrichment culture for 4 h.     

Factors affecting the survival of Salmonella and Escherichia coli in anaerobically fermented pig waste

The survival of Salmonella typhimurium was investigated in acidogenic, anaerobically fermented pig wastes and in synthetic media, each containing volatile fatty acids (VFA). Salm. typhimurium survived at pH 6.8, but not at pH 4.0, when incubated at 37 degrees C for 24 h in either fermented or synthetic medium containing VFA. The minimum inhibiting concentration of VFA for Salm. typhimurium after 48 h incubation at 30 degrees C at pH 4.0 was 0.03 mol/l and for Escherichia coli it was 0.09 mol/l. Fermented pig wastes in a digester, maintained at pH 5.9, were inoculated with Salm. typhimurium and then incubated at 37 degrees C for 24 h. The pH was adjusted to either 4.0 or 5.0 and after a further 48 h at 30 degrees C, Salm. typhimurium survived at pH 5.0 but not at pH 4.0. It was concluded that pH is critical in determining the survival of this organism in acidogenic anaerobically fermented pig waste.     

Molecular typing of Salmonella enterica serovar Sofia in Australia by pulsed‐field gel electrophoresis and repetitive element PCR typing

Aims: In this study, we used two molecular fingerprinting methods to investigate the genetic and clonal relationship shared by Australian Salmonella Sofia isolates. Methods and Results: A total of 84 Australian Salm. Sofia isolates from various states in Australia were typed using pulsed‐field gel electrophoresis (PFGE) (XbaI and SpeI) and repetitive element PCR (REP1R‐I primer). The previous problem of DNA degradation of Salm. Sofia strains was solved by modifying the lysis solution used to treat the bacterial plugs, allowing Salm. Sofia to be subtyped using PFGE. Molecular typing of isolates resulted in the generation of eight XbaI, six SpeI and five REP1 pattern profiles. Individual typing methods showed low discrimination index values (<0·5), indicating the poor discriminatory ability of the methods. However, the combination of the typing methods was able to improve the discrimination of isolates, further dividing them into 16 subtypes and raising the index value to 0·721. Conclusions: The combination of typing methods was shown to be the best approach to fingerprint Salm. Sofia. The Australian Salm. Sofia isolates only showed limited genetic diversity and probably share a clonal relationship. A majority of the Salm. Sofia isolates were not geographically restricted with the predominant pattern subtype observed amongst the isolates from various states. Significance and Impact of the Study: We have successfully devised a PFGE protocol that counteracts DNase activity of Salm. Sofia, enabling typing of this serovar.