Herd-level diagnosis for Salmonella enterica subsp. enterica serovar Dublin infection in bovine dairy herds

Herd-level sensitivities of bacteriological and serological methods were compared in 79 bovine dairy herds, recently infected with Salmonella enterica subsp. enterica serovar Dublin. All farms experienced clinical signs of salmonellosis for the first time and had no history of vaccination against salmonellosis. At the start of the study, infection with serovar Dublin was confirmed with at least one positive bacteriologic culture for serovar Dublin from a clinical case (gold standard for herd infection).Bacteriological culture was done on samples of dung-pits, drinking water, bulk-milk filters, and faeces of animals with current or earlier clinical signs of salmonellosis. Blood samples of all animals and bulk-milk samples were tested using an ELISA.Herd-level sensitivity (HSe) of culture of dung-pits, drinking water, bulk-milk filters, and faeces of animals with current or earlier signs of salmonellosis was 45, 5, 7, and 38%, respectively. HSe for serology of all animals was 100%. If blood samples of all calves 4-6 months old were examined, at least one calf was seropositive on 91% of the infected farms. If serology was performed on samples of animals with current or earlier signs of salmonellosis, at least one animal was seropositive on 80% of the infected farms. HSe for bulk-milk samples was 54%. However, if clinical signs of salmonellosis were observed only in lactating animals, sensitivity of bulk-milk serology was 79%.Interesting combinations of methods were the combination of serology of bulk milk with either serology of animals with current or earlier signs of salmonellosis (HSe=91%), or serology of all calves of 4-6 months old (HSe=99%).     

Risk factors for Salmonella enterica subsp. enterica infection in senegalese broiler-chicken flocks

Our objective was to assess the association of managerial practices, general hygiene and Salmonella infection in Senegalese broiler flocks. Seventy broilers farms were studied from January 2000 to December 2001 around Dakar. A questionnaire was submitted to the farmers and samples of fresh broiler droppings were taken. A 28.6% of the flocks were infected by Salmonella (mainly Hadar and Brancaster serovars). Salmonella infection of the previous flock (OR=6.82) and of day-old chicks (OR=3.73), frequent poultry farmers’ visits (OR=5.38) and keeping sick birds inside the farm (OR=5.32) increased the risk of Salmonella infection. But, using antibiotics on day-old chicks (OR=0.17) and a detergent for cleaning (OR=0.16) decreased the risk.     

Typing of Salmonella enterica subsp. enterica serovar Mbandaka isolates

Recently, Salmonella enterica subsp. enterica serovar Mbandaka (S. Mbandaka) has gained some importance in the epidemiology of salmonellosis in Poland. Since biotyping, resistance typing, and plasmid profiling were insufficient for strain differentiation, genome macrorestriction by means of pulse-field gel electrophoresis (PFGE) was applied and proved to be the method of choice in S. Mbandaka epidemiological studies. XbaI and BcuI macrorestriction produced 15 and 14 pulse-field profiles (PFP), respectively, but in the case of each enzyme one profile was prevalent. When macrorestriction profiles were combined, a total 24 patterns were found. Based on the similarity of the profiles, four clonal lineages were identified. One clonal lineage contained the majority of poultry, feed and human isolates. Poultry was concluded to be an important source of S. Mbandaka for humans in Poland. Complementary use of various typing techniques improved efficacy of epidemiological studies giving possibility to subdivide S. Mbandaka into 35 types and the index of discrimination reached 0.947.     

Mitsuokella jalaludinii inhibits growth of Salmonella enterica serovar Typhimurium

Salmonella continues to be a significant human health threat, and the objective of this study was to identify microorganisms with the potential to improve porcine food-safety through their antagonism of Salmonella. Anaerobic culture supernatants of 973 bacterial isolates from the gastrointestinal tract and feces of swine were screened for their capacity to inhibit the growth of Salmonella enterica serovar Typhimurium. Growth inhibition of 1000-fold or greater was observed from 16 isolates, and 16S rRNA sequencing identified the isolates as members of the genera Mitsuokella, Escherichia/Shigella, Anaerovibrio, Selenomonas, and Streptococcus. Four isolates were identified as Mitsuokella jalaludinii, and the mechanism of Salmonella Typhimurium growth inhibition by M. jalaludinii was further investigated. M. jalaludinii stationary phase culture supernatants were observed to significantly inhibit growth, and featured the production of lactic, succinic, and acetic acids. Aerobic and anaerobic S. Typhimurium growth was restored when the pH of the culture supernatants (pH 4.6) was increased to pH 6.8. However, S. Typhimurium growth in fermentation acid-free media was the same at pH 4.6 and pH 6.8 - indicating a synergistic effect between fermentation acid production and low pH as the cause of S. Typhimurium growth inhibition. Furthermore, exposure of S. Typhimurium to M. jalaludinii culture supernatants inhibited Salmonella invasion of HEp-2 cells by 10-fold. The results identify M. jalaludinii as a possible inhibitor of Salmonella growth and invasion in swine, and thus a potential probiotic capable of improving food safety.     

Risk factors for Salmonella enterica subsp. enterica shedding by market-age pigs in French farrow-to-finish herds

Fattening-pigs carriers of Salmonella enterica are believed to be a main source of carcass and pork contamination at the later steps of the meat process. We did a prospective study in 2000-2001 in 105 French farrow-to-finish pig farms. In each farm, a batch of contemporary fattening pigs housed in the same room was followed throughout the fattening period. Salmonella shedding was assessed on environmental samples of faecal material (taken by means of pairs of gauze socks) analysed by classical bacteriological methods. 36.2% of the batches studied had at least one contaminated environmental sample and therefore were classified as Salmonella-shedding batches. Logistic regression was used to assess the association between managerial and hygiene practices and health status and the shedding risk at the end of the finishing period. Emptying the pit below the slatted floor after the previous batch of sows was removed and frequent removal of sow dung during the lactation period were protective. Presence of residual Salmonella contamination of the floor and pen partitions in the fattening rooms before loading the growing pigs also was a risk factor. The risk for Salmonella shedding at the end of the fattening period was increased when dry feed (versus wet feed) was provided during the fattening period. Lastly, Lawsonia intracellularis seroconversion and PRRSV seropositivity during the fattening period also was a risk factor for Salmonella shedding.     

Phenotypic and genetic characterization of Salmonella enterica subsp. enterica serovar Typhimurium isolated from pigs in Rio Grande do Sul, Brazil

This study aimed to investigate the relatedness of porcine Salmonella enterica subsp. enterica (S.) serovar Typhimurium strains isolated in Southern Brazil. Sixty-six isolates from pigs belonging to three commercial companies were submitted to phage typing, XbaI-macrorestriction (PFGE), IS200 hybridization, rep-PCR, antimicrobial susceptibility testing, and PCR assay targeting the spvR region. All strains presented a unique rep-PCR pattern and 63 strains had a common IS200 profile. One pulse-type (XA) was the most prevalent (39/66 strains) and included strains of phage types DT177, DT192, DT194 and RDNC. The spvR region was detected in three strains, which harboured plasmids of 90 kb. High rates of tetracycline, sulfonamide and streptomycin resistance were found. Isolates from farms located in different geographic regions but associated to the same commercial companies clustered together and presented a common resistance profile. Results suggested that clonal groups of S. Typhimurium are present in pig commercial companies in Southern Brazil.     

Phenotypic and genetic characterization of Salmonella enterica subsp. enterica serovar Typhimurium isolated from pigs in Rio Grande do Sul,Brazil

This study aimed to investigate the relatedness of porcine Salmonella enterica subsp. enterica (S.) serovar Typhimurium strains isolated in Southern Brazil. Sixty-six isolates from pigs belonging to three commercial companies were submitted to phage typing, XbaI-macrorestriction (PFGE), IS200 hybridization, rep-PCR, antimicrobial susceptibility testing, and PCR assay targeting the spvR region. All strains presented a unique rep-PCR pattern and 63 strains had a common IS200 profile. One pulse-type (XA) was the most prevalent (39/66 strains) and included strains of phage types DT177, DT192, DT194 and RDNC. The spvR region was detected in three strains, which harboured plasmids of 90 kb. High rates of tetracycline, sulfonamide and streptomycin resistance were found. Isolates from farms located in different geographic regions but associated to the same commercial companies clustered together and presented a common resistance profile. Results suggested that clonal groups of S. Typhimurium are present in pig commercial companies in Southern Brazil.     

Risk factors associated with Salmonella enterica subsp. enterica contamination of chicken carcases in Senegal

The objective of this study was to identify the risk factors for Salmonella spp. contamination of Senegalese chicken carcases during slaughtering. One hundred and twenty traditional slaughterhouses were studied from January 2000 to December 2002 in and around Dakar. A questionnaire was administered to the slaughterers and samples of breast skin were taken to assess the Salmonella spp. status of chicken carcases. Results showed that 43.3% of the chicken batches were contaminated with Salmonella spp., with Salmonella Hadar and Salmonella Brancaster as the two main serovars. Salmonella spp. contamination of the live birds before slaughtering was related to contamination of the carcases after slaughtering. Feed withdrawal before slaughtering and thorough cleaning and disinfection procedures decreased the risk of Salmonella contamination. One individual worker for each slaughtering stage was also associated with a decreased risk of Salmonella contamination. Using scalding water for plucking increased the risk of contamination. These results will help slaughterers to produce safer products for local consumers.     

Risk factors associated with Salmonella enterica subsp. enterica contamination of chicken carcases in Senegal

This study was to identify the risk factors for Salmonella spp. contamination of Senegalese chicken carcases during slaughtering. One hundred and twenty traditional slaughterhouses were studied from January 2000 to December 2002 in and around Dakar. A questionnaire was answered by the slaughterers, and samples of breast skin were taken to assess the Salmonella status of chicken carcases. Results showed that 43.3% of the batches were contaminated with Salmonella, indicating Salmonella Hadar and Salmonella Brancaster as the two main serovars. Salmonella contamination of the carcases after slaughtering was related to contamination of the live birds. Feed withdrawal before slaughtering and thorough cleaning and disinfection procedures decreased the risk of contamination. One individual worker for each slaughtering stage was also associated with a decreasing risk of contamination. Using scalding water for plucking the chicken carcases increased contamination risk. These results will help slaughters to produce safer products for local consumers.     

Molecular analysis of Salmonella enterica subsp. enterica serovar Agona isolated from slaughter pigs

Salmonella enterica subsp. enterica (S.) serovar Agona plays an important role in Brazil as causative agent of salmonellosis in food-producing animals - in particular, pigs and poultry - as well as in humans. A total of 45 S. Agona isolates collected from slaughter pigs at three different slaughterhouses in Southern Brazil was investigated in this study for their phenotypic and genotypic relatedness. For this, the antimicrobial susceptibility patterns and the phage types were determined. Molecular analysis included the determination of plasmid profiles as well as the analysis of XbaI- and BlnI-generated macro-restriction patterns. Moreover, a novel typing method called subtracted restriction fingerprinting (SRF) was successfully applied to the S. Agona isolates. Based on all properties determined, a dominant clonal group comprising 33 of the 45 isolates was identified. Members of this group were susceptible to all antimicrobials tested, did not carry plasmids, shared the same phage type and were closely related or even indistinguishable by their EcoRI-PauI SRF patterns as well as their XbaI and BlnI macro-restriction patterns. Members of this clonal group were identified at all 3 slaughterhouses at variable frequencies and originated from pig herds raised in 15 different cities in Southern Brazil which were located up to 450 km apart from each other. Since the S. Agona-carrying slaughter pigs were from various integrated production lines, the results of this study suggest that a specific clonal group of S. Agona had entered numerous pig production lines. This observation supports the requirement for the establishment of monitoring and control programmes in Brazil which should also include molecular techniques to better trace the dissemination of S. Agona and other Salmonella serovars in pigs and other food-producing animals.     

Phenotypic and genotypic differentiation of porcine Salmonella enterica subsp. enterica serovar Derby isolates

Sixty-two Salmonella enterica subsp. enterica serovar Derby isolates from slaughter pigs and meat products isolated in Southern Brazil were analyzed for their genomic relationships and for the presence of antimicrobial resistance genes. Twenty-four S. Derby isolates were indistinguishable by their subtracted restriction fingerprinting (SRF) pattern, XbaI- and BlnI-macrorestriction patterns, phage type, plasmid profile, and resistance pattern. In contrast to the BlnI-macrorestriction patterns, the XbaI-macrorestriction patterns were in good agreement with the results of SRF analysis and phage typing. Among the four phage types detected, PT10 and PT21 were the most common. The combination of all typing methods revealed a great diversity among the S. Derby isolates. All strains carried plasmids and the 60 resistant isolates showed at least tetracycline resistance. The resistance genes found were sul1 and/or sul2 (sulfonamide resistance), aadA2 (streptomycin/spectinomycin resistance), tet(A) (tetracycline resistance), tet(B) (tetracycline/minocycline resistance), bla(TEM) (ampicillin resistance), and dfrA14 (trimethoprim resistance). A correlation of the geno- and phenotypic characteristics with the origin of the isolates revealed a substantial temporal variation in the occurrence of specific S. Derby isolates in different independent pig production lines in Southern Brazil. The large number of resistant isolates underlined the potential risk that S. Derby isolates can pose to human health when they enter the food chain.     

Phage types, ribotypes and tetracycline resistance genes of Salmonella enterica subsp. enterica serovar Typhimurium strains isolated from different origins in Italy

The tetracycline resistance (tet) gene patterns of 52 tetracycline resistant Salmonella enterica subsp. enterica (S.) serovar Typhimurium isolates collected from animals, food of animal origin, and humans in Italy, were investigated to evaluate whether the tet gene patterns could be used for strain differentiation in addition to phage typing and ribotyping. The detection of tet genes was performed by specific PCR assays. Ribotyping was performed automatically using PvuII as restriction enzyme. Ten different ribotyping patterns were detected. All isolates were positive for at least one of the tet genes studied and six different tet gene patterns were observed. Ribotyping and tet gene patterns showed discriminatory indices of 0.741 and 0.812, respectively. Multiple tet genes were commonly found among tetracycline resistant S. typhimurium isolates from various sources. The resulting tet gene patterns allowed further discrimination of strains which were otherwise indistinguishable by their phage type, ribotype and origin. Thus, the analysis of tet gene patterns might represent an additional tool for the differentiation of S. typhimurium isolates.     

Comparison of Salmonella enterica subspecies enterica serovar Typhimurium isolates from dairy cattle and humans using in vitro assays of virulence

Salmonella enterica subspecies enterica serovar Typhimurium (Salmonella typhimurium) can infect and cause disease in a wide range of host species however there have been suggestions that this serovar may have genes involved with host range or specificity [Tsolis, R.M., Townsend, S.M., Miao, E.A., Miller, S.I., Ficht, T.A., Adams, L.G., Baumler, A.J., 1999. Identification of a putative S. enterica serotype Typhimurium host range factor with homology to IpaH and YopM by signature-tagged mutagenesis. Infect. Immun. 67 (12), 6385-6393]. Our goal in this study was to determine if in vitro virulence assays would support this suggestion. Twelve human and 10 bovine isolates of S. typhimurium from a single county in California were evaluated using in vitro virulence assays of adhesion and invasion. The resulting data was combined with results from previously reported genotypic and phenotypic testing of the isolates and statistical analysis performed using multivariate general linear models. Human isolates had higher adhesion values in each of the statistical models tested (p<0.05) but no statistical differences were found in the invasion values of human and bovine source isolates. Both adhesion and invasion values differed between the two largest groups of isolates segregated on the basis of pulsed-field gel patterns. The findings suggest there may be genetically defined in vitro virulence attributes in S. typhimurium that are associated with host species.     

Salmonella enterica serovar Typhimurium and Enteritidis infection of pigs and cytokine signalling in palatine tonsils

Pigs are considered as one of the major sources of zoonotic strains of Salmonella enterica for humans. Out of many S. enterica serovars, S. Typhimurium dominates in pigs, however, in several countries in Central Europe, S. Enteritidis is also quite frequent in pig herds. In this study we therefore compared the colonisation of pigs with S. Typhimurium and S. Enteritidis. We found that 3 weeks after infection S. Enteritidis 147 colonised the intestinal tract in higher quantities but was shed in faeces in lower quantities than S. Typhimurium 17C10. In a second experiment we found out that S. Enteritidis 147 and its SPI-1 and SPI-4 mutants increased proinflammatory cytokine (IL-1β and IL-8) signalling in the ileum 5 days post infection. On the other hand, independent of SPI-1 or SPI-4, S. Enteritidis 147 suppressed expression of IL-18, MCP1, TLR2, CD86, IL-7, IL-10 and IL-15 in the palatine tonsils. The suppression of cytokine signalling may facilitate the initial colonisation of the palatine tonsils by Salmonella. Moreover, immune suppression may also influence pig resistance to opportunistic pathogens and Salmonella infection in pigs thus may become an issue not only in terms of pork contamination but also in terms of affecting the immunological status of pig herds.     

Pulsed field gel electrophoresis for animal Salmonella enterica serovar Typhimurium isolates in Taiwan

Salmonella enterica subspecies enterica serovar Typhimurium is a common pathogen for humans and animals. In order to trace the clonal relationship and to find the circulating strains between human and animal isolates, chromosomal DNAs from 87 serovar Typhimurium strains isolated from animals (pigs were the majority) were subjected to XbaI and SpeI digestion and pulsed field gel electrophoresis (PFGE). For the 87 animal isolates, 38 PFGE pattern combinations were obtained. As the subtyping results from animal isolates were compared with those from the 45 human isolates, it was found that 14 of the animal isolates and 13 of the human isolates shared a common PFGE pattern combination, i.e., pattern XgSf (or called X5S4). When these human and animal isolates were subjected to antibiotic susceptibility test using 11 antibiotics, it was found that strains of pattern XgSf (X5S4) belong to a common antibiogram pattern which is tetracycline, gentamicin, ampicillin, streptomycin and chloramphenicol resistant. Since most of the animal and human strains in pattern XgSf were originally isolated from various areas over different years, strains of this PFGE pattern may be the most epidemic strains which circulating between human and animal sources.     

Spatial distribution of antibodies to Salmonella enterica serovar Typhimurium O antigens in bulk milk from Texas dairy herds

Environmental factors that enhance either the survivability or dispersion of Salmonella enterica serovar Typhimurium (S. Typhimurium) could result in a spatial pattern of disease risk. The objectives of this study were to: (1) describe herd status based on antibody response to Salmonella Typhimurium as estimated from bulk tank milk samples and (2) to describe the resulting geographical patterns found among Texas dairy herds. Eight hundred and fifty-two bulk milk samples were collected from georeferenced dairy farms and assayed by an indirect enzyme-linked immunosorbent assay (ELISA) using S. Typhimurium lipopolysaccharide (LPS). ELISA signal-to-noise ratios for each bulk tank milk sample were calculated and used for geostatistical analyses. Best-fit parameters for the exponential theoretical variogram included a range of 438.8 km, partial sill of 0.060 and nugget of 0.106. The partial sill is the classical geostatistical term for the variance that can be explained by the herd's location and the nugget is the spatially random component of the variance. We have identified a spatial process in bulk milk tank titers for S. Typhimurium in Texas dairy herds and present a map of the expected smoothed surface. Areas with higher expected titers should be targeted in further studies on controlling Salmonella infection with environmental modifications.     

Herd-level risk factors for Salmonella enterica subsp. enterica in U.S. market pigs

Midwest U.S. herds (n = 63) were studied to identify risk factors for harboring Salmonella enterica among slaughter-weight pigs. Samples collected on farms (feces) and at slaughter (distal colonic content, cecal content and ileocolic lymph nodes) were cultured using conventional means. Approximately 15 pigs were studied per herd, for a total of 3754 samples. The proportion of pigs positive in one or more samples was calculated for each herd. Herd characteristics were described by a combination of interview and written survey. Logistic regression was used to detect relationships between the detection of Salmonella and potential herd-level risk factors. The mean individual pig prevalence was 5% for feces, 4% for distal colonic content, 15% for ileocolic lymph nodes, and 17% for cecal contents. One or more Salmonella isolates were detected in at least one sample type in every herd. The five most common serovars were S. Agona, S. Derby, S. Schwarzengrund, S. Typhimurium and S. Senftenberg, with 25 additional serovars detected. Salmonella prevalence estimates were positively correlated among all samples except distal colonic content and ileocolic lymph nodes. Pigs with culture positive fecal samples were at increased odds of being detected positive for each of the slaughter-collected samples examined, namely distal colonic content (OR = 30.5), ileocolic lymph nodes (OR = 12.9) and cecal content (OR = 23.2). Herds with positive fecal sample(s) had increased odds of having positive cecal content (OR > 1.5), distal colonic content (OR = 15.3) and ileocolic lymph nodes (OR = 12.7). Pigs from herds with at least some bowl drinkers had eight-fold higher odds of testing Salmonella positive than did pigs from herds with only nipple drinkers. Pigs from herds with only dry feeders had five-fold higher odds of testing Salmonella positive when compared with pigs from herds with combinations of wet/dry style feeders. Interventions at these two points should be considered when designing growing pig facilities to reduce Salmonella shedding.     

Risk factors for Salmonella enterica subsp. enterica contamination in French broiler-chicken flocks at the end of the rearing period.

Broiler-chicken are often Salmonella carriers. However, these bacteria are responsible for major food-borne human infection, in which poultry-meat products are frequently implicated. In order to prevent Salmonella spread during the slaughtering process, control measures should be implemented at the farm level to reduce the prevalence before slaughtering. The objective of this study was to identify the risk factors for Salmonella contamination in French commercial broiler flocks at the end of the rearing period. A prospective study was carried out in 1996 and 1997 on 86 broiler flocks located in western France. The Salmonella status of the flocks was assessed by means of litter swabs and dust samples analyzed with classical bacteriological methods. Sixty flocks (70%) had at least one contaminated environmental sample and were classified as Salmonella-contaminated flocks. Logistic regression was used to assess association of managerial practices, general hygiene and results of environmental Salmonella recovery, with the odds that the flock itself would be Salmonella-contaminated at the end of the rearing period. Salmonella contamination of the house before placing day-old chicks and the Salmonella contamination of day-old chicks were significantly related to Salmonella contamination of the flock at the end of the rearing period. The risk for Salmonella contamination of the flock was increased when feed trucks parked near the entrance of the change room and when feed meal, instead of small pellets, was provided at the start.     

Insertional mutation of marA vitiates inducible multiple antimicrobial resistance in Salmonella enterica subsp. enterica serovar Choleraesuis

marA has been shown to mediate a multiple antimicrobial resistance (MAR) phenotype following induction in some members of the Enterobacteriaceae. When Salmonella Choleraesuis was exposed to inducing agents they displayed higher minimal inhibitory concentrations (MIC) to multiple antimicrobial agents and an increase in marA expression as determined by northern hybridization analysis. The objective of the present study was to determine if mutation of marA vitiated multiple antimicrobial resistance inducibility in S. Choleraesuis. A loss-of-function mutation of marA in a single S. Choleraesuis isolate was created by insertion of the dihydrofolate reductase (DHFR) gene cassette within marA using double homologous recombination. This mutation was complemented with an expression plasmid possessing marA under the control of an IPTG-inducible promoter. Mutation and complementation of marA was verified using polymerase chain reaction, Northern hybridization, and Western blotting assays. Minimum inhibitory concentrations (MICs) of tetracycline, chloramphenicol, nalidixic acid, and rifampin were determined against induced and uninduced wildtype, marA-disrupted and marA-complemented strains using a microbroth dilution assay. Minimum inhibitory concentrations against induced wildtype and marA-complemented strains increased four- to eight-fold for all antimicrobials tested when compared to the uninduced strains while the MICs of the induced marA-disrupted mutant remained the same. However, this increase was abrogated when the cells were grown in the presence of the efflux pump inhibitor compound EPI phe-arg-naphthylamide. The results indicate that a functional marA is solely required for an inducible multiple antimicrobial resistance phenotype in S. Choleraesuis.