Molecular typing of Salmonella enterica serovar Enteritidis isolates from food-producing animals in Japan by multilocus variable-number tandem repeat analysis: evidence of clonal dissemination and replacement

Background

Salmonella enterica serovar Enteritidis is a zoonotic pathogen. Human infections are associated with contaminated eggs and egg products. In Japan, since 1989, the incidence of food-borne disease caused by S. Enteritidis has increased and a pandemic has occurred; however, little is known about changes that occurred before and after this pandemic event in the dominant lineage of isolates from food-producing animals. This study aimed to determine the S. Enteritidis lineages in Japan over the last few decades by using multilocus variable-number tandem repeat analysis (MLVA).

Findings

MLVA was used to analyse 79 S. Enteritidis isolates collected from chickens (n = 63), cattle (n = 12), pigs (n = 2), and goats (n = 2) during 1975–2009. The S. Enteritidis isolates showed 14 different MLVA allele combinations, which were classified into two major clusters (A and C) and a minor cluster (B). All the 62 isolates in cluster A were isolated after 1988, whereas 13 of the 17 isolates belonging to cluster B and C were isolated before 1989.

Conclusions

The MLVA results showed that cluster C was predominant before 1989, and isolates in cluster A disseminated since 1989 and replaced the previous dominant clone, suggesting that isolates of cluster A originated from imported S. Enteritidis infection.     

6-hydroxydopamine-mediated release of norepinephrine increases faecal excretion of Salmonella enterica serovar Typhimurium in pigs

Salmonella enterica serovar Typhimurium is an animal and zoonotic pathogen of worldwide importance. In pigs, transport and social stress are associated with reactivation and spread of Salmonella Typhimurium infection. The stress-related catecholamine norepinephrine (NE) has been reported to activate growth and virulence factor expression in Salmonella; however the extent to which NE contributes to stress-associated salmonellosis is unclear. We studied the impact of releasing NE from endogenous stores during Salmonella Typhimurium infection of pigs by administration of 6-hydroxydopamine (6-OHDA), which selectively destroys noradrenergic nerve terminals. Treatment of pigs with 6-OHDA 7 or 16 days post-oral inoculation with Salmonella Typhimurium produced elevated plasma NE levels and transiently, but significantly, increased faecal excretion of the challenge strain. Oral administration of NE to Salmonella Typhimurium-infected pigs also transiently and significantly increased shedding; however pre-culture of the bacteria with NE did not alter the outcome of infection. Salmonella has been proposed to sense and respond to NE via a homologue of the adrenergic sensor kinase QseC. A ΔqseC mutant of Salmonella Typhimurium was consistently excreted in lower numbers than the parent strain post-oral inoculation of pigs, though not significantly so. 6-OHDA treatment of pigs infected with the ΔqseC mutant also increased faecal excretion of the mutant strain, albeit to a lesser extent than observed upon 6-OHDA treatment of pigs infected with the parent strain. Our data support the notion that stress-related catecholamines modulate the interaction of enteric bacterial pathogens with their hosts.     

Evaluation of PCR inhibitory effect of enrichment broths and comparison of DNA extraction methods for detection of Salmonella Enteritidis using real-time PCR assay

The best enrichment broth and DNA extraction scheme was determined for rapid and sensitive detection of Salmonella Enteritidis in steamed pork using real-time PCR. The inhibitory effect of commonly used Salmonella enrichment broths, Rappaport-Vassiliadis (RV) and Muller-Kauffmann tetrathionate with novobiocin (MKTTn), on real-time PCR was confirmed. The inhibition of PCR was statistically significant (p < 0.05) in RV and MKTTn, as compared with buffered peptone water (BPW) or phosphate-buffered saline. The inhibitory effect of the selective enrichment media was successfully removed by using a modified DNA extraction, PrepMan Ultra Reagent with an additional washing step or the DNeasy Tissue Kit. In three experiments, when applied to detection of Salmonella Enteritidis in steamed pork, the real-time PCR coupled with single 24 h enrichment with BPW performed better than double 48 h enrichment with BPW plus RV or MKTTn. The simple real-time PCR assay using BPW proved to be a rapid and sensitive test for detection of low concentrations of Salmonella Enteritidis in steamed pork samples as compared with the conventional culture method.     

Characterization of Salmonella enterica serovar Typhimurium and Salmonella enterica serovar 4,[5],12:i:- isolates from pigs presenting with diarrhea in Korea

Between 2011 and 2012, a total of 896 pig fecal samples were collected from nine provinces in Korea, and 50 salmonella enterica susp. enterica serovar Typhimurium (S. Typhimurium) was isolated. The characteristics of the 50 strains were analyzed, and 4 strains were identified as Salmonella enterica subsp. enterica serovar 4,[5],12:i:-. Salmonella 4,[5],12:i:- could not be distinguished from S. Typhimurium through phage typing, antimicrobial resistance testing or multiple-locus variable-number tandem repeat analysis (MLVA). However, among the four Salmonella 4,[5],12:i:- strains, one (KVCC-BA1400078) was identified as a Salmonella 4,[5],12:i:- clone isolated from humans in the United States, and another (KVCC-BA1400080) was identified as DT193, which has been primarily isolated from humans and animals in European countries. The presence of Salmonella 4,[5],12:i:- in Korea poses a significant threat of horizontal transfer between pigs and humans.     

Identification of Salmonella spp. isolates from chicken abattoirs by multiplex-PCR

The present study was carried out to report the occurrence Salmonella spp., Salmonella Enteritidis, and Salmonella Typhimurium in chicken abattoirs. Samples of feces; feathers; scald, evisceration, and chiller water; and rinse water of non-eviscerated, eviscerated, and chilled carcass were collected from six chicken abattoirs. Salmonella isolates were identified by a multiplex-PCR using three sets of primers targeting the invA, pefA, and sefA gene sequences from Salmonella spp., S. Typhimurium and S. Enteritidis, respectively. Salmonella spp. was detected in 10% (29/288) of the samples, whereas serovars Enteritidis and Typhimurium were identified in 62% (7/288), respectively. The results indicate the need to improve hygiene and sanitary standards in poultry slaughter lines, besides the education of food handlers and information to consumers.     

Structural characterization and serological specificities of lipopolysaccharides from Salmonella enterica serovar Gallinarum biovar Pullorum standard, intermediate and variant antigenic type strains

The structure and serological specificities of the lipopolysaccharides (LPSs) from Salmonella enterica serovar Gallinarum biovar Pullorum were studied to provide an improved basis for the distinction between antigenic types and the development of improved diagnostic tests. The structure of the LPS O-polysaccharide (O-PS) from S. Pullorum standard, intermediate and variant antigenic type strains was determined by mass spectrometry, nuclear magnetic resonance spectroscopy and chemical analysis. The LPS of the three strains shared a common structural repeating oligosaccharide unit containing d-mannose, l-rhamnose, d-galactose and d-tyvelose (1:1:1:1). The O-PS of the variant type LPS contained an additional d-glucose residue linked to the O-4 position of the d-galactose residue. The O-PS of the intermediate type LPS was partially the same as that of the variant LPS, however, the molar ratio of the d-glucose component was lower with respect to the other glycose components. Serological specificities of the three antigenic type LPSs were examined with anti-S. Pullorum LPS monoclonal antibodies (Mabs). On immunoblots, Mabs to the standard type O-PS reacted with high molecular mass (HMM) and low molecular mass (LMM) LPS from the standard strain, and with LMM but not HMM LPS from the variant strain. Monoclonal antibodies to the variant type O-PS reacted with HMM but not LMM LPS from the variant strain, and did not react with HMM or LMM LPS from the standard strain. On ELISA, the standard, intermediate and variant antigenic type strains were differentiated by the relative reactivity with the anti-LPS O-PS Mabs. Several of the anti-LPS O-PS Mabs were specific for S. Pullorum and other serogroup D1 Salmonella, and are potentially useful for the development of improved diagnostic tests for these organisms.     

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.     

Dissemination and tracking of Salmonella spp. in integrated broiler operation

Controlling Salmonella in integrated broiler operation is complicated because there are numerous potential sources of Salmonella contamination, including chicks, feed, rodents, wild poultry operations, and the processing plant. The objective of this study was to investigate the distribution of Salmonella through all phases of two integrated broiler operations and to determine the key areas related to the control of all known sources of infection. Two different Salmonella serotypes were observed at integrated broiler chicken company A. S. enteritidis, the predominant company A isolate, was consistently found in the breeder farm, hatcheries, broiler farms, and chicken slaughterhouse. At company B, a total of six different serotypes, S. heidelberg, S. senftenberg, S. enteritidis, S. blockley, S. gallinarum, and S. virchow, were detected. Although S. heidelberg was not found in the broiler farms, it was consistently found in the breeder farm, hatcheries, and chicken slaughterhouse. In addition, S. enteritidis was found in the hatcheries, broiler farm, and chicken slaughterhouse. In order to obtain the genetic clonality, 22 S. enteritidis isolates were digested with XbaI and analyzed by pulsed-field gel electrohporesis (PFGE). A difference in the PFGE pattern was found to be related to the origin of the integrated broiler operation. These data support the critical need to control Salmonella in breeder farms and hatcheries, and demonstrate important points related to the control of infection in large-scale poultry operations of Korea.     

A questionnaire-based,retrospective field study of persistence of Salmonella Enteritidis and Salmonella Typhimurium in Danish broiler houses

A questionnaire-based, retrospective field study was conducted in 78 Danish broiler houses (analytical units) on 42 farms. In spring 1997, all these broiler houses had been infected with Salmonella Enteritidis, phage type 8, and/or Salmonella Typhimurium, definitive-type 66, by day-old chicks delivered from the same hatchery. Because these two salmonella types had not been detected in the Danish broiler sector before and the broiler houses were infected in a period of low salmonella prevalence, the study could focus on farm-related factors, without being distorted by other salmonella infections.

The same person visited all 78 broiler houses, and farm-related factors were listed both from personal observations and by interrogating the person in charge of attending to the broilers. Additional factors (noted at the visits) were recorded in a later telephone interview. Altogether, 187 variables were analyzed.

Broiler houses were divided into single-infection houses (which had one or both of the salmonella types in only one crop), and multiple-infection houses (where infection occurred in multiple crops). This was done both generally and separately for each of the salmonella types.

Factors associated with house status—but that were beyond the control of the farmer—were the salmonella type and the percentage of salmonella positive samples in the first crop. Factors which could be controlled by the farmer and which were associated with reduced prevalence of multiple-infection houses included antiseptic soap and water for washing hands in the anteroom, hygiene barriers when removing dead broilers, gravel alongside the broiler house, systematic checks of indoor rodent-bait depots, and combined surface and pulse-fogging disinfection.     

Analysis of Salmonella enterica serotype Enteritidis isolated from human and chickens by repetitive sequence-PCR fingerprinting, antibiotic resistance and plasmid profiles

A total of 22 Salmonella enterica serotype Enteritidis (S. Enteritidis) strains isolated from human and chicken were subjected to DNA fingerprinting by repetitive sequence PCR using ERIC and BOX primers, antibiotic resistance and plasmid patterns. Both ERIC and BOX PCR amplification data revealed a highly genetic homogeneity between isolates from human and chicken except one isolate, which originated from chicken and showed a different DNA band pattern from others. Eleven of 22 S. Enteritidis isolates (50%) were resistant to more than one antibiotics and characterized by 5 resistance patterns. The most common pattern was penicillin resistant (63.6%). Only one isolate from chicken showed a multiple drug resistance patterns to 4 antibiotics. All 22 S. Enteritidis isolates harbored more than two plasmids with eight different plasmid profiles including two to six plasmids with approximate molecular size ranging from 1.9 to 21 kb. A band of 15 kb size was detected in all isolates tested, however, the band sizes smaller than 15 kb were found only in isolates from chicken.     

Effect of Drinking-Water Administration of Experimental Chlorate Ion Preparations on Salmonella enterica serovar Typhimurium Colonization in Weaned and Finished Pigs

Foodborne disease caused by Salmonella is of public health and economic significance. In order to assess the practical effectiveness of a new intervention strategy, experimental chlorate preparations (ECP) were administered via the drinking water to weaned and finished pigs that had been orally challenged the previous day with 10(9)-10(10) colony-forming units of Salmonella serovar Typhimurium. After 24 or 36 h ad libitum access to 0X, 1X or 2X ECP treatment (where X is the concentration estimated to deliver a minimal daily effective dose), the pigs were euthanized and gut contents and lymph tissue collected at necropsy were cultured for the challenge Salmonella. Drinking water administration of ECP effectively reduced (p < 0.05) caecal Salmonella concentrations and, with the weaned pigs, tended (p < or = 0.10) to reduce rectal Salmonella concentrations. No negative effects of ECP treatment on water intake and animal wellbeing were observed and only marginal effects on gut fermentation characteristics occurred. The bactericidal effect of administering ECP in drinking water was relatively rapid, with reductions in caecal Salmonella concentrations occurring within 24 h. These results suggest that ECP administered to pigs just days before slaughter may reduce gut concentrations of Salmonella; however, the impacts of such reductions on slaughter hygiene have yet to be determined.     

链球菌、金黄色葡萄球菌、沙门菌和大肠杆菌多重PCR检测方法的建立及应用

根据NCBI上已收录的链球菌ef-tu基因、金黄色葡萄球菌nuc基因、沙门菌hut基因和大肠杆菌23SrRNA基因的序列,设计并合成4对特异性引物,通过优化多重PCR的反应条件,建立了能够同时检测4种细菌混合感染的多重PCR诊断方法。特异性分析结果表明,应用该方法可以从链球菌、金黄色葡萄球菌、沙门菌和大肠杆菌以及4种细菌的混合物中扩增出4条大小分别为197、278、495和652bp的特异性条带,其他对照组的检测结果均为阴性;敏感性分析表明,该方法对4种病原菌基因组DNA的检出量分别为链球菌25.6pg、金黄色葡萄球菌33.2pg、沙门菌35.7pg、大肠杆菌52.1pg;人工模拟感染样本检测表明,该方法能从混合感染的病料中特异地检测出4种病原菌。本试验建立的多重PCR方法具有特异性强,敏感度高,稳定性好的特点,可以有效的检测链球菌、金黄色葡萄球菌、沙门菌和大肠杆菌的混合感染。     

Systemic and mucosal immunity induced by attenuated Salmonella enterica serovar Typhimurium expressing ORF7 of porcine reproductive and respiratory syndrome virus

Oral administration of attenuated Salmonella vaccine may provide valuable advantages such as low cost, easy preparation, and safety. Attenuated Salmonella vaccines also serve as carriers of foreign antigens and immunomodulatory cytokines. Presently, an attenuated Salmonella enterica serovar Typhimurium strain was used as a carrier for open reading frame 7 (ORF7) protein of porcine reproductive and respiratory syndrome virus (PRRSV), a swine pathogen of significant global economic importance. Initially, an attenuated S. enterica serovar Typhimurium expressing ORF7 gene derived from PRRSV Korean isolate was constructed. Following oral administration of a single dose of the attenuated Salmonella vaccine expressing PRRSV ORF7, humoral and cell-mediated immune responses specific for ORF7 were induced at both systemic and mucosal sites including spleen, mesenteric lymph node, Peyer's patch, and laminar propria, as evaluated by determining serum ORF7-specific IgG and mucosal IgA responses, as well as Th1- and Th2-type cytokine production from antigen-stimulated T cells. The induced humoral responses were sustained for at least 12 weeks post-immunization. In particular, the immunized mice displayed immune responses to both the foreign ORF7 antigen and Salmonella itself. The results indicate the value of attenuated S. enterica serovar Typhimurium as an oral carrier of PRRSV antigenic proteins to induce effective systemic and mucosal immunity.     

Evaluation of Salmonella enterica serovar Typhimurium and Choleraesuis slyA mutant strains for use in live attenuated oral vaccines

SlyA protein plays a key role in virulence in Salmonella enterica. In this study, we evaluated the ability of the slyA mutant strain of S. enterica serovar Choleraesuis (S. choleraesuis) to protect against swine salmonellosis. Using a murine model infected with S. enterica serovar Typhimurium (S. typhimurium), we showed that the Salmonella strain with a deletion of slyA could be used as a highly immunogenic, effective and safe vaccine in mice. Based on these data, a slyA mutant of S. enterica serovar Choleraesuis strain RF-1 was constructed, and the ability of this mutant to protect immunized pigs from S. choleraesuis infection was examined. As with the S. typhimurium slyA mutant, immunization of pigs with the S. choleraesuis slyA mutant strain provided significant protection against subsequent challenge by the wild-type RF-1. These results demonstrate that SlyA is a potential target in the development of a novel live attenuated vaccine against S. enterica.     

Isolation and identification of Salmonella spp. from red foxes (Vulpes vulpes) and badgers (Meles meles) in northern Italy

Background

Salmonella spp. have been isolated from a wide range of wild animals. Opportunistic wild carnivores such as red foxes (Vulpes vulpes) and badgers (Meles meles) may act as environmental indicators or as potential sources of salmonellosis in humans. The present study characterizes Salmonella spp. isolated from the intestinal contents of hunted or dead red foxes (n = 509) and badgers (n = 17) in northern Italy.

Findings

Thirty-one strains of Salmonella belonging to 3 Salmonella enterica subspecies were isolated. Fourteen different serovars of S. enterica subsp. enterica were identified, among which were serovars often associated with human illness.

Conclusions

Wild opportunistic predators can influence the probability of infection of both domestic animals and humans through active shedding of the pathogen to the environment. The epidemiological role of wild carnivores in the spread of salmonellosis needs to be further studied.     

A survey of Salmonella spp and Campylobacter spp in dairy goat faeces and bulk tank milk in the Murcia region of Spain

This study was designed to investigate the occurrence of Salmonella spp and Campylobacter spp in faeces samples from 222 healthy Murciano-Granadina dairy goats reared on 12 farms in Spain and in samples of bulk tank milk from 11 of those herds. Neither Salmonella spp nor Campylobacter spp were isolated from any of the samples. Our results suggest that, under the management practices applied to this breed in Spain, Murciano-Granadina goats are not likely to be a significant reservoir for these food-borne pathogens.     

Application of a multiplex PCR assay for Campylobacter fetus detection and subspecies differentiation in uncultured samples of aborted bovine fetuses

Campylobacter (C.) fetus (epsilonproteobacteria) is an important veterinary pathogen. This species is currently divided into C. fetus subspecies (subsp.) fetus (Cff) and C. fetus subsp. venerealis (Cfv). Cfv is the causative agent of bovine genital Campylobacteriosis, an infectious disease that leads to severe reproductive problems in cattle worldwide. Cff is a more general pathogen that causes reproductive problems mainly in sheep although cattle can also be affected. Here we describe a multiplex PCR method to detect C. fetus and differentiate between subspecies in a single step. The assay was standardized using cultured strains and successfully used to analyze the abomasal liquid of aborted bovine fetuses without any pre-enrichment step. Results of our assay were completely consistent with those of traditional bacteriological diagnostic methods. Furthermore, the multiplex PCR technique we developed may be easily adopted by any molecular diagnostic laboratory as a complementary tool for detecting C. fetus subspecies and obtaining epidemiological information about abortion events in cattle.     

Evaluation of efficacy of a new live Salmonella Typhimurium vaccine candidate in a murine model

Efficacy of a new live Salmonella Typhimurium vaccine candidate attenuated by two virulence gene deletions was evaluated in this study. Live form of the vaccine was orally administered and formalin-inactivated form was intramuscularly (IM) administered. 105 female BALB/c mice were used and divided into 5 groups, A to E, containing 21 mice per group. Serum IgG and secretory IgA titers and levels of serum IFN-γ, IL-4, TNF-α, IL-12 of the immunized groups, especially group C (oral prime-oral booster) significantly increased. In addition, all animals in groups C showed no clinical symptoms and survived the virulent challenges, whereas all or some of mice from the other groups died of the challenge. These indicate that the vaccine candidate can be an effective tool for prevention of Salmonella infections by inducing robustly protective immune responses and leading to the production of inflammatory cytokines. Booster immunization, especially via oral administration, is necessary to optimize its protection.     

Improvement of bacterial clearance and relief of clinical signs of Salmonella enterica serovar Typhimurium infection in pigs through upregulation of Th 1-specific responses by administration of a combination of two silicate minerals,biotite and bentonite

Biotite and bentonite are phyllosilicate minerals that were originally used inindustrial applications. Several beneficial activities of them have recently beenreported, especially regulation of the immune system and antimicrobial effects. Therefore,we investigated the immune-enhancing and bacterial clearance effects of a biotite andbentonite mixture (BBM) on experimental infection of Salmonella entericaserovar Typhimurium (S. Typhimurium) to determine whether the BBM couldbe used as an alternative antibiotic. We administered 1% or 2% BBM as a feed supplement.We then evaluated the bacterial clearance effects of the BBM against S.Typhimurium. We also evaluated the immune-enhancing effect of the BBM through severalimmunological experiments that included examination of the lysozyme activity,CD4⁺/CD8⁺ T lymphocyte ratio and the T-helper type 1 (Th 1)cytokine profile. The clinical signs of S. Typhimurium and the number ofviable bacteria in feces and tissues were significantly decreased in both BBM groups,especially in the 2% BBM group. The BBM also markedly enhanced the lysozyme activity,CD4⁺/CD8⁺ T lymphocyte ratio and expression levels of IFN-γ andIL-12 in S. Typhimurium-challenged pigs. Therefore, the BBM could be agood candidate as an alternative antibiotic that improves Th 1-specific immune responsesand the bacterial clearance effect.