Auxotrophic, plasmid-cured Salmonella enterica serovar typhimurium for use as a live vaccine in calves

Calves were vaccinated orally, subcutaneously or intraperitoneally with a smooth, plasmid-cured strain of Salmonella enterica serovar typhimurium, strain 81. Oral vaccination was not effective, as only 1/5 calves survived challenge with virulent S. typhimurium. Strain 81 was attenuated for calves, as only a slight rise in rectal temperatures was detected after vaccination. The organism was excreted by some calves in the faeces, but no signs of diarrhoea were observed after vaccination. After parenteral vaccination, strain 81 was able to reach the intestines, gastric associated lymphoid tissues and other internal lymphoid tissues and remained viable for up to 14 days in the bovine host. After oral challenge with a virulent strain, 9/10 vaccinated calves survived challenge as opposed to 4/10 control calves (p<0.5). Diarrhoea was present in all calves of the control groups, but in only 4/10 of the vaccinated calves. The clinical reactions of the vaccinated calves were milder than in the control calves, as the rises in rectal temperatures were lower, diarrhoea was less severe, and the challenge strain was present in fewer organs from vaccinated calves than control calves. This study showed that parenterally administered Salmonella vaccines can induce both mucosal and systemic immunity, and it is postulated that this capability of strain 81 is related to its colonisation of lymphoid tissues and other systemic and intestinal tissues. This study confirmed that plasmid-cured strains were attenuated in the bovine host and conferred significant protection after parenteral vaccination, but not oral vaccination.     

Distribution and hybridization patterns of the insertion element IS900 in clinical isolates of mycobacterium paratuberculosis

Reference strains and 31 clinical isolates of M. paratuberculosis, mainly from goats, were analysed for restriction fragment length polymorphism (RFLP). Restriction digests of bacterial DNA were hybridized with a repetitive insertion sequence, IS900, to obtain banding patterns for comparison of strains. Twenty-five of the 31 field-strains hybridized with IS900, and five hybridization patterns were identified. It was not possible to identify specific patterns for goat strains of M. paratuberculosis. Four hybridization patterns were similar, whereas the fifth pattern of a sheep strain diverged considerably in position and number of band. Six goat strains failed to hybridize with IS900, and the absence of IS900 was verified by the polymerase chain reaction and hybridization with an oligonucleotide probe. The six IS900-negative goat strains had diverging phenotypic properties, and the identification of these strains is discussed. The present study shows that M. paratuberculosis strains infecting goats are genetically similar to cattle strains and that IS900 is a specific genetic element for identification of M. paratuberculosis.     

Herd-level risk factors for faecal shedding of Salmonella enterica in Spanish fattening pigs

Herd-level risk factors for faecal shedding of Salmonella enterica were investigated in a cross-sectional study on Spanish finishing units. For this purpose, 10 faecal samples were collected from 10 different pens containing pigs close to market weight in a total of 232 fattening units. The total sample size was proportionally distributed according to the fattener census in each of the regions and provinces of the country in order to ensure a sample representative of the entire swine population. All samples were individually examined by culture of 25 g of faecal material. Data regarding characteristics and management of each fattening unit were collected by means of a questionnaire. Logistic regression was used to detect relationships between the detection of faecal shedding of S. enterica and potential herd-level risk factors. The feeding of pelleted feed was associated with an increased risk of culture-positive faecal samples (OR = 2.28; 95% CI: 1.22, 4.26). The odds of a farm being Salmonella positive were associated with its size. Fattening units that slaughtered more than 3500 pigs per year had a higher risk for Salmonella faecal shedding (OR = 1.78; 95% CI: 0.96, 3.31). Interventions at these two points should be considered when designing or managing growing pig facilities to reduce Salmonella faecal shedding by fatteners.     

Age at primary infection with Salmonella enterica serovar Typhimurium in the chicken influences persistence of infection and subsequent immunity to re-challenge

Salmonella enterica remains one of the most important food-borne pathogens of humans and is often acquired through consumption of infected poultry meat or eggs. Control of Salmonella infections in chicken is therefore an important public health issue. Infection with S. enterica serovar Typhimurium results in a persistent enteric infection without clinical disease in chickens of more than 3 days of age, and represents a source for contamination of carcass at slaughter and entry into the human food chain. Data presented indicate a profound effect of age at initial exposure on the persistence of infection and a lesser effect on the development of effective immunity to re-challenge. The percentage of birds positive for Salmonella was high until 8–9 weeks of age, regardless of the age at which the birds were infected (1, 3 or 6 weeks). The birds infected at 3 and 6 weeks of age produced a more rapid and higher antibody response (IgY and IgA) than those infected at 1 week of age, but in all cases infection persisted for a considerable period despite the presence of high antibody levels. Following a re-challenge infection with S. Typhimurium, all three previously-infected groups had fewer bacteria in the gut, spleen and liver compared with age-matched birds receiving a parallel primary infection. However, the birds primary infected at 3 and 6 weeks of age cleared infection more rapidly than those infected at a younger age. Interestingly older-primed birds had higher specific T lymphocyte proliferative responses and specific circulating levels of IgY antibody at time of re-challenge. Although birds initially infected at 1 week of age and those that were previously uninfected produced a stronger antibody response following re-challenge, they were slower to clear Salmonella from the gut than the older-primed groups which expressed a stronger T lymphocyte response. The data presented indicate that clearance of Salmonella from the gut is age-dependent and we propose that this relates to the increased competence of the enteric T cell response. The findings that Salmonella persists beyond 8–9 weeks, irrespective of age at exposure, has implications for the broiler sector and indicates the need to remain Salmonella free throughout the rearing period. Moreover, the re-challenge data demonstrates that infection at a young age is less effective in producing protective immunity than in older chickens. This feature of the development of protective immunity needs to be considered when developing vaccines for the broiler sector of the poultry industry.     

Salmonella enterica level in French pig farms effluents: Experimental and field data

The study aimed to (1) validate the mini-MSRV-MPN method to quantify Salmonella enterica in pig slurry, (2) estimate the effect and interaction on temperature, time and initial Salmonella load on the survival of the 2 strains of Salmonella typhimurium (PF 1690 and DT104 100/706/037) during slurry storage and (3) identify Salmonella contaminated finishing pig batches and assess the level of contamination of their slurry. The mini-MSRV-MPN method was compared to direct isolations on brilliant green agar supplemented with rifampicin to quantify Salmonella in pig slurry. Doelhert uniform shell design was used to study the effect of different parameters on the survival of the 2 strains of Salmonella in pig slurry. Environmental samples of faecal material and a sample of the slurry of 50 batches of finishing pigs were analysed by bacteriological classical method to identify Salmonella. Quantification was performed on pools of faeces and in slurry using the mini-MSRV-MPN technique. This method proved to be suited to quantify Salmonella in pig slurry. Temperature, time of slurry storage and their interaction influenced Salmonella decrease. 12 batches of pigs tested Salmonella positive. Quantification was possible in 5 batches of faecal samples (2.4–350 MPN g^− 1 of Salmonella). Quantification was achieved in 2 out of 6 positive samples of slurry (1.6 and 110 MPN mL^− 1 of Salmonella).     

Herd prevalence of Salmonella enterica infections in Danish slaughter pigs determined by microbiological testing

As a part of a nationwide programme to survey and control salmonella in pig herds, a microbiological survey of 1363 pig herds was performed in Denmark. A total of 13 468 slaughter pigs were examined at slaughter by culture of 5 g of caecal contents. Overall, 30 different serotypes of Salmonella enterica were isolated from 832 pigs (6.2%). The predominant serotype was S. Typhimurium, comprising 536 (64.4%) of the isolates. Four hundred and forty-eight isolates of S. Typhimurium were examined by phage typing, resulting in detection of 17 different phage types (definitive types, DT) with DT12 being the most frequent (49.1%).

Salmonella enterica was found in 302 herds (22.2%), S. Typhimurium was found in 61.1% of these. 279 (23.1%) large herds (producing more than 2600 slaughter pigs per year) were found to be salmonella positive compared with 23 (14.7 %) small herds (annual production of 500 to 550 slaughter pigs). Practical constraints in the study design did not allow for a firm conclusion on the interplay among herd size, geographical location and occurrence of salmonella.

In 284 of 302 infected herds (94.0%) only one serotype was detected. Infections with two different serovars were seen in 18 herds (6.0%).     

Proteomic analysis of intestinal mucosa responses to Salmonella enterica serovar typhimurium in naturally infected pig

Salmonella enterica serovar typhimurium (S. typhimurium) is one of the most frequent Salmonella serotypes isolated from European pigs. Despite the advances in understanding the mechanisms involved in host–pathogen interactions and host cell responses to S. typhimurium, the global change that occurs in naturally exposed populations has been poorly characterized. Here, we present a proteomics study on intestinal mucosa of pigs naturally infected with S. typhimurium, in order to better understand the pathogenesis of salmonellosis and the pathways which might be affected after infection. Samples were analyzed by 2D-DIGE and 44 different proteins exhibited statistically significant differences. The data set was analyzed by employing the Ingenuity Pathway Analysis and the physiological function most significantly perturbed were immunological and infectious disease, cellular assembly and organization and metabolism. The pathways implicated in the porcine immune response to S. typhimurium were gluconeogenesis and Rho GDI/RhoA signaling, and our results suggest that keratins and the intermediate filaments could play an important role in the damage of the mucosa and in the success of infection. The role of these findings in salmonellosis has been discussed, as well as the importance of analyzing naturally infected animals to have a complete picture of the infection. Also, we compared the results found in this work with those obtained in a similar study using experimentally infected animals.     

Deletion of sodCI and spvBC in Salmonella enterica serovar Enteritidis reduced its virulence to the natural virulence of serovars Agona, Hadar and Infantis for mice but not for chickens early after infection

Salmonella enterica serovars Typhimurium, Enteritidis, Dublin, Choleraesuis or Gallinarum can colonise liver and spleen in particular hosts while infections with serovars Infantis, Agona, Hadar, etc. are usually limited to gastrointestinal tract. Reasons for this behavior are unknown, although it has been shown that sodCI and spv genes exhibit a strict distribution between more and less virulent serovars and they influence Salmonella virulence. However to what extent the presence or absence of these genes is associated with the increased virulence of serovars which possess them has never been addressed experimentally. In this study we therefore first confirmed the exclusive association of spvB and sodCI genes with the former group of serovars. In the next step we removed these two genes from S. Enteritidis genome and compared the virulence of such a mutant with the virulence of S. Infantis, S. Agona and S. Hadar for chickens and highly sensitive Balb/C mice. Single strain infection showed that the deletion of these two genes from S. Enteritidis resulted in the reduction of its virulence for mice but not for chickens. Mixed infection further confirmed these observations and indicated that in mice but not in chickens the virulence of sodCI and spv mutant was reduced to the natural virulence of serovars Infantis, Agona and Hadar. Although sodCI and spv genes do not influence S. Enteritidis virulence for chickens directly, they may be of an indirect effect through the increased persistence of S. Enteritidis in mice and increased probability of the reintroduction of S. Enteritidis into poultry flocks.     

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.     

Passive protection against Salmonella enterica serovar Enteritidis infection from maternally derived antibodies of hens vaccinated with a ghost vaccine

This study evaluated maternal immunity against Salmonella enterica serovar Enteritidis acquired through the egg yolk. Two-hundred 19-week-old specific pathogen free (SPF) broiler breeders which were randomly divided into two groups of equal size were injected with S. Enteritidis ghosts (5 × 10⁹ colony forming units in 0.1 ml per hen) and phosphate-buffered saline (PBS, 0.01 mol⋅l⁻¹, pH 7.4) twice, respectively, with an interval of 2 weeks. An indirect enzyme-linked immunosorbent assay (ELISA) was applied to detect specific antibodies against S. Enteritidis. S. Enteritidis-specific antibody levels in the vaccinated group increased over time and were significantly higher than those of the control group on days 28 (P < 0.001) and 35 (P < 0.001) post-vaccination. Ten 7-day-old chicks from hens that were vaccinated with a S. Enteritidis ghost vaccine were challenged at 14 days of age with 5 × 10⁹ CFU of S. Enteritidis DH091 (homologous to the vaccine strain), 8/10 (80%) chicks from vaccinated hens survived, whereas 3/10 (30%) chicks from unvaccinated hens survived. The chicks acquired high levels of serum antibodies against S. Enteritidis. These results reveal that maternal antibodies in chicks acquired from vaccinated hens through eggs can confer a significant protection against S. Enteritidis infection.     

Genetic diversity,anti‐microbial resistance,plasmid profile and frequency of the Vi antigen in Salmonella Dublin strains isolated in Brazil

Salmonella Dublin is strongly adapted to cattle causing enteritis and/or systemic disease with high rates of mortality. However, it can be sporadically isolated from humans, usually causing serious disease, especially in patients with underlying chronic diseases. The aim of this study was to molecularly type S. Dublin strains isolated from humans and animals in Brazil to verify the diversity of these strains as well as to ascertain possible differences between strains isolated from humans and animals. Moreover, the presence of the capsular antigen Vi and the plasmid profile was characterized in addition to the anti‐microbial resistance against 15 drugs. For this reason, 113 S. Dublin strains isolated between 1983 and 2016 from humans (83) and animals (30) in Brazil were typed by PFGE and MLVA. The presence of the capsular antigen Vi was verified by PCR, and the phenotypic expression of the capsular antigen was determined serologically. Also, a plasmid analysis for each strain was carried out. The strains studied were divided into 35 different PFGE types and 89 MLVA‐types with a similarity of ≥80% and ≥17.5%, respectively. The plasmid sizes found ranged from 2 to >150 kb and none of the strains studied presented the capsular antigen Vi. Resistance or intermediate resistance was found in 23 strains (20.3%) that were resistant to ampicillin, ciprofloxacin, chloramphenicol, imipenem, nalidixic acid, piperacillin, streptomycin and/or tetracycline. The majority of the S. Dublin strains studied and isolated over a 33‐year period may descend from a common subtype that has been contaminating humans and animals in Brazil and able to cause invasive disease even in the absence of the capsular antigen. The higher diversity of resistance phenotypes in human isolates, as compared with animal strains, may be a reflection of the different anti‐microbial treatments used to control S. Dublin infections in humans in Brazil.     

Analysis of the immunoproteome of Mycoplasma mycoides subsp. mycoides small colony type reveals immunogenic homologues to other known virulence traits in related Mycoplasma species

Contagious bovine pleuropneumonia (CBPP) caused by Mycoplasma mycoides subsp. mycoides small colony type (MmmSC) has been eradicated in the developed world, but it is still present in many countries of sub-Saharan Africa. After initially successful control measures in the 1960s it has been spreading due to a lack of money, fragmentation of veterinary services, uncontrolled cattle movement, insufficient vaccine efficacy and sensitivity of current diagnostic tests.In this study we used two-dimensional polyacrylamide gel electrophoresis followed by immunoblot with sera from MmmSC-infected animals and MALDI-ToF mass spectrometry to identify novel immunogenic proteins as candidate molecules for improved diagnostics and vaccines. We identified 24 immunogens recognized by pooled sera from experimentally infected cattle. Furthermore, a serum from an animal with acute clinical disease as well as severe pathomorphological lesions recognized 13 additional immunogens indicating variation in the antibody responses to CBPP amongst cattle. Most immunogens showed compelling similarity to protein/gene sequences in the two ruminant pathogens M. capricolum subsp. capricolum and M. mycoides subsp. mycoides large colony type both belonging to the mycoides cluster. Three of these proteins, namely glycerol-3-phosphate oxidase, adenylosuccinate synthase, and glyceraldehyde-3-phosphate dehydrogenase, had no compelling homologue in the other distantly related bovine pathogen M. agalactiae. In addition, translation elongation factor Tu, heat shock protein 70, pyruvate dehydrogenase, and FKBP-type peptidyl-prolyl isomerase, which have been found to mediate adhesion to host tissue in other mycoplasmas were shown to be expressed and recognized by sera. These proteins have potential for the development of improved diagnostic tests and possibly vaccines.     

Occurrence and Characterization of Escherichia coli O157 Isolated from Cattle in Norway

Faecal samples from 504 imported beef cattle were screened to investigate the occurrence of Escherichia coli O157. The results were compared with those from a previous screening of Norwegian dairy cattle, and the occurrence was found to be higher in the imported beef cattle. The E. coli O157 isolates from the previous and present studies were characterized for the genes encoding for shigatoxin 1 (stx ₁), shigatoxin 2 (stx ₂), the intimin protein (eae) and the flagellar protein H7 (fliC) using PCR analysis, pulsed-field gel electrophoresis (PFGE) with the restriction enzyme XbaI, and bacteriophage lambda RFLP analysis using the PvuII restriction enzyme. The isolates from the dairy and beef cattle could be distinguished by the profiles of the toxin genes and by PFGE patterns. Whether the importation of animals in itself should be regarded as a risk factor for the occurrence of E. coli O157, or whether other management factors contribute to the differences in carrier rates compared to the previous study on domestic cattle, is discussed.