Transport to the Slaughterhouse Affects the Salmonella Shedding and Modifies the Fecal Microbiota of Finishing Pigs.
ABSTRACT: Contaminated pork is a significant source of foodborne Salmonellosis. Pork is contaminated at the slaughterhouse and the intestinal content is the predominant source of Salmonella for carcass contamination. The prevalence of Salmonella-positive pigs increases significantly when the time of transport to the slaughterhouse is longer than two hours. The hypothesis behind this study is that transport to the slaughterhouse increases the load of Salmonella in feces and determines a shift of the fecal microbiota in finishing pigs. Fecal samples were collected in a pig herd positive for Salmonella spp., the day before the transport and at the slaughterhouse. Salmonella loads were estimated by the most probable number (MPN) technique, according to the ISO/TS 6579-2:2012/A1. Moreover, the fecal bacteria composition was assessed by sequencing the V3-V4 hypervariable regions of the 16S rRNA gene. Our study showed that the load of Salmonella increases after transport, confirming that this phase of the production chain is a critical point for the control of Salmonella contamination. A lower richness and an increased beta-diversity characterized the fecal microbiota composition of Salmonella-positive animals after transport. In this stage, a natural Salmonella infection causes a disruption of the fecal microbiota as observed in challenge studies.
Project description:Salmonella spp. are estimated to cause 1.2 million cases of human foodborne illness each year in the United States, and pigs can often be asymptomatically colonized with Salmonella spp. (>50% of farms). Recent reports state that 18.3% of Salmonella enterica serovar Typhimurium isolates are resistant to ?3 antimicrobial classes, and multidrug-resistant (MDR) strains are associated with an increased hospitalization rate and other complications. Chlortetracycline is commonly used in swine production to prevent/treat various diseases; therefore, chlortetracycline treatment of pigs unknowingly colonized with MDR Salmonella may have collateral effects on Salmonella spp. (and other gut bacteria). In this study, we determined the effect of in-feed chlortetracycline (400?g/ton) on shedding and colonization of pigs challenged with the MDR S Typhimurium strain DT104 (n = 11/group). We also assessed the impact on the fecal microbiota over the 12-day experimental period and on the ileum, cecum, and tonsil microbiota at 7?days postinoculation (dpi). In MDR S Typhimurium-inoculated pigs, chlortetracycline administration significantly increased fecal shedding at 2?dpi (+1.4 log10 CFU/g; P?<?0.001) and enhanced tonsil colonization (+3.1 log10 CFU/g; P?<?0.001). There were few major alterations detected in the gut or tonsillar microbiota of pigs treated with MDR S Typhimurium and/or chlortetracycline. The tonsillar transcriptome was largely unaffected despite increased colonization by MDR S Typhimurium following inoculation of the chlortetracycline-treated pigs. These results highlight the idea that chlortetracycline administration can enhance shedding and colonization of MDR S Typhimurium in pigs, which could increase the risk of environmental dissemination of MDR Salmonella strains.IMPORTANCE Salmonella spp. are an important cause of foodborne illness in North America, and pork products are associated with sporadic cases and outbreaks of human salmonellosis. Isolates of Salmonella may be resistant to multiple antibiotics, and infections with multidrug-resistant (MDR) Salmonella spp. are more difficult to treat, leading to increased hospitalization rates. Swine operations commonly use antimicrobials, such as chlortetracycline, to prevent/treat infections, which may have collateral effects on pig microbial populations. Recently, we demonstrated that chlortetracycline induces the expression of genes associated with pathogenesis and invasion in MDR Salmonella enterica serovar Typhimurium in vitro In our current study, we show increased tonsillar colonization and fecal shedding of the MDR S Typhimurium strain DT104 from pigs administered chlortetracycline. Therefore, pigs unknowingly colonized with multidrug-resistant Salmonella spp. and receiving chlortetracycline for an unrelated infection may be at a greater risk for disseminating MDR Salmonella spp. to other pigs and to humans through environmental or pork product contamination.
Project description:Salmonella is the second most commonly reported zoonotic gastrointestinal pathogen in the European Union, and a significant proportion of the cases are linked to the consumption of contaminated pork. Reduction of Salmonella at the farm level helps to mininimise the contamination pressure at the slaughterhouse, and therefore the number of Salmonella bacteria entering the food chain. Cleaning and disinfection (C&D) between batches of pigs is an intervention measure that has potential to reduce the transmission of Salmonella contamination within farms. In this study, two pig finisher buildings in each of 10 Salmonella positive farms were sampled pre-C&D, post-C&D, post-restocking with the following batch of pigs, and shortly before these pigs were sent to slaughter. The incoming batch of pigs was also sampled before it reached the study building (pre-restocking). At each visit, pooled and individual faecal samples were collected and Salmonella isolation was carried out according to an ISO 6579:2002 Annex D-based method. One building on each farm (intervention) was cleaned and disinfected according to a rigorous protocol consisting of several steps and a Defra-approved disinfectant used at the General Orders concentration, whilst the other building (control) was cleaned and disinfected as per normal farm routine. At the post-C&D visit, Enterobacteriaceae and total bacterial counts were determined to evaluate residual faecal contamination and general hygiene levels. Rodent specialists visited the farms before and after C&D and rodent carcasses were collected for Salmonella testing. The intervention buildings were significantly less likely (p = 0.004) to be positive for Salmonella after C&D. The pre-restocking pigs had the highest likelihood (p<0.001) of being Salmonella positive (often with multiple serovars) and there was no significant difference between intervention and control buildings in Salmonella prevalence at the post-restocking visit (p = 0.199). However, the pigs housed in the intervention buildings were significantly less likely (p = 0.004) to be positive for Salmonella at slaughter age. Multivariable analysis suggested that cleaning all fixtures of buildings, leaving the pens empty for 2-3 days and using an effective disinfectant are factors significantly improving the likelihood of removing Salmonella contamination during C&D. Signs of rodents were recorded in all farms, but rodent activity and harbourage availability decreased between visits. All the rats tested were Salmonella negative. S. Typhimurium or its monophasic variants were isolated from 6 mouse carcasses in 3 farms where the same serovars were isolated from pigs. This study demonstrates that an appropriate C&D programme significantly reduces the likelihood of residual contamination in Salmonella positive pig buildings, and suggests a significant reduction in the prevalence of Salmonella in the pigs in appropriately cleaned and disinfected buildings when sampled before slaughter. Due to a high prevalence of infection in replacement pigs, control of Salmonella in pig farms is challenging. Rodents may also contribute to the carry-over of infection between batches. C&D is a useful measure to help reduce the number of infected pigs going to the slaughterhouse, but should be supplemented by other control measures along the pig breeding and production chain.
Project description:Pork meat is estimated to be responsible for 10-20% of human salmonellosis cases in Europe. Control strategies at the farm could reduce contamination at the slaughterhouse. One of the targeted sectors of production is maternity, where sows could be Salmonella reservoirs. The aim of this study was to assess the dynamics of shedding of Salmonella in terms of variation in both shedding prevalence and strains excreted during gestation in Quebec's maternity sector. The evolution of the fecal microbiota of these sows during gestation was also assessed to detect bacterial populations associated with these variations. A total of 73 sows both at the beginning and the end of the gestation were randomly selected and their fecal matter was analyzed. Salmonella detection was conducted using a method that includes two selective enrichment media (MSRV and TBG). Nine isolates per positive samples were collected. Among the 73 sows tested, 27 were shedding Salmonella. Sows in the first third of their gestation shed Salmonella significantly more frequently (21/27) than those in the last third (6/46) (?2P < 0.05). The shedding status of 19 of the sows that were previously sampled in the first third of their gestation was followed, this time in the last third of their gestation, which confirmed reduction of shedding. Using 16S rRNA gene sequencing and qPCR, significant differences between the fecal flora of sows at the beginning and the end of the gestation, shedding Salmonella or not and with different parity number were detected. Using MaAsLin, multiple OTUs were found to be associated with the time of gestation, the status of Salmonella excretion and parity number. Some of the identified taxa could be linked to the reduction of the shedding of Salmonella at the end of gestation. In this study, we showed that the level of Salmonella shedding was variable during gestation with significantly higher shedding at the beginning rather than at the end of gestation. We also observed for the first time a significant change in the microbiota during sow gestation and identified interesting taxa which could be linked to a reduced Salmonella shedding.
Project description:Livestock-associated methicillin-resistant <i>Staphylococcus aureus</i> (LA-MRSA) ST398 was recovered from infections in humans exposed to animals, raising public health concerns. However, contact with food producing chain as a means of transmission of LA-MRSA to humans remains poorly understood. We aimed to assess if pork production chain is a source of MRSA ST398 for human colonization and infection. MRSA from live pigs, meat, the environment, and slaughterhouse workers were analyzed by Pulsed-Field Gel Electrophoresis (PFGE), <i>spa</i>, MLST typing, SNPs and for antibiotic resistance and virulence gene profiles. We compared core and accessory genomes of MRSA ST398 isolated from slaughterhouse and hospital. We detected MRSA ST398 (t011, t108, t1451) along the entire pork production chain (live pigs: 60%; equipment: 38%; meat: 23%) and in workers (40%). All MRSA ST398 were multidrug resistant, and the majority carried genes encoding biocide resistance and enterotoxins. We found 23 cross-transmission events between live pigs, meat, and workers (6-55 SNPs). MRSA ST398 from infection and slaughterhouse environment belonged to the same clonal type (ST398, t011, SCC<i>mec</i> V), but differed in 321-378 SNPs. Pork production chain can be a source of MRSA ST398 for colonization of human slaughterhouse workers, which can represent a risk of subsequent meat contamination and human infection.
Project description:Nontyphoidal Salmonella spp. are a leading cause of human food poisoning and can be transmitted to humans via consuming contaminated pork. To reduce Salmonella spread to the human food chain, bacteriophage (phage) therapy could be used to reduce bacteria from animals' preslaughter. We aimed to determine if adding a two-phage cocktail to feed reduces Salmonella colonization in piglets. This first required spray drying phages to allow them to be added as a powder to feed, and phages were spray dried in different excipients to establish maximum recovery. Although laboratory phage yields were not maintained during scale up in a commercial spray dryer (titers fell from 3 × 10<sup>8</sup> to 2.4 × 10<sup>6</sup> PFU/g respectively), the phage titers were high enough to progress. Spray dried phages survived mixing and pelleting in a commercial feed mill, and sustained no further loss in titer when stored at 4°C or barn conditions over 6 months. Salmonella-challenged piglets that were prophylactically fed the phage-feed diet had significantly reduced Salmonella colonization in different gut compartments (<i>P</i> < 0.01). 16S rRNA gene sequencing of fecal and gut samples showed phages did not negatively impact microbial communities as they were similar between healthy control piglets and those treated with phage. Our study shows delivering dried phages via feed effectively reduces Salmonella colonization in pigs. <b>IMPORTANCE</b> Infections caused by Salmonella spp. cause 93.8 million cases of human food poisoning worldwide, each year of which 11.7% are due to consumption of contaminated pork products. An increasing number of swine infections are caused by multidrug-resistant (MDR) Salmonella strains, many of which have entered, and continue to enter the human food chain. Antibiotics are losing their efficacy against these MDR strains, and thus antimicrobial alternatives are needed. Phages could be developed as an alternative approach, but research is required to determine the optimal method to deliver phages to pigs and to determine if phage treatment is effective at reducing Salmonella colonization in pigs. The results presented in this study address these two aspects of phage development and show that phages delivered via feed prophylactically to pigs reduces Salmonella colonization in challenged pigs.
Project description:Salmonella colonization and infection in production animals such as pigs are a cause for concern from a public health perspective. Variations in susceptibility to natural infection may be influenced by the intestinal microbiota. Using 16S rRNA compositional sequencing, we characterized the fecal microbiome of 15 weaned pigs naturally infected with Salmonella at 18, 33, and 45?days postweaning. Dissimilarities in microbiota composition were analyzed in relation to Salmonella infection status (infected, not infected), serological status, and shedding pattern (nonshedders, single-point shedders, intermittent-persistent shedders). Global microbiota composition was associated with the infection outcome based on serological analysis. Greater richness within the microbiota postweaning was linked to pigs being seronegative at the end of the study at 11?weeks of age. Members of the Clostridia, such as Blautia, Roseburia, and Anaerovibrio, were more abundant and part of the core microbiome in nonshedder pigs. Cellulolytic microbiota (Ruminococcus and Prevotella) were also more abundant in noninfected pigs during the weaning and growing stages. Microbial profiling also revealed that infected pigs had a higher abundance of Lactobacillus and Oscillospira, the latter also being part of the core microbiome of intermittent-persistent shedders. These findings suggest that a lack of microbiome maturation and greater proportions of microorganisms associated with suckling increase susceptibility to infection. In addition, the persistence of Salmonella shedding may be associated with an enrichment of pathobionts such as Anaerobiospirillum. Overall, these results suggest that there may be merit in manipulating certain taxa within the porcine intestinal microbial community to increase disease resistance against Salmonella in pigs. IMPORTANCE Salmonella is a global threat for public health, and pork is one of the main sources of human salmonellosis. However, the complex epidemiology of the infection limits current control strategies aimed at reducing the prevalence of this infection in pigs. The present study analyzes for the first time the impact of the gut microbiota in Salmonella infection in pigs and its shedding pattern in naturally infected growing pigs. Microbiome (16S rRNA amplicon) analysis reveals that maturation of the gut microbiome could be a key consideration with respect to limiting the infection and shedding of Salmonella in pigs. Indeed, seronegative animals had higher richness of the gut microbiota early after weaning, and uninfected pigs had higher abundance of strict anaerobes from the class Clostridia, results which demonstrate that a fast transition from the suckling microbiota to a postweaning microbiota could be crucial with respect to protecting the animals.
Project description:Salmonella enterica serovar Typhimurium is an animal welfare and public health concern due to its ability to parasite livestock and potentially contaminate pork products. To reduce Salmonella shedding and the risk of pork contamination, antibiotic therapy is used and can contribute to antimicrobial resistance. Here we hypothesized that immune system education by the microbiota can play a role in intestinal resilience to infection. We used amoxicillin (15mg/Kg) to modulate the intestinal microbiome of 10 piglets, paired with same age pigs that received a placebo (n = 10) from 0 to 14 days of age. Animals were euthanized at 4-weeks old. Each pig donated colon sections for ex vivo culture (n = 20 explants/pig). Explants were inoculated with S. Typhimurium, PBS or LPS (n = 6 explants/pig/group, plus technical controls). The gut bacteriome was characterized by sequencing of the 16S rRNA at 7, 21 days of age, and upon in vitro culture. Explants response to infection was profiled through high-throughput mRNA sequencing. In vivo antibiotic treatment led to ?-diversity differences between groups at all times (P<0.05), while ?-diversity did not differ between amoxicillin and placebo groups on day 21 and at euthanasia (P<0.03 on day 7). Explant microbiomes were not different from in vivo. In vitro challenge with S. Typhimurium led to lower necrosis scores in explants from amoxicillin-treated pigs, when compared to explants placebo-treated pigs (P<0.05). This was coupled with the activation of immune-related pathways in explants from amoxicillin-treated pigs (IL-2 production, NO production, BCR activation), when compared to placebo-treated pigs. In addition, several DNA repair and intestinal wound healing pathways were also only activated in explants from amoxicillin-treated pigs. Taken together, these findings suggest that immune education by the amoxicillin-disturbed microbiota may have contributed to mitigate intestinal lesions following pathogen exposure.
Project description:Salmonella Typhimurium infections in pigs often result in the development of carriers that intermittently excrete Salmonella in very low numbers. During periods of stress, for example transport to the slaughterhouse, recrudescence of Salmonella may occur, but the mechanism of this stress related recrudescence is poorly understood. Therefore, the aim of the present study was to determine the role of the stress hormone cortisol in Salmonella recrudescence by pigs. We showed that a 24 h feed withdrawal increases the intestinal Salmonella Typhimurium load in pigs, which is correlated with increased serum cortisol levels. A second in vivo trial demonstrated that stress related recrudescence of Salmonella Typhimurium in pigs can be induced by intramuscular injection of dexamethasone. Furthermore, we found that cortisol, but not epinephrine, norepinephrine and dopamine, promotes intracellular proliferation of Salmonella Typhimurium in primary porcine alveolar macrophages, but not in intestinal epithelial cells and a transformed cell line of porcine alveolar macrophages. A microarray based transcriptomic analysis revealed that cortisol did not directly affect the growth or the gene expression or Salmonella Typhimurium in a rich medium, which implies that the enhanced intracellular proliferation of the bacterium is probably caused by an indirect effect through the cell. These results highlight the role of cortisol in the recrudescence of Salmonella Typhimurium by pigs and they provide new evidence for the role of microbial endocrinology in host-pathogen interactions.
Project description:Leptospirosis, brucellosis, and Q fever (coxiellosis) are bacterial zoonoses that cause acute febrile illness in people as well as reproductive losses in pigs. Pig keeping is an increasingly important livelihood to millions of smallholder farmers in Uganda because of exponential increases in demand for pork. The prevalence of leptospirosis and Q fever in pigs is unknown, and the few studies of porcine brucellosis have estimated a range of seroprevalence. Therefore, we undertook a prevalence survey of leptospirosis, brucellosis, and Q fever in pigs using quantitative real-time PCR to determine the potential importance of these zoonoses to the growing pig sector in Uganda. Six hundred forty-nine pigs were sampled in 2015-2016 at an urban pork slaughterhouse. Ten percent of pigs (<i>n</i> = 68) had leptospiral DNA in either their kidney or reproductive tissue. In adjusted analyses, variables predictive of leptospiral status included female sex (odds ratio [OR]: 2.37, <i>P</i> < 0.01) and pigs sampled in March 2016 (OR: 2.23, <i>P</i> = 0.02) and October 2016 (OR: 0.30, <i>P</i> = 0.04). DNA fingerprinting revealed circulation of at least four distinct serovars in these pigs. <i>Brucella</i> spp. and <i>Coxiella burnetii</i> DNA were not detected in any sampled pig. This is the first report of widespread circulation of pathogenic <i>Leptospira</i> spp. in pigs in Uganda, suggesting that leptospirosis likely has a greater impact on the health of pigs than was previously recognized. Pig farmers, pig traders, and slaughterhouse workers may be at greatest occupational risk because of their direct contact with infective leptospires in aborted fetuses, bodily fluids, and other tissues.
Project description:Hepatitis E virus (HEV) is the causative agent of acute hepatitis E. Genotype 3 (G3) and 4 (G4) HEV have recently been identified in and isolated from swine as the main HEV genotypes worldwide. However, there is limited information on HEV infection status among pigs in Taiwan, especially pigs in the stage before transportation to the slaughterhouse. To determine the frequency of HEV infection among pigs in Taiwan, we detected and quantified HEV RNA contained in 295 fecal specimens collected from 6-month-old pigs bred in 30 pig farms located in 8 counties. We found that 25.1% (74/295) of the fecal specimens were positive for HEV RNA by a quantitative real-time reverse transcription-polymerase chain reaction, and the copy number ranged from 2.3?×?103 to 2.08?×?107 copies/g. Amplification of a 338 bp sequence in ORF2 was achieved in 16 of 74 HEV RNA-positive samples, and their nucleotide sequences were determined. Two HEV sequences appeared to belong to subtype 3a of G3 and the remaining 14 HEV sequences belonged to subtype 4b of G4 (G4b). The entire genome sequence of two G4b HEVs was obtained by next-generation sequence analyses, and the phylogenetic analyses indicated that unique G4b HEVs were circulating in pig farms in Taiwan. In the present study, we found that both G3 and G4 HEVs were circulating in Taiwanese pig farms and G4b was the predominant subtype. In addition, the relatively high detection frequency of HEV RNA in the 6-month-old pigs indicated that Taiwanese pigs just before transportation to the slaughterhouse are at risk of carrying HEVs, and thus thorough cooking or heating of pork meat or organs is needed before consumption in Taiwan and possibly in other countries as well.