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One of the commonly used methods to control on-farm Salmonella outbreaks in the poultry industry is vaccination. Vaccine diluents may affect vaccine efficacy but there are limited studies on how different diluents affect the colonization of live, attenuated vaccines. In this study, buffered peptone water, Marek's diluent and water were used as the diluents to reconstitute the commercially available Salmonella Typhimurium vaccine (Vaxsafe® ST). The diluent reconstituted vaccine was then given to day-old layer hens to test its effects on gut colonization. The vaccine reconstituted in Marek's diluent and buffered peptone water had a significantly higher colonization rate at 7 days post-vaccination in liver, spleen, oesophagus, crop, and proventriculus. At 14 days post-vaccination, there was a significantly higher vaccine load in the upper digestive tract of chickens that received vaccine reconstituted in Marek's diluent and buffered peptone water. The Marek's diluent and buffered peptone water reconstituted vaccine had a higher detection rate in the chicken's liver and spleen. Colonisation with water as a diluent was higher than the others especially in the lower parts of the intestine at 7 days post-vaccination. There was non-significant variation in the antibody titers at 7- and 14-days post-vaccination. In general, the results demonstrated that the use of nutrient-rich diluents in reconstituting live, attenuated Salmonella vaccine can enhance vaccine colonization. This study establishes a foundation for future work aimed at optimising strategies for vaccine reconstitution, crucial for eliciting a robust immune response in the host.

Among the Salmonella reduction strategies in poultry production, one option is to use a Salmonella vaccine. The aim of vaccinating layer flocks is to reduce the shedding of wild-type Salmonella in the poultry environment, thereby reducing the contamination of poultry products (eggs and meat). Nutritive diluent and a higher dose of vaccine may enhance its colonization potential in the gut of chickens. In this study, a commercially available live attenuated vaccine (Vaxsafe® ST) was reconstituted in different media and delivered orally to day-old chicks at three different doses (107, 108, and 109 CFU/chick). Gut colonization of the vaccine strain and the effects of vaccination on gut microbiota were assessed in commercial-layer chickens. The vaccine diluent and dosage minimally affected microbiota alpha diversity. Microbiota beta diversity was significantly different (P < 0.05) based on the vaccine diluent and dose, which indicated that the vaccinated and unvaccinated chickens had different gut microbial communities. Differences were noted in the abundance of several genera, including Blautia, Colidextribacter, Dickeya, Enterococcus, Lactobacillus, Pediococcus, and Sellimonas. The abundance of Colidextribacter was significantly lower in chickens that received vaccine reconstituted in Marek's and water diluents, while Lactobacillus abundance was significantly lower in the water group. The highest vaccine dose (109 CFU/chick) did not significantly alter (P > 0.05) the abundance of microbial genera. Chicken age affected the microbiota composition more significantly than the vaccine dose and diluent. The abundance of Lactobacillus, Blautia, Caproiciproducens, Pediococcus, and Colidextribacter was significantly higher on day 14 compared with day 7 post-vaccination. The Salmonella Typhimurium vaccine load in the caeca was not significantly affected by diluent and vaccine dose; however, it was significantly lower (P < 0.0001) on day 14 compared with day 7 post-vaccination. Overall, the S. Typhimurium vaccine minimally affected the gut microbiota structure of layer chicks, whereas changes in microbiota were more significant with chicken age.

ABSTRACT: The spin-chill process at poultry processing plants involves the immersion of chicken carcasses in cold water (

Abstract Salmonella Typhimurium is a human pathogen associated with eggs and egg-derived products. In Australia, it is recommended that eggs should be refrigerated to prevent condensation that can enhance bacterial penetration across the eggshell. Except for the United States, the guidelines on egg refrigeration are not prescriptive. In the current study, in-vitro and in-vivo experiments were conducted to understand the role of egg storage temperatures (refrigerated vs ambient) on bacterial load and the virulence genes expression of Salmonella Typhimurium. The in-vitro egg study showed that the load of Salmonella Typhimurium significantly increased in yolk and albumen stored at 25 °C. The gene expression study showed that ompR, misL, pefA, spvA, shdA, bapA, and csgB were significantly up-regulated in the egg yolk stored at 5 °C and 25 °C for 96 h; however, an in-vivo study revealed that mice infected with egg yolk stored at 25 °C, developed salmonellosis from day 3 post-infection (p.i.). Mice fed with inoculated egg yolk, albumen, or eggshell wash stored at refrigerated temperature did not show signs of salmonellosis during the period of the experiment. Data obtained in this study highlighted the importance of egg refrigeration in terms of improving product safety.

Abstract Chemical decontamination during processing is used in many countries to mitigate the Campylobacter load on chicken meat. Chlorine is a commonly used sanitizer in poultry processing to limit foodborne bacterial pathogens but its efficacy is limited by high bacterial loads and organic material. Acidified sodium chlorite (ASC) is a potential alternative for poultry meat sanitization but little is known about its effects on the cellular response of Campylobacter. In this study, the sensitivity of C. jejuni isolates to ASC was established. RNAseq was performed to characterize the transcriptomic response of C. jejuni following exposure to either chlorine or ASC. Following chlorine exposure, C. jejuni induced an adaptive stress response mechanism. In contrast, exposure to ASC induced higher oxidative damage and cellular death by inhibiting all vital metabolic pathways and upregulating the genes involved in DNA damage and repair. The transcriptional changes in C. jejuni in response to ASC exposure suggest its potential as an effective sanitizer for use in the chicken meat industry.

Abstract Eggs and raw or undercooked egg-containing food items are frequently identified as the bacterial source during epidemiolocal investigation of Salmonella outbreaks. Multi-locus variable number of tandem repeats analysis (MLVA) is a widely used Salmonella typing method enabling the study of diversity within populations of the same serotype. In vivo passage, however, has been linked with changes in MLVA type and more broadly the Salmonella genome. We sought to investigate whether in vivo passage through layer hens had an effect on MLVA type as well as the bacterial genome and whether any mutations affected bacterial virulence. Layer hens were infected with either Salmonella Typhimurium DT9 (03-24-11-11-523) as part of a single infection or were co-infected with an equal amount of Salmonella Mbandaka. Salmonella shedding in both single and co-infected birds was variable over the course of the 16-week experiment. Salmonella Typhimurium and Salmonella Mbandaka were identified in feces of co-infected birds. Salmonella colonies isolated from fecal samples were subtyped using MLVA. A single change in SSTR-6 was observed in Salmonella Typhimurium strains isolated from co-infected birds. Isolates of Salmonella Typhimurium of both the parent (03-24-11-11-523) and modified (03-24-12-11-523) MLVA type were sequenced and compared with the genome of the parent strain. Sequence analysis revealed that in vivo passaging resulted in minor mutation events. Passaged isolates exhibited significantly higher invasiveness in cultured human intestinal epithelial cells than the parent strain. The microevolution observed in this study suggests that changes in MLVA may arise more commonly and may have clinical significance.

Abstract Human cases of salmonellosis are frequently liked with the consumption of contaminated table eggs. Recently, there has been an increase in consumer demand for cage-free eggs precipitating the need for a greater understanding of Salmonella dynamics in free-range production systems. A longitudinal study was conducted to determine the points in production where birds are most likely to be exposed to Salmonella and where the risk of egg contamination is highest. In this study, two free-range flocks were sampled from hatch to the end of production. At hatch, all chicks were Salmonella negative and remained negative during rearing. During production, the proportion of positive samples was low on both farms. Salmonella positive samples were detected intermittently for Flock A. Dust, nest box, and egg belt swabs had the highest proportion of positive samples and highest overall loads of Salmonella. The egg grading floor was swabbed at different points following the processing of eggs from Flock A. Only the suction cups that handle eggs prior to egg washing tested positive for Salmonella. Swabs collected from machinery handling eggs after washing were Salmonella negative. During production, positive samples from Flock B were observed at only single time point. Dust has been implicated as a source of Salmonella that can lead to flock to flock contamination. Bulk dust samples were collected and tested for Salmonella. The proportion of positive dust samples was low and is likely due to physical parameters which are not likely to support the survival of Salmonella in the environment.

Broiler chickens frequently become colonized by Campylobacter species. As a consequence, Campylobacter, can enter the poultry meat supply chain and represents a significant risk for human public health. A number of on-farm biosecurity and processing measures are used to mitigate the load of Campylobacter on chicken meat. In many countries, chlorine is commonly used as a biocide in processing plants to reduce bacterial loads on poultry carcasses but there is limited evidence of its effectiveness on Campylobacter. In this study, 116 Campylobacter isolates (89 C. jejuni and 27 C. coli) were isolated from poultry meat carcasses prior to the inside/outside wash step and used in in vitro assays exploring the efficacy of chlorine. A high proportion of isolates exhibited MIC and MBC values of 128 ppm but organic material present in the broth likely affected this result. Thus, additional bactericidal assays (time kill and chlorine inactivation) were used to characterize the response of C. jejuni isolates to different concentrations of chlorine. At 106 CFU, C. jejuni was found to be highly sensitive to concentrations of chlorine and was inhibited at low concentrations (0.2–2.0 ppm). At a higher bacterial load (108 CFU), variation in the response of different C. jejuni isolates was observed. One isolate was growth inhibited at 1.8 ppm while another required 16 ppm. At 108 CFU, C. jejuni could be resuscitated following exposure to chlorine highlighting a potential limitation of chlorine use. Analysis of UV leakage indicated that high chlorine concentrations resulted in increased 280 nm absorbance values suggesting bacterial membrane damage. Scanning electron and transmission electron microscopy were performed to characterize the morphological effects of chlorine exposure. Some effects of chlorine exposure included changes in shape (coccoid, or elongated), cellular degeneration, and shriveled bacterial cells. Interestingly, C. jejuni cells with normal morphology were also observed in the chlorine exposed group and represent a population of cells that could be resuscitated. This study is useful for the chicken meat industry and provides data for future optimization of chlorine use in reducing Campylobacter loads.

This study evaluated the impact of vaccine diluents (peptone or water) on the protective effects of Salmonella Typhimurium (S. Typhimurium) vaccine. Vaccinated broilers were challenged with different doses of wild-type S. Typhimurium through dust. At the time of cull, vaccine load was highest in caeca and lowest in spleen. Wild-type S. Typhimurium was detectable after 24 hrs only in the vaccinated birds challenged with 108 CFU and positive control. S. Typhimurium load was lower in the organs of the groups challenged with 104 and 106 compared to the 108 CFU group. The caecal microbiota alpha diversity of the vaccinated or vaccinated and challenged chickens differed from the positive and negative control groups. Beta diversity of the positive control clustered separately from all other treatment groups, showing that vaccine caused minimal changes in gut microbiota structure. The vaccinated and/or wild-type challenged chickens showed significantly higher abundance of Anaerostignum, Lachnoclostridium, Intestinimonas, Colidextribacter, Monoglobus, Acetanaerobacterium and Subdoligranulum. Outcomes from this study demonstrate that the vaccine effectively protected broiler chickens from S. Typhimurium infection and helped maintain a more stable gut microbiota structure, reducing the impact of S. Typhimurium on gut health. Vaccine diluent did not affect gut microbiota composition.

Salmonella Typhimurium is among the most common causes of bacterial foodborne gastrointestinal disease in humans. Food items containing raw or undercooked eggs are frequently identified during traceback investigation as the source of the bacteria. Layer hens can become persistently infected with Salmonella Typhimurium and intermittently shed the bacteria over the course of their productive lifetime. Eggs laid in a contaminated environment are at risk of potential exposure to bacteria. Thus, mitigating the bacterial load on farms aids in the protection of the food supply chain. Layer hen producers use a multifaceted approach for reducing Salmonella on farms, including the all-in-all-out management strategy, strict biosecurity, sanitization, and vaccination. The use of live attenuated Salmonella vaccines is favored because they elicit a broader host immune response than killed or inactivated vaccines that have been demonstrated to provide cross-protection against multiple serovars. Depending on the vaccine, two to three doses of Salmonella Typhimurium vaccines are generally administered to layer hens within the first few weeks. The productive life of a layer hen, however, can exceed 70 weeks and it is unclear whether current vaccination regimens are effective for that extended period. The objective of this review is to highlight layer hen specific challenges that may affect vaccine efficacy.

Salmonella remains one of the most common causes of bacterial foodborne gastrointestinal disease in humans. Raw eggs or food items containing undercooked eggs are frequently identified as the source of Salmonella. Salmonella Typhimurium contamination of table eggs most commonly occurs when they are laid in a contaminated environment. Several control strategies, including vaccination, are widely used to mitigate the total Salmonella load. It is unclear, however, whether live attenuated Salmonella vaccines are efficacious over the life span of a layer hen. Live attenuated Salmonella vaccines have been favored due to their ability to illicit a strong humoral immune response. The lifespan of a layer hen ranges between 60 and 80 weeks and the long term efficacy of attenuated vaccine strains has not been investigated. In this study, commercial brown layer chicks were vaccinated at day old, 6 weeks of age, and again at 10 weeks of age with the Bioproperties VaxsafeTM STM1 aroA mutant vaccine. Birds were challenged at 18 weeks of age with Salmonella Typhimurium DT9 (MLVA 03 15 08 11 550). Feces and eggs were monitored for S. Typhimurium for 40 weeks post-infection. Birds produced a strong immune response following the final dose which was administered intramuscularly. The serum antibody response to S. Typhimurium DT9 infection did not differ between challenged groups. Fecal shedding and egg contamination was highly variable and did not differ significantly between vaccinated and unvaccinated birds that had been challenged with S. Typhimurium DT9. Total bacterial load in feces was quantified using qPCR. No significant difference was detected between unvaccinated and vaccinated birds after challenge.

It is becoming increasingly clear that many diseases are the result of infection from multiple genetically distinct strains of a pathogen. Such multi-strain infections have the capacity to alter both disease and pathogen dynamics. Infection with multiple strains of human cytomegalovirus (HCMV) is common and has been linked to enhanced disease. Suggestions that disease enhancement in multi-strain infected patients is due to complementation have been supported by trans-complementation studies in mice during co-infection of wild type and gene knockout strains of murine CMV (MCMV). Complementation between naturally circulating strains of CMV has, however, not been assessed. In addition, many models of multi-strain infection predict that co-infecting strains will compete with each other and that this competition may contribute to selective transmission of more virulent pathogen strains. To assess the outcome of multi-strain infection, C57BL/6 mice were infected with up to four naturally circulating strains of MCMV. In this study, profound within-host competition was observed between co-infecting strains of MCMV. This competition was MCMV strain specific and resulted in the complete exclusion of certain strains of MCMV from the salivary glands of multi-strain infected mice. Competition was dependent on Ly49H(+) natural killer (NK) cells as well as the expression of the ligand for Ly49H, the MCMV encoded product, m157. Strains of MCMV which expressed an m157 gene product capable of ligating Ly49H were outcompeted by strains of MCMV expressing variant m157 genes. Importantly, within-host competition prevented the shedding of the less virulent strains of MCMV, those recognized by Ly49H, into the saliva of multi-strain infected mice. These data demonstrate that NK cells have the strain specific recognition capacity required to meditate within-host competition between strains of MCMV. Furthermore, this within-host competition has the capacity to shape the dynamics of viral shedding and potentially select for the transmission of more virulent virus strains.