Correlating bacterial shedding with fecal corticosterone levels and serological responses from layer hens experimentally infected with Salmonella Typhimurium

Salmonella Enteriditis and Salmonella Typhimurium are commonly isolated during egg-related outbreaks of salmonellosis and represent a significant international public health issue. In Australia, Salmonella Typhimurium is the most common serovar identified in egg product related foodborne outbreaks. While a number of studies have investigated Salmonella shedding and host responses to infection, they have been conducted over a short time period. The present study sought to characterise bacterial shedding and host responses to infection in hens infected with only Salmonella Typhimurium or co-infected with both Salmonella Typhimurium and Salmonella Mbandaka over a 16 week period. Salmonella shedding was quantified using the most probable number and qPCR methods and was highly variable over the course of the experiment. On day 1, fecal corticosterone metabolites in birds infected with Salmonella Typhimurium (674.2 ± 109.3 pg/mg) were significantly higher than control (238.0 ± 12.62 pg/mg) or co-infected (175.4 ± 8.58 pg/mg) birds. The onset of lay occurred between weeks 6-8 post-infection (pi) and Fecal corticosterone metabolite (FCM) concentrations increased in both control and co-infected birds. Antibody responses to infection were monitored in both serum and yolk samples. Salmonella Typhimurium specific antibody was lower in co-infected animals than monoinfected animals. Bacterial loads in internal organs were characterised to determine persistence. Spleen, liver and caecal tonsils were positive for bacteria in both groups, indicating that Salmonella was not cleared from the birds and internal organ colonization could serve as a reservoir for continued bacterial shedding.Pardeep Sharma, Vivek V. Pande, Talia S. Moyle, Andrea R. McWhorter and Kapil K. Chousalka

In vivo passage of Salmonella Typhimurium results in minor mutations in the bacterial genome and increases in vitro invasiveness

International audienceAbstractEggs 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

A long-term efficacy trial of a live, attenuated Salmonella Typhimurium vaccine in layer hens

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.Andrea R. McWhorter and Kapil K. Chousalka

The eggshell quality of table eggs and how this affects food safety

The longitudinal effect of age and production system was evaluated for egg quality parameters and egg microbial load by following 2 flocks (Hy Line brown commercial layer), one conventional cage production system (CC) and one free range (FR). A horizontal study of the effect of flock age (22, 39, 55 and 79 wk) of the same strain of bird from the same CC was also evaluated for egg quality parameters and egg microbial load. From the CC and FR production systems, eggs were collected at the age of 25, 35, 45, 55, 65 and 75 wk with one extra collection at 85 wk from CC. For the eggshell and egg internal quality parameters, a significant main effect (

Salmonella Typhimurium in the Australian egg industry: multidisciplinary approach to addressing the public health challenge and future directions

In Australia, numerous egg related human Salmonella Typhimurium outbreaks have prompted significant interest amongst public health authorities and the egg industry to jointly address this human health concern. Nationwide workshops on Salmonella and eggs were conducted in Australia for egg producers and regulatory authorities. State and National regulators representing Primary Production, Communicable Disease Control, Public Health and Food Safety and Food Standards Australia and New Zealand. All attendees participated in discussions aimed at evaluating current evidence based information, issues related to quality egg production and how to ensure safe eggs in the supply chain, identifying research gaps and practical recommendations. The perceptions from egg producers and regulatory authorities from various states were recorded during the workshops. We presented the issues discussed during the workshops including Salmonella in the farm environment, Salmonella penetration across egg shell, virulence in humans, food/egg handling in the supply chain and intervention strategies. We also discussed the perceptions from egg producers and regulators. Recommendations placed emphasis on future research needs, communication between industry and regulatory authorities and education of food handlers. Communication between regulators and industry is pivotal to control egg borne S. Typhimurium outbreaks and collaborative efforts are required to design effective and appropriate control strategies.Kapil K. Chousalkar, Margaret Sexton, Andrea McWhorter, Kylie Hewson, Glen Martin, Craig Shadbolt & Paul Goldsmit

Effects of different Papua New Guinea sweetpotato varieties on performance and level of enteric pathogens in chickens

Published: 23 April 2019In the last decade, research has targeted the evaluation of local feed ingredients for use in monogastric diets to alleviate the high cost of production of livestock at smallholder levels in Papua New Guinea (PNG). The PNG smallholder poultry production system involves many families who rear multiple batches of meat birds every year. This study was conducted to evaluate the levels of enteric pathogens in the caeca of broilers fed with sweetpotato diets with varying levels of non-starch polysaccharides (NSP). Selection of a sweetpotato variety for use in broiler diets should be based on the total NSP content. In particular, varieties with low soluble NSPs are economical to use as Apparent Metabolizable Energy (AME) values are within the desired range for poultry and there is minimal need to include enzymes to improve NSP digestibility. The use of varieties with a low total NSP is also advantageous as the numbers of Clostridium perfringens was lower in broilers fed with these sweetpotato varieties. The level of Campylobacter and Salmonella levels were high in the ceca of birds fed with the sweetpotato varieties with high total NSP. These levels can be reduced with the inclusion of enzymes. This information will assist in the efficient use of local varieties of sweetpotato in PNG by small holder poultry farmers for sustainable poultry production and the commercial industry.Janet Pandi, Phil Glatz, Rebecca Forder and Kapil Chousalka

The Relationship of Eggshell Structure to Eggshell Penetration by 'Salmonella' Typhimurium in Table Eggs

This project sought to correlate external shell features with underlying shell structures and examine if these structures are indicative of increased incidence of eggshell penetration by Salmonella Typhimurium. Eggs are an important nutrient and protein source in most cultures around the world, and the importance of food safety in intensive food production industries is likely to increase. Microbial infection of eggs is the source of approximately 49% of Salmonella food poisoning cases in Australia each year. The most significant source of these infections in Australia is Salmonella Typhimurium. Eggshell translucency is a phenomenon observed when an egg is candled over a light source; the underlying cause and incidence of eggshell translucency form the basis of our investigations of shell structures. In order to image eggshell structure, both Computed Tomography and Scanning Electron Microscopy were used to examine different structural components of the shell

Challenges in vaccinating layer hens against salmonella typhimurium

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.Siyuan Jia, Andrea R. McWhorter, Daniel M. Andrews, Gregory J. Underwood and Kapil K. Chousalka

PCR-based detection of genes encoding virulence determinants in Staphylococcus aureus from bovine subclinical mastitis cases

The present study was carried out to genotypically characterize Staphylococcus aureus (S. aureus) isolated from bovine mastitis cases. A total of 37 strains of S. aureus were isolated during processing of 552 milk samples from 140 cows. The S. aureus strains were characterized phenotypically, and were further characterized genotypically by polymerase chain reaction using oligonucleotide primers that amplified genes encoding coagulase (coa), clumping factor (clfA), thermonuclease (nuc), enterotoxin A (entA), and the gene segments encoding the immunoglobulin G binding region and the X region of protein A gene spa. All of the isolates yielded an amplicon with a size of approximately 1,042 bp of the clfA gene. The amplification of the polymorphic spa gene segment encoding the immunoglobulin G binding region was observed in 34 isolates and X-region binding was detected in 26 isolates. Amplification of the coa gene yielded three different products in 20, 10, and 7 isolates. The amplification of the thermonuclease gene, nuc, was observed in 36 out of 37 isolates. All of the samples were negative for the entA gene. The phenotypic and genotypic findings of the present strategies might provide an understanding of the distribution of the prevalent S. aureus clones among bovine mastitis isolates, and might aid in the development of steps to control S. aureus infections in dairy herds