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Globally, Campylobacter spp. are the most common food-associated bacterial cause of human gastrointestinal disease. Campylobacteriosis is primarily associated with the consumption of contaminated chicken meat. Chemical decontamination of chicken carcasses during processing is one of the most effective interventions to mitigate Campylobacter contamination. Following exposure to sanitizers, however, sublethally injured populations of bacteria may persist. The risk that sublethally injured Campylobacter pose for public health is unknown. Furthermore, the virulence potential of sublethally injured Campylobacter jejuni during prolonged storage in relation to host pathogenesis and the host immune response has not been well established. Therefore, we evaluated the effects of sublethally injured C. jejuni on the host, after storage in chicken meat juice. C57BL/6 mice were infected with two C. jejuni chicken meat isolates or the ATCC 33291 strain that had been stored in the chicken meat juice, after exposure to chlorine or acidified sodium chlorite (ASC). Although chlorine exposure was unable to reduce intestinal colonization by C. jejuni, exposure to ASC significantly reduced the intestinal colonization and tissue translocation in mice. The expression of pro- and anti-inflammatory cytokine genes for interleukin-6 (

Over the past decade, the scientific community has witnessed significant advances in egg quality and safety research. Table eggs are rarely implicated in the transmission of viral foodborne disease, but nontyphoidal Salmonella spp. have been a significant focus of egg safety research. Cold storage also helps limit bacterial replication on and in eggs. However, in many countries, there are no prescriptive guidelines on egg storage in the retail market. The risk of foodborne virus transmission via table egg is low, but given the increase in cage-free egg production, there is an increased risk of fungal contamination. Probiotics or vitamins supplementation can improve the egg defence by altering the caecal microbiota, although how indicator bacteria related or influenced by egg quality are is yet to be investigated.

Chicken meat is frequently contaminated with zoonotic bacterial pathogens such as Campylobacter spp and Salmonella spp. These two bacterial genera are commonly linked with cases of human gastrointestinal disease, thus mitigating their presence in the poultry meat supply chain is paramount. Here, the efficacy of two sanitizers, peroxyacetic acid (PAA) and acidified sodium chlorite (ASC), was tested using whole chicken carcasses obtained either prior to the inside/outside wash or the post-immersion spin chill steps of processing. Two concentrations of PAA (100 and 200 ppm) and ASC (450 and 900 ppm) were tested, and both significantly reduced total viable bacteria and Campylobacter counts per carcass. Both sanitizers also reduced the prevalence of Salmonella on whole carcasses from both processing steps. Log reduction of both the total viable and Campylobacter counts were, however, temperature and processing stage dependent. The efficacy of both PAA and ASC were also compared with sodium hypochlorite. No significant difference between the three sanitizers was observed for the reduction of TVC, Campylobacter or Salmonella using carcasses obtained at either processing step. These results demonstrate that PAA or ASC could be implemented as a replacement or used in addition to sodium hypochlorite to effectively reduce bacteria on whole carcasses.

Reptiles are carriers of Salmonella and can intermittently shed bacteria in their faeces. Contact with snakes and lizards is a source of human salmonellosis. Here, two populations of reptiles, wild and captive were surveyed for Salmonella. One hundred thirty wild-caught reptiles were sampled for Salmonella including 2 turtle, 9 snake and 31 lizard species. Fifty-two of 130 (40%) animals were Salmonella positive: one of 5 (20%) turtles, 7 of 14 (50%) snakes and 44 of 111 (39.6%) lizards. One hundred twenty-two reptiles were sampled from a zoo collection including 1 turtle, 6 tortoise, 9 lizard, 14 snake and 1 crocodile species. Forty-two of 122 (34.4%) captive reptiles sampled were Salmonella positive. Salmonella was most commonly isolated from lizards and snakes. Fifteen serotypes were identified from zoo and 19 from wild-caught reptiles and most were members of subspecies enterica (I), salamae (II), arizonae (IIIa) or diarizonae (IIIb). Antimicrobial susceptibility testing was conducted on all Salmonella isolates; only two exhibited resistance, a Salmonella subsp. (II) ser. 21:z10 :z6 (Wandsbek) isolate cultured from a wild-caught reptile and a Salmonella Typhimurium DT120 isolated from a captive snake. The invasive capacity of reptile-associated Salmonella strains into cultured human intestinal epithelial (Caco2) and mouse macrophages cell lines (J774A.1) was also investigated. All isolates were invasive into both cell lines. Significant (P ≤ 0.001) variability in invasiveness into polarized Caco2 cells was observed. Salmonella Eastbourne exhibited the highest invasiveness into Caco2 cells and Salmonella Chester the lowest, with mean per cent recoveries of 19.99 ± 0.32 and 1.23 ± 0.30, respectively. Invasion into J774A.1 macrophages was also variable but was not significant. Salmonella subsp. II ser. 17:g,t:- (Bleadon) exhibited the highest invasiveness into J774A.1 with a mean per cent recovery of 10.19 ± 0.19. Thus, reptile-associated Salmonellae are likely to have different capacities to cause disease in humans.

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.

This study evaluated the antibacterial activity of commercially available organic acid water additives against Salmonella enterica isolates and examined the susceptibility of Salmonella Typhimurium biofilms to these products. Three commercial organic acid products (A, B, and C) were evaluated for minimum inhibitory and bactericidal concentrations against isolates of S. enterica serovars. Three- and five-day-old S. Typhimurium biofilms were formed at 22 ± 2°C using an MBEC™ assay system and exposed for 30 min or 90 min at 0.2% and 0.4% concentrations. No significant difference among serovars for inhibitory and bactericidal concentrations was detected. Two products (A and C) significantly reduced viable cells from biofilms of both ages in a dose- and time-dependent manner. Increased biofilm age did not enhance resistance towards organic acid treatments. None of the products completely eliminated biofilm cells at any concentration or exposure time. Product composition, exposure time, and concentration of organic acid products were important factors in reducing viable biofilm cells. This study has expanded our understanding about the susceptibility of Salmonella biofilms to commercial organic acid products. These findings have implications in the usage, development, and optimization of organic acid products.

In Australia, numerous egg-related human Salmonella typhimurium outbreaks have prompted significant interest among 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 represented 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 of 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 eggshell, 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 the 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.

Raw egg-based sauces, such as mayonnaise and aioli, are frequently identified as sources of Salmonella during outbreaks of human cases of foodborne gastrointestinal disease. In this study, we surveyed aioli and mayonnaise recipes from different popular food websites to identify potential risk factors that may lead to the survival of Salmonella Typhimurium. In laboratory experiments, different ratios of food acids were used to determine if lemon juice, vinegar, or a combination of both restricted Salmonella Typhimurium culturability. We found that as long as the pH was below 4.2, bacterial culturability was limited. The use of whole egg alone or in combination with egg yolk was also investigated. Sauce preparations containing whole egg exhibited higher pH and supported Salmonella Typhimurium culturability longer than those containing yolk only. Ten restaurant prepared sauces were also obtained to further characterize the effect of preparation variability. Sauce preparations with a pH ≤ 3.8 did not support bacterial culturability after 4 h incubation at any temperature. The higher the pH the longer Salmonella Typhimurium remained culturable. Based on this study, it is recommended that raw egg-based foods are acidified, then stored at room temperature for at least 4 h prior to consumption.

Chicken meat can potentially become contaminated with bacteria at the processing plant. In Australia, there is currently a lack of knowledge on the parameters and indications of use of non-chlorine based treatments in the chicken meat processing plants. Chlorine is widely used as a sanitizer in Australian chicken meat processing plants but due to occupational health and safety concerns and consumer perception, there is a need to identify alternative sanitizers. This study aimed to assess the efficacy of four different sanitizers in reducing the microbial load from naturally contaminated chicken meat carcasses collected from the processing plants in South Australia. There was a significant variation in a load of Campylobacter and total viable count (TVC) between samples collected from two different processing plants and within carcass batches collected from the same plant that was tested during the study. All sanitizers generally reduced the load of Campylobacter on chicken meat carcasses. Treatment with acidified sodium chlorite significantly reduced the level of Salmonella enterica&nbsp, serovars at all temperatures tested during this study. These findings are helpful to the industry for selection of the appropriate sanitizers. Findings are also useful for the regulatory authorities in Australia for providing approval for the use of sanitizers.

Single-aged caged layer hen flocks were monitored for Salmonella over the course of their lifetime. Chicks from both flocks were Salmonella negative at hatch and remained negative during rearing. Pullets were transported to production farms at 15 weeks of age. Pre-population dust swabs collected from both production sheds had a high percentage of Salmonella positive samples (80 and 90%). Flocks were sampled at regular intervals until 70-72 weeks of age. The proportion of Salmonella positive samples and mean load detected on eggs was low on both farms. Analysis of dust samples revealed that Salmonella persisted in dust over 8 weeks. Dust total moisture content and water activity appears to influence bacterial persistence. On egg grading equipment, only suction cups prior to egg washing were Salmonella positive (mean proportion Salmonella positive samples 0.13 ± 0.07; mean load of 18.6 ± 12.31 MPN/ml). An egg washing experiment demonstrated that while washing reduced the total Salmonella load from eggshell surfaces, no effect was observed for shell pores. These results demonstrate that despite environmental contamination on farm, Salmonella contamination of eggs is low and is further minimized by washing.

In Australia and other parts of the world, contaminated eggs or egg-containing food products are common vehicles for human Salmonella outbreaks. Recently, an uncommon serotype, Salmonella Hessarek, has emerged in foodborne salmonellosis due to the consumption of contaminated eggs and egg products. Limited research is available on the behaviour of Salmonella Hessarek in eggs. Therefore, this study was performed to understand the penetration ability and transcriptional behavior of Salmonella Hessarek in table eggs stored at different temperatures. The assay revealed that the penetration ability of Salmonella Hessarek was significantly (P

Despite food safety recommendations, raw egg-based foods, such as mayonnaise, are frequently identified as the source of Salmonella during outbreaks. Acidification and storage temperature have been linked with reduced bacterial culturability. Raw egg-based sauces stored at 25 °C have historically been linked with faster decline of Salmonella culturability than preparations stored at 5 °C. This study aimed to determine whether reduced culturability in acidified mayonnaise correlated with reduced in vitro bacterial motility, invasiveness and viability as well as disease-causing capacity in BALB/c mice. Acidification of mayonnaise and incubation at 25 °C for 4 h significantly reduced culturability of Salmonella Typhimurium DT9 but was dependent on initial bacterial load. Bacteria recovered from acidified mayonnaise exhibited reduced invasiveness into polarized cultured intestinal epithelial cells and 12 h post inoculation were no longer invasive suggesting a reduced capacity to cause disease. To confirm this, BALB/c mice were inoculated with Salmonella Typhimurium contaminated mayonnaise stored at 5 °C or 25 °C for 12, 24, 48, 72, and 96 h. Mice inoculated with mayonnaise incubated at 5 °C for 12 and 24 h exhibited mild to moderate disease symptoms; all other mayonnaise treatment groups did not exhibit disease symptoms. In acidified mayonnaise, Salmonella Typhimurium DT9 exhibited a global downregulation of metabolism, stress response, and virulence genes upon addition to mayonnaise. After 4 h of incubation at both 5 °C and 25 °C, however, the vast majority of genes were upregulated which was maintained over the 96-hour experiment suggesting that bacteria were severely stressed. Salmonella Typhimurium DT9 cells were isolated from mayonnaise samples and ATP production was quantified. At both 5 °C and 25 °C, ATP production decreased in acidified mayonnaise preparations. At 25 °C, ATP production decreased more rapidly than at 5 °C. After 24 h, ATP production of bacteria in mayonnaise stored at 25 °C was not significantly different from the dead control group. Thus, the current recommendation of only serving freshly prepared raw egg-sauces or refrigerating immediately after preparation, could be placing consumers at higher risk for contracting salmonellosis.

This study was conducted to examine the antimicrobial resistance (AMR) of Salmonella spp. isolated from commercial caged layer flocks in New South Wales and South Australia. All Salmonella isolates (n=145) were subjected to phenotypic and genotypic characterisation of AMR and carriage of integrons. The majority of Salmonella isolates (91.72%) were susceptible to all antimicrobials tested in this study. Limited resistance was observed to amoxicillin and ampicillin (5.51%), tetracycline (4.13%), cephalothin (2.06%) and trimethoprim (0.68%). None of the isolates were resistant to cefotaxime, ceftiofur, ciprofloxacin, chloramphenicol, gentamycin, neomycin or streptomycin. A low frequency of Salmonella isolates (4.83%) harboured antimicrobial resistance genes and a class 1 integron. The most commonly detected AMR genes among the Salmonella isolates were blaTEM (2.07%), tet A (1.38%) and dhfrV (0.69%). Overall, Salmonella enterica isolates exhibited a low frequency of AMR and represent a minimal public health risk associated with the emergence of multidrug resistant Salmonella spp. from the Australian layer industry.

Background Salmonella vaccination is one of the control measure that farmers can use to reduce bacterial shedding in their flocks. This study aimed to examine the efficacy of the Vaxsafe® ST (Strain STM-1) attenuated live vaccine administered as ocular and oral doses followed by an intramuscular (IM) dose in rearing, in reducing contamination by Salmonellae of both eggs and the environment in the commercial multi-age cage layer sheds. A randomised controlled trial was conducted up to 26 weeks post last vaccine on two different multi-age caged egg farms. Results No clinical symptoms were observed following IM administration of STM-1 during rearing. Following the first two STM-1 doses, both vaccinated and unvaccinated birds exhibited antibody titres below the positive cut-off value, however after IM administration of STM-1, antibody titres in the vaccinated group were above the cut-off value. Wild type Salmonella Typhimurium was not detected during the rearing of pullets. During production, the antibody titres were significantly higher in the vaccinated group at all sampling points during this trial. There was no significant difference in the prevalence of Salmonella (detected by culture and PCR method) between the vaccinated and unvaccinated groups on the egg belt and faeces in early lay. Wild-type Salmonella spp. were consistently found in dust samples. Quantitative PCR (qPCR) assay was able to differentiate between the live vaccine strain and wild type Salmonella. The load of wild-type Salmonella in shed environment was relatively low (1.3 log10 ± 0.48 CFU/m2 of surface area). Conclusion Given that Salmonella Typhimurium and other serovars are able to survive/persist in the shed environment (such as in dust), regular cleaning and or removal of dust from shed is important. Use of the Vaxsafe® ST vaccine in multi-age flocks is “not an ultimate intervention” for reduction of Salmonella Typhimurium because of the complexities involved in achieving control, such as the efficacy of cleaning of sheds, the lack of resting periods between batches and the possible carry over of contamination from existing flocks. Hence implementation of more than one or several interventions strategies is essential. Electronic supplementary material The online version of this article (10.1186/s12866-018-1201-0) contains supplementary material, which is available to authorized users.

Vaccination of chicks with Salmonella (S.) Typhimurium aroA deletion mutants has previously been shown to inhibit intestinal colonization of wild-type S. Typhimurium strains. In Australia, Bioproperties VaxSafe™ STM1 strain is the only licensed and commercially available S. Typhimurium vaccine. This vaccine is a live attenuated aroA deletion mutant. Currently, it is recommended that the first dose of the STM1 vaccine is administered through coarse spray. It is unclear whether this mode of administration effectively permits intestinal colonization. Furthermore, it is not known whether the STM1 strain prevents or inhibits Salmonella colonization of chicks following this first dose. This study investigated both in vitro and in vivo colonization parameters. Invasiveness was assessed using an in vitro invasion assay into sections of ileum and caecum collected from day-old chicks. The S. Typhimurium definitive types (DT) 9 and 44 exhibited the greatest invasion into both intestinal segments. STM1 was invasive but was significantly less so than both isolates of S. Typhimurium. In dual and triple infections, no competitive microbial interactions between STM1 and wild-type Salmonella were observed. In vivo colonization inhibition was also tested. Vaccinated and nonvaccinated day-old chicks were challenged with S. Typhimurium DT9. Both STM1 and S. Typhimurium DT9 were found in spleen, liver, ileum, caecum and caecal contents from day 2 postinfection. No significant exclusion effect was observed in vaccinated and challenged chicks.

In Australia, the egg industry is periodically implicated during outbreaks of Salmonella food poisoning. Salmonella enterica serovar Typhimurium and other nontyphoidal Salmonella spp., in particular, are a major concern for Australian public health. Several definitive types of Salmonella Typhimurium strains, but primarily Salmonella Typhimurium definitive type 9 (DT9), have been frequently reported during egg-related food poisoning outbreaks in Australia. The aim of the present study was to generate a pathogenicity profile of nontyphoidal Salmonella isolates obtained from Australian egg farms. To achieve this, we assessed the capacity of Salmonella isolates to cause gastrointestinal disease using both in vitro and in vivo model systems. Data from in vitro experiments demonstrated that the invasion capacity of Salmonella serovars cultured to stationary phase (liquid phase) in LB medium was between 90- and 300-fold higher than bacterial suspensions in normal saline (cultured in solid phase). During the in vivo infection trial, clinical signs of infection and mortality were observed only for mice infected with either 10 3 or 10 5 CFU of S . Typhimurium DT9. No mortality was observed for mice infected with Salmonella serovars with medium or low invasive capacity in Caco-2 cells. Pathogenicity gene profiles were also generated for all serovars included in this study. The majority of serovars tested were positive for selected virulence genes. No relationship between the presence or absence of virulence genes by PCR and either in vitro invasive capacity or in vivo pathogenicity was detected. Our data expand the knowledge of strain-to-strain variation in the pathogenicity of Australian egg industry-related Salmonella spp.

In Australia, Salmonella Typhimurium definitive type 9 is frequently isolated during foodborne outbreaks of salmonellosis. Multiple-locus variable number tandem repeat analysis (MLVA) trace back investigations frequently identify isolate distribution patterns that may be epidemiologically linked to disease outbreaks. In this study, the in vitro virulence potential of S. Typhimurium DT9 isolates possessing different MLVA patterns (03 15 07 11 550, 03 24 11 10 523, 03 15 08 11 550 and 03 14 08 11 550) isolated from either humans or layer hens was assessed using a human colon carcinoma cell line. Four strains per MLVA from each host for a total of 32 isolates were included in these experiments. Bacteria were grown to stationary phase and added to cells at a multiplicity of infection of 100. Across all isolates, mean percent recovery ranged from 7.1 ± 1.1 to 33.3 ± 7.1%. The layer hen isolate, KC900 (MLVA profile 03 15 08 11 550), exhibited the greatest invasion with a mean percent recovery of 33.3 ± 7.1%. Overall, layer hen isolates of S. Typhimurium DT9 had significantly higher invasion into Caco2 cells than human isolates (p = .0021). RAPD and enterobacterial repetitive intergenic consensus genomic fingerprinting was also performed. Irrespective of source, the SalmonellaDT9 isolates included in this study exhibited similar fingerprint patterns.

Undercooked eggs and egg-related products are frequently implicated as primary sources of Salmonella during food poisoning outbreaks. Globally, Salmonella Enteriditis is most commonly identified from reported salmonellosis cases, but it is not endemic in commercial poultry industry in Australia. Salmonella Typhimurium has filled this niche and over the past decade has been the most frequently isolated serovar during Australian outbreaks. In 2015, the Department of Health recorded 16,952 notified Salmonella cases; of these many were epidemiologically linked with the consumption of raw egg products. Eggs can become contaminated with S. Typhimurium during lay when they come into contact with Salmonella-positive fecal material. Small defects in the egg shell may subsequently influence bacterial penetration across the shell leading to contamination of egg internal contents. Farm-specific Salmonella intervention strategies are not sufficient for bacterial control and combined efforts through the entire supply chain are essential to limiting Salmonella.

Raw or undercooked eggs and egg products are frequently identified as the source of Salmonella following outbreaks of foodborne gastrointestinal disease. Some Salmonella serovars, such as Salmonella Enteriditis, have a high tropism for the oviduct of laying hens. Oviduct colonization with S. Enteriditis can result in both internal and external contamination of an egg. While oviduct invasion is not limited to S. Enteriditis, the invasive capacities of other serovars is not widely known. In this study, the in vitro invasive ability of eighteen Salmonella isolates of representative serovars into different segments of the oviduct was assessed. All Salmonella isolates tested were invasive and the highest bacterial invasion was observed in segments of the isthmus and vagina. S. Bredeney consistently exhibited the lowest invasion into all sections of the oviduct. Interestingly, the S. Typhimurium definitive types included in this study did not exhibit significantly greater invasion capacity than other serovars. In this study, the genomic capacity of the selected isolates of representative Salmonella serovars to colonize the layer hen oviduct was also investigated. Previous studies have identified several genes upregulated during oviduct colonization by S. Enteriditis. Single gene comparison of 107 genes from eleven Salmonella isolates was conducted to determine whether these oviduct colonization genes were present within each bacterial genome. The degree of homology with corresponding sequences in S. Enteriditis P125109 was also determined for each gene. Genes encoding the O-antigen as well as phage and virulence plasmid genes were among the most highly variable and may serve specific roles in oviduct invasion.

Members of Salmonella enterica are frequently involved in egg and egg product related human food poisoning outbreaks worldwide. In Australia, Salmonella Typhimurium is frequently involved in egg and egg product related foodborne illness and Salmonella Mbandaka has also been found to be a contaminant of the layer farm environment. The ability possessed by Salmonella Enteritidis to colonize reproductive organs and contaminate developing eggs has been well-described. However, there are few studies investigating this ability for Salmonella Typhimurium. The hypothesis of this study was that the Salmonella Typhimurium can colonize the gut for a prolonged period of time and that horizontal infection through feces is the main route of egg contamination. At 14 weeks of age hens were orally infected with either S. Typhimurium PT 9 or S. Typhimurium PT 9 and Salmonella Mbandaka. Salmonella shedding in feces and eggs was monitored for 15 weeks post-infection. Egg shell surface and internal contents of eggs laid by infected hens were cultured independently for detection of Salmonella spp. The mean Salmonella load in feces ranged from 1.54 to 63.35 and 0.31 to 98.38 most probable number/g (MPN/g) in the S. Typhimurium and S. Typhimurium + S. Mbandaka group, respectively. No correlation was found between mean fecal Salmonella load and frequency of egg shell contamination. Egg shell contamination was higher in S. Typhimurium + S. Mbandaka infected group (7.2% S. Typhimurium, 14.1% S. Mbandaka) compared to birds infected with S. Typhimurium (5.66%) however, co-infection had no significant impact on egg contamination by S. Typhimurium. Throughout the study Salmonella was not recovered from internal contents of eggs laid by hens. Salmonella was isolated from different segments of oviduct of hens from both the groups, however pathology was not observed on microscopic examination. This study investigated Salmonella shedding for up to 15 weeks p.i which is a longer period of time compared to previously published studies. The findings of current study demonstrated intermittent but persistent fecal shedding of Salmonella after oral infection for up to 15 weeks p.i. Further, egg shell contamination, with lack of internal egg content contamination and the low frequency of reproductive organ infection suggested that horizontal infection through contaminated feces is the main route of egg contamination with S. Typhimurium in laying hens.

Syndecan-1 (Sdc1) is a matrix receptor shown to associate via its extracellular domain with the alpha(v)beta(3) and alpha(v)beta(5) integrins, potentially regulating cell adhesion, spreading, and invasion of cells expressing these integrins. Using Sdc1 deletion mutants expressed in human mammary carcinoma cells, we identified the active site within the Sdc1 core protein and derived a peptide inhibitor called synstatin (SSTN) that disrupts Sdc1's interaction with these integrins. Because the alpha(v)beta(3) and alpha(v)beta(5) integrins are critical in angiogenesis, a process in which a role for Sdc1 has been uncertain, we used human vascular endothelial cells in vitro to show that the Sdc1 regulatory mechanism is also required for integrin activation on these cells. We found Sdc1 expressed in the vascular endothelium during microvessel outgrowth from aortic explants in vitro and in mouse mammary tumors in vivo. Moreover, we show that SSTN blocks angiogenesis in vitro or when delivered systemically in a mouse model of angiogenesis in vivo, and impairs mammary tumor growth in an orthotopic mouse tumor model. Thus, Sdc1 is a critical regulator of these two important integrins during angiogenesis and tumorigenesis, and is inhibited by the novel SSTN peptide.

We have developed a murine cytomegalovirus (MCMV)-vectored vaccine expressing the mouse zona-pellucida-3 gene (rMCMV-ZP3), which successfully induces infertility in experimentally inoculated laboratory or wild-derived mice. However, the future success of this vector as a fully disseminating vaccine in free-living mice may be compromised by pre-existing immunity since there is a high prevalence of naturally acquired MCMV infection in these mice. To evaluate the effect of prior immunity to MCMV on vaccine efficacy, we constructed two new biologically effective recombinant MCMV vectors expressing the mouse ZP3 protein from two MCMV strains (N1 and G4) derived from free-living mice. In wild mice, mixed MCMV infection is common and could be acquired either by simultaneous coinfection or sequential infection with different MCMV strains. Interestingly, while coinfection with both wild-type and rMCMV-ZP3 via the intraperitoneal route reduced the impact of the rMCMV-ZP3, prior infection with the same wild-type strain as that used to construct the rMCMV-ZP3 abrogated the immunocontraceptive effects of either N1-ZP3 or G4-ZP3. However, prior infection with G4 28 days before the introduction of N1-ZP3 had a reduced influence on the efficacy of the rMCMV-ZP3. Thus, the strain of virus and the timing of prior infection are factors that may influence the efficacy of the rMCMV-ZP3. Given that mixed infection of mice with MCMV is common, it is possible that prior immunity acquired by natural mucosal infection may have less a less inhibitory effect on the immunocontraceptive outcome.

Murine cytomegalovirus (MCMV) is widely used to model human cytomegalovirus (HCMV) infection. However, it is known that serially passaged laboratory strains of HCMV differ significantly from recently isolated clinical strains of HCMV. It is therefore axiomatic that clinical models of HCMV using serially passaged strains of MCMV may not be able to fully represent the complexities of the system they are attempting to model and may not fully represent the complex biology of MCMV. To determine whether genotypic and phenotypic differences also exist between laboratory strains of MCMV and wild derived strains of MCMV, we sequenced the genomes of three low-passage strains of MCMV, plus the laboratory strain, K181. We coupled this genetic characterization to their phenotypic characteristics. In contrast to what is seen with HCMV (and rhesus CMV), there were no major genomic rearrangements in the MCMV genomes. In addition, the genome size was remarkably conserved between MCMV strains with no major insertions or deletions. There was, however, significant sequence variation between strains of MCMV, particularly at the genomic termini. These more subtle genetic differences led to considerable differences in in vivo replication with some strains of MCMV, such as WP15B, replicating preferentially in otherwise-MCMV-resistant C57BL/6 mice. CBA mice were no more resistant to MCMV than C57BL/6 mice and for some MCMV strains appeared to control infection less well than C57BL/6 mice. It is apparent that the previously described host resistance patterns of inbred mice and MCMV are not consistently applicable for all MCMV strains.

This study examined the eggshell biofilm forming ability of Salmonella enterica isolates recovered from egg farms. Multicellular behaviour and biofilm production were examined at 22 and 37°C by Congo red morphology and the crystal violet staining assay. The results indicated that the biofilm forming behaviour of Salmonella isolates was dependent on temperature and associated with serovars. Significantly greater biofilm production was observed at 22°C compared with 37°C. The number of viable biofilm cells attached to eggshells after incubation for 48 h at 22°C was significantly influenced by serovar. Scanning electron microscopic examination revealed firm attachment of bacterial cells to the eggshell surface. The relative expression of csgD and adrA gene was significantly higher in eggshell biofilm cells of S. Mbandaka and S. Oranienburg. These findings demonstrate that Salmonella isolates are capable of forming biofilm on the eggshell surface and that this behaviour is influenced by temperature and serovar.

The role of gap junctional intercellular communication during inflammatory processes is not well understood. In particular, changes in the expression and function of vascular endothelial connexins (gap junction proteins) in response to inflammatory agents has not been fully investigated. In this study, we used intercellular dye transfer methods to assess interendothelial communication in aortic segments isolated from mice treated with or without intraperitoneal lipopolysaccharide (LPS), a potent inflammatory mediator. LPS treatment resulted in a 49% decrease in endothelial dye coupling 18 h after injection. Western blots indicated that LPS treatment also caused a reduction in endothelial connexin40 (Cx40) levels to 33% of control levels. Connexin37 (Cx37) levels decreased only slightly after LPS treatment to 79% of control levels. We also examined endothelial communication in aortic segments isolated from Cx37–/– and Cx40–/– mice. LPS treatment caused a significantly greater decrease in dye transfer in endothelium isolated from Cx37–/– animals compared with endothelium from Cx40–/– animals (71 vs. 26% decrease). LPS injection caused a reduction in Cx40 levels in Cx37–/– endothelium, whereas LPS actually increased Cx37 levels in Cx40–/– endothelium. These results suggest that LPS mediates changes in endothelial gap junction-mediated communication, at least in part, through modulation of Cx40 and Cx37 levels.

Mice lacking both connexin37 (Cx37) and connexin40 (Cx40), gap junction proteins expressed in vascular endothelium, die perinatally with pronounced vascular abnormalities. Early vasculogenesis proceeds normally, but by E18.5 Cx37(-/-)Cx40(-/-) animals display vessel dilatation and congestion as well as localized hemorrhages in skin, testis, intestines, and lungs. Abnormal vascular channels are present in the testis, often forming cavernous hemangioma-like defects. Unusually large, distended vessels are also present in the submucosa and lamina propria of the intestine. Ablation of Cx40 has a greater effect on endothelial dye-transfer than ablation of Cx37, and the effect of Cx40 ablation is age-dependent. Only in embryonic aortas lacking both Cx37 and Cx40 is there a complete loss of endothelial coupling. Surprisingly, elimination of Cx40 results in a large drop in aortic endothelial Cx37 on western blots, and deletion of Cx37 also reduces endothelial Cx40 levels. In contrast, in the medial layer, both Cx37 and Cx43 increase when Cx40 is ablated. These studies indicate that Cx37 and Cx40 are collectively critical for endothelial communication and provide evidence of an important role for gap junctions in vascular development. In addition, Cx37 and Cx40 appear to be mutually dependent on each other for normal expression in vascular endothelium.

Infection with multiple genetically distinct strains of pathogen is common and can lead to positive (complementation) or negative (competitive) within-host interactions. These interactions can alter aspects of the disease process and help shape pathogen evolution. Infection of the host with multiple strains of cytomegalovirus (CMV) occurs frequently in humans and mice. Profound, NK-cell-mediated (apparent) competition has been identified in C57BL/6 mice, and prevented the replication and shedding of certain co-infecting CMV strains. However, the frequency of such strong competition has not been established. Other within-host interactions such as complementation or alternative forms of competition remain possible. Moreover, high rates of recombination in both human CMV and murine CMV (MCMV) suggest prolonged periods of viral co-replication, rather than strong competitive suppression. An established model was employed to investigate the different possible outcomes of multi-strain infection in other mouse strains. In this study, co-replication of up to four strains of MCMV in the spleen, liver and salivary glands was observed in both MCMV-susceptible and MCMV-resistant mice. In the absence of apparent competition, no other forms of competition were unmasked. In addition, no evidence of complementation between viral strains was observed. Importantly, co-replication of MCMV strains was apparent for up to 90 days in the salivary glands. These data indicated that competition was not the default outcome of multi-strain CMV infection. Prolonged, essentially neutral, co-replication may be the norm, allowing for multi-strain transmission and prolonged opportunities for recombination.

There are over 2,500 Salmonella enterica serovars that circulate globally. Of these, serovars those classified into subspecies I are the most common cause of human salmonellosis. Many subspecies I Salmonella serovars are routinely isolated from egg farm environments but are not frequently associated with causing disease in humans. In this study, virulence profiles were generated for ten strains of Salmonella enterica isolated directly from egg farm environments to investigate their potential public health risk. Three virulence parameters were assessed including in vitro invasion, in vivo pathogenicity and characterization of genomic variation within five specific pathogenicity islands. These ten Salmonella strains exhibited significant differences in invasion into the human intestinal epithelial cell line, Caco2. Low, moderate, and high invasion patterns were observed and the degree of invasion was dependent on bacterial growth in a nutritive environment. Interestingly, two Salmonella strains, S. Adelaide and S. Bredeney had consistently low invasion. The S. Typhimurium definitive types and S. Virchow exhibited the greatest cell invasion following growth in Luria Bertani broth. Only the S. Typhimurium strains caused disease in BALB/c mice, yet the majority of serovars were consistently detected in feces over the 21 day experiment. Genomic comparison of the five specific pathogenicity islands has shown that variation in virulence is likely multifactorial. Sequence variability was observed primarily in strains with low virulence. In particular, genes involved in forming the structures of the SPI-1 and SPI-2 type 3 secretion systems as well as multiple effector proteins were among the most variable. This variability suggest that serovars with low virulence are likely to have both invasion and within host replication defects that ultimately limit their pathogenicity.

The herpesvirus lifestyle results in a long-term interaction between host and invading pathogen, resulting in exquisite adaptation of virus to host. We have sequenced the genomes of nine strains of murine cytomegalovirus (a betaherpesvirus), isolated from free-living mice trapped at locations separated geographically and temporally. Despite this separation these genomes were found to have low levels of nucleotide variation. Of the more than 160 open reading frames, almost 90% had a dN/dS ratio of amino acid substitutions of less than 0.6, indicating the level of purifying selection on the coding potential of MCMV. Examination of selection acting on individual genes at the codon level however indicates some level of positive selection, with 0.03% of codons showing strong evidence for positive selection. Conversely, 1.3% of codons show strong evidence of purifying selection. Alignments of both genome sequences and coding regions suggested that high levels of recombination have shaped the MCMV genome.

Cx37 mice. (A, B) Transverse sections of a newborn wild-type mouse pup with normalatrial septation. The section in A is superior to the section in B. An arrow indicates the septum secundum, whereas arrowheads indicate the septum primum.The septum primum and septum secundum overlap, with the septum primum forming the flap of the foramen ovale at birth. (C, D) Sections of newbornCx40

Vascular endothelial cells are coupled by gap junctions that permit cell-to-cell transfer of small molecules, including signals that may be important for vasomotor responses. Connexin37 (Cx37) and connexin40 (Cx40) are the predominant gap-junction proteins present in mouse endothelium. We examined the effect of eliminating Cx37, Cx40, or both, on interendothelial communication in mouse aorta. Intercellular transfer of biocytin and [2-(4-nitro-2,1,3-benzoxadiazol-7-yl)aminoethyl]trimethylammonium (NBD-TMA) was used to assess gap-junction-mediated coupling. Ablation of Cx40 generally had a greater effect on dye-transfer than ablation of Cx37. The effect of Cx40 ablation on dye-transfer was age dependent. There was a 27-fold reduction in biocytin transfer in embryonic Cx40-/- aortic endothelium, a much larger change than in aortas of 6-7-week-old Cx40-/- animals, which showed a 3.5-fold reduction. By contrast, there was no reduction in biocytin transfer in embryonic Cx37-/- endothelium. Embryonic aortas lacking both Cx37 and Cx40 showed a complete loss of endothelial dye-transfer. Surprisingly, elimination of Cx40 resulted in up to a 17-fold drop in endothelial Cx37 on western blots, whereas deletion of Cx37 reduced endothelial Cx40 up to 4.2-fold. By contrast, in the medial layer, both Cx37 and Cx43 increased approximately fourfold in Cx40-/- aortas. Declines in non-ablated endothelial connexins were not mediated by changes in connexin mRNA levels, suggesting a post-transcriptional effect. Our results indicate that Cx37 and Cx40 are the only functional connexins expressed in mouse aortic endothelium and are collectively crucial for endothelial communication. Furthermore, Cx37 and Cx40 are codependent on each other for optimal expression in vascular endothelium.

Killed avian influenza virus (AIV) vaccines have been used to control H5N1 infections in countries where the virus is endemic. Distinguishing vaccinated from naturally infected birds (DIVA) in such situations however, has become a major challenge. Recently, we introduced the recombinant ectodomain of the M2 protein (M2e) of H5N1 subtype as a novel tool for an ELISA based DIVA test. Despite being antigenic in natural infection the monomer form of the M2e used in ELISA had limited antigenicity and consequently poor diagnostic capability. To address this shortcoming, we evaluated the use of four tandem copies of M2e (tM2e) for increased efficiency of M2e antibody detection. The tM2e gene of H5N1 strain from Indonesia (A/Indonesia/CDC540/2006) was cloned into a pMAL- p4x expression vector and expressed in E.coli as a recombinant tM2e-MBP or M2e-MBP proteins. Both of these, M2e and tM2e antigens reacted with sera obtained from chickens following live H5N1 infection but not with sera from vaccinated birds. A significantly stronger M2e antibody reaction was observed with the tM2e compared to M2e antigen. Western blotting also supported the superiority of tM2e over M2e in detection of specific M2e antibodies against live H5N1 infection. Results from this study demonstrate that M2e tetramer is a better antigen than single M2e and could be more suitable for an ELISA based DIVA test.

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., Author Summary Infection of the host with multiple strains of a pathogen is common and occurs with the herpesvirus, human cytomegalovirus (HCMV). However the effects of multi-strain infection on the host and the pathogen remain poorly studied. Here we show, in a mouse model, that infection of C57BL/6 mice with multiple strains of murine CMV (MCMV) results in profound within-host competition. Competition between the strains of MCMV is dependent on Ly49H+ natural killer (NK) cells. The NK cell activation receptor Ly49H receptor targets certain genotypes of the viral protein, m157. During multi-strain infection, strains of MCMV encoding an m157 capable of binding Ly49H are excluded from the salivary gland and the saliva of C57BL/6 mice, allowing for the shedding of only non-Ly49H binding strains of MCMV in the saliva. This within-host competition could therefore have significant impacts on the circulation of MCMV strains, as only the most virulent MCMV strains were present in the saliva.

International audience; 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.