Effect of Salmonella vaccination of breeder chickens on contamination of broiler chicken carcasses in integrated poultry operations

While measures to control carcass contamination with Salmonella at the processing plant have been implemented with some success, on-farm interventions that reduce Salmonella prevalence in meat birds entering the processing plant have not translated well on a commercial scale. We determined the impact of Salmonella vaccination on commercial poultry operations by monitoring four vaccinated and four nonvaccinated breeder (parental) chicken flocks and comparing Salmonella prevalences in these flocks and their broiler, meat bird progeny. For one poultry company, their young breeders were vaccinated by using a live-attenuated Salmonella enterica serovar Typhimurium vaccine (Megan VAC-1) followed by a killed Salmonella bacterin consisting of S. enterica serovar Berta and S. enterica serovar Kentucky. The other participating poultry company did not vaccinate their breeders or broilers. The analysis revealed that vaccinated hens had a lower prevalence of Salmonella in the ceca (38.3% versus 64.2%; P < 0.001) and the reproductive tracts (14.22% versus 51.7%; P < 0.001). We also observed a lower Salmonella prevalence in broiler chicks (18.1% versus 33.5%; P < 0.001), acquired from vaccinated breeders, when placed at the broiler farms contracted with the poultry company. Broiler chicken farms populated with chicks from vaccinated breeders also tended to have fewer environmental samples containing Salmonella (14.4% versus 30.1%; P < 0.001). There was a lower Salmonella prevalence in broilers entering the processing plants (23.4% versus 33.5%; P < 0.001) for the poultry company that utilized this Salmonella vaccination program for its breeders. Investigation of other company-associated factors did not indicate that the difference between companies could be attributed to measures other than the vaccination program.     

Public Health Assessment of Salmonella enterica Serovar Enteritidis Inactivated-Vaccine Treatment in Layer Flocks

Although there have been several reports on the efficacy assessment of a Salmonella enterica serovar Enteritidis vaccine against intestinal and parenchymatous organ diseases of laying hens, no public health risk characterization of its long-term effect on eggs has been reported. In this study, we attempted to assess the public health effect of an inactivated S. enterica serovar Enteritidis vaccine against serovar Enteritidis contamination of chicken eggs. We analyzed serovar Enteritidis isolation test results from four windowless farms in which inactivated-vaccine administration was initiated based on the sanitary monitoring program of a farm. When flocks with and without S. enterica serovar Enteritidis vaccine treatments were mixed, the application of an inactivated serovar Enteritidis vaccine decreased the most probable number (MPN) of bacteria by at least 100-fold in broken (liquid) egg samples positive for serovar Enteritidis, although a statistical difference between those MPNs could not be obtained. The isolation frequency after the vaccine application was less than 1/10 (P < 0.01). No S. enterica serovar Enteritidis bacteria were isolated approximately 1 year after all of the chickens had received the inactivated serovar Enteritidis vaccine. It was suggested that an adequate administration of an inactivated serovar Enteritidis vaccine reduced the contamination risk of eggs (the number of isolated serovar Enteritidis cells and detection frequency) compared to the contamination risk of eggs laid by nonvaccinated hens.     

<Emphasis Type="Italic">allB</Emphasis>, allantoin utilisation and <Emphasis Type="Italic">Salmonella enterica</Emphasis> serovar Enteritidis and Typhimurium colonisation of poultry and mice

Natural variation in the presence or the absence of STM0517-0529 genes allowing allantoin utilisation has been described in field isolates of the multidrug resistant Salmonella enterica serovar Typhimurium belonging to the phage type DT104. Interestingly, S. enterica subspecies enterica serovar Typhimurium DT104 is quite frequent in pigs and cattle, but rarely present in egg-laying hens. Taking into account the different mode of allantoin metabolism in birds and mammals, we were interested in whether the absence of STM0517-0529 genes may disable this clone in poultry colonisation. We have therefore constructed the allB (also designated as STM0523) mutants in S. enterica subspecies enterica serovar Typhimurium and S. enterica subspecies enterica serovar Enteritidis, and with these, we infected mice, newly hatched chickens and adult egg-laying hens to show that the defect in allantoin utilisation does not influence S. enterica virulence for mice or adult hens, but slightly decreases virulence of S. enterica for chickens. The decrease in virulence of the allB mutant was relatively minor as it could be observed only after a mixed infection model, consistent with a lower prevalence, but not a total absence of such clones in poultry flocks.     

Cytokine signaling in splenic leukocytes from vaccinated and non-vaccinated chickens after intravenous infection with Salmonella enteritidis

In order to design a new Salmonella enterica vaccine, one needs to understand how naive and immune chickens interact differently when exposed to S. enterica. In this study we therefore determined the immune response of vaccinated and non-vaccinated chickens after intravenous infection with Salmonella enterica serovar Enteritidis (S. Enteritidis). Using flow cytometry we showed that 4 days post infection (DPI), counts of CD4 and B-lymphocytes did not change, CD8 and γδ T-lymphocytes decreased and macrophages and heterophils increased in the spleen. When vaccinated and non-vaccinated chickens were compared, only macrophages and heterophils were found in significantly higher counts in the spleens of the non-vaccinated chickens. The non-vaccinated chickens also expressed higher anti-LPS antibodies than the vaccinated chickens. The expression of interleukin (IL)1β, IL6, IL8, IL18, LITAF, IFNγ and iNOS did not exhibit any clear pattern in the cells sorted from the spleens of vaccinated or non-vaccinated chickens. Only IL17 and IL22 showed a differential expression in the CD4 T-lymphocytes of the vaccinated and non-vaccinated chickens at 4 DPI, both being expressed at a higher level in the non-vaccinated chickens. Due to a similar IFNγ expression in the CD4 T-lymphocytes in both the vaccinated and non-vaccinated chickens, and a variable IL17 expression oscillating around IFNγ expression levels, the IL17∶IFNγ ratio in CD4 T-lymphocytes was found to be central for the outcome of the immune response. When IL17 was expressed at higher levels than IFNγ in the non-vaccinated chickens, the Th17 immune response with a higher macrophage and heterophil infiltration in the spleen dominated. However, when the expression of IL17 was lower than that of IFNγ as in the vaccinated chickens, the Th1 response with a higher resistance to S. Enteritidis infection dominated.     

Dynamics of Salmonella enterica and antimicrobial resistance in the Brazilian poultry industry and global impacts on public health

Non-typhoidal Salmonella enterica is a common cause of diarrhoeal disease; in humans, consumption of contaminated poultry meat is believed to be a major source. Brazil is the world’s largest exporter of chicken meat globally, and previous studies have indicated the introduction of Salmonella serovars through imported food products from Brazil. Here we provide an in-depth genomic characterisation and evolutionary analysis to investigate the most prevalent serovars and antimicrobial resistance (AMR) in Brazilian chickens and assess the impact to public health of products contaminated with S. enterica imported into the United Kingdom from Brazil. To do so, we examine 183 Salmonella genomes from chickens in Brazil and 357 genomes from humans, domestic poultry and imported Brazilian poultry products isolated in the United Kingdom. S. enterica serovars Heidelberg and Minnesota were the most prevalent serovars in Brazil and in meat products imported from Brazil into the UK. We extended our analysis to include 1,259 publicly available Salmonella Heidelberg and Salmonella Minnesota genomes for context. The Brazil genomes form clades distinct from global isolates, with temporal analysis suggesting emergence of these Salmonella Heidelberg and Salmonella Minnesota clades in the early 2000s, around the time of the 2003 introduction of the Enteritidis vaccine in Brazilian poultry. Analysis showed genomes within the Salmonella Heidelberg and Salmonella Minnesota clades shared resistance to sulphonamides, tetracyclines and beta-lactams conferred by sul2, tetA and bla_CMY-2 genes, not widely observed in other co-circulating serovars despite similar selection pressures. The sul2 and tetA genes were concomitantly carried on IncC plasmids, whereas bla_CMY-2 was either co-located with the sul2 and tetA genes on IncC plasmids or independently on IncI1 plasmids. Long-term surveillance data collected in the UK showed no increase in the incidence of Salmonella Heidelberg or Salmonella Minnesota in human cases of clinical disease in the UK following the increase of these two serovars in Brazilian poultry. In addition, almost all of the small number of UK-derived genomes which cluster with the Brazilian poultry-derived sequences could either be attributed to human cases with a recent history of foreign travel or were from imported Brazilian food products. These findings indicate that even should Salmonella from imported Brazilian poultry products reach UK consumers, they are very unlikely to be causing disease. No evidence of the Brazilian strains of Salmonella Heidelberg or Salmonella Minnesota were observed in UK domestic chickens. These findings suggest that introduction of the Salmonella Enteritidis vaccine, in addition to increasing antimicrobial use, could have resulted in replacement of salmonellae in Brazilian poultry flocks with serovars that are more drug resistant, but less associated with disease in humans in the UK. The plasmids conferring resistance to beta-lactams, sulphonamides and tetracyclines likely conferred a competitive advantage to the Salmonella Minnesota and Salmonella Heidelberg serovars in this setting of high antimicrobial use, but the apparent lack of transfer to other serovars present in the same setting suggests barriers to horizontal gene transfer that could be exploited in intervention strategies to reduce AMR. The insights obtained reinforce the importance of One Health genomic surveillance.     

The Salmonella PathogeniCity Island (SPI) 1 contributes more than SPI2 to the colonization of the chicken by Salmonella enterica serovar Typhimurium

Background  

Salmonella enterica serovar Typhimurium (Typhimurium) is an important pathogen that infects a broad range of hosts. In humans, Typhimurium causes a gastroenteritis characterized by vomiting, diarrhea, and abdominal pains. Typhimurium infection occurs mainly through the ingestion of contaminated food including poultry, pork, eggs, and milk. Chickens that are asymptomatic carriers of Typhimurium constitute a potential reservoir for infection. The type three secretion systems encoded by Salmonella pathogeniCity islands (SPI) 1 and 2 are major virulence factors of Salmonella. However, only a few studies have investigated their role during the infection of chickens.     

Vaccination with Recombinant RNA Replicon Particles Protects Chickens from H5N1 Highly Pathogenic Avian Influenza Virus

Highly pathogenic avian influenza viruses (HPAIV) of subtype H5N1 not only cause a devastating disease in domestic chickens and turkeys but also pose a continuous threat to public health. In some countries, H5N1 viruses continue to circulate and evolve into new clades and subclades. The rapid evolution of these viruses represents a problem for virus diagnosis and control. In this work, recombinant vesicular stomatitis virus (VSV) vectors expressing HA of subtype H5 were generated. To comply with biosafety issues the G gene was deleted from the VSV genome. The resulting vaccine vector VSV*ΔG(HA) was propagated on helper cells providing the VSV G protein in trans. Vaccination of chickens with a single intramuscular dose of 2×10⁸ infectious replicon particles without adjuvant conferred complete protection from lethal H5N1 infection. Subsequent application of the same vaccine strongly boosted the humoral immune response and completely prevented shedding of challenge virus and transmission to sentinel birds. The vaccine allowed serological differentiation of infected from vaccinated animals (DIVA) by employing a commercially available ELISA. Immunized chickens produced antibodies with neutralizing activity against multiple H5 viruses representing clades 1, 2.2, 2.5, and low-pathogenic avian influenza viruses (classical clade). Studies using chimeric H1/H5 hemagglutinins showed that the neutralizing activity was predominantly directed against the globular head domain. In summary, these results suggest that VSV replicon particles are safe and potent DIVA vaccines that may help to control avian influenza viruses in domestic poultry.     

A Virulent Bioluminescent and Fluorescent Dual-Reporter Marek's Disease Virus Unveils an Alternative Spreading Pathway in Addition to Cell-to-Cell Contact

Marek''s disease virus (MDV) is a growing threat for the poultry industry. Unfortunately, despite successful vaccination against the disease, MDV remains in circulation within vaccinated flocks, leading to the selection of increasingly virulent pathotypes. Detailed knowledge of the virus biology and the host-virus interaction is required to improve the vaccine efficiency. In the present study, I engineered an original, dual-reporter MDV to track and quantify virus replication in vitro and in vivo.     

Poultry Body Temperature Contributes to Invasion Control through Reduced Expression of Salmonella Pathogenicity Island 1 Genes in Salmonella enterica Serovars Typhimurium and Enteritidis

Salmonella enterica serovars Typhimurium (S. Typhimurium) and Enteritidis (S. Enteritidis) are foodborne pathogens, and outbreaks are often associated with poultry products. Chickens are typically asymptomatic when colonized by these serovars; however, the factors contributing to this observation are uncharacterized. Whereas symptomatic mammals have a body temperature between 37°C and 39°C, chickens have a body temperature of 41°C to 42°C. Here, in vivo experiments using chicks demonstrated that numbers of viable S. Typhimurium or S. Enteritidis bacteria within the liver and spleen organ sites were ≥4 orders of magnitude lower than those within the ceca. When similar doses of S. Typhimurium or S. Enteritidis were given to C3H/HeN mice, the ratio of the intestinal concentration to the liver/spleen concentration was 1:1. In the avian host, this suggested poor survival within these tissues or a reduced capacity to traverse the host epithelial layer and reach liver/spleen sites or both. Salmonella pathogenicity island 1 (SPI-1) promotes localization to liver/spleen tissues through invasion of the epithelial cell layer. Following in vitro growth at 42°C, SPI-1 genes sipC, invF, and hilA and the SPI-1 rtsA activator were downregulated compared to expression at 37°C. Overexpression of the hilA activators fur, fliZ, and hilD was capable of inducing hilA-lacZ at 37°C but not at 42°C despite the presence of similar levels of protein at the two temperatures. In contrast, overexpression of either hilC or rtsA was capable of inducing hilA and sipC at 42°C. These data indicate that physiological parameters of the poultry host, such as body temperature, have a role in modulating expression of virulence.     

The Early Innate Response of Chickens to Salmonella enterica Is Dependent on the Presence of O-Antigen but Not on Serovar Classification

Salmonella vaccines used in poultry in the EU are based on attenuated strains of either Salmonella serovar Enteritidis or Typhimurium which results in a decrease in S. Enteritidis and S. Typhimurium but may allow other Salmonella serovars to fill an empty ecological niche. In this study we were therefore interested in the early interactions of chicken immune system with S. Infantis compared to S. Enteritidis and S. Typhimurium, and a role of O-antigen in these interactions. To reach this aim, we orally infected newly hatched chickens with 7 wild type strains of Salmonella serovars Enteritidis, Typhimurium and Infantis as well as with their rfaL mutants and characterized the early Salmonella-chicken interactions. Inflammation was characterized in the cecum 4 days post-infection by measuring expression of 43 different genes. All wild type strains stimulated a greater inflammatory response than any of the rfaL mutants. However, there were large differences in chicken responses to different wild type strains not reflecting their serovar classification. The initial interaction between newly-hatched chickens and Salmonella was found to be dependent on the presence of O-antigen but not on its structure, i.e. not on serovar classification. In addition, we observed that the expression of calbindin or aquaporin 8 in the cecum did not change if inflammatory gene expression remained within a 10 fold fluctuation, indicating the buffering capacity of the cecum, preserving normal gut functions even in the presence of minor inflammatory stimuli.     

Virulence potential of five major pathogeniCity islands (SPI-1 to SPI-5) of Salmonella enterica serovar Enteritidis for chickens

Background  

Salmonella is a highly successful parasite of reptiles, birds and mammals. Its ability to infect and colonise such a broad range of hosts coincided with the introduction of new genetic determinants, among them 5 major pathogeniCity islands (SPI1-5), into the Salmonella genome. However, only limited information is available on how each of these pathogeniCity islands influences the ability of Salmonella to infect chickens. In this study, we therefore constructed Salmonella Enteritidis mutants with each SPI deleted separately, with single individual SPIs (i.e. with the remaining four deleted) and a mutant with all 5 SPIs deleted, and assessed their virulence in one-day-old chickens, together with the innate immune response of this host.     

rpoS-Regulated Core Genes Involved in the Competitive Fitness of Salmonella enterica Serovar Kentucky in the Intestines of Chickens

Salmonella enterica serovar Kentucky has become the most frequently isolated serovar from poultry in the United States over the past decade. Despite its prevalence in poultry, it causes few human illnesses in the United States. The dominance of S. Kentucky in poultry does not appear to be due to single introduction of a clonal strain, and its reduced virulence appears to correlate with the absence of virulence genes grvA, sseI, sopE, and sodC1. S. Kentucky''s prevalence in poultry is possibly attributable to its metabolic adaptation to the chicken cecum. While there were no difference in the growth rate of S. Kentucky and S. Typhimurium grown microaerophilically in cecal contents, S. Kentucky persisted longer when chickens were coinfected with S. Typhimurium. The in vivo advantage that S. Kentucky has over S. Typhimurium appears to be due to differential regulation of core Salmonella genes via the stationary-phase sigma factor rpoS. Microarray analysis of Salmonella grown in cecal contents in vitro identified several metabolic genes and motility and adherence genes that are differentially activated in S. Kentucky. The contributions of four of these operons (mgl, prp, nar, and csg) to Salmonella colonization in chickens were assessed. Deletion of mgl and csg reduced S. Kentucky persistence in competition studies in chickens infected with wild-type or mutant strains. Subtle mutations affecting differential regulation of core Salmonella genes appear to be important in Salmonella''s adaptation to its animal host and especially for S. Kentucky''s emergence as the dominant serovar in poultry.     

Prevalence and Fimbrial Genotype Distribution of Poultry Salmonella Isolates in China (2006 to 2012)

In this study, a total of 323 Salmonella enterica strains were isolated from 3,566 rectal swab samples of 51 poultry farms in seven regions of 12 provinces of China between 2006 and 2012. The prevalences of Salmonella sp. carriage were 12.4% in geese (66 positive/533 samples), 10.4% in turkeys (32/309), 9.8% in chickens (167/1,706), 6.8% in ducks (41/601), and 4.1% in pigeons (17/417), respectively. These isolates belonged to 20 serovars, in which the most frequent serovars were S. enterica serovar Gallinarum biovar Pullorum (herein, S. Pullorum) (55 isolates, 17.0%), S. enterica serovar Typhimurium (50 isolates, 15.5%), and S. enterica serovar Enteritidis (39 isolates, 12.1%). Overall, S. Typhimurium was the most commonly detected serovar; among the individual species, S. Pullorum was most commonly isolated from chickens, S. Enteritidis was most common in ducks, S. Typhimurium was most common in geese and pigeons, and S. enterica serovar Saintpaul was most common in turkeys. PCR determination of 20 fimbrial genes demonstrated the presence of bcfD, csgA, fimA, stdB, and sthE genes and the absence of staA and stgA genes in these isolates, and other loci were variably distributed, with frequency values ranging from 11.8 to 99.1%. These 323 Salmonella isolates were subdivided into 41 different fimbrial genotypes, and of these isolate, 285 strains (88.2%) had 12 to 14 fimbrial genes. Our findings indicated that the Salmonella isolates from different poultry species were phenotypically and genetically diverse and that some fimbrial genes are more frequently associated with serovars or serogroups.     

Identification by Subtractive Hybridization of Sequences Specific for Salmonella enterica Serovar Enteritidis

Salmonella enterica serovar Enteritidis, a major cause of food poisoning, can be transmitted to humans through intact chicken eggs when the contents have not been thoroughly cooked. Infection in chickens is asymptomatic; therefore, simple, sensitive, and specific detection methods are crucial for efforts to limit human exposure. Suppression subtractive hybridization was used to isolate DNA restriction fragments present in Salmonella serovar Enteritidis but absent in other bacteria found in poultry environments. Oligonucleotide primers to candidate regions were used in polymerase chain reactions to test 73 non-Enteritidis S. enterica isolates comprising 34 different serovars, including Dublin and Pullorum, two very close relatives of Enteritidis. A primer pair to one Salmonella difference fragment (termed Sdf I) clearly distinguished serovar Enteritidis from all other serovars tested, while two other primer pairs only identified a few non-Enteritidis strains. These primer pairs were also useful for the detection of a diverse collection of clinical and environmental Salmonella serovar Enteritidis isolates. In addition, five bacterial genera commonly found with Salmonella serovar Enteritidis were not detected. By treating total DNA with an exonuclease that degrades sheared chromosomal DNA but not intact circular plasmid DNA, it was shown that Sdf I is located on the chromosome. The Sdf I primers were used to screen a Salmonella serovar Enteritidis genomic library and a unique 4,060-bp region was defined. These results provide a basis for developing a rapid, sensitive, and highly specific detection system for Salmonella serovar Enteritidis and provide sequence information that may be relevant to the unique characteristics of this serovar.     

Population dynamics of Salmonella enterica serotypes in commercial egg and poultry production

Fresh and processed poultry have been frequently implicated in cases of human salmonellosis. Furthermore, increased consumption of meat and poultry has increased the potential for exposure to Salmonella enterica. While advances have been made in reducing the prevalence and frequency of Salmonella contamination in processed poultry, there is mounting pressure on commercial growers to prevent and/or eliminate these human pathogens in preharvest production facilities. Several factors contribute to Salmonella colonization in commercial poultry, including the serovar and the infectious dose. In the early 1900s, Salmonella enterica serovars Pullorum and Gallinarum caused widespread diseases in poultry, but vaccination and other voluntary programs helped eradicate pullorum disease and fowl typhoid from commercial flocks. However, the niche created by the eradication of these serovars was likely filled by S. Enteritidis, which proliferated in the bird populations. While this pathogen remains a significant problem in commercial egg and poultry production, its prevalence among poultry has been declining since the 1990s. Coinciding with the decrease of S. Enteritidis, S. Heidelberg and S. Kentucky have emerged as the predominant serovars in commercial broilers. In this review, we have highlighted bacterial genetic and host-related factors that may contribute to such shifts in Salmonella populations in commercial poultry and intervention strategies that could limit their colonization.     

Dimethyl Adenosine Transferase (KsgA) Deficiency in Salmonella enterica Serovar Enteritidis Confers Susceptibility to High Osmolarity and Virulence Attenuation in Chickens

Dimethyl adenosine transferase (KsgA) performs diverse roles in bacteria, including ribosomal maturation and DNA mismatch repair, and synthesis of KsgA is responsive to antibiotics and cold temperature. We previously showed that a ksgA mutation in Salmonella enterica serovar Enteritidis results in impaired invasiveness in human and avian epithelial cells. In this study, we tested the virulence of a ksgA mutant (the ksgA::Tn5 mutant) of S. Enteritidis in orally challenged 1-day-old chickens. The ksgA::Tn5 mutant showed significantly reduced intestinal colonization and organ invasiveness in chickens compared to those of the wild-type (WT) parent. Phenotype microarray (PM) was employed to compare the ksgA::Tn5 mutant and its isogenic wild-type strain for 920 phenotypes at 28°C, 37°C, and 42°C. At chicken body temperature (42°C), the ksgA::Tn5 mutant showed significantly reduced respiratory activity with respect to a number of carbon, nitrogen, phosphate, sulfur, and peptide nitrogen nutrients. The greatest differences were observed in the osmolyte panel at concentrations of ≥6% NaCl at 37°C and 42°C. In contrast, no major differences were observed at 28°C. In independent growth assays, the ksgA::Tn5 mutant displayed a severe growth defect in high-osmolarity (6.5% NaCl) conditions in nutrient-rich (LB) and nutrient-limiting (M9 minimum salts) media at 42°C. Moreover, the ksgA::Tn5 mutant showed significantly reduced tolerance to oxidative stress, but its survival within macrophages was not impaired. Unlike Escherichia coli, the ksgA::Tn5 mutant did not display a cold-sensitivity phenotype; however, it showed resistance to kasugamycin and increased susceptibility to chloramphenicol. To the best of our knowledge, this is the first report showing the role of ksgA in S. Enteritidis virulence in chickens, tolerance to high osmolarity, and altered susceptibility to kasugamycin and chloramphenicol.     

Pathogenic Role of SEF14, SEF17, and SEF21 Fimbriae in Salmonella enterica Serovar Enteritidis Infection of Chickens

Very little is known about the contribution of surface appendages of Salmonella enterica serovar Enteritidis to pathogenesis in chickens. This study was designed to clarify the role of SEF14, SEF17, and SEF21 fimbriae in serovar Enteritidis pathogenesis. Stable, single, defined sefA (SEF14), agfA (SEF17), and fimA (SEF21) insertionally inactivated fimbrial gene mutants of serovar Enteritidis were constructed. All mutant strains invaded Caco-2 and HT-29 enterocytes at levels similar to that of the wild type. Both mutant and wild-type strains were ingested equally well by chicken macrophage cell lines HD11 and MQ-NCSU. There were no significant differences in the abilities of these strains to colonize chicken ceca. The SEF14⁻ strain was isolated in lower numbers from the livers of infected chickens and was cleared from the spleens faster than other strains. No significant differences in fecal shedding of these strains were observed.