Reduced Uterine Tissue Damage during Chlamydia muridarum Infection in TREM-1,3-Deficient Mice.

Genital infections with Chlamydia trachomatis can lead to uterine and oviduct tissue damage in the female reproductive tract. Neutrophils are strongly associated with tissue damage during chlamydial infection, while an adaptive CD4 T cell response is necessary to combat infection. Activation of triggering receptor expressed on myeloid cells-1 (TREM-1) on neutrophils has previously been shown to induce and/or enhance degranulation synergistically with Toll-like receptor (TLR) signaling. Additionally, TREM-1 can promote neutrophil transepithelial migration. In this study, we sought to determine the contribution of TREM-1,3 to immunopathology in the female mouse genital tract during Chlamydia muridarum infection. Relative to control mice, trem1,3-/- mice had no difference in chlamydial burden or duration of lower-genital-tract infection. We also observed a similar incidence of hydrosalpinx 45 days postinfection in trem1,3-/- compared to wild-type (WT) mice. However, compared to WT mice, trem1,3-/- mice developed significantly fewer hydrometra in uterine horns. Early in infection, trem1,3-/- mice displayed a notable decrease in the number of uterine glands containing polymorphonuclear cells and uterine horn lumens had fewer neutrophils, with increased granulocyte colony-stimulating factor (G-CSF). trem1,3-/- mice also had reduced erosion of the luminal epithelium. These data indicate that TREM-1,3 contributes to transepithelial neutrophil migration in the uterus and uterine glands, promoting the occurrence of hydrometra in infected mice.

Altering the Microbiome Inhibits Tumorigenesis in a Mouse Model of Oviductal High-Grade Serous Carcinoma.

Studies have shown bacteria influence the initiation and progression of cancers arising in sites that harbor rich microbial communities, such as the colon. Little is known about the potential for the microbiome to influence tumorigenesis at sites considered sterile, including the upper female genital tract. The recent identification of distinct bacterial signatures associated with ovarian carcinomas suggests microbiota in the gut, vagina, or elsewhere might contribute to ovarian cancer pathogenesis. Here, we tested whether altering the microbiome affects tumorigenesis in a mouse model of high-grade serous carcinoma (HGSC) based on conditional oviduct-specific inactivation of the Brca1, Trp53, Rb1, and Nf1 tumor suppressor genes. Cohorts of control (n = 20) and antibiotic-treated (n = 23) mice were treated with tamoxifen to induce tumor formation and then monitored for 12 months. The antibiotic cocktail was administered for the first 5 months of the monitoring period in the treatment group. Antibiotic-treated mice had significantly fewer and less advanced tumors than control mice at study endpoint. Antibiotics induced changes in the composition of the intestinal and vaginal microbiota, which were durable in the fecal samples. Clustering analysis showed particular groups of microbiota are associated with the development of HGSC in this model. These findings demonstrate the microbiome influences HGSC pathogenesis in an in vivo model that closely recapitulates the human disease. Because the microbiome can modulate efficacy of cancer chemo- and immunotherapy, our genetically engineered mouse model system may prove useful for testing whether altering the microbiota can improve the heretofore poor response of HGSC to immunotherapies. SIGNIFICANCE: This study provides strong in vivo evidence for a role of the microbiome in ovarian cancer pathogenesis.

IL-1α Is Essential for Oviduct Pathology during Genital Chlamydial Infection in Mice.

Chlamydia trachomatis infection of the female genital tract can lead to irreversible fallopian tube scarring. In the mouse model of genital infection using Chlamydia muridarum, IL-1R signaling plays a critical role in oviduct tissue damage. In this study, we investigated the pathologic role of IL-1α, one of the two proinflammatory cytokines that bind to IL-1R. Il1a-/- mice infected with C. muridarum cleared infection at their cervix at the same rate as wild-type (WT) mice, but were significantly protected from end point oviduct damage and fibrosis. The contribution of IL-1α to oviduct pathology was more dramatic than observed in mice deficient for IL-1ß. Although chlamydial burden was similar in WT and Il1a-/- oviduct during peak days of infection, levels of IL-1ß, IL-6, CSF3, and CXCL2 were reduced in Il1a-/- oviduct lysates. During infection, Il1a-/- oviducts and uterine horns exhibited reduced neutrophil infiltration, and this reduction persisted after the infection resolved. The absence of IL-1α did not compromise CD4 T cell recruitment or function during primary or secondary chlamydial infection. IL-1α is expressed predominantly by luminal cells of the genital tract in response to infection, and low levels of expression persisted after the infection cleared. Ab-mediated depletion of IL-1α in WT mice prevented infection-induced oviduct damage, further supporting a key role for IL-1α in oviduct pathology.

The role of GtxA during Gallibacterium anatis infection of primary chicken oviduct epithelial cells.

Gallibacterium anatis (G. anatis), one of the major pathogens causing reproductive tract disorders in laying hens, leads to a reduction in egg production and increased mortality, caused by either single or mixed infections with other pathogens. As a specific virulence factor of G. anatis, the role of GtxA in layers' salpingitis remains unclear. In this study, we explored the effect of GtxA on G. anatis infection by comparing wild strain Yu-PDS-RZ-1-SLG (RZ) and its GtxA deleted counterpart RZΔgtxA in primary chicken oviduct epithelial cells (COEC). Their adherence, invasion, cytoxicity, and ability to induce apoptosis and and cytokine secretion were evaluated and the cytotoxicity and cytokine secretion of the recombinant GtxA protein and its N-terminal adenylate cyclase and C-terminal RTX hemolysin domain were also analyzed. We found that the adhesion ability of RZΔgtxA was significantly lower than that of parental strain RZ, and its toxicity to COEC was weakened; Meanwhile, apoptosis was inhibited and the expression of IL-6, IL-2, TNF-α and IFN-γ were dramatically reduced in COEC infected by RZΔgtxA. In contrast, the recombinant protein GtxA inhibited the proliferation of oviduct cells and induced obvious cytotoxicity, and the expression of IL-6, TNF-α and IFN-γ were up-regulated in COEC interacted with recombinant proteins. Our study indicates that GtxA promotes G. anatis adherence to cells, changes cells permeability and expression of inflammatory factors, resulting in cell damage and apoptosis.

Comparison of microbiota in the cloaca, colon, and magnum of layer chicken.

Anatomically terminal parts of the urinary, reproductive, and digestive systems of birds all connect to the cloaca. As the feces drain through the cloaca in chickens, the cloacal bacteria were previously believed to represent those of the digestive system. To investigate similarities between the cloacal microbiota and the microbiota of the digestive and reproductive systems, microbiota inhabiting the colon, cloaca, and magnum, which is a portion of the chicken oviduct of 34-week-old, specific-pathogen-free hens were analyzed using a 16S rRNA metagenomic approach using the Ion torrent sequencer and the Qiime2 bioinformatics platform. Beta diversity via unweighted and weighted unifrac analyses revealed that the cloacal microbiota was significantly different from those in the colon and the magnum. Unweighted unifrac revealed that the cloacal microbiota was distal from the microbiota in the colon than from the microbiota in the magnum, whereas weighted unifrac revealed that the cloacal microbiota was located further away from the microbiota in the magnum than from the microbiota inhabiting the colon. Pseudomonas spp. were the most abundant in the cloaca, whereas Lactobacillus spp. and Flavobacterium spp. were the most abundant species in the colon and the magnum. The present results indicate that the cloaca contains a mixed population of bacteria, derived from the reproductive, urinary, and digestive systems, particularly in egg-laying hens. Therefore, sampling cloaca to study bacterial populations that inhabit the digestive system of chickens requires caution especially when applied to egg-laying hens. To further understand the physiological role of the microbiota in chicken cloaca, exploratory studies of the chicken's cloacal microbiota should be performed using chickens of different ages and types.

High phylogenetic diversity of Gallibacterium anatis is correlated with low biosecurity measures and management practices on poultry farms.

Gallibacterium anatis is considered one of the most common bacterial causative agents of reproductive tract disorders in poultry. In this study, phylogenetic analysis of partial rpoB sequences and biotyping using MALDI-TOF MS was done in order to investigate the genetic diversity of Gallibacterium isolates from 13 farms with different biosecurity measures and management practices. Sampling was done as a part of regular monitoring, except for Farms 9-13 that were included in the study to represent extensive production systems with lowest biosecurity levels. Pharyngeal and cloacal swabs were taken from live birds, while swabs from trachea, liver, peritoneum and oviduct were taken during necropsies. After cultivation and identification, strains from each farm were randomly selected for sequencing and biotyping. Both results showed high level of heterogeneity among the isolates originating from farms with low biosecurity levels, unlike isolates from farms with higher biosecurity levels and proper management that were more closely related and clustered together. Such correlation was statistically significant. Low biosecurity levels enable horizontal transmission of the pathogens, as well as gene transfer. The results confirm the importance of adequate biosecurity measures and management on poultry farms as they greatly affect the genetic diversity of the pathogens. Therefore, implementation of basic biosecurity measures could help control the heterogeneity of Gallibacterium strains, which would alleviate control of the infection prevalence on farms through immunoprophylaxis, and consequently improve poultry production. Also, the genetic diversity of G. anatis on poultry farms could be a good bioindicator of management practices and biosecurity measures used. RESEARCH HIGHLIGHTS High correlation between low biosecurity and high diversity of Gallibacterium anatis. Diversity of Gallibacterium is a good bioindicator of management practices on farms.

Characterization of microbial communities in the chicken oviduct and the origin of chicken embryo gut microbiota.

The transferred microbiota from mother to baby constitutes the initial infant gastrointestinal microbiota and has an important influence on the development and health of infants in human. However, the reproductive tract microbiota of avian species and its inheritance have rarely been studied. We aimed to characterize the microbial community in the chicken reproductive tract and determine the origin of the chicken embryo gut microbiota. Microbiota in four different portions of chicken oviduct were determined using 16S rRNA metagenomic approach with the IonTorrent platform. Additionally, we analyzed the mother hen's magnum and cloaca, descendent egg, and embryo gut microbiota. The microbial composition and relative abundance of bacterial genera were stable throughout the entire chicken reproductive tract, without significant differences between the different parts of the oviduct. The chicken reproductive tract showed a relatively high abundance of Lactobacillus species. The number of bacterial species in the chicken reproductive tract significantly increased following sexual maturation. Core genera analysis detected 21 of common genera in the maternal magnum and cloaca, descendent egg shell, egg white, and embryo gut. Some elements of the maternal oviduct microbiota appear to be transferred to the embryo through the egg white and constitute most of the embryo gut bacterial population.

TLR3 deficiency exacerbates the loss of epithelial barrier function during genital tract Chlamydia muridarum infection.

PROBLEM: Chlamydia trachomatis infections are often associated with acute syndromes including cervicitis, urethritis, and endometritis, which can lead to chronic sequelae such as pelvic inflammatory disease (PID), chronic pelvic pain, ectopic pregnancy, and tubal infertility. As epithelial cells are the primary cell type productively infected during genital tract Chlamydia infections, we investigated whether Chlamydia has any impact on the integrity of the host epithelial barrier as a possible mechanism to facilitate the dissemination of infection, and examined whether TLR3 function modulates its impact. METHOD OF STUDY: We used wild-type and TLR3-deficient murine oviduct epithelial (OE) cells to ascertain whether C. muridarum infection had any effect on the epithelial barrier integrity of these cells as measured by transepithelial resistance (TER) and cell permeability assays. We next assessed whether infection impacted the transcription and protein function of the cellular tight-junction (TJ) genes for claudins1-4, ZO-1, JAM1 and occludin via quantitative real-time PCR (qPCR) and western blot. RESULTS: qPCR, immunoblotting, transwell permeability assays, and TER studies show that Chlamydia compromises cellular TJ function throughout infection in murine OE cells and that TLR3 deficiency significantly exacerbates this effect. CONCLUSION: Our data show that TLR3 plays a role in modulating epithelial barrier function during Chlamydia infection of epithelial cells lining the genital tract. These findings propose a role for TLR3 signaling in maintaining the integrity of epithelial barrier function during genital tract Chlamydia infection, a function that we hypothesize is important in helping limit the chlamydial spread and subsequent genital tract pathology.

Chlamydia muridarum plasmid induces mouse oviduct pathology by promoting chlamydial survival and ascending infection and triggering host inflammation.

BACKGROUND: Infection with plasmid-free Chlamydia trachomatis and Chlamydia muridarum fails to induce severe pathology, however, the mechanisms underlying this phenotype are unclear. OBJECTIVES: To elucidate the mechanisms of chlamydial plasmid-mediated pathology in mouse oviducts. MATERIALS & METHODS: BALB/c mice were intravaginally infected with either plasmid-competent or plasmid-free C. muridarum strains. To compare the survival and ascending infection of these strains, vaginal swabs and genital tract tissues were collected and cultured with HeLa cells to monitor the recovery of live organisms. In addition, Chlamydia strains were intrabursally inoculated into the oviducts of mice to assess pathogenicity. Cytokine levels in the vaginal swabs collected from both the plasmid-competent and plasmid-free C. muridarum-infected mice were detected using Bio-Plex Pro Mouse Cytokine, Chemokine, and Growth Factor Assays. RESULTS: The plasmid-competent C. muridarum strain induced hydrosalpinx formation in mouse oviducts following intravaginal inoculation, however, this was not the case for the plasmid-free C. muridarum strain. The lack of hydrosalpinges in response to the plasmid-free C. muridarum strain correlated with its significantly reduced ability to survive and disseminate to the upper genital tract. Furthermore, the plasmid-free C. muridarum failed to induce hydrosalpinx formation in mice, even when the strain was intrabursally injected into oviducts. A comparison of the cytokine levels in mouse vaginal secretions showed that the plasmid-free C. muridarum strain induced less IL-15, LIF, MIP-2, IL-1α, IL-1ß, TNF-α, and RANTES. CONCLUSION: C. muridarum plasmid contributes to oviduct pathology by promoting bacterial survival and ascending infection, and triggering host inflammatory responses.

The Genital Tract Virulence Factor pGP3 Is Essential for Chlamydia muridarum Colonization in the Gastrointestinal Tract.

The cryptic plasmid is essential for Chlamydia muridarum dissemination from the genital tract to the gastrointestinal (GI) tract. Following intravaginal inoculation, a C. muridarum strain deficient in plasmid-encoded pGP3 or pGP4 but not pGP5, pGP7, or pGP8 failed to spread to the mouse gastrointestinal tract, although mice infected with these strains developed productive genital tract infections. pGP3- or pGP4-deficient strains also failed to colonize the gastrointestinal tract when delivered intragastrically. pGP4 regulates pGP3, while pGP3 does not affect pGP4 expression, indicating that pGP3 is critical for C. muridarum colonization of the gastrointestinal tract. Mutants deficient in GlgA, a chromosome-encoded protein regulated by pGP4, also consistently colonized the mouse gastrointestinal tract. Interestingly, C. muridarum colonization of the gastrointestinal tract positively correlated with pathogenicity in the upper genital tract. pGP3-deficient C. muridarum strains did not induce hydrosalpinx or spread to the GI tract even when delivered to the oviduct by intrabursal inoculation. Thus, the current study not only has revealed that pGP3 is a novel chlamydial colonization factor in the gastrointestinal tract but also has laid a foundation for investigating the significance of gastrointestinal Chlamydia.

Uterotubal junction prevents chlamydial ascension via innate immunity.

Ascension to the oviduct is necessary for Chlamydia to induce tubal infertility. Using the Chlamydia muridarum induction of hydrosalpinx mouse model, we have demonstrated a significant role of the uterotubal junction in preventing chlamydial ascending infection. First, delivery of C. muridarum to either side of the uterotubal junction resulted in significant reduction in live organisms from the tissues on the opposite sides. However, the recovery yields remained similar among different sections of the uterine horn. These observations suggest that the uterotubal junction may function as a barrier between the uterine horn and oviduct. Second, deficiency in innate immunity signaling pathways mediated by either MyD88 or STING significantly compromised the uterotubal junction barrier function, permitting C. muridarum to spread freely between uterine horn and oviduct. Finally, transcervical inoculation of C. muridarum led to significantly higher incidence of bilateral hydrosalpinges in the STING-deficient mice while the same inoculation mainly induced unilateral hydrosalpinx in the wild type mice, suggesting that the STING pathway-dependent uterotubal junction plays a significant role in preventing tubal pathology. Thus, we have demonstrated for the first time that the uterotubal junction is a functional barrier for preventing tubal infection by a sexually transmitted agent, providing the first in vivo evidence for detecting chlamydial infection by the STING pathway.

In vitro adherence and invasion of primary chicken oviduct epithelial cells by Gallibacterium anatis.

Gallibacterium anatis (G. anatis) has been suggested to have a causal role in salpingitis and peritonitis in egg-laying chickens, leading to decreased egg production and increased mortality worldwide. Adherence and invasion of epithelial cells are thought to play a role in the pathogenesis of G. anatis infection. The purpose of this article was to study adherence and invasion of G. anatis using two G. anatis strains of different virulence (Yu-PDS-RZ-1-SLG strain, highly virulent and F149T strain, non-virulent) via infection of the primary chicken oviduct epithelial cells (PCOECs).The results showed that Yu-PDS-RZ-1 -SLG strain was able to attach to PCOECs at higher levels than that of F149T strain, but no invasion was observed with either strain. However, cell debris and cell apoptosis were observed after being exposed to G. anatis Yu-PDS-RZ-1-SLG for 90min, whereas G. anatis F149T did not cause cell damage, and adherence was prevented by trypsin treatment of bacterial cells. Cytokines were detected by ELISA after infection, and the results showed that the expression of IL-6, TNF-α, and IFN-γ levels was higher in virulent strain infection than that of the avirulent group. Results also indicated that the highly virulent strain G. anatis displayed an increased level of adherence. Changes in cytokine profiles in this study suggested that the production of cytokines might influence the microenvironment of oviduct and promote adherence, serving as a possible mechanism inducing cell damage.

Effects of inhibitors of transcription factors, nuclear factor-κB and activator protein 1, on the expression of proinflammatory cytokines and chemokines induced by stimulation with Toll-like receptor ligands in hen vaginal cells.

The goal of this study was to determine whether nuclear factor-κB (NFκB) and activator protein 1 (AP-1) were the responsible transcription factors for the induction of proinflammatory cytokines in hen vaginal cells stimulated by different Toll-like receptor (TLR) ligands. Cultured vaginal cells were treated with or without poly I:C (TLR3 ligand; dsRNA virus), lipopolysaccharide (LPS) (TLR4 ligand; gram-negative bacteria), flagellin (TLR5 ligand; bacterial flagellum), R848 (TLR7 ligand; ssRNA virus), and CpG-oligodeoxynucleotide (CpG-ODN) (TLR21 ligand; bacteria and DNA virus) in the presence or absence of different doses of BAY11-7085 (NFκB inhibitor) and tanshinone IIA (AP-1 inhibitor). Then, gene expressions of IL1B, IL6, and CXCLi2 were examined by real-time PCR analysis. The results showed that the induction of the expression of IL1B, IL6 and CXCLi2 by poly I:C, LPS, and CpG-ODN were suppressed by Bay11-7085, but not by tanshinone IIA. IL1B expression was upregulated by flagellin and R848, and the increase in its expression was suppressed by Bay11-7085, but not by tanshinone. These results suggest that NFκB is the responsible transcription factor for the expression of proinflammatory cytokines and chemokines, including I IL1B, IL6, and CXCLi2 in response to the ligands of TLR3, 4, and 21, and IL1B in response to the ligands of TLR5 and 7 in the vaginal cells.

Salmonella Enteritidis flagellar mutants have a colonization benefit in the chicken oviduct.

Egg borne Salmonella Enteritidis is still a major cause of human food poisoning. Eggs can become internally contaminated following colonization of the hen's oviduct. In this paper we aimed to analyze the role of flagella of Salmonella Enteritidis in colonization of the hen's oviduct. Using a transposon library screen we showed that mutants lacking functional flagella are significantly more efficient in colonizing the hen's oviduct in vivo. A micro-array analysis proved that transcription of a number of flagellar genes is down-regulated inside chicken oviduct cells. Flagella contain flagellin, a pathogen associated molecular pattern known to bind to Toll-like receptor 5, activating a pro-inflammatory cascade. In vitro tests using primary oviduct cells showed that flagellin is not involved in invasion. Using a ligated loop model, a diminished inflammatory reaction was seen in the oviduct resulting from injection of an aflagellated mutant compared to the wild-type. It is hypothesized that Salmonella Enteritidis downregulates flagellar gene expression in the oviduct and consequently prevents a flagellin-induced inflammatory response, thereby increasing its oviduct colonization efficiency.

Colonization of internal organs by Salmonella serovars Heidelberg and Typhimurium in experimentally infected laying hens housed in enriched colony cages at different stocking densities.

Contaminated eggs produced by infected commercial laying flocks are often implicated as sources of human infections with Salmonella Enteritidis, but Salmonella serovars Heidelberg and Typhimurium have also been associated with egg-transmitted illness. Contamination of the edible contents of eggs is a consequence of the colonization of reproductive tissues in systemically infected hens. In recent years, the animal welfare implications of diverse poultry housing and management systems have been vigorously debated, but the food safety significance of laying hen housing remains uncertain. The present study evaluated the effects of 2 different bird stocking densities on the invasion of internal organs by Salmonella serovars Heidelberg and Typhimurium in groups of experimentally infected laying hens housed in colony cages enriched with perching and nesting areas. Laying hens were distributed at 2 different stocking densities (648 and 973 cm2/bird) into colony cages and (along with a group housed in conventional cages at 648 cm2/bird) orally inoculated with doses of 107 cfu of 2-strain cocktails of either Salmonella Heidelberg or Salmonella Typhimurium. At 5 to 6 d post-inoculation, hens were euthanized and samples of internal organs (cecum, liver, spleen, ovary, and oviduct) were removed for bacteriologic culturing. The overall frequency of Salmonella isolation from ceca after inoculation with strains of serovar Heidelberg (83.3%) was significantly (P < 0.001) greater than the corresponding value for strains of serovar Typhimurium (53.8%), whereas Salmonella was recovered significantly more often from both livers (85.2% vs. 53.7%; P < 0.0001) and spleens (78.7% vs. 56.5%; P = 0.0008) after inoculation with strains of serovar Typhimurium than strains of serovar Heidelberg. However, there were no significant differences (P > 0.05) between stocking densities or cage systems in the frequencies of isolation of either Salmonella serovar from any of the five sampled tissues. These results contrast with prior studies, which reported increased susceptibility to internal organ invasion by Salmonella Enteritidis among hens in conventional cages at higher stocking densities.

Two strains of Mycoplasma synoviae from chicken flocks on the same layer farm differ in their ability to produce eggshell apex abnormality.

Mycoplasma synoviae (Ms) is considered to be an economically important poultry pathogen. Although the full economic costs of infection in layer chickens are still under debate, the prevalence of Ms is known to be high in some countries and earlier reports have shown a correlation between infection and Eggshell Apex Abnormality (EAA). This work is a continuation of an earlier study of a clinical case of EAA on a layer hen farm where the presence of two different strains of Ms, based on the sequence of the 5' end of the vlhA gene, was demonstrated. Both strains could be detected in the trachea but only one (designated strain PASC8) appeared able to colonize the oviduct, while the other (designated TRACH) was not found in the oviduct and has not been related to EAA. The PASC8 partial vlhA gene sequence differs from that of the TRACH in having a 39 nucleotide deletion in the proline rich region and three point mutations in the RIII region. Based on this information an experimental infection was performed in SPF chickens using groups infected with either the PASC8 or the TRACH strain and a non-infected control group. Both Ms strains were detected in the trachea of infected birds, but only the PASC8 strain was found in the oviduct. Furthermore, EAA developed only in the group infected with PASC8 strain. Compared to the control group, both strains produced an adverse impact on egg production: a decrease in the numbers laid and in their average weight (P<0.05) This work demonstrates a difference in oviduct tropism between two Ms strains and a possible relationship to the production of EAA in experimental conditions.

In vitro invasive capacity of Salmonella strains into sections of the layer hen oviduct.

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.

Tissue colonization and circulating T lymphocytes in laying hens upon oral challenge with Salmonella enterica serovars.

Evaluating the potential of Salmonella serovars for tissue colonization and egg contamination in laying hens is critical due to widespread consumption of poultry and egg-containing products. The 2009 FDA Egg Rule was implemented to target the eradication of Salmonella enterica Enteritidis (SE) from layers; however, other Salmonella serovars, such as Heidelberg (SH) and Typhimurium (ST), have also been associated with poultry-related outbreaks. We conducted this study to see if serovars other than SE could colonize in laying hens, cause egg contamination, and modulate circulating T-cell populations. Laying hens were orally gavaged with 107 colony forming units (CFU) of SE, SH, or ST and assessed for colonization in spleen, ovaries, and oviduct 10 d postchallenge. Splenic colonization was similar for all the serovars; however, colonization of ovaries and oviducts was significantly higher with SH compared to SE and ST. Furthermore, SH challenge resulted in egg contamination, while SE and ST did not result in contaminated eggs. Phenotypic evaluation of peripheral blood lymphocytes showed significant reduction in CD4 cells in SH-challenged birds and lower CD8α and CD8ß cells in SE-challenged birds compared to controls. Our data showed that non-SE serovars have equal or higher potential to colonize reproductive tissues of laying hens and may be accompanied by altered lymphocyte populations.

Development and evaluation of a multi-locus sequence typing scheme for Mycoplasma synoviae.

Reproducible molecular Mycoplasma synoviae typing techniques with sufficient discriminatory power may help to expand knowledge on its epidemiology and contribute to the improvement of control and eradication programmes of this mycoplasma species. The present study describes the development and validation of a novel multi-locus sequence typing (MLST) scheme for M. synoviae. Thirteen M. synoviae isolates originating from different poultry categories, farms and lesions, were subjected to whole genome sequencing. Their sequences were compared to that of M. synoviae reference strain MS53. A high number of single nucleotide polymorphisms (SNPs) indicating considerable genetic diversity were identified. SNPs were present in over 40 putative target genes for MLST of which five target genes were selected (nanA, uvrA, lepA, ruvB and ugpA) for the MLST scheme. This scheme was evaluated analysing 209 M. synoviae samples from different countries, categories of poultry, farms and lesions. Eleven clonal clusters and 76 different sequence types (STs) were obtained. Clustering occurred following geographical origin, supporting the hypothesis of regional population evolution. M. synoviae samples obtained from epidemiologically linked outbreaks often harboured the same ST. In contrast, multiple M. synoviae lineages were found in samples originating from swollen joints or oviducts from hens that produce eggs with eggshell apex abnormalities indicating that further research is needed to identify the genetic factors of M. synoviae that may explain its variations in tissue tropism and disease inducing potential. Furthermore, MLST proved to have a higher discriminatory power compared to variable lipoprotein and haemagglutinin A typing, which generated 50 different genotypes on the same database.

In-feed supplementation of trans-cinnamaldehyde reduces layer-chicken egg-borne transmission of Salmonella enterica serovar enteritidis.

Salmonella enterica serovar Enteritidis is a major foodborne pathogen in the United States, causing gastroenteritis in humans, primarily through consumption of contaminated eggs. Chickens are the reservoir host of S. Enteritidis. In layer hens, S. Enteritidis colonizes the intestine and migrates to various organs, including the oviduct, leading to egg contamination. This study investigated the efficacy of in-feed supplementation with trans-cinnamaldehyde (TC), a generally recognized as safe (GRAS) plant compound obtained from cinnamon, in reducing S. Enteritidis cecal colonization and systemic spread in layers. Additionally, the effect of TC on S. Enteritidis virulence factors critical for macrophage survival and oviduct colonization was investigated in vitro. The consumer acceptability of eggs was also determined by a triangle test. Supplementation of TC in feed for 66 days at 1 or 1.5% (vol/wt) for 40- or 25-week-old layer chickens decreased the amounts of S. Enteritidis on eggshell and in yolk (P<0.001). Additionally, S. Enteritidis persistence in the cecum, liver, and oviduct in TC-supplemented birds was decreased compared to that in controls (P<0.001). No significant differences in feed intake, body weight, or egg production in birds or in consumer acceptability of eggs were observed (P>0.05). In vitro cell culture assays revealed that TC reduced S. Enteritidis adhesion to and invasion of primary chicken oviduct epithelial cells and reduced S. Enteritidis survival in chicken macrophages (P<0.001). Follow-up gene expression analysis using real-time quantitative PCR (qPCR) showed that TC downregulated the expression of S. Enteritidis virulence genes critical for chicken oviduct colonization (P<0.001). The results suggest that TC may potentially be used as a feed additive to reduce egg-borne transmission of S. Enteritidis.