In vivo and in vitro evaluation of tissue colonization and survival capacity of Salmonella Oranienburg in laying hens.

Salmonella enterica serovar Oranienburg (SO) was linked to a human salmonellosis outbreak in the Midwest in 2015 and 2016 from consumption of eggs. However, unlike Salmonella enterica serovar Enteritidis (SE), little is known regarding the potential of SO to colonize in laying hens and contaminate eggs. We used in vivo and in vitro models to evaluate tissue colonization and survival capacity of SO. Twenty eight-week-old laying hens were each challenged with an oral dose of approximately 107 (n = 92) or 109 (n = 96) colony-forming units (CFU) in 1 mL saline and evaluated after 1, 2, and 4 wk. Standard microbiological methods with pre-enrichment and enrichment in selective media were used for detection of SO in tissues, egg shell wash, internal egg contents, and excreta. Peak colonization of spleen (86.9%), ovaries (31.6%), upper oviduct (15.8%), and lower oviduct (34.3%) was detected between 1 and 2 wk post-infection (pi), while at 4 wk SO was only recovered from spleens (25%). Salmonella enterica serovar Oranienburg was not recovered from internal egg contents. However, the presence of SO on egg shells was seen when there were traces of excreta. Shedding in excreta was found in 92 and 100% birds gavaged with 107 and 109 CFU at 2 wk pi, respectively. The invasion and proliferation of SO in ovarian granulosa cells (GC) was compared to that of SE, and while the invasion of SO into GC was comparable to SE, proliferation of SO was significantly lower (P < 0.05). The infective potential of SO was also assessed by enumerating survival in egg white over 4 wk under refrigerated conditions, resulting in 65% survival at 4 wk. Overall, our data suggested that SO infection in layers did not result in egg contamination via vertical transmission, and colonization of egg-forming tissues was limited to 2 wk pi. Survival within GC and egg white demonstrates the ability of SO to withstand antibacterial factors and the potential of SO to penetrate the yolk.

Differential antibacterial response of chicken granulosa cells to invasion by Salmonella serovars.

In the United States, Salmonella enterica ser. Enteritidis (SE) is among the leading bacterial cause of foodborne illness via consumption of raw or undercooked eggs. The top Salmonella serovars implicated in U.S. foodborne outbreaks associated with chicken consumption include SE, Typhimurium (ST), Heidelberg (SH), Montevideo, Mbandka, Braenderup, and Newport. While enforcement actions target the eradication of SE from layer hens, there is a growing concern that other serovars could occupy this niche and be a cause of egg-transmitted human salmonellosis. Therefore, we tested the invasion and survival of SE, SH, ST, and Salmonella enterica ser. Hadar (S. Hadar) at 4 and 20 h post infection (hpi) in chicken ovarian granulosa cells (cGC); a cellular layer which surrounds the previtelline layer and central yolk in egg-forming follicles. We also evaluated cGC transcriptional changes, using an antibacterial response PCR array, to assess host response to intracellular SalmonellaWe observed that invasion of cGC by SE, SH, and ST was significantly higher than invasion by S. Hadar, with ST showing the highest level of invasion. The Bacterial Survival Index, defined as the ratio of intracellular bacteria at 20 and 4 h, were 18.94, 7.35, and 15.27 for SE, SH, and ST, respectively, with no significant difference in survival between SE or ST compared to SH. Evaluation of cGC anti-Salmonella gene responses indicated that at 4 hpi there was a significant decrease in Toll-like receptor (TLR)-4 mRNA in cGC infected with SE, whereas TLR5 and myeloid differentiation primary response gene 88 were significantly down regulated across all serovars. At 4 hpi, invasion by Salmonella serovars resulted in significant upregulation of several antimicrobial genes, and proinflammatory cytokines and chemokines (PICs). At 20 hpi, all the serovars induced PICs with SH being the strongest inducer. Additionally, SE, SH and ST differentially induced signal transduction pathways. Although only a single strain from each serovar was tested, cGC presents a useful ex vivo cell culture model to assess the virulence potential of Salmonella serovars.

Salmonella Enteritidis infection slows steroidogenesis and impedes cell growth in hen granulosa cells.

Infection by Salmonella Enteritidis (SE) causes decreased egg production in laying hens. Immunoresponse, steroidogenesis, and cell proliferation by chicken granulosa cells (cGCs) are of particular interest because these changes are involved in follicular growth, atresia, and ovulation. To elucidate the possible mechanisms underlying these changes, transcriptional alterations in cGCs at distinct stages of follicular maturity were studied. Luteinizing hormone (LH)-and follicle-stimulating hormone (FSH) were applied to the cGCs isolated from hierarchical and prehierarchical follicles, respectively, to imitate the effects of gonadotropin during in vitro SE infection. Results showed that the expression of Toll-like receptor 15 was dependent on the follicular maturity, with mature cells having a more significant and progressively stronger immunoresponse. Attenuated responses to LH and FSH as well as retardant steroidogenesis due to down-regulated LH receptor, FSH receptor, and the P450 side-chain cleavage system were observed and may have led to delayed hierarchical follicular growth. Deteriorated cell viability of prehierarchical follicles may occur, as the proliferation of stimulator heparin-binding epidermal growth factor was reduced significantly. Furthermore, the infection led to a higher probability of cGCs from the smaller follicles undergoing apoptosis than those from F1 follicles. Collectively, the data provide evidence of a tendency toward pathogen elimination in F1 follicles by induction of a strong immune response and cell apoptosis in smaller follicles to avoid bacterial transovarian infection. It is our speculation that slowed steroidogenesis and impeded follicular growth may play essential roles in decreased ovulation rate as well as further decreased egg production during SE infection.

A time-course study of gene responses of chicken granulosa cells to Salmonella Enteritidis infection.

Consumption of eggs contaminated with Salmonella Enteritidis (SE) has been recognized as one of the important causes of human foodborne salmonellosis. Chicken granulosa cells (cGCs) comprise the last tissue layer surrounding the yolk in preovulatory follicles and are a preferred site for SE invasion. To understand the cGC response to SE infection, we conducted an in vitro time-course study to identify cGC transcriptional changes using chicken whole genome microarrays. The expression of 135 (4h postinfection) and 120 cGC genes (48 h postinfection) were altered (P<.01) compared to uninfected cells. Many of the altered genes were related to immune response, physiological processes, signal transduction, and transcription. Furthermore, we also found that the Jak-STAT pathway, which is essential in the regulation of cellular cytokines and growth factors, was highly active in this study. Among the genes identified by microarray, the mRNA levels of TLR15, IL-6, CXCLi1, CXCLi2, and K203 were shown to be upregulated by real-time RT-PCR (qRT-PCR). In contrast, the mRNA levels of RASD1 and HB-EGF decreased according to both microarray and qRT-PCR analyses. These results suggest that during the SE infection, cGCs recruit cells of the innate immune responses; the infection may also induce suppression of cGC cell proliferation, which alters follicular development and ovulation.

Cloning of Salmonella enterica serovar Enteritidis fimbrial protein SefA as a surface protein in Escherichia coli confers the ability to attach to eukaryotic cell lines.

The gene for the Salmonella enterica serovar Enteritidis fimbrial protein SefA was cloned into an Escherichia coli surface expression vector and confirmed by Western blot assay. E. coli clones expressing SefA attached to avian ovary granulosa cells and HEp-2 cells, providing evidence for the involvement of SefA in the ability of Salmonella to attach to eukaryotic cells.

Escherichia coli derived factors modulate human granulosa cell steroidogenesis.

AIM: In order to identify bacterial compounds which directly affect granulosa cell steroidogenesis, the effect of culture-supernatants and extracts from Escherichia coli were tested in an in vitro granulosa cell culture model. METHODS: Samples were drawn from the culture-supernatant of E. coli ATCC25922 cultured in Luria-broth medium. Bacterial extract was prepared by incubation of freshly harvested bacteria in buffer. The bacterial culture-supernatants and extracts were added to human granulosa cells in culture. The granulosa cells collected from the follicular aspirates from women undergoing in-vitro fertilization were cultured for 17-53 h and progesterone or oestradiol was assayed in the spent culture medium. RESULTS: The E. coli culture-supernatant stimulated the basal granulosa cell progesterone production demonstrating its maximum activity reached after 200-240 min of bacterial growth. The heat denaturated bacterial extract as well as its low-molecular-weight fraction (<10 kDa) stimulated both the basal and the hCG-stimulated progesterone production; the oestradiol production was slightly inhibited. Stimulation of progesterone production was time dependent increasing from 125 +/- 18% of control within the first 3 h to 205 +/- 35% within 17-53 h. The high-molecular-weight fraction (>30 kDa) of the bacterial extract inhibited progesterone production. The inhibitory activity was significantly diminished by heat denaturation. CONCLUSIONS: The present study demonstrates the existence of various compounds which are secreted by E. coli and could also be extracted out of E. coli bacteria. These bacterial compounds modulate ovarian steroidogenesis. Further studies are needed to clarify how far these compounds contribute to menstrual disturbance observed in chronic pelvic inflammation.

Invasion of Salmonella enteritidis in the tissues of reproductive organs in laying Japanese quail: an immunocytochemical study.

The aim of this study was to determine whether Salmonella enteritidis (SE) inoculated into the peritoneal cavity would colonize tissues of reproductive organs in Japanese quail hens. Quail hens regularly laying were intraperitoneally inoculated with 5 x 10(7) or 5 x 10(8) SE cells, and the ovary, oviduct, kidney, spleen, liver, and large intestine were excised 24 or 48 h after the treatment. Paraffin sections of these organs were immunostained for SE. Invasion of SE was found in the tissues of the ovarian stroma, the follicular wall including superficial and theca layers, and occasionally in the granulosa layer and yolk. The SE immunoreaction product frequently was found in the fibroblast-like and macrophage-like cells in the stroma and surface layer of follicles. The SE immunoreaction products were identified on the mucosal surface, in the mucosal epithelium, and in the stromal tissues in all segments of the oviduct. Many of the bacteria were contained in the cytoplasm of mucosal epithelial cells and stromal cells in those tissues. The SE immunoreactions were also found in the tissues of kidney, spleen, and liver and in the large intestine. These results suggest that SE organisms introduced into the peritoneal cavity can invade and colonize the tissues of ovary and oviduct and may be responsible in the production of contaminated eggs.

Antigonadotrophic effect of Mycobacterium tuberculosis.

The present investigation has been carried out to study if the Mycobacterium tuberculosis has any direct effect on granulosa cell progesterone production. Granulosa cells from pregnant mare serum gonadotropin (PMSG)-treated immature rats were incubated in replicates with and without the ultrasonicated Mycobacterium tuberculosis in the presence and absence of human chorionic gonadotrophin (hCG). Progesterone production and granulosa cell viability were assayed. The mycobacterial lysate significantly inhibited the basal production of progesterone. The lysate completely blocked the stimulatory effect of hCG. Any cytotoxic effect of the lysate was ruled out as none of the treatments decreased the viability of the granulosa cells as compared with the control values.

In vitro attachment and invasion of chicken ovarian granulosa cells by Salmonella enteritidis phage type 8.

The attachment and invasion of chicken ovarian granulosa cells by Salmonella enteritidis was examined in vitro. The attachment was inhibited by preincubation of granulosa cells with anti-chicken fibronectin antibody (approximately 70% reduction in attachment) or preincubation with a 14-kDa fimbrial protein isolated from S. enteritidis (68% reduction in attachment). Treatment of bacterial cells with the tetrapeptide RGDS before addition to granulosa cells resulted in inhibition of attachment (60% inhibition when 2 x 10(7) CFU of bacteria was treated with 500 microg of peptide). Treatment with the peptide GRGD resulted in similar magnitude of inhibition, indicating that extracellular matrix proteins play significant roles in the interaction of S. enteritidis with granulosa cells. In contrast, treatment of the bacterial cells with the peptide GRAD did not result in significant inhibition of attachment to the granulosa cells. S. enteritidis was found to attach specifically to fibronectin, collagen IV, and laminin-coated microtiter plate wells, with the rank order of attachment as follows: fibronectin > laminin > collagen IV. Light and transmission electron micrographs of S. enteritidis invasion of granulosa cells showed organisms with or without a surrounding membrane in the cytoplasm of granulosa cells. In some instances, dividing bacterial cells were observed in the cytoplasm. Results of this study demonstrated that S. enteritidis interacts with granulosa cells in a specific manner and can invade and multiply in these cells. The granulosa cell layer of the preovulatory follicles may be a preferred site for the colonization of the chicken ovaries by invasive strains of S. enteritidis.

Mechanism of transovarian transmission of Salmonella enteritidis in laying hens.

To understand the mechanism of transovarian transmission of Salmonella enteritidis in laying hens, experiments were conducted to examine the isolation of S. enteritidis from the preovulatory follicles of experimentally infected hens. Salmonella enteritidis was isolated from the preovulatory follicles in 16 birds (from follicle membrane alone in 10 birds, from the follicle yolk alone in 4 birds, and from both membrane and yolk in 2 birds). In addition, 83 S. enteritidis isolates of the major phage types prevalent in United States were tested for attachment to hen ovarian granulosa cells and HEp-2 cells. Salmonella enteritidis demonstrated three different patterns of attachment to granulosa cells, namely, local, diffuse, and aggregative; whereas, only local attachment pattern was observed on HEp-2 cells. The total number of S. enteritidis isolates that demonstrated any pattern of attachment was significantly greater on the granulosa cells than on HEp-2 cells (P < .05). Salmonella enteritidis isolates of phage Types 8 and 28 demonstrated similar patterns of attachment on granulosa cells derived from the mature and developing follicles of the hen ovary. This suggest that S. enteritidis can colonize the preovulatory follicles at different stages of development. Preincubation of bacteria with the tetrapeptide arg-gly-asp-ser, the amino acid sequence known to mediate the interaction of adhesive proteins with cells, abrogated the local attachment of bacteria to granulosa cells. These results suggest that S. enteritidis can colonize the preovulatory follicles by interacting with the ovarian granulosa cells and that adhesive proteins may be involved in this process.

Isolation of Chlamydia trachomatis during IVF-ET.

The Authors isolated Chlamydia Trachomatis from the granulosa and from the spermatozoa of an infertile couple taking part in the IVF-ET program at the Department of Obstetrics and Gynaecology of the University of Pisa.