Comparative transcriptome analysis reveals PERP upregulated during Salmonella Enteritidis challenge in laying ducks.

Salmonella Enteritidis (SE) can be transmitted to eggs through cecum or the ovary from infected layers and causes food poisoning in humans. The mechanism of cecal transmission has been extensively studied. However, the mechanism and route of transovarian transmission of SE remain unclear. In this study, the ducks were orally inoculated with SE, and the ovarian follicles and stroma were collected to detect SE infection. The immune responses were triggered and the innate and adaptive immune genes (TLR4, NOD1, AvßD7, and IL-1ß) were upregulated significantly during the SE challenge. Moreover, the ovary tissues (small follicle and stroma) of susceptible and resistant-laying ducks were performed by RNA sequencing. We obtained and identified 23 differentially expressed genes (DEGs) between susceptible and resistant-laying ducks in both small follicle and stroma tissues ( p < 0.05). The DEGs were predominately identified in the p53 signaling pathway. The expression of key genes (p53, MDM2, PERP, caspase-3, and Bcl-2) involved in the signaling pathway was significantly higher in granulosa cells (dGCs) from SE-infected ducks than those from uninfected ducks. Moreover, the overexpression of PERP resulted in further induction of p53, MDM2, caspase-3, and Bcl-2 during SE infection in dGCs. Whereas, an opposite trend was observed with the knockdown of PERP. Besides, it is further revealed that the PERP could enhance cell apoptosis, SE adhesion, and SE invasion in SE-infected dGCs overexpression. Altogether, our results demonstrate the duck PERP involved in the ovarian local immune niche through p53 signaling pathway in dGCs challenged with SE.

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.

Pathogenesis of Gallibacterium anatis in a natural infection model fulfils Koch's postulates: 1. Folliculitis and drop in egg production are the predominant effects in specific pathogen free layers.

Pathogenicity of Gallibacterium anatis was studied in specific pathogen free layers in a controlled environment, applying the intranasal route for experimental infection. At 30 weeks, 37 hens were infected with 0.4 ml of 1.53 × 10(8) colony-forming units/ml suspension of G. anatis strain 07990 whereas equal numbers of hens were left uninfected for control. Following experimental infection, clinical signs and the number and weight of the eggs were recorded daily until 5 weeks post infection. Three birds from each group were killed at 3, 7, 10, 28 and 38 days post infection (d.p.i.) for necropsy and sampling for bacteriological and histopathological examinations. Additionally, necropsy examination was performed on all remaining birds at 38 d.p.i. G. anatis infection was found to have an immediate and severe effect on egg production, showing early and persistent colonization in respiratory and reproductive organs as well as in the gut of infected layers. In birds killed at various time points, G. anatis infection caused focal necrosis in the liver (1/37), folliculitis (2/37), pericarditis (3/37), haemorrhagic follicles (2/37), ruptured follicles (20/37), yolk in the body cavity (2/37) and egg peritonitis (1/37). The inflammation of the ovaries could be further confirmed by histopathological examination. Recovery of G. anatis from yolk at 10 d.p.i. indicates the potential of vertical transmission. Altogether, lesions reflect typical findings of G. anatis infection reported in natural cases. Thus, for the first time, lesions and the consecutive disease caused by G. anatis infection have been reproduced experimentally in a natural infection model.

Naturally occurring mastitis disrupts developmental competence of bovine oocytes.

We examined the effects of naturally occurring mastitis on bovine oocyte developmental competence in vitro. Specifically, we investigated the effects of intramammary infection on the ovarian pool of oocytes (i.e., follicle-enclosed oocytes) and their ability to undergo in vitro maturation, fertilization, and further development to the blastocyst stage. Culled Holstein cows (n=50) from 9 commercial dairy farms in Israel were allotted to 3 groups according to somatic cell count (SCC) records of the last 3 monthly milk tests as well as of quarter samples collected before slaughter: (1) low SCC (n=7), (2) medium SCC (n=16), or (3) high SCC (n=27). Means of SCC values differed among low-, medium-, and high-SCC groups: 148,000, 311,000 and 1,813,000 cell/mL milk, respectively. Milk yield and days in milk did not differ among the 3 groups. Bacterial isolates included coagulase-negative staphylococci, Escherichia coli, Streptococcus dysgalactiae, or no bacteria found. Ovaries were collected at the abattoir and brought to the laboratory. Cumulus oocyte complexes were recovered separately from each cow and subjected individually to in vitro maturation and fertilization, followed by 8d in culture. The number of aspirated oocytes did not differ among groups, with a range of 17 to 21 oocytes per cow. The proportion of oocytes that cleaved into 2- to 4-cell-stage embryos (86.1 ± 3.4%) did not differ among groups. In contrast, mean percentages of embryos developed to the blastocyst stage on d 7 and 8 after fertilization were less in both medium- and-high SCC groups than in the low-SCC group (5.6 ± 2.3 and 4.1 ± 1.8 vs. 18.1 ± 4.6%, respectively). Additional analysis indicated that cleavage and blastocyst-formation rates did not differ among the bacterial types in the low-, medium-, and high-SCC groups. These are the first results to demonstrate that naturally occurring mastitis disrupts the developmental competence of the ovarian pool of oocytes, (i.e., oocytes at the germinal vesicle stage). The disruption was associated with elevation of SCC rather than bacterial type. The results may provide a partial explanation for the low fertility of cows that have contracted mastitic pathogens before insemination.

In vitro invasion of laying hen ovarian follicles by Salmonella Enteritidis strains.

Salmonella is the major foodborne bacterial pathogen worldwide. Among numerous serotypes, Salmonella Enteritidis (SE) is one of the most common Salmonella serotypes responsible for human infections in the United States. The main source of SE outbreaks is foods associated with raw or undercooked chicken eggs. Salmonella Enteritidis is the only serotype that routinely contaminates eggs. The transovarian transmission of SE and subsequent contamination of the eggs before egg shell formation is considered to be the main route of egg contamination by SE. To evaluate whether invasion of ovarian follicles is an important step during the production of eggs contaminated by SE, we used an in vitro invasion assay to determine ovarian follicle invasion by 5 SE strains. After inoculating the freshly collected ovarian follicles, all 5 SE strains were able to invade into the follicles after 2 h of incubation at 37°C. The mean percentage of SE invasion ranged from 0.016 to 0.034% and no significant difference was found among the SE strains. For Escherichia coli K-12 strain, which was used as a negative control, the mean percentage of invasion was 0.0003%. The in vitro follicle invasion by SE strains demonstrated in this study may reflect the ability of the strains to invade ovarian follicles in laying hens once SE cells reach ovaries through various routes.

Pretreatment with pancaspase inhibitor (Z-VAD-FMK) delays but does not prevent intraperitoneal heat-killed group B Streptococcus-induced preterm delivery in a pregnant mouse model.

Caspases and apoptosis are thought to play a role in infection-associated preterm-delivery. We have shown that in vitro treatment with pancaspase inhibitor Z-VAD-FMK protects trophoblasts from microbial antigen-induced apoptosis. Objective. To examine whether in vivo administration of Z-VAD-FMK would prevent infection-induced preterm-delivery. Methods. We injected 14.5 day-pregnant-mice with heat-killed group B streptococcus (HK-GBS). Apoptosis within placentas and membranes was assessed by TUNEL staining. Calpain expression and caspase-3 activation were assessed by immunohistochemistry. Preterm-delivery was defined as expulsion of a fetus within 48 hours after injection. Results. Intrauterine (i.u.) or intraperitoneal (i.p.) HK-GBS injection led to preterm-delivery and induced apoptosis in placentas and membranes at 14 hours. The expression of calpain, a caspase-independent inducer of apoptosis, was increased in placenta. Treatment with the specific caspase inhibitor Z-VAD-FMK (i.p.) prior to HK-GBS (i.p.) delayed but did not prevent preterm-delivery. Conclusion. Caspase-dependent apoptosis appears to play a role in the timing but not the occurrence of GBS-induced preterm delivery in the mouse.

The relationship between uterine pathogen growth density and ovarian function in the postpartum dairy cow.

In cattle, the first postpartum dominant follicle grows slower and produces less oestradiol in animals with high numbers of bacteria contaminating the uterine lumen. However, only bacteria that are uterine pathogens are correlated with severe clinical disease and an increased inflammatory response. It is unknown whether the effect on the ovary in relation to uterine bacterial contamination is associated with the presence of recognised uterine pathogens. Therefore, the present study examined the relationship between pathogenic bacteria in the postpartum uterine lumen, follicle growth and function and the formation of a competent corpus luteum. In addition, peripheral plasma concentrations of immune mediators were quantified. Swabs were collected from the uterine lumen of cattle on day 7 postpartum. Bacteria were cultured and identified and bacterial growth was scored semi-quantitatively. Animals were categorized into high or low recognized uterine pathogen contamination groups based on the number of colonies. Ovarian structures were monitored by daily transrectal ultrasonography and blood samples were collected. In animals with high numbers of uterine pathogens on day 7 postpartum, the diameter of the first postpartum dominant follicle was smaller and plasma oestradiol concentrations were lower. In addition, these animals had smaller corpora lutea, which produced less progesterone. Furthermore, animals with a high day 7 uterine pathogen growth density had higher peripheral concentrations of acute phase proteins. Thus, contamination of the uterus with recognized uterine pathogens is associated with ovarian dysfunction during the postpartum period. Furthermore, infection results in an increase in the production of inflammatory mediators.

Presence of naturally occurring Campylobacter and Salmonella in the mature and immature ovarian follicles of late-life broiler breeder hens.

Campylobacter and Salmonella are known to cause acute bacterial gastroenteritis in humans. Raw poultry products have been implicated as a significant source of these infections. Five trials were conducted to determine whether Campylobacter and Salmonella spp. exist naturally in the mature and immature ovarian follicles of late-life broiler breeder hens. Broiler breeder hens ranging from 60 to 66 wk of age were obtained from four different commercial breeder operations. For each trial, the hens were removed from the commercial operation and held overnight at the University of Georgia processing facility. The hens were euthanized, defeathered, and aseptically opened. To reduce the possibility of cross-contamination between samples, first the mature and immature ovarian follicles, then the ceca, were aseptically removed. Individual samples were placed in sterile bags, packed on ice, and transported to the laboratory for evaluation. Overall, Campylobacter was found in 7 of 55 immature follicles, 12 of 47 mature follicles, and 41 of 55 ceca. Campylobacter was found in at least one of each sample of mature follicles and in ceca in each of the five trials. Salmonella was found in 0 of 55 immature follicles, 1 of 47 mature follicles, and 8 of 55 ceca. In this study, the recovery rate of Salmonella from late-life broiler breeder hen ovarian follicles was relatively low. However, the recovery rate of Campylobacter from the hen ovarian follicles was reasonably high, suggesting that these breeder hens could be infecting fertile hatching eggs. Determining how Campylobacter contaminated these ovarian follicles and how many chicks could be colonized from this source are the next steps in helping to elucidate a better understanding of this ecology and the control of Campylobacter in poultry production.

Ovarian laying hen follicular maturation and in vitro Salmonella internalization.

Transovarian transmission of paratyphoid Salmonella is well documented and occurs at a low incidence in chickens. However, the exact mechanism of follicular invasion is not well understood. The following study investigates the ability of Salmonella to invade ovarian follicles at different stages of follicular maturity in vitro. Ovarian follicles were collected from Leghorn hens and separated into three stages of maturity: (1) large yellow follicles or F follicles (LYF), (2) small yellow follicles (SYF), and (3) small white follicles (SWF). All follicles were incubated at 37 degrees C in RPMI 1640 medium. Follicles were incubated with 1 x 10(6) CFU/mL of Salmonella typhimurium and Salmonella enteritidis sensitive to gentamicin for 2 h. Samples were then removed from the bacterial culture, and placed in medium containing gentamicin sulfate for 5 h to kill any S. typhimurium or S. enteritidis, which had not invaded the follicular membrane. After the 5 h incubation, follicles were stomached in phosphate buffered saline. Serial dilutions were made of each follicle and viable S. typhimurium and S. enteritidis cells were enumerated on brilliant green agar. Two identical trials were conducted. Data suggest that Salmonella may differentially invade ovarian follicles depending on maturity of the follicle, and that SWF may be more susceptible to S. typhimurium and S. enteritidis invasion than either the SYF or the LYF.

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.

Effect of unilateral, intraovarian infusions of bacteria on ovarian morphology in gilts.

The aim of this study was to investigate whether unilateral, intraovarian infusions of bacteria might have induced morphological changes in the contralateral ovary. Eleven sexually matured gilts with controlled estrous cycle were used. The animals were randomly divided into two groups: I (Gr. I, treated; n = 4), and II (Gr. II, control; n = 7). In Gr. I, 1 ml of bacterial suspension (10(3) colony forming units/ml of saline of Escherichia coli, Staphylococcus aureus and Corynebacterium pyogenes, in proportion 1:1:1) was infused into the hilus of one ovary from the 15th to the 19th day of the estrous cycle. At the same time, 1 ml of saline was infused into the hilus of the contralateral ovary and into both ovaries of the control gilts. On the 7th day of the next cycle, the ovaries were dissected out. There were no significant differences in the number of follicles and corpora lutea (CL) as well as in weight and size between the bacteria-infused, contralateral and control ovaries. The microscopic observations of the bacteria-infused ovaries revealed the presence of focal infiltrations of neutrophils in the softened stroma, especially around dilated blood vessels filled with erythrocytes. In the contralateral ovaries, the number of regularly distributed neutrophils in the softened stroma was greater than that found in the bacteria-treated ovaries. CL of the bacteria-infused ovaries had more numerous, dilated blood vessels than CL observed in the contralateral gonads. More neutrophils were found in CL of both ovaries in Gr. I as compared to those observed in Gr. II. In Gr. II, single neutrophils were found also in the stroma where the tip of the cannula was inserted. This study revealed that in gilts, unilateral, intraovarian administration of bacteria did not change the number of ovarian structures, the weight and size of the bacteria-infused and contralateral ovary, but induced inflammatory changes in both ovaries.

[The characteristics of embryogenesis and oogenesis in Adalia bipunctata L. and Harmonia axyridis Pall. beetles in all-female families]. / Osobennosti émbriogeneza i oogeneza zhukov Adalia bipunctata L. i Harmonia axyridis Pall. v bessamtsovykh sem'iakh.

We describe abnormal embryonic development leading to death of 50% of embryos in all-female lines of Adalia and Harmonia infected with an androcide Spiroplasma. The arrest of embryonic development takes place at different stages throughout embryogenesis. Oogenesis of the infected beetles proceeds without any significant morphological changes.

Simplified preparation of a specific S. enteritidis antigen for ELISA and other immunological techniques.

This study was conducted to prepare a specific S. enteritidis antigen (FG-Antigen) for the serological detection of S. enteritidis infections in chicken flocks. This antigen (FG-Antigen) consistent mainly of the flagellar fraction H:g and partly of the fimbrial fraction SEF14 from a S. enteritidis-phage type 4 strain. The initial steps followed in the preparation of this antigen were conducted based on a previously described procedure, which involved the application of heat at 60 degrees C. The purification process (filtration and concentration) enabled the exclusion of the cross-reaction causing LPS antigens from the preparation and allowed the retention of S. enteritidis-specific antigens composed of fimbria and H:g fractions. As a result, no cross-reaction with S. typhimurium nor with S. gallinarum was exhibited by the prepared FG-antigen. To characterize and determine its specificity, the following laboratory tests were conducted: indirect ELISA, immunoblotting and a SEF14 agglutination test. In these examinations, rabbit and chicken reference sera as well as chicken field sera and absorbed hyperimmune sera against H:g-carrying serovars were used.

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.

Expression of an endogenous murine leukemia virus-related proviral sequence is androgen regulated and primarily restricted to the epididymis/vas deferens and oviduct/uterus.

A cDNA representing a 5.2-kb defective, endogenous murine leukemia proviral sequence (EPI-EPS) was isolated from a C57BL/6 mouse cDNA epididymal library. Northern blot analysis demonstrated that EPI-EPS was predominantly expressed in the C57BL/6 mouse epididymis and vas deferens with 10-fold lower expression in the seminal vesicle, kidney, and submandibular gland. Analysis of tissues from other inbred strains of mice as well as the wild mouse, Mus musculus musculus, showed a similar pattern of tissue expression. EPI-EPS expression was also highly androgen regulated in both the reproductive and nonreproductive tissues of the C57BL/6 strain. However, a differential response to testosterone replacement was observed between tissues. Expression of EPI-EPS mRNA in the epididymis and vas deferens exhibited only a partial recovery to precastration levels after testosterone replacement; in the kidney and submandibular gland there was a complete recovery of EPI-EPS expression. Finally, EPI-EPS expression was also highly restricted in the female tissues, with expression limited to the oviduct and uterus. EPI-EPS, however, was not estrogen regulated in the female. These results suggest that a proviral sequence, EPI-EPS, is expressed in M. m. musculus and several inbred strains of mice due to its integration near a highly tissue-specific and androgen-regulated genetic locus.

Virus p30-related protein in follicular fluids and C virus-like particles on the cell membrane of the human oocyte.

Fluids from 208 follicles were analyzed with respect to the concentration of estradiol and progesterone and to the presence of RD 114 retrovirus p30-related protein (rp), assuming that such a protein indicates the presence of C-virus particles in the follicular fluids. The results obtained showed that 60-80% of the follicular fluids within the same range of steroid hormone levels contained virus p30 rp. Electron microscopy showed 80-nm virus-like particles, more or less well preserved, in about half of the number of oocytes examined. Negative staining of purified follicular fluid particles showed ring-like structures with a size of 100-200 mm. It is concluded that the presence of virus p30 rp in follicular fluids is correlated neither to a certain steroid hormone concentration nor to the oocyte capability to become fertilized and cleave. The observed expression of an endogenous virus genome during oocyte maturation strengthens the hypothesis that this type of genome is active at early developmental stages.

Secretion of an inhibitor of follicle-stimulating hormone binding to receptor by the bacteria Serratia, including a strain isolated from porcine follicular fluid.

Bacterial growth in contaminated porcine follicular fluid (PFF) was associated with increased concentrations of a large molecular weight (Mr greater than 6000) inhibitor (FSH-BI) of 125I-FSH binding to calf testis membranes (Sluss and Reichert, 1983). We undertook to identify the bacteria and to determine if the inhibitor was a secretory product. Only one of 39 pure bacterial colonies isolated from PFF generated FSH-BI. The bacterium was tentatively identified as Serratia liquifaciens and was subsequently shown to also secrete FSH-BI when grown in synthetic culture media. Serratia liquifaciens from PFF secreted FSH-BI in a minimal culture medium containing only glucose as a carbon source. Other bacteria, including strains of Pseudomonas and Streptococcus did not secrete FSH-BI in either sterile PFF or synthetic culture media. Six strains of Serratia, obtained from the American Type Culture Collection, also secreted FSH-BI. FSH-BI secreted by Serratia liquifaciens was inactivated by heat (T 1/2 = 30 min at 60 degrees C), exposure to pH 2 (2 h at 25 degrees C) and was insoluble in ether, 75% acetone or 40% ammonium sulfate. Protease activity, using a casein substrate, was undetected in doses of FSH-BI which effectively (50%) inhibited 125I-FSH binding. Initial studies suggested that FSH-BI was due to effects on membranes rather than on the radioligand. These data demonstrate that Serratia liquifaciens isolated from PFF secretes a substance of Mr greater than 6000 which inhibits receptor binding of 125I-hFSH. Furthermore, the FSH-BI appears to be secreted constitutively by all (7) strains of Serratia tested.

[Failures at in vitro fertilization: electron microscopic studies of unfertilized oocytes and undivided ova]. / Les échecs de la fécondation in vitro: étude au microscope électronique des ovocytes non fécondés et des oeufs non divisés.

We have examined by electron microscopy the non fertilized oocytes and the non divided eggs after assay of in vitro fertilization. We have observed three blocking points; 1) absence of fusion of the gametes, either because of oocyte immaturity or over-maturity, either because of spermatozoa phagocytosis by follicle cells transformed in phagocytic cells, in presence of intra or extra-cellular bacteria; 2) fusion of the gametes but absence of spermatic nucleus decondensation; 3) fusion of the gametes and pronuclei formation but absence of amphimixy. Two causes are evident: failure of oocyte maturation in vivo or in vitro, and bacterial contamination problems after female or male infra-clinical infections.