The role of the oviduct environment in embryo survival.

CONTEXT: Declining fertility is an issue in multiple mammalian species. As the site of fertilisation and early embryo development, the oviduct plays a critical role in embryo survival, yet there is a paucity of information on how the oviduct regulates this process. AIMS: We hypothesised that differences in steroid hormone signalling and/or immune function would be observed in a model of poor embryo survival, the peripubertal ewe. METHODS: We examined expression of steroid hormones in systemic circulation, oviductal expression of oestrogen receptorαand genes important in steroid hormone signalling, and immune function in pregnant and cyclic peripubertal and adult ewes on day 3 after oestrus. KEY RESULTS: Concentrations of progesterone, but not oestradiol, were decreased in the peripubertal ewe compared to the adult ewe. Oestrogen receptorαprotein expression was increased in the peripubertal ewe, but pathway analysis of gene expression revealed downregulation of the oestrogen signalling pathway compared to the adult ewe. Differential expression of several genes involved in immune function between the peripubertal and adult ewe was consistent with an unfavourable oviductal environment in the peripubertal ewe lamb. Oestradiol concentration was positively correlated with the expression of multiple genes involved in the regulation of immune function. CONCLUSIONS: Differences in the immune environment of the oviduct, potentially linked to differential modulation by steroid hormones, may partially underly the poor fertilisation and early embryo survival observed in the peripubertal ewe. IMPLICATIONS: A unfavourable oviductal environment may play an important role in limiting reproductive success.

A novel, stain-free, natural auto-fluorescent signal, Sig M, identified from cytometric and transcriptomic analysis of infectivity of Cryptosporidium hominis and Cryptosporidium parvum.

Cryptosporidiosis is a worldwide diarrheal disease caused by the protozoan Cryptosporidium. The primary symptom is diarrhea, but patients may exhibit different symptoms based on the species of the Cryptosporidium parasite they are infected with. Furthermore, some genotypes within species are more transmissible and apparently virulent than others. The mechanisms underpinning these differences are not understood, and an effective in vitro system for Cryptosporidium culture would help advance our understanding of these differences. Using COLO-680N cells, we employed flow cytometry and microscopy along with the C. parvum-specific antibody Sporo-Glo™ to characterize infected cells 48 h following an infection with C. parvum or C. hominis. The Cryptosporidium parvum-infected cells showed higher levels of signal using Sporo-Glo™ than C. hominis-infected cells, which was likely because Sporo-Glo™ was generated against C. parvum. We found a subset of cells from infected cultures that expressed a novel, dose-dependent auto-fluorescent signal that was detectable across a range of wavelengths. The population of cells that expressed this signal increased proportionately to the multiplicity of infection. The spectral cytometry results confirmed that the signature of this subset of host cells closely matched that of oocysts present in the infectious ecosystem, pointing to a parasitic origin. Present in both C. parvum and C. hominis cultures, we named this Sig M, and due to its distinct profile in cells from both infections, it could be a better marker for assessing Cryptosporidium infection in COLO-680N cells than Sporo-Glo™. We also noted Sig M's impact on Sporo-Glo™ detection as Sporo-Glo™ uses fluoroscein-isothiocynate, which is detected where Sig M also fluoresces. Lastly, we used NanoString nCounter® analysis to investigate the transcriptomic landscape for the two Cryptosporidium species, assessing the gene expression of 144 host and parasite genes. Despite the host gene expression being at high levels, the levels of putative intracellular Cryptosporidium gene expression were low, with no significant difference from controls, which could be, in part, explained by the abundance of uninfected cells present as determined by both Sporo-Glo™ and Sig M analyses. This study shows for the first time that a natural auto-fluorescent signal, Sig M, linked to Cryptosporidium infection can be detected in infected host cells without any fluorescent labeling strategies and that the COLO-680N cell line and spectral cytometry could be useful tools to advance the understanding of Cryptosporidium infectivity.

Characterization of local and peripheral immune system in pregnant and nonpregnant ewes.

Modulation of the immune system is known to be important for successful pregnancy but how immune function might differ between the lymph nodes draining the reproductive tract and peripheral lymph nodes is not well understood. Additionally, if immune system changes in response to the presence of an embryo during early pregnancy, and if this response differs in local versus peripheral immune tissue, has not been well characterized. To address these questions, we examined expression of genes important for immune function using NanoString technology in the ampulla and isthmus of the oviduct, endometrium, lymph nodes draining the reproductive tract (lumbo-aortic and medial iliac) as well as a peripheral lymph node (axillary), the spleen, and circulating immune cells from ewes on day 5 of the estrous cycle or pregnancy. Concentrations of estradiol and progesterone in plasma were also determined. Principal component analysis revealed separation of the local from the peripheral lymph nodes (MANOVA P = 3.245e-08, R2 = 0.3) as well as separation of tissues from pregnant and nonpregnant animals [lymph nodes (MANOVA P = 2.337e-09, R2 = 0.5), reproductive tissues (MANOVA P = 2.417e-14, R2 = 0.47)]. Nine genes were differentially (FDR < 0.10) expressed between lymph node types, with clear difference in expression of these genes between the lumbo-aortic and axillary lymph nodes. Expression of these genes in the medial iliac lymph node was not consistently different to either the axillary or the lumbo-aortic lymph node. Expression of IL10RB was increased (FDR < 0.05) by 24% in the reproductive tissue of the pregnant animals compared to nonpregnant animals. Analysis of gene categories revealed that expression of genes of the T-cell receptor pathway in reproductive tract tissues was associated (P < 0.05) with pregnancy status. In conclusion, assessment of gene expression of reproductive and immune tissue provides evidence for a specialization of the local immune system around the reproductive tract potentially important for successful establishment of pregnancy. Additionally, differences in gene expression patterns in reproductive tissue from pregnant and nonpregnant animals could be discerned as early as day 5 of pregnancy. This was found to be associated with expression of genes important for T-cell function and thus highlights the important role of these cells in early pregnancy.

Molecular Epidemiology of Shiga Toxin-Producing Escherichia coli (STEC) on New Zealand Dairy Farms: Application of a Culture-Independent Assay and Whole-Genome Sequencing.

New Zealand has a relatively high incidence of human cases of Shiga toxin-producing Escherichia coli (STEC), with 8.9 STEC cases per 100,000 people reported in 2016. Previous research showed living near cattle and contact with cattle feces as significant risk factors for STEC infections in humans in New Zealand, but infection was not linked to food-associated factors. During the 2014 spring calving season, a random, stratified, cross-sectional study of dairy farms (n = 102) in six regions across New Zealand assessed the prevalence of the "Top 7" STEC bacteria (serogroups O157, O26, O45, O103, O111, O121, and O145) in young calves (n = 1,508), using a culture-independent diagnostic test (PCR/MALDI-TOF). Twenty percent (306/1,508) of calves on 75% (76/102) of dairy farms were positive for at least one of the "Top 7" STEC bacteria. STEC carriage by calves was associated with environmental factors, increased calf age, region, and increased number of calves in a shared calf pen. The intraclass correlation coefficient (ρ) indicated strong clustering of "Top 7" STEC-positive calves for O157, O26, and O45 serogroups within the same pens and farms, indicating that if one calf was positive, others in the same environment were likely to be positive as well. This finding was further evaluated with whole-genome sequencing, which indicated that a single E. coli O26 clonal strain could be found in calves in the same pen or farm, but different strains existed on different farms. This study provides evidence that would be useful for designing on-farm interventions to reduce direct and indirect human exposure to STEC bacteria.IMPORTANCE Cattle are asymptomatic carriers of Shiga toxin-producing E. coli (STEC) bacteria that can cause bloody diarrhea and kidney failure in humans if ingested. New Zealand has relatively high numbers of STEC cases, and contact with cattle feces and living near cattle are risk factors for human infection. This study assessed the national prevalence of STEC in young dairy cattle by randomly selecting 102 farms throughout New Zealand. The study used a molecular laboratory method that has relatively high sensitivity and specificity compared to traditional methods. "Top 7" STEC was found in 20% of calves on 75% of the farms studied, indicating widespread prevalence across the country. By examining the risk factors associated with calf carriage, potential interventions that could decrease the prevalence of "Top 7" STEC bacteria at the farm level were identified, which could benefit both public health and food safety.