Purulent vaginal discharge diagnosed in pasture-based Holstein-Friesian cows at 21 days postpartum is influenced by previous lactation milk yield and results in diminished fertility.

In a subset of dairy cows, prolonged pathological uterine inflammation results in purulent vaginal discharge (PVD), which can have negative consequences for both fertility and milk production. However, unlike for intensive systems, analysis of the effects of PVD in predominantly pasture-based herds is limited. The objective of this study was to assess the effect of PVD in spring-calving, pasture-based dairy cows on production and reproduction indices, stratified according to previous full-lactation milk yield. We assessed clinical disease as defined by vaginal mucus score (VMS) in 440 Holstein-Friesian cows from 5 farms. Cows were categorized as healthy (VMS 0) or having PVD (VMS 1-3) at 21 d postpartum. We recorded 305-d milk, milk protein, and milk fat yields (kg) before and after disease diagnosis, as well as fertility data, such as services per conception and the calving-conception period (CCP). Using SAS 9.4 (SAS Institute Inc., Cary, NC), we analyzed data using PROC MIXED, PROC PHREG, and PROC LOGISTIC to determine the least squares means differences and hazard and odds ratios between the groups, respectively. Overall, a 60% prevalence of PVD was recorded at 21 d postpartum. Milk yield and milk constituents were similar between all VMS categories and between healthy cows and cows with PVD. Although cows in the 4 VMS categories had statistically similar CCP, cows with PVD had a significantly longer CCP than healthy cows on average (9 d). The hazard ratio for cows with PVD was 0.66, indicating a 34% higher risk of a prolonged CCP than healthy cows. Odds ratio analysis determined that cows with PVD were 3 times more likely not to conceive at all, twice as likely not to conceive at first service, twice as likely not to conceive by 100 d postpartum, and 3 times more likely to fail to conceive before 150 d postpartum compared with healthy cows. Cows were retrospectively categorized as having low or high milk yield, based on whether they were above or below the median 305-d milk yield of the study population (6,571 kg) in the lactation before vaginal mucus scoring. Based on a univariate odds ratio, high-yield cows were 1.6 times more likely to present with PVD in the subsequent lactation. The number of services per conception did not differ between healthy and PVD cows in the low- and high-yield groups. In the high-yield group, cows with PVD were 4.9 times more likely not to conceive, 2.7 times more likely to require multiple services to conceive, 2.1 times more likely to remain not pregnant by 100 d postpartum, and 4.4 times more likely to remain not pregnant by 150 d postpartum. The CCP was also significantly longer in cows with PVD than their healthy counterparts (115.9 ± 4.9 and 104 ± 7.4 d, respectively). In conclusion, PVD significantly increased the CCP in all cows, but to a greater extent in cows with a high milk yield in the lactation before disease diagnosis.

A preliminary analysis of the variation in circulating 25-hydroxycholecalciferol concentrations in peri-partum spring-calving dairy cows.

Vitamin D has a well-established role in regulating the intestinal absorption of minerals but its association with immunity has not been extensively explored in livestock. Although an optimal circulating concentration of 30 ng/ml 25-hydroxycholecalciferol (25(OH)D) is proposed for immune function, it is unknown if this vitamin D concentration is sufficient, particularly for cows under a pasture-based, spring-calving dairy production system. The objectives of this retrospective analysis were to assess circulating vitamin D concentrations in a total of 843 bio-banked serum samples from Holstein-Friesian dairy cows enrolled from 12 spring-calving, pasture-based dairy farms in Ireland. Mean 25(OH)D concentrations were 36.3 ng/ml at calving, 30.7 ng/ml at 7 days post-partum (DPP), and 38.3 ng/ml at 21 DPP. However, mean concentrations masked significant inter-farm and inter-individual variation (P < 0.05). In fact, the proportion of cows with vitamin D insufficiency of < 30 ng/ml was found to be 33.8%, 55.5% and 19.5% at each time point, respectively. In addition, 25(OH)D concentrations correlated positively with immune cell populations (monocytes and lymphocytes) and negatively with blood urea and non-esterified fatty acids (NEFA) at 7 DPP. This is the first report of 25(OH)D concentrations in pasture-based peripartum dairy cows and we show a high degree of variation across farms and between individual animals. Sub-optimal concentrations of vitamin D in some post-partum cows may predispose cattle to multiple metabolic or infectious diseases, and therefore further work is now warranted.

Use of a cow-side oestrus detection test for fertility management in Kenyan smallholder dairy herds.

Background: The use of artificial insemination (AI) has great potential to improve smallholder dairy herds in Africa, however poor success and, in some situations, high costs in Kenya, have been discouraging.  Effective AI requires accurate oestrus detection and the measurement of progesterone (P4) can be used to indicate oestrus as well as non-pregnancy.  A cow-side progesterone lateral flow test, P4 Rapid, was evaluated as an aid to detect oestrus and non-pregnancy in Kenyan dairy cows, and assessed for association with AI efficiency.  Methods: A total of 527 cows were enrolled in the study, from two counties in central and southern Kenya.  Cattle in the test group (n = 308) were presented when suspected to be in oestrus and tested with the P4 Rapid (low P4 = oestrus, medium P4 = inconclusive, high P4 = not in oestrus/pregnant).  Cattle with low P4 were inseminated.  Cattle in the control group (n = 219) were inseminated when oestrus behaviour was detected i.e. standard practice. Results: Of the total P4 Rapid tests performed (n = 745), 1.5% were inconclusive, with the true accuracy of the test between 87-97%.  Conception rates were not significantly higher in the test group (83.9%) compared to the control group (77.9%). Abortion rates were not significantly different between the control (9.5%) and test groups (8.2%).  In the test group, 6.2% (19/308) cows showed a medium or high P4 level on day 0 and nine of these were subsequently found to have been already pregnant. Conclusions: The data indicated that the P4 Rapid test can be a useful tool to assist farmer decision-making in the confirmation of correct timing for AI, and importantly may avoid unnecessary inseminations in pregnant animals, thus reducing the risk of AI-induced abortion.

Delayed differentiation of vaginal and uterine microbiomes in dairy cows developing postpartum endometritis.

Bacterial overgrowth in the uterus is a normal event after parturition. In contrast to the healthy cow, animals unable to control the infection within 21 days after calving develop postpartum endometritis. Studies on the Microbial Ecology of the bovine reproductive tract have focused on either vaginal or uterine microbiomes. This is the first study that compares both microbiomes in the same animals. Terminal Restriction Fragment Length Polymorphism of the 16S rRNA gene showed that despite large differences associated to individuals, a shared community exist in vagina and uterus during the postpartum period. The largest changes associated with development of endometritis were observed at 7 days postpartum, a time when vaginal and uterine microbiomes were most similar. 16S rRNA pyrosequencing of the vaginal microbiome at 7 days postpartum showed at least three different microbiome types that were associated with later development of postpartum endometritis. All three microbiome types featured reduced bacterial diversity. Taken together, the above findings support a scenario where disruption of the compartmentalization of the reproductive tract during parturition results in the dispersal and mixing of the vaginal and uterine microbiomes, which subsequently are subject to differentiation. This differentiation was observed early postpartum in the healthy cow. In contrast, loss of bacterial diversity and dominance of the microbiome by few bacterial taxa were related to a delayed succession at 7DPP in cows that at 21 DPP or later were diagnosed with endometritis.

Uterine diseases in cattle after parturition.

Bacterial contamination of the uterine lumen is common in cattle after parturition, often leading to infection and uterine disease. Clinical disease can be diagnosed and scored by examination of the vaginal mucus, which reflects the presence of pathogenic bacteria such as Escherichia coli and Arcanobacterium pyogenes. Viruses may also cause uterine disease and bovine herpesvirus 4 (BoHV-4) is tropic for endometrial cells, causing a rapid cytopathic effect. The elimination of pathogens by the innate immune system is dependent on pattern recognition receptors binding pathogen-associated molecules. Uterine epithelial and stromal cells express receptors such as Toll-like Receptor 4 that binds E. coli lipopolysaccharide. The infertility associated with uterine disease is caused by damage to the endometrium and disruption of ovarian cyclic activity. Bacteria modulate endometrial prostaglandin secretion, and perturb ovarian follicle growth and function. Understanding the molecular basis of uterine disease will lead to novel approaches to treating infertility.

Expression and function of Toll-like receptor 4 in the endometrial cells of the uterus.

Prostaglandins have a central role in many endocrine functions in mammals, including regulation of the life span of the corpus luteum by prostaglandin F(2alpha) (PGF) and prostaglandin E2 (PGE), which are secreted by the uterine endometrium. However, the uterus is readily infected with bacteria such as Escherichia coli, which disrupt luteolysis. Immune cells detect E. coli by Toll-like receptor 4 (TLR4) binding its pathogenic ligand, lipopolysaccharide (LPS), although signaling requires accessory molecules such as CD14. The objective of this study was to determine the effect of E. coli or LPS on the function of bovine endometrial cells, and whether purified populations of epithelial and stromal cells express the molecules involved in LPS recognition. In addition, because the female sex hormones estradiol and progesterone modify the risk of uterine infection, their effect on the LPS response was investigated. Endometrial explants produced prostaglandins in response to LPS, with an increased ratio of PGE to PGF. Addition of LPS or E. coli to stromal and epithelial cells stimulated production of PGE and PGF and increased their cyclooxygenase 2 mRNA expression. The production of prostaglandins was abrogated by an LPS antagonist. In addition, estradiol and progesterone inhibited the production of PGE and PGF in response to LPS, indicating a role for steroid hormones in the response to bacterial infection. For the first time, Toll-like receptor 4 mRNA and CD14 mRNA and protein were detected in bovine endometrial stromal and epithelial cells by RT-PCR and flow cytometry. In conclusion, epithelial and stromal cells detect and respond to bacteria, which modulate their endocrine function.

Clinical evaluation of postpartum vaginal mucus reflects uterine bacterial infection and the immune response in cattle.

Bacteria contaminate the uterus of most dairy cattle after parturition and endometritis causes infertility. An endometritis score can be ascribed based on the vaginal mucus character and odour but it is not clear if the clinical score reflects the number of uterine bacteria or the inflammatory response. The present study tested the hypothesis that clinical evaluation of endometritis reflects the number of bacteria present in the uterus, and the acute phase protein response. Swabs (n = 328) were collected from the uterine lumen of dairy cattle, 21 and 28 days postpartum, vaginal mucus was scored for character and odour, and blood samples collected for acute phase protein measurement. Bacteria were identified following aerobic and anaerobic culture, and the bacterial growth density was scored semi-quantitatively. When bacteria were categorised by their expected pathogenic potential in the uterus, purulent or fetid odour vaginal mucus was associated with the growth density of pathogenic bacteria but not opportunist contaminants. When bacteria were analysed independently, Arcanobacterium pyogenes, Proteus and Fusobacterium necrophorum growth densities were associated with mucopurulent or purulent vaginal mucus. The bacterial growth densities for A. pyogenes, Escherichia coli, non-hemolytic Streptococci, and Mannheimia haemolytica were associated with a fetid mucus odour. Peripheral plasma concentrations of alpha(1)-acid glycoprotein were higher if there was a fetid compared with a normal vaginal mucus odour (1.50 +/- 0.09 mg/mL versus 1.05 +/- 0.02 mg/mL, P < 0.001), but did not differ significantly between vaginal mucus character scores. The evaluation of the character and odour of vaginal mucus reflects the number of bacteria in the uterus, and the acute phase protein response.

Monitoring clinical outcomes, pathological changes and shedding of Chlamydophila abortus following experimental challenge of periparturient ewes utilizing the natural route of infection.

Enzootic abortion of ewes (EAE) caused by Chlamydophila abortus is an important disease resulting in significant lamb loss in most sheep producing countries. Ewes are considered to be naturally infected with C. abortus via the oral-nasal route and may become persistent carriers, shedding during subsequent oestrous cycles and at lambing. The aim of this study was to monitor the clinical outcomes, pathological changes and shedding of C. abortus in 18 periparturient orally infected sheep for two breeding seasons. In the first season, C. abortus was detected by real-time PCR (rt-PCR) in 13/18 conjunctival swabs at oestrus. Three out of the 15 pregnant ewes gave birth to 1 live and 1 dead lamb, and 2 of them aborted. Following parturition/abortion, C. abortus was detected in 12/15 vaginal swabs and in all the collected foetal membranes. However, only those membranes containing high copy numbers of the bacterium displayed the EAE typical lesions. In the second season, none of the 13 pregnant ewes aborted, and 5 of them gave birth to dead or weak lambs. C. abortus was not detected in conjunctival or vaginal swabs at oestrus or parturition. The bacterium was detected at low levels in 36% of the foetal membranes, but with no evidence of histopathological lesions. These results indicate that C. abortus can be detected in a large proportion of animals during the first pregnancy after oral infection. However, this proportion is reduced at the subsequent breeding season, confirming the occurrence of a chronic low level persistent infection in post-abortion/lambing ewes.

Amniotic and allantoic fluids from experimentally infected sheep contain immunoglobulin specific for Chlamydophila abortus.

Chlamydophila abortus, the aetiological agent of enzootic abortion of ewes (EAE), replicates in trophoblast cells leading to their destruction and dissemination of the bacterium to foetal organs. To further understand the pathogenesis of EAE, amniotic and allantoic fluids were collected from experimentally infected pregnant ewes at 30 (7 samples from each fluid), 35 (8 samples from each fluid), 40 (10 samples from each fluid) and 43 (6 amniotic fluids and 7 allantoic fluids) days post-infection to determine pathogen numbers and other markers of infection. Whilst experimentally infected ewes had characteristic placental lesions, only two amniotic and seven allantoic fluid samples were positive for C. abortus by real-time PCR. In contrast, all amniotic and allantoic fluids were positive for immunoglobulin. Immunoglobulins were generally detected earlier in allantoic fluid than in amniotic fluid and the numbers of samples containing immunoglobulins increased as infection progressed. IgG in amniotic and allantoic fluids was shown to be specific for C. abortus, and reacted with the major outer membrane proteins, polymorphic outer membrane protein and macrophage infectivity potentiator protein. A comparison of two-dimensional immunoblots using purified IgG from the allantoic fluid, amniotic fluid, ewe serum and foetal serum of a C. abortus infected animal at 40 days post infection indicated a pattern of reactivity intermediate between that of the ewe serum and the foetal serum. Results suggest that a maternal source of immunoglobulin is predominant at 30 days post-infection but that foetal derived antibodies may be contributed at a later stage.

Bacterial lipopolysaccharide induces an endocrine switch from prostaglandin F2alpha to prostaglandin E2 in bovine endometrium.

Escherichia coli infection of the endometrium causes uterine disease after parturition and is associated with prolonged luteal phases of the ovarian cycle in cattle. Termination of the luteal phase is initiated by prostaglandin F(2alpha) (PGF) from oxytocin-stimulated endometrial epithelial cells. Compared with normal animals, the peripheral plasma of animals with E. coli infection of the endometrium had higher concentrations of lipopolysaccharide (LPS) and prostaglandin E(2) (PGE) but not PGF. Endometrial explants accumulated predominantly PGE in the culture medium in response to LPS, and this effect was not reversed by oxytocin. Endometrial cells expressed the Toll-like receptor 4/CD14/MD-2 receptor complex necessary to detect LPS. Epithelial and stromal cells treated with LPS had higher steady-state media concentrations of PGE rather than PGF. Arachadonic acid is liberated from cell membranes by phospholipase 2 (PLA2) enzymes and converted to prostaglandins by synthase enzymes. Treatment of epithelial and stromal cells with LPS did not change the levels of PGE or PGF synthase enzymes. However, LPS stimulated increased levels of PLA2 group VI but not PLA2 group IV C immunoreactive protein in epithelial cells. Endometrial cells expressed the E prostanoid 2 and E prostanoid 4 receptors necessary to respond to PGE, which regulates inflammation as well as being luteotropic. In conclusion, LPS detection by endometrial cells stimulated the accumulation of PGE rather than PGF, providing a mechanism to explain prolonged luteal phases in animals with uterine disease, and this PGE may also be important for regulating inflammatory responses in the endometrium.

The effect of Escherichia coli lipopolysaccharide and tumour necrosis factor alpha on ovarian function.

PROBLEM: Pelvic inflammatory disease and metritis are important causes of infertility in humans and domestic animals. Uterine infection with Escherichia coli in cattle is associated with reduced ovarian follicle growth and decreased estradiol secretion. We hypothesized that this effect could be mediated by the bacterial lipopolysaccharide (LPS) or cytokines such as tumour necrosis factor alpha (TNFalpha). METHOD OF STUDY: In vitro, bovine ovarian theca and granulosa cells were treated with LPS or TNFalpha and steroid secretion measured. In vivo, the effect of LPS or TNFalpha intrauterine infusion was determined by ovarian ultrasonography and measurement of hormones in cattle. RESULTS: Lipopolysaccharide reduced granulosa cell estradiol secretion, whilst TNFalpha decreased theca and granulosa cell androstenedione and estradiol production, respectively. In vivo, fewer animals ovulated following intrauterine infusion with LPS or TNFalpha. CONCLUSION: Lipopolysaccharide and TNFalpha suppress ovarian cell function, supporting the concept that pelvic inflammatory disease and metritis are detrimental for bovine ovarian health.

Ovarian follicular cells have innate immune capabilities that modulate their endocrine function.

Oestrogens are pivotal in ovarian follicular growth, development and function, with fundamental roles in steroidogenesis, nurturing the oocyte and ovulation. Infections with bacteria such as Escherichia coli cause infertility in mammals at least in part by perturbing ovarian follicle function, characterised by suppression of oestradiol production. Ovarian follicle granulosa cells produce oestradiol by aromatisation of androstenedione from the theca cells, under the regulation of gonadotrophins such as FSH. Many of the effects of E. coli are mediated by its surface molecule lipopolysaccharide (LPS) binding to the Toll-like receptor-4 (TLR4), CD14, MD-2 receptor complex on immune cells, but immune cells are not present inside ovarian follicles. The present study tested the hypothesis that granulosa cells express the TLR4 complex and LPS directly perturbs their secretion of oestradiol. Granulosa cells from recruited or dominant follicles are exposed to LPS in vivo and when they were cultured in the absence of immune cell contamination in vitro they produced less oestradiol when challenged with LPS, although theca cell androstenedione production was unchanged. The suppression of oestradiol production by LPS was associated with down-regulation of transcripts for aromatase in granulosa cells, and did not affect cell survival. Furthermore, these cells expressed TLR4, CD14 and MD-2 transcripts throughout the key stages of follicle growth and development. It appears that granulosa cells have an immune capability to detect bacterial infection, which perturbs follicle steroidogenesis, and this is a likely mechanism by which ovarian follicle growth and function is perturbed during bacterial infection.