Natali Krekeler

Uropathogenic virulence factor FimH facilitates binding of uteropathogenic Escherichia coli to canine endometrium

Pyometra is a potentially life-threatening condition in bitches and is often caused by Escherichia coli infection. Both pathogenic and non-pathogenic E. coli strains commonly carry the genes for type 1 fimbriae that mediate bacterial adhesion onto host epithelium. To investigate whether the type 1 fimbrial adhesin, FimH, facilitates the binding of uropathogenic E. coli to canine endometrium, the fimH gene was insertionally inactivated in a pathogenic E. coli strain. The ability of E. coli to bind to canine endometrial epithelial cells was determined in vitro using canine uterine biopsies. Binding of the fimH mutant was only 0.3% of that of the wild type. Complementation of the mutation restored the phenotype to that of the parent. This study has developed an in vitro model that allows quantitative and qualitative assessment of bacterial binding to canine endometrium and has demonstrated that the fimH gene plays a role in adherence of pathogenic E. coli to canine endometrium.

The role of Type 1, P and S fimbriae in binding of Escherichia coli to the canine endometrium

Escherichia coli (E. coli) is the most commonly isolated infectious agent causing pyometra in bitches. Many E. coli strains isolated from the uteri of infected dogs carry several adhesin genes (fimH, papGIII and sfa). The objective of this study was to investigate the role of each adhesin gene product, acting alone or expressed in combination, in the bacterial binding to canine endometrium. E. coli strain P3, which was isolated from a uterus of a bitch naturally affected with pyometra, was shown by PCR to carry all three known fimbrial adhesin genes fimH, papGIII and sfa. Knockout (KO) mutants of this wildtype (P3-wt) strain were generated using insertional inactivation. Adhesion assays on anoestrous uteri of three post-pubertal bitches were undertaken. Overall, the number of bacteria adhering to canine endometrial biopsies was comparable between strains and no significant difference in the number of bound bacteria was found between the P3-wt strain and the single or double KO-strains. However, the triple knockout strain displayed less binding to the canine endometrium compared with the P3-wt strain. This study shows that a pathogenic E. coli strain (P3) isolated from the uterus of a bitch with pyometra was able to fully compensate for the loss of two of its three known adhesin genes. It was necessary to inactivate all three known adhesin genes in order to see a significant decrease in binding to canine endometrium.

Calcium, parathyroid hormone, oxytocin and pH profiles in the whelping bitch.

Despite the high prevalence of primary uterine inertia in whelping bitches, the underlying pathogenesis remains unclear. The objectives were to i) determine serum concentrations of total calcium, ionized calcium (iCa), parathyroid hormone (PTH), and blood pH in normally whelping bitches throughout the peri-parturient period; and ii) investigate relationships among iCa, PTH, and acid-base status, and the role that they and oxytocin may have in the underlying pathogenesis of canine uterine inertia. Bitches were randomly selected from a population of German Shepherd Dog bitches with a history of uncomplicated parturition (Group 1; n=10), and from a population of Labrador bitches with a clinical history of an increased incidence of uterine inertia and stillbirths (Group 2; n=20). Jugular blood samples were collected daily from -4 d to the onset of whelping (t=0 h), and then every 4h until the last pup was born. Overall, bitches from Group 2 had higher mean+/-SEM serum concentrations of PTH (4.72+/-2.45 pmol/L, P<0.001), lower iCa (1.31+/-0.08 pmol/L, P<0.05), and higher venous pH (7.41+/-0.03, P<0.005) than bitches from Group 1 (2.9+/-1.44 pmol/L, 1.38+/-0.06 mmol/L, and 7.33+/-0.02, respectively) during the periparturient period. However, there was no significant difference between Groups 1 and 2 for serum oxytocin concentrations during the periparturient period (45.5+/-40 and 65.5+/-82 pg/mL). We inferred that low iCa resulting from a rising pH and decreasing PTH during the periparturient period may have contributed to decreased uterine contractility and increased risk of stillbirths. Therefore, manipulating the cationic/anionic difference in diets of pregnant bitches, similar to the bovine model for hypocalcamia, may reduce the incidence of stillbirths in the bitch.

Periparturient characteristics of mares and their foals on a New Zealand Thoroughbred stud farm

Abstract AIMS: To describe selected periparturient variables in a sample of Thoroughbred mares and their foals on a commercial stud farm in New Zealand. METHODS: Data were collected for 969 foaling records, from 592 mares, by foaling attendants from one Thoroughbred stud farm in the Waikato region of New Zealand for the breeding seasons 2008–2013, inclusive, and collated retrospectively. The associations between mare- and foal-level variables and gestation length, time of birth (expressed as the number of hours after midday), fetal membrane retention time, time for the foal to stand and suckle after birth, and foal mortality were quantified using linear and logistic regression models. RESULTS: Mean gestation length for mares in this study was 350 (min 296, max 429) days, and was shorter for filly foals (348 (min 296, max 429) days) compared with colt foals (350 (min 310, max 404) days) (p=0.001). Overall, 517/968 (53%) foalings occurred between 19:00 and 01:00 and 212/968 (22%) foalings occurred during daylight. Overall, 815/855 (95%) expelled their fetal membranes within 4 hours of parturition. The mean interval from birth to standing and to suckling was shorter (p<0.001) for filly foals than colts by 13 (95% CI=9–17) and 17 (95% CI=8–26) minutes, respectively. Dystocia was reported for 81/968 (8.4%) foalings with relative fetal oversize being the most common cause (36/81 (44%) cases). Perinatal foal death was recorded for 26/966 (2.7%) foalings. The odds of foal mortality decreased with increasing length of gestation (p=0.027), and were greater for colt than filly foals (p=0.042) and when dystocia was reported (p<0.001). CONCLUSIONS: Gestation length was influenced by the sex of the foal, as was the time taken for the foal to stand and suckle after birth. One fifth of mares foaled during daylight hours. The majority of mares in this study population expelled their fetal membranes within 4 hours of foaling. CLINICAL RELEVANCE: Accurate measures for periparturient behaviour can be important in the management of mares and newborn foals. Clinically relevant differences appear to exist between mares and foals in New Zealand and those in the Northern Hemisphere for some key parameters, likely as a result of differences in management and environment. The results of this study suggest that mares should be considered to have retained their membranes if not passed within 4 hours of parturition.

Effect of ovarian hormones on the healthy equine uterus: a global gene expression analysis

The physiological changes associated with the varying hormonal environment throughout the oestrous cycle are linked to the different functions the uterus needs to fulfil. The aim of the present study was to generate global gene expression profiles for the equine uterus during oestrus and Day 5 of dioestrus. To achieve this, samples were collected from five horses during oestrus (follicle >35 mm in diameter) and dioestrus (5 days after ovulation) and analysed using high-throughput RNA sequencing techniques (RNA-Seq). Differentially expressed genes between the two cycle stages were further investigated using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. The expression of 1577 genes was found to be significantly upregulated during oestrus, whereas 1864 genes were expressed at significantly higher levels in dioestrus. Most genes upregulated during oestrus were associated with the extracellular matrix, signal interaction and transduction, cell communication or immune function, whereas genes expressed at higher levels in early dioestrus were most commonly associated with metabolic or transport functions, correlating well with the physiological functions of the uterus. These results allow for a more complete understanding of the hormonal influence on gene expression in the equine uterus by functional analysis of up- and downregulated genes in oestrus and dioestrus, respectively. In addition, a valuable baseline is provided for further research, including analyses of changes associated with uterine inflammation.

Pyometra in the queen To spay or not to spay

Practical relevance: Pyometra is a commonly occurring uterine disease in cats that often leads to loss of breeding potential and, in some cases, can be life threatening. An increased incidence of cystic endometrial hyperplasia (CEH) and pyometra is seen with age. Most queens present with uterine lesions after 5–7 years of age (average 7.6 years, range 1–20 years). Clinical signs most commonly occur within 4 weeks of the onset of oestrus in queens that are either mated, spontaneously ovulate or are induced to ovulate (mechanical stimulation or hormone induction). The disease is most often observed in dioestrus. Clinical challenges: Queens with pyometra often go undiagnosed as there may be few or only very mild clinical signs and laboratory changes. For example, the classic sign of mucopurulent bloody vulvar discharge often goes unnoticed. Abdominal ultrasound is the best tool for diagnosis of pyometra and for monitoring response to therapy. Patient group: Classically, middle-aged/older nulliparous intact queens present with pyometra. However, so-called ‘stump pyometra’ can occur if ovarian tissue is left behind during ovariectomy or ovariohysterectomy (ovarian remnant syndrome). Queens treated with exogenous steroid hormones such as high doses of megestrol acetate or medroxyprogesterone acetate for oestrus prevention can also develop CEH and pyometra. Evidence base: There has been little published to date on CEH, endometritis and pyometra in the queen and most of the currently available information has been extrapolated from studies carried out in the bitch. The queen and the bitch have very different reproductive physiology; thus, further research and investigation into the precise aetiopathogenesis of these disease processes of the uterus in the queen is warranted. Audience: This review is aimed at clinicians working in small animal practice, especially those in countries where surgical sterilisation is not practised as commonly as in the United States, Canada or Australasia, and who will therefore see a greater proportion of intact queens.

Evaluation of pregnancy and foaling rates after reduction of twin pregnancy via transvaginal ultrasound-guided aspiration in mares

OBJECTIVE To assess pregnancy and live foaling rates after reduction of twin pregnancy via transvaginal ultrasound-guided aspiration (TUA) in mares and evaluate effects of gestational period, localization of conceptuses, fluid aspiration volume, and combination of TUA with embryonic or fetal puncture on these outcomes. DESIGN Clinical trial. ANIMALS 44 mares pregnant with twins (25 to 62 days of gestation). PROCEDURES TUA was performed in all mares and combined with embryonic or fetal puncture in 13. Follow-up ultrasonographic examinations were performed by referring veterinarians. Effects of gestational period and TUA-related variables on pregnancy and foaling rates were assessed. RESULTS Singleton pregnancy (32/44 [73%]), persistent twin pregnancy (3/44 [7%]), or loss of both conceptuses (9/44 [20%]) was confirmed 5 to 7 days after TUA. Two mares with persistent twin pregnancy underwent another TUA, and 1 underwent prostaglandin F(2α)-induced abortion; these were excluded from subsequent analyses. Eighteen of 24 mares reevaluated 3 to 4 weeks after TUA were pregnant with 1 conceptus. Twenty of 41 (49%) mares delivered live singleton foals. Gestational period of TUA did not affect early pregnancy rates. Foaling rate for mares that underwent TUA after day 42 (0/5) was lowest of all groups and was significantly lower than that of mares that underwent TUA between days 31 and 35 (9/14). Three of 13 mares that underwent TUA with embryonic or fetal puncture delivered live foals, compared with 17 of 28 that underwent TUA alone. Effects of other variables were nonsignificant. CONCLUSIONS AND CLINICAL RELEVANCE TUA was effective for reduction of twin pregnancy in mares, with best results achieved before gestational day 43.

Success rates of various techniques for reduction of twin pregnancy in mares

Twinning is an important cause of pregnancy loss in mares and is often associated with economic loss. Most twin pregnancies can be expected to result in early gestational death, late-term abortions, or delivery of small foals with retarded growth. These foals can be more susceptible to infections and slower to develop after birth, compared with singletons. 1,2 Abortion in late gestation and the birth of twins often involve dystocia and trauma to the reproductive tract and are associated with poor rebreeding potential. 3 Prior to 1997, reported incidence rates of twinning-associated spontaneous abortion in mares ranged from 112 of 516 (22%) 4 to 689 of 1,788 (38.5%). 5 Results of a 9-year study 6 undertaken between 1988 and 1997 in the United Kingdom have shown that the rate of spontaneous abortion in mares was as low as 75 of 1,252 (6%). Although the direct effect of increased availability of ultrasound scanning on twin-associated spontaneous abortions has not been studied, it is possible that routine early scanning practices have contributed to this reduction. A report in 1973 indicated that only 9 of 62 (15%) twin pregnancies produced 2 live foals and that foals born alive were usually emaciated. 4 In that study, 4 13 of 31 (42%) foals born of twin pregnancies had died by 7 days of age, compared with 15 of 1,665 (0.9%) singleton foals. Mares that give birth to twins are also likely to have an oversized uterus, which delays postpartum involution and has been associated with reduced live foaling rates in subsequent breeding seasons. 1,7 Undetected and unmanaged twin pregnancies have important implications, with negative effects on animal health and economic loss being the principle detriments. Reduction of twin pregnancies without subsequent loss of the remaining foal is particularly important in animals with high economic value, mares with poor subsequent breeding potential, cases where the diagnosis is made late in gestation or late in the breeding season, and situations where a stallion is not available for rebreeding. Twin births in mares can and should be avoided,

Effect of Simulated Stages of the Canine Oestrous Cycle on Escherichia coli Binding to Canine Endometrium

Pyometra, a prevalent infectious uterine disease that affects intact middle-aged bitches, is typically associated with Escherichia coli. Our hypotheses were (i) that bacterial adhesion to canine endometrium differs between different stages of the oestrous cycle and (ii) that the adhesin FimH facilitates this adhesion. Twelve post-pubertal, ovariectomized greyhound bitches were treated with exogenous hormones to simulate different stages of the oestrous cycle. Tissue samples from each uterus were incubated with a pathogenic E. coli strain carrying the fimH gene, but no other adhesin genes (P4-wt)--or an E. coli strain in which fimH was insertionally inactivated (P4-∆fimH::kan)--or with phosphate-buffered saline as a negative control. After washing, tissue samples were homogenized for quantification of adherent bacteria. The differences in binding to canine endometrium at different stages of the oestrous cycle were not significant. However, the mean difference in binding of the P4-wt and the P4-∆fimH::kan across all stages of the simulated oestrous cycle was significant (p < 0.001 by paired t-test on geometric means). Individual differences in numbers of P4-wt bacteria bound between dogs might suggest genetic variations or epigenetic differences in FimH receptor expression by the endometrium, unrelated to the stage of the oestrous cycle.

Treatment of Retained Fetal Membranes in the Mare—A Practitioner Survey

Retained fetal membranes (RFM) is a common post-partum problem in mares for which the treatment is highly variable. The aim of this study was (i) to investigate the different treatments used by equine practitioners for RFM and (ii) to determine if there is a difference between treatments used by reproductive specialists and general equine practitioners. Information regarding treatment of RFM was sought from veterinary practitioners via a survey and this was compared to recommendations in the current literature. The survey was sent out to equine veterinarians and mixed practitioners with a high equine case load. Most treatments of RFM were in line with current recommendations, while some obsolete practices are still routinely performed by a small number of practitioners. Treatment recommendations for RFM have changed over the last few decades, but there are no universally accepted guidelines. The vast variety of treatments reported by practitioners in the present survey reflect this lack of guidance. More extensive research is needed in this area to establish evidence-based, uniformly agreed upon protocols.