P.J. Wright

Uropathogenic virulence factors in isolates of Escherichia coli from clinical cases of canine pyometra and feces of healthy bitches.

Escherichia coli is commonly isolated in canine pyometra, but little is known of the virulence factors that may be involved in the precipitation of this disease. The aim of this study was to compare the prevalence of uropathogenic virulence factor (UVF) genes in E. coli isolates from canine pyometra and from feces of healthy bitches to evaluate their role in the pathogenesis of pyometra. E. coli from 23 cases of canine pyometra and from the feces of 24 healthy bitches were analyzed, by polymerase chain reaction, for UVF genes associated with canine and human urinary tract infections (UTIs). The prevalences of UVFs in E. coli from canine pyometra were similar to that in canine and human uropathogenic E. coli. The prevalence of pap was greater (P=0.036) for E. coli from pyometra (52%) than for fecal isolates (21%), and the papGIII allele was present in all pap-containing isolates. The prevalences of genes for alpha-haemolysin and cytotoxic necrotising factor 1 were not significantly higher (P=0.075) in E. coli from pyometra than from feces. The proportion of pyometra strains with >or=3 UVFs was higher (P=0.039) than that of fecal strains, suggesting that possession of >or=3 UVF genes enhances the pathogenicity of the strain. Our findings demonstrate that E. coli associated with canine pyometra are similar to uropathogenic strains, and that operons that encode P fimbriae, alpha-haemolysin and cytotoxic necrotising factor 1 probably enhance the virulence and pathogenicity of the strain in the canine genital tract.

Pharmacologic Manipulation of Fertility

The professional application of agents to the manipulation of fertility of cows requires basic and applied knowledge of the physiologic mechanisms that are affected and of the pharmacologic agents that are used. In all areas of the pharmacologic manipulation of fertility, the achievement is less than the ideal, and further research is required to improve the efficiency of treatments. The induction of estrus in acyclic animals can involve a reduction in the depth of anestrus, pretreatment with progestagen to ensure estrous behavior and the formation of a normal corpus luteum, and then treatment with exogenous gonadotropin. Responsiveness to treatment can be variable and reflects the depth of anestrus of the animals. Improved treatment regimens require a knowledge of the basic mechanisms involved with the depth of anestrus, a means of assessing the depth of anestrus, and an understanding of the hormonal requirements of ovarian follicles for development and maturation in animals at different depths of anestrus. The optimal precision in the synchronization of estrus (and ovulation) in cyclic animals requires the synchronization of both follicular waves and the end of progestational phase. The end of progestational phase can be synchronized effectively using prostaglandin F2a (or analogs), or by treatment with progestagens with or without luteolytic agents. Procedures to synchronize follicular waves need to be established. The induction of superovulation can be achieved readily using gonadotropins prior to estrus synchronization using prostaglandin F2a. The responses to standard treatments in terms of ovulation rates and yield of transferable embryos are highly variable. The development of procedures to reduce this variability requires an understanding of the intra-ovarian mechanisms involved in recruitment of follicles for a wave of follicular growth, in the selection of dominant follicles for further development, and in the mechanisms controlling follicular atresia. Cystic ovarian disease can be treated effectively using HCG or GnRH (follicular cysts) or prostaglandin F2a (luteal cysts). The basic mechanisms resulting in failure of estrogen positive feedback on LH secretion (that results in cystic follicles) remain to be determined. Small but significant increases in pregnancy rates can be achieved treating cows with prostaglandin during the post-partum period, with prostaglandin to induce estrus for insemination, with GnRH or HCG at estrus, and with GnRH or progestagen treatment during diestrus. Beneficial effects of treatment have been shown in some trials but not in others.(ABSTRACT TRUNCATED AT 400 WORDS)

Evidence for differential regulation of multiple transcripts of the gonadotropin releasing hormone receptor in the ovine pituitary gland; effect of estrogen

The number of pituitary gonadotropin-releasing hormone receptors (GnRH-R) varies across the estrous cycle. We report that there is variable expression of the differently-sized GnRH-R transcripts in cyclic ewes and in an experimental model. During the follicular phase of the cycle, and compared to the luteal phase, there was increased expression of the 1.5, 2.3 and 3.7 kilobase (kb) transcripts with no change in the levels of the 5.6 or the 1.2 kb transcripts. Steady state levels of mRNA for luteinising hormone beta and common alpha subunit were also increased in the follicular phase of the cycle. In hypothalamo-pituitary disconnected ovariectomised ewes given pulsatile GnRH replacement, injection of estrogen increased the 1.5, 2.3 and 3.7 kb, while the levels of the 5.6 and 1.2 kb transcripts were not altered. We conclude that the differential regulation of GnRH-R mRNA occurs through a direct effect of E on the pituitary.

Cytology of the canine reproductive system.

The methods for semen collection, its laboratory examination, and the interpretation of findings are presented in this article. The lack of comprehensive data for normal dogs and the lack of data associating actual percentages of spermatozoa with specific abnormalities with fertility or infertility are highlighted. Consequently, there is a need for standardization and completeness of semen examination procedures, especially in studies destined for publication. Collection and analysis of prostatic samples then is discussed, and the distinguishing cytological features of benign prostatic hyperplasia, prostatic adenocarcinoma, prostatis (including prostatic abscessation), and prostatic cysts are presented. This is followed by an assessment of the clinical usefulness of vaginal cytology, particularly to assist in the management of normal canine reproduction and in the diagnosis of reproductive disorders. The ways in which vaginal smears can facilitate the diagnosis of the stage of the estrous cycle and the diagnosis of abnormalities of the cycle and other disorders of reproduction are presented. Further consideration is given to its use to estimate the time of ovulation retrospectively and estimate the time of whelping prospectively. Finally, two specific diseases that can affect dogs and bitches are reviewed, namely, canine brucellosis and transmissible venereal tumor.

The influence of nutrient status of post-partum ewes on ovarian cyclicity and on the oestrous and ovulatory responses to ram introduction

Abstract We have assessed, in post-partum Merino ewes (n = 120) lambing during the ovulatory season, the influence of nutrient status on the onset and cessation of ovarian cyclicity and on the oestrous and ovulatory responses to ram introduction. Two flocks were studied from mid-pregnancy onwards; flock R + + included vasectomized rams (8%) and flock R - + was isolated from rams until Day 1 of the study when the ewes were 21–37 days post partum. Within each flock, from mid-pregnancy until Day 7, three groups of ewes (n = 20/group) received normal or restricted nutrition, or restricted nutrition with a supplement of lupins and oats from Day - 14 to Day 7. After Day 7 all ewes received similar normal nutrition. All ewes received progestagen from a pessary from Day - 7 to Day 1. On Day 1 the rams were removed from flock R + + and new vasectomized rams (10%) were introduced into both flocks. Compared with ewes of normal nutrient status, the ewes of reduced nutrient status had a longer (P 0.05) incidence of ovulation (as assessed from plasma progesterone on Day 13) (15/19 vs 19/20) (flock R - +). Short-term nutrient supplementation did not affect these responses. The occurrence of oestrus within 14 days of ram introduction was similar (P>0.05) in the ewes receiving normal nutrition in flock R - + and flock R + + (14/20, 14/20). These results demonstrate that reduced nutrient status of post-partum ewes can (a) delay the onset of ovarian cyclicity post partum; (b) inhibit the occurrence of ram-induced oestrus (but not ovulation); and (c) hasten the onset of cessation of oestrous cycles.

The efficacy of ram introduction, GnRH administration, and immunisation against androstenedione and oestrone for the induction of oestrus and ovulation in anoestrous post-partum ewes

Abstract Four treatments, designed to increase plasma concentrations of LH, were assessed for efficacy at inducing oestrus and ovulation in anoestrous progestagen-pretreated Merino ewes that were 20–37 days post partum during the ovulatory season. The treatments involved the introduction of rams to ewes that had been isolated from rams, GnRH administration, and immunisation against androstenedione or oestrone. Ram introduction resulted in a higher (P 16 19 ewes, flock R-+) than in ewes having continuous contact with rams ( 4 20 ewes, flock R++). Treatment with GnRH (125 ng/h s.c. for 7 days) inhibited the ram effect on the number of ewes showing oestrus, but not on the number of ewes ovulating. GnRH treatment, and immunisation against androstenedione or against oestrone did not induce oestrus or ovulation. Immunisation against oestrone inhibited the occurrence of ovulation in ewes in both flocks (R++, R-+). Pregnancy rates were lower (P

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.

Restoration of LH output and 17β-oestradiol responsiveness in acutely ovariectomised holstein dairy cows pre-treated with a GnRH agonist (deslorelin) for 10 days

The objectives of the study were firstly to identify the role of the ovary in maintaining plasma luteinising hormone (LH) concentrations in cows treated with an implant of a potent GnRH agonist (deslorelin), and secondly to characterise the changes in LH following ovariectomy (OVX) in the same animals. Oestrus was synchronised in mature Holstein dairy cows and deslorelin implants were inserted 17 days later into two-third of the cows. A further 10 days later (day 0) all cows had bilateral OVX performed. A control group (CON; n=4) received no treatment and had blood samples collected at 15-min intervals for 8h on the day prior to OVX (day -1) and similarly on days 4 and 10. One group (DES_IN; n=4) had implants in place for the duration of the study while another group had implants removed (DES_OUT; n=4) at the time of OVX. DES_IN cows were sampled hourly at each sampling session (days -1, +4 and +10), whereas DES_OUT cows were sampled similarly to CON except on day -1 when hourly samples were collected. Predictable post-operative increases in mean LH (0.61 ng/ml versus 1.79 ng/ml; P<0.01) and LH pulse amplitude (0.66 ng/ml versus 1.56 ng/ml; day -1 versus day +10; P<0.01) occurred after CON cows were ovariectomised. Smoothed LH means showed a delayed effect of time compared to arithmetic means. Pulse frequency was unchanged following OVX in CON cows. A comparison of all cows that had been treated with deslorelin from day -1 showed a significant elevation of smoothed mean LH compared to untreated cows (0.80 ng/ml versus 0.34 ng/ml; DES_IN and DES_OUT versus CON; P<0.05). DES_IN cows had a 54% reduction in mean LH from day -1 to +4 following OVX (1.05 ng/ml versus 0.48 ng/ml; P<0.01) indicating the probable involvement of the ovary in the maintenance of elevated basal LH. No further reduction was detected by day +10. The LH response to an intramuscular (IM) injection of 500 microg 17beta-oestradiol (E2) on day +11 varied significantly between treatment groups (P<0.01). CON cows showed a typical LH surge, reaching maximum concentrations (10.3 ng/ml) at 17.3h post-injection. Even though low amplitude LH pulsatility had been restored in DES_OUT cows by day +4, there was an inconsistent response to E2 on day +12; one cow had an apparently normal surge yet, others showed only attenuated responses. Pulse amplitude in DES_OUT cows was lower at days +4 and +10 compared to CON (P<0.05). DES_IN cows did not produce any surge after E2. Mean LH prior to OVX (day -1) remained unchanged following the 500 microg oestradiol injection (0.38 ng/ml versus 0.45 ng/ml pre-E2 versus post-E2 compared to 1.05 ng/ml pre-OVX). The results of this experiment implicated ovarian involvement in maintaining elevated basal LH output in cows that were chronically treated with a GnRH agonist. Individual cows varied in their LH surge response to exogenous E2 given 12 days after implant removal, even though LH pulse amplitude and frequency had been restored.

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.

The induction of parturition in the bitch using sodium cloprostenol.

The objectives of this studies were to determine a continuous low-dose treatment regimen for the administration of sodium cloprostenol to the bitch that did not cause polydipsia, and whether this treatment would induce normal and timed parturition in bitches during late pregnancy. Non-pregnant greyhound bitches (n=18) received sodium cloprostenol subcutaneously, via a miniosmotic pump, at dose rates of 0.875 to 4.5 microg/kg/24 h, for 7 days (Days 0 to 7). Daily water intake was measured from Day -2 to Day 9. Polydipsia was observed in bitches treated with the higher dose rates but not in bitches treated with the lowest dose rate of 0.875 microg/kg/24 h. In the second experiment, pregnant greyhound bitches received sodium cloprostenol at dose rates of 1 (n=4), 2 (n=1) and 3 microg/kg/24 h (n=1), on Day 57 of pregnancy. Polydipsia was observed in bitches treated at the higher dose rates of 2 and 3 microg/kg/24 h, but not in the bitches treated at the lower dose rate of 1 microg/kg/24 h. These treatments resulted in the successful induction of parturition. Parturition was associated with a decrease in plasma progesterone concentrations, a reduction in body temperature, and an increase in plasma concentrations of 13,14-dihydro-15-keto prostaglandin F2alpha. The first puppy was born 37.7 +/- 2.9 h after the start of treatment (range 28 to 46 h). The duration of whelping was approximately 15.7 +/- 2.2 h (range 10 to 24 h). The litter size was 9.2 +/- 0.8 pups (range 6 to 12 pups), and the puppy survival rate was 6.0 +/- 0.8 per litter (range 4 to 9 pups). This study demonstrated that the administration of sodium cloprostenol in continuous low dose for 24 h is an effective treatment for the induction of parturition in bitches during late pregnancy. This treatment resulted in the birth of healthy pups, with minimal or no side effects to the bitch.