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.     

International disease surveillance. International disease monitoring, October to December 2004

To assess whether the rates of antimicrobial susceptibility in bacteria isolated from the urine of dogs with urinary tract infections are similar to those of bacteria isolated from bitches with pyometra, the antimicrobial resistance of Escherichia coli isolated from the two groups were determined and compared. The samples were collected in Sweden between April 2002 and March 2003, and potential changes over time were assessed by comparing the results with corresponding data from 1991 to 1993. Among 80 isolates of E coli from cases of pyometra, the proportions that were resistant to the antimicrobials used in canine practice were generally low (ampicillin 10 per cent, enrofloxacin 4 per cent, gentamicin 0 per cent, streptomycin 5 per cent, sulfamethoxazole 8 per cent, tetracycline 4 per cent and trimethoprim 2 per cent) and similar to the proportions reported previously. Significantly lower proportions of resistance were recorded among the pyometra isolates than among 92 isolates from urine samples submitted by animal hospitals to ampicillin (P=0.04), streptomycin (P=0.002) and tetracycline (P=0.03), but there were no differences between the pyometra isolates and 113 isolates from urine samples submitted by animal clinics.     

Uterine, cervical and vaginal microflora of the normal bitch throughout the reproductive cycle

Samples for microbiological culture were collected from the uterus of bitches using transcervical uterine cannulation (31 samples, 23 bitches) and from the uterus, cervix and vagina post mortem (19 bitches) at all stages of the reproductive cycle. Samples were cultured for aerobic and anaerobic bacteria and for aerobic mycoplasmas. Bacteria were always found in the uterus during prooestrus and oestrus (12 positive in 12 cultures) and rarely at other stages of the reproductive cycle: during anoestrus (one in 14) and other stages (none in 24). When microorganisms were detected at three sites post mortem, those found in the cervix and vagina were always of the same species as those found in the uterus. In six out of 13 instances, microorganisms were found in the cervix or vagina when none were found in the uterus. The mean number of isolates, number of bacteria seen in uterine cytology and bacterial growth were greater (P < 0–005) during oestrus and pro-oestrus than at other stages. Bacteria isolated from the uterus, in order of frequency, were Escherichia coli, Haemophilus species, α-haemo-lytic streptococci, Corynebacterium species, Streptococcus canis, Alcaligenes faecalis, Bac-teroides species, Pasteurella species and Proteus mirabilis. No mycoplasmas were cultured from the samples. This study indicates that the uterus of the normal bitch has a uterine microflora during pro-oestrus and oestrus that is similar to that of the vagina and cervix.     

Use of a low dose prostaglandin F2 alpha in bitches

The dosage of Prostaglandin F2 alpha used until the present (100, 250 and 1000 micrograms/kg bw), in order to treat pyometra in the bitch, was accompanied with side effects such as salivation, vomiting and diarrhea. In the present work, the efficiency of low dose Prostaglandin (20 micrograms/kg bw) was examined in two different groups of patients: Group 1: Included 9 bitches pregnant for a period of 5-7 weeks duration. Initially the bitches were treated 3 or 4 times per day with Prostaglandin F2 alpha. In these cases abortion took place within 4 to 11 days. Group 2: 12 dogs, suffering from pyometra, were treated 3 times per day with PGF2 alpha for 8 days. In 9 dogs the pyometra resolved and the bitches came in estrus 2-5 months after treatment. 7 bitches have been mated and 6 of these gave birth to healthy litters. During a follow-up period of at least 10 months there has not been a reoccurrence of pyometra. In 3 out of the 12 dogs the uteri were still enlarged after 8 days of treatment. These bitches underwent ovariohysterectomy and a cystic hyperplasia of the endometrium was diagnosed histologically. The low dose (20 micrograms/kg BW) Prostaglandin F2 alpha induced in all dogs the expulsion of the uterine contents. Side effects during the treatment were not observed.     

Aerobic Bacteria Occurring in the Vagina of Bitches with Reproductive Disorders

A retrospective survey was performed of aerobic bacterial species found in the vagina of 203 bitches with genital disorders, e.g. infertility, vaginitis, pyometra and puppy death. Escherichia coli, beta-hemolytic streptococci, Staphylococcus intermedius and Pasteurella multocida were the species most often isolated. From bitches with pyometra E. coli in pure culture was the most frequent isolate. In contrast, the majority of infertile bitches gave rise to mixed cultures, and no specific bacterial species was consistently associated with infertility. Thus, bacterial sampling from infertile bitches was concluded to be of low diagnostic value. Bacterial species isolated from the bitches having vaginitis were present in pure culture in 26.9% of the samples while nonspecific mixed cultures were obtained from 34.6% of the samples from these bitches. E. coli was the most frequently isolated bacterial species from bitches with dead puppies. However, in such cases it is important to relate the vaginal bacterial findings to autopsy findings and the results of bacteriological cultures of the pups.     

Treatment of spontaneous pyometra in 22 bitches with a combination of cabergoline and cloprostenol

Twenty-two bitches with ultrasonographically diagnosed spontaneous pyometra were treated with a combination of 5 microg/kg cabergoline per day and 5 mug/kg cloprostenol every third day, and potentiated sulphonamide twice a day. Bitches with either open-cervix or closed-cervix pyometra showed a rapid clinical improvement, associated with a reduction in plasma progesterone concentration, increased vulval discharge and a reduction in the diameter of the uterus. The haematological profiles of 21 of the bitches returned to normal within six days of treatment, and their biochemical profiles returned to normal within nine days; 19 of the bitches were managed successfully by a 10-day period of treatment. Two bitches required a further three days of treatment, and in one bitch with a partial uterine torsion the treatment was not successful. Adverse effects of the treatment were limited to the 60 minutes immediately after the administration of prostaglandin, and included retching, vomiting, mild abdominal straining, diarrhoea and panting. The incidence of adverse effects was reduced after each successive dose of prostaglandin. Eleven of the 21 successfully treated bitches were mated at the next oestrus, and seven became pregnant; their litters were smaller than the published breed averages. In four of the bitches the pyometra recurred after the next oestrus.     

Efficacy and toxicity of tamoxifen citrate for prevention and termination of pregnancy in bitches

Five groups of bitches were given tamoxifen citrate (1 mg/kg of body weight) orally twice daily for 10 days. Drug administration commenced during late proestrus, day 4 of estrus, day 2 of diestrus, day 15 of diestrus, or day 30 of diestrus (n = 4/group). Nineteen of the bitches accepted natural mating by 1 or more of 3 stud dogs of known fertility (1 bitch did not). Twenty days after cessation of drug administration, ovarian, uterine, and hepatic specimens were obtained from each bitch in 4 of the groups. Pregnancy proceeded to natural termination in bitches of the remaining group (diestrous day 30). Pregnancy was not detected in any bitch of the proestrus, estrous, or early diestrous groups. Of 4 bitches of each of the remaining groups (diestrous day 15 and diestrous day 30), 2 aborted fetuses and/or resorbed placental remnants; the other 2 bitches in each of these groups had normal-appearing fetuses (diestrous day-15 group) or clinically normal pups (diestrous day-30 group). Of the 20 bitches given tamoxifen citrate, 5 developed endometritis with or without pyometra, and 4 of these had ovarian cysts. Although tamoxifen citrate is effective for preventing or terminating pregnancy in the bitch, the regimen used in the study reported here was associated with high frequency of pathologic changes in the reproductive tract.     

Pyometra

Pyometra is a relatively uncommon diestral uterine disorder seen primarily in older bitches and queens. The incidence of pyometra appears to be increasing, especially in younger animals, as a result of increasing use of estrogen and progesterone for mismating and for certain medical disorders. The clinical signs of pyometra and abnormalities on physical examination are dependent on the patency of the cervix and how quickly the client recognizes the problem. Ovariohysterectomy is still the treatment of choice; however, prostaglandin F2 alpha as a medical alternative in the management of pyometra is gaining more and more acceptance. The goal of prostaglandin F2 alpha therapy is to salvage the reproductive tract in the young breeding bitch or queen in the hopes of obtaining future litters. The results have been very promising, especially for bitches or queens with open-cervix pyometra. Prostaglandin F2 alpha appears to offer the owner a reasonable alternative in the management of pyometra.     

Histological characteristics of canine deciduoma induced by intrauterine inoculation of E. coli suspension.

Canine deciduoma could be induced in the diestrous uterus by an intrauterine inoculation of a culture suspension of E. coli originally isolated from naturally occurring canine pyometra. These deciduomas had the same histological findings as those of naturally occurring canine pyometra with so called "Swiss cheese endometrium". This suggests a possibility that the canine pyometra is a kind of naturally occurring decidual reaction (deciduoma) induced by one of several triggers such as bacterial infection.     

Canine bacteruria

Urine from 237 dogs submitted for euthanasia was collected directly by needle aspiration from the urinary bladder. The bacteriological findings were correlated with the sex and age of the dogs. E. coli was the most frequent species found. The overall incidence of bladder infection was 6–2% for males and 26–6% for females. Bladder infection can be categorized on the basis of occurrence in either very young or in older dogs. The infection incidence increased linearly in bitches older than 4 years, ending at a 50% frequency in those more than 10 years old. Pyometra frequency also increased after 4 years of age and this pattern may indicate a common causative factor for urinary tract infection and pyometra. This investigation shows that subclinical urinary tract infection is common in dogs and is related to age in bitches.     

Is Mechanically Induced Cystic Endometrial Hyperplasia (CEH) a Suitable Model for Study of Spontaneously Occurring CEH in the Uterus of the Bitch?

Cystic endometrial hyperplasia (CEH) was experimentally reproduced by the intraluminal insertion of a silk wire in the uterus of metestral bitches to obtain a model for the study of spontaneously occurring CEH in the bitch. This mechanically reproduced CEH corresponded histologically very well to spontaneously occurring CEH. With respect to sex hormone receptor expressions, however, there was no similarity. In the mechanically induced CEH, progesterone receptor expression was reduced in the epithelial cells (surface epithelium and endometrial glands) and slightly increased in the stromal fibroblasts and myometrium as compared with the normal metestrus uterus. The oestrogen receptor expression in mechanically induced CEH was reduced in the epithelial cells (surface epithelium and endometrial glands) and more or less unchanged in the stromal fibroblast and myometrium as compared with the normal metestrus uterus. This is in contrast to the increased sex hormone receptor expressions in all uterine cell types, observed in spontaneously occurring CEH. The mechanically induced CEH model corresponds immunohistochemically more to early placentation and to pyometra in the bitch. Thus this experimental CEH model is not suitable for pathogenic studies of spontaneously occurring CEH in the bitch.     

Measurement of C‐reactive protein and Prostaglandin F2α Metabolite Concentrations in Differentiation of Canine Pyometra and Cystic Endometrial Hyperplasia/Mucometra

Canine pyometra is a dioestrus period disease in which systemic inflammatory response syndrome (SIRS) is a common outcome due to the response of the body to the bacterial infection. The purpose of this study was i) to differentiate canine pyometra and cystic endometrial hyperplasia (CEH)/mucometra by measuring serum C‐reactive protein (CRP) and prostaglandin F_2α metabolite (PGFM) concentrations in blood and ii) to compare serum concentrations of CRP and PGFM in bitches with a pathological uterus (pyometra or CEH/mucometra) to concentrations in bitches with a healthy uterus. Mean CRP concentrations were found significantly higher (p < 0.001) in dogs with pyometra compared to those with CEH/mucometra or healthy uterus. However, no statistical difference could be detected between the groups for mean PGFM concentrations. Mean white blood cell count (WBC), alkaline phosphatase (ALP) and total protein concentrations were found significantly higher (p < 0.001) in dogs with pyometra. Escherichia coli was the most frequently isolated microorganism from dogs with pyometra (64.3%). Edwardsiella spp. was detected in a single case of pyometra for the first time. In conclusion, our results demonstrate that serum CRP concentrations were increased in dogs with pyometra and thus we conclude that serum CRP concentration but not PGFM might be useful as a marker to differentiate a case of CEH/mucometra from pyometra in female dogs. To the authors' knowledge, this is the first report in which Edwardsiella spp. has been isolated in the canine uterus.     

Prostaglandin F2 alpha treatment of canine pyometra

Immediate and long-term outcomes of prostaglandin F2 alpha treatment for canine pyometra were studied in 10 bitches. Examination of pretreatment uterine biopsy specimens, taken for histopathologic diagnosis and classification of disease severity, revealed either type III or IV pyometra. Dinoprost tromethamine (0.25 or 0.5 mg/kg of body weight, SC) was given once daily for 3 days. Bitches were bred at the first posttreatment estrus and monitored for a minimum of one year. When pure cultures of Escherichia coli (n = 3) or Staphylococcus aureus (n = 1) were obtained from the vagina, these bacteria also were found in the uterus. Pretreatment WBC counts often did not reflect the severity of histopathologic findings in the uterus, but posttreatment WBC counts were useful in monitoring response to treatment. Four bitches produced a litter within one year of treatment. Four bitches (40%) had recurrence of pyometra within one year of treatment, and these same bitches had another recurrence after an additional prostaglandin treatment. Three additional bitches had a recurrence by 27 months after therapy, establishing a total recurrence rate of 77% (7/9). Results suggested that subclinical disease may persist after treatment, with clinical recurrence during diestrus. Despite the high recurrence rate, it was concluded that this treatment is a practical treatment for canine pyometra when reproduction is desired.     

Laboratory diagnosis of canine pyometra.

The principal laboratory test used to confirm the pyometra diagnosis in the bitch has been the determination of the total white blood cell count in venous blood. A marked elevation is known to be a characteristic of the disease.In the present study, the white blood cell count was determined as well as the γ-globulin level. An elevation of the γ-globulin level and the total white blood cell count was very characteristic to the pyometra patients. The increase in the number of white blood cells nor the high γ-globulin level cannot be regarded specific for pyometra, therefore it was regarded important to find out a more specific test for pyometra.When sonicated E. coli bacteria were tested against sera from pyometra patients in electroimmunodiffusion, the precipitation was almost always detected when E. coli had been isolated from the uterus. This technique provides a quick method in detecting the causative E. coli infection.The present study suggests that whenever laboratory tests are used to confirm the pyometra diagnosis by the total white blood cell count, it is advantageous to analyze the total γ-globulin level in the serum as well as specific antibodies against a common E. coli antigen.Because of the reliability of the glutaraldehyde coagulation test and the simple technique, this can be suggested as the method of choice for an average small animal practice.     

Unconjugated oestrogen and progesterone concentrations in the blood of bitches with false pregnancy and pyometra.

The changes in total unconjugated oestrogen and progesterone concentrations in the peripheral blood of one bitch throughout a false pregnancy were measured. The levels of these hormones detected in 11 bitches when examined during a false pregnancy, and in 13 other bitches with pyometa are also reported. Oestrogen and progesterone concentrations were within normal limits in both of these conditions where single samples were obtained, but the bitch with false pregnancy studied in detail showed a raised oestrogen level during metoestrus. The significance of these results is discussed in relation to previous studies on the aetiology of false pregnancy and pyometra.     

Investigating uterine disease in the bitch: Uterine cannulation for cytology, microbiology and hysteroscopy

A technique for transcervical cannulation of the uterus of the bitch for the collection of samples for cytology and microbiology was developed. This technique involved passing a cannula into the uterus with the aid of an endoscope. The uterus was cannulated successfully in mature bitches (over 10 kg) 110 times in 144 attempts. Success of cannulation was influenced by the experience of the operator and ease of cannulation by the stage of the reproductive cycle, the size of the bitch and the type of cannula. Complications associated with the procedure were endometritis, vaginitis and vaginal tears. Uterine cannulation was generally possible in all bitches studied at all stages of the reproductive cycle. Techniques for the collection of samples from the uterus for cytology and microbiology and for the processing of samples for uterine cytology are outlined. Hysteroscopy was performed on seven occasions in post partum bitches and was found to be possible at least until day 17 after whelping.     

Nonenteric Escherichia coli isolates from dogs: 674 cases (1990-1998)

OBJECTIVE: To determine nonenteric sites associated with Escherichia coli isolates in dogs and the antimicrobial susceptibilities of the isolates. DESIGN: Retrospective study. SAMPLE POPULATION: 17,000 canine specimens. PROCEDURE: Medical records of 17,000 canine specimens submitted for bacteriologic culture were examined and the number of isolations of E coli was determined. For these cases, records were further examined with respect to body system involvement, sex, concurrent infection with other species of bacteria, and antimicrobial susceptibility. RESULTS: 674 E coli isolates (424 from urine, 62 from the skin, 52 from the respiratory tract, 45 from the ear, 43 from the female reproductive tract, 25 from the male reproductive tract, and 23 from other organ systems) were identified. There was a significantly higher proportion of isolates from urine specimens from spayed females than from sexually intact females or males. Escherichia coli was isolated in pure culture from 65.9% of the specimens. Most E coli isolates were susceptible to norfloxacin (90%), enrofloxacin (87.5%), gentamicin (90.7%), and amikacin (85.9%). CONCLUSIONS AND CLINICAL RELEVANCE: Most nonenteric E coli infections in dogs involve the urinary tract. Amikacin, gentamicin, norfloxacin, and enrofloxacin have the highest efficacy against canine E coli isolates. For E coli isolates from dogs, in vitro susceptibility to commonly used antimicrobial agents has remained fairly stable during the past decade.     

Follow-up examinations of bitches after conservative treatment of pyometra with the antigestagen aglepristone

The aim of this study was to determine the therapeutic success of the medical treatment of canine pyometra with the antigestagen aglepristone and to document the recurrence rate in relation to the time interval after treatment with antigestagens. In 48 (92.8%) of the 52 treated bitches, healing could be achieved within the first 3 weeks after the treatment had been started. One bitch died as a result of renal insufficiency; in three bitches there was no emptying of the uterus, so ovariohysterectomy became necessary. In these three patients, ovarian and endometrial cysts were present. Forty-one bitches could be followed up for 3 months. Four animals developed a recurrence (9.8%). In three bitches ovarian cysts and cystic endometrial hyperlasia could be found intra operationem. The development of 37 bitches could be followed for at least 1 year. Seven animals developed a pyometra again (18.9%). Two received a repeated treatment with aglepristone and have been free from recurrence for over 12 months. In 37 animals data on the subsequent sex cycles are available. In 22 bitches next heat started at the expected time, in seven animals heat started too early. In eight bitches the period of anoestrus was prolonged. Five of the six bred bitches delivered at least one litter. The presented data show that treatment of pyometra by aglepristone results in a high healing rate. The recurrence rate can be minimized by the selection of bitches without ovarian cysts and cystic endometrial hyperplasia.