Morphologic evaluation of acute endometritis in mares with differing resistance to uterine infections

The study was designed to determine differences between normal mares and mares with endometrial pathology in the inflammatory response after bacterial challenge. Six normal mares (biopsy category I) and 4 mares with pathological endometrial changes (biopsy category II) were given an intrauterine infusion of β-hemolytic streptococci on the second day of estrus. All mares had a similar kind of inflammatory response after the bacterial inoculation as assessed by rectal and vaginal examinations. There were no significant differences in the amount of discharge, uterine tone, uterine size and cervical relaxation between the groups. Leukocytic response, as determined by endometrial smears and biopsies, was of the same magnitude in both groups. Two mares from the pathological group were not able to eliminate the infection, but had vaginal discharge and bacteriologically positive uterine swabs until the end of the experiment. It is concluded that the inability of some mares to clear uterine infections cannot be explained by a deficient inflammatory response.     

Results of bacteriological and cytological examinations of the endometrium of subfertile mares in stud farms in Serbia

Uterine microbiology, antimicrobial susceptibility and endometrial cytology were investigated in a total of 51 mares with fertility problems from 16 different stud farms in Serbia. Uterine cultures were performed after collection with a double guarded uterine swab, and endometrial cytology was evaluated after collection of endometrial cells with a special device (cytology brush). In 21 of 51 mares, at least one bacterial species was isolated from the uterus; the most frequent were Streptococcus equi subsp. zooepidemicus (13 isolates) and E. coli (four isolates). All isolates of Streptococcus equi subsp. zooepidemicus were susceptible to penicillin. Results from endometrial cytology were inconsistent; in 17 animals with positive bacteriological culture, cytology was not altered. It can be concluded that in Serbia, as in many other contries, Streptococcus equi subsp. zooepidemicus is the main cause for equine endometritis. It can be easily diagnosed by uterine culture but endometrial cytology does not always prove the existence of an endometrial infection with this agent.     

Comparison of Clinical Signs,Endometrial Culture,Endometrial Cytology,Uterine Low-Volume Lavage,and Uterine Biopsy and Combinations in the Diagnosis of Equine Endometritis

Endometritis is the most common cause of infertility in mares; however, many mares fail to be diagnosed despite availability of many diagnostic tests. Our objective was to compare different diagnostic methods and establish a cutoff value for the number of polymorphonuclear cells in cytology samples. Fifty-four mares were classified positive for endometritis based on endometrial biopsy (“gold standard”), and in a later analysis, the mare was reclassified as positive for endometritis if two or more of the following five criteria on a checklist were present (“new gold standard” [NGS]): (1) abnormal clinical findings, (2) abnormal gross character of low-volume lavage (LVL) fluid, (3) positive endometrial cytology, (4) bacterial growth on culture of the LVL pellet, and (5) histologic evidence of inflammation on endometrial biopsy. Kappa (κ) coefficient and percentages were calculated for sensitivity and positive predictive value (PPV) using SAS 9.3 software. Endometritis was diagnosed in 35/44 (79.5%) mares by biopsy. Based on the endometritis checklist, 33/51 (64.7%) mares were positive for endometritis. The character of LVL was 45% sensitive, whereas culture was 22% sensitive, when compared with endometrial biopsy. One percent of neutrophil-to-epithelial cell ratio was the most sensitive cutoff value (93.3%) when using a guarded swab (κ = 0.64). Endometrial biopsy was the most sensitive diagnostic method when compared against the NGS (sensitivity = 86%). Abnormal clinical findings and positive cytology showed moderate agreement with the NGS (κ = 0.41 and κ = 0.38, respectively). These studies demonstrate the importance of combining clinical findings and laboratory data when evaluating mares for endometritis.     

Technique and interpretation of cervical and endometrial cytology in the mare

This paper illustrates and describes the findings obtained from cervical and uterine cytology smears in the mare. Cervical/endometrial cytology is a simple technique that provides valuable information to the clinician regarding the existence of an acute or active endometritis. The most frequent observation is the presence of neutrophils. Chronically infected/inflamed mares with some degree of inflammatory cellular infiltration can also be identified on the cytology smear by the presence of neutrophils, macrophages and lymphocytes. Microorganisms are sometimes found with either acute or chronic endometritis.

This technique is not a substitute for uterine culture and biopsy. The correct identification of bacteria should be based on the cultural characteristics of the microorganisms.     

Endometritis in the mare caused by a Coryneform organism--a case report and experimental studies

Persistent purulent endometritis in a mare was attributed to an unclassified species of Corynebacterium. Following intrauterine infusions of 20% betadine for 5 days the purulent vulval discharge ceased and the mare appeared clinically normal. Based on histological examination of endometrial biopsy samples, the severe acute inflammatory reaction had largely resolved 2 days after therapy. Three maiden mares considered resistant to bacterial endometritis received single intrauterine inoculations of 1.8 X 10(9) colony-forming units of the Corynebacterium species. The uterine response was followed by vaginal speculum examinations, uterine cultures and cytology, and endometrial histology. After an acute inflammatory reaction, each mare had recovered completely within 2 weeks. Most rapid recovery occurred in the mare in estrus at the time of inoculation. Subsequent secondary infections were detected in two mares. The uncertainty of correlations between results obtained by various diagnostic techniques emphasized the problems associated with each. This report illustrates the concept that endometritis in individual mares may relate more to as yet unidentified "mare factors" controlling uterine defense than to primary invasion by bacteria.     

Diagnosing appendicular osteosarcoma with ultrasound-guided fine-needle aspiration: 36 cases

OBJECTIVES: To determine the ability to obtain diagnostic cytology samples from appendicular bone lesions using ultrasound-guided needle aspirations. Secondary objectives were to compare cytological evaluations with histopathological results and to determine the utility of staining malignant mesenchymal cells for the presence of alkaline phosphatase. METHODS: Aspirations from 36 aggressive appendicular bone lesions with histological diagnoses were included in the study. Ultrasound was used to guide the needle to the medullary cavity or the adjacent soft tissue mass. The smears stained with Wright-Giemsa and nitroblue tetrazolium chloride/5-bromo-4-chloro-3-indoyl phosphate toluidine salt (NBT/BCIP) were examined. RESULTS: A diagnostic sample was obtained in 32 of the 36 cases. Of the 32 diagnostic samples, cytology indicated sarcoma, with a sensitivity of 97 per cent (confidence interval: 83 to 100 per cent) and a specificity of 100 per cent (confidence interval: 16 to 100 per cent). When a diagnosis of sarcoma was made on cytology, alkaline phosphatase staining indicated osteosarcoma, with a sensitivity of 100 per cent (confidence interval: 87 to 100 per cent). CLINICAL SIGNIFICANCE: The results of this study indicate that ultrasound-guided needle aspiration of aggressive bone lesions is a viable technique for identifying malignant mesenchymal cells and for diagnosing sarcomas. It is cost-effective and minimally invasive. Furthermore, identifying alkaline-phosphatase-negative malignant mesenchymal cells from a bone aspiration may rule out osteosarcoma, whereas alkaline-phosphatase-positive malignant mesenchymal cells are suggestive of osteosarcoma.     

Assessment of the breeding prognosis of mares using paired endometrial biopsy techniques

Paired endometrial biopsy samples were taken from 530 subfertile mares, before and after treatment (where indicated) and a period of sexual rest. Prognoses were made after each biopsy (Categories 1A-4A before treatment and Categories 1B-4B after treatment), using histopathological criteria similar to those described by Kenney and Doig (1986). Eighty-seven per cent of the mares were assigned to first biopsy prognosis Category 3A. The second biopsy prognosis produced a more even population distribution (10, 47, 40 and 3 per cent respectively for Category 1B, 2B, 3B and 4B mares). First biopsy Category 3A and 4A mares produced mean foaling rates of 51 and 40 per cent respectively. None of the 14 second biopsy Category 4B mares delivered a live foal (12 were barren, one aborted and one suffered early foetal death). This study supports the hypothesis that a breeding prognosis assigned on the basis of two endometrial biopsy samples, taken before and after specific uterine treatment, where indicated, which takes into consideration the mare's ability to respond to treatment, is likely to be more accurate than one assigned after a single biopsy sample.     

Equine mastitis--a review of 28 cases

Mastitis was diagnosed in 28 mares. Cytological evaluation of milk samples showed large numbers of neutrophils in 72 per cent and bacteria in 33 per cent. Aerobic bacteria were cultured from 71 per cent of samples. Streptococcus zooepidemicus was the most common isolate (37 per cent). Gram-negative species accounted for 42 per cent. Determination of the probability for antimicrobial susceptibility of bacterial isolates from mares with mastitis indicated that a trimethoprim-sulphonamide combination would be active against more than 75 per cent of isolates while penicillin would be active against less than 60 per cent. Systemic antibiotics, frequent milking, hotpacks and/or hydrotherapy and, if possible, infusion of an intramammary antibiotic preparation is recommended for treatment of equine mastitis.     

The Effects of Treatment with N-Acetyl Cysteine on Clinical Signs in Persistent Breeding-Induced Endometritis Susceptible Mares

Persistent breeding-induced endometritis (PBIE) is a major cause of infertility in mares. Endometrial inflammation that persists until embryonic descent ultimately results in early embryonic death. A poor endometrial biopsy grade (IIb or III) has been identified as a risk factor for PBIE. Intrauterine fluid accumulation (>2 cm in depth), pathologic endometrial edema, and elevated intrauterine neutrophil levels are all clinical features of PBIE. Commonly applied treatment options include uterine lavage and oxytocin therapy. N-acetyl cysteine (NAC), a mucolytic used to treat bacterial endometritis in mares, has anti-inflammatory properties and was investigated as a potential treatment for PBIE. A randomized, blinded, cross-over design clinical trial used NAC before breeding in PBIE-susceptible mares (n = 9). Intrauterine infusion of 3.3% NAC was performed 12 hours before insemination, and endometrial cytology and endometrial biopsy samples were obtained at 12 and 60 hours after insemination. Endometrial biopsies were evaluated for the degree of inflammation present. Clinical signs of endometrial edema and intrauterine fluid volumes were assessed by transrectal ultrasound at 12 and then every 24 hours after breeding. Data were analyzed using repeated measures analysis of variance and a Mann Whitney Wilcoxon Test. Treatment with NAC did not improve clinical signs in PBIE-affected mares. However, endometrial biopsies from mares treated with NAC displayed more diffuse and severe neutrophil infiltration than control cycles. Further research using a larger population of mares is required to evaluate the effects of NAC treatment on the endometrium of PBIE-susceptible mares.     

Effects of immunostimulation with Propionibacterium acnes (EQSTIM) in mares cytologically positive for endometritis

Thirty-seven mares, barren for at least one year, were diagnosed to have endometrial inflammation by cytological examination and/or bacterial culture. Sixteen of these mares (the control group) were treated with prostaglandin and reevaluated cytologically within 10 days of behavioral estrus. Fifteen out of sixteen (15/16) (93.8%) remained positive for endometrial inflammation. Twenty-one mares (immunologically treated group) were treated with Propionibacterium acnes by injecting 1 ml per each 113.3 kilograms body weight intravenously on days 1, 3, and 7 following the diagnosis of endometrial inflammation. They also received prostaglandin. Twelve of the immunologically treated mares were examined cytologically as were the control mares. One of twelve examined (1/12) (08.3%) was positive cytologically for endometrial inflammation when examined within 10 days after behavioral estrus. The other nine mares in the treated group were bred on the first behavioral estrus following treatment. Nine of nine (9/9) were determined to be pregnant to 60 days gestation. The other treated mares were bred following the cytological evaluation. Eighteen of the twenty-one treated mares (18/21) (85 7%) were confirmed pregnant to 60 days gestation.     

Streptomycin-resistant Escherichia coli as a marker of vulvovestibular contamination of endometrial culture swabs in the mare.

To investigate the vulvovestibular contamination of endometrial culture swabs in the mare, a liquid culture of a streptomycin-resistant strain of Escherichia coli was applied to the vulvovestibular area of mares and used as a marker of contamination of endometrial culture swabs. Prior to taking endometrial swabs, the perineal area was washed with soap, rinsed with water, and dried. Endometrial culture swabs were taken from mares that were in anestrus or diestrus and from mares that were in estrus. When a manual transvaginal swabbing technique was used, 22 of 24 endometrial swab specimens from 12 mares were contaminated with the experimental bacterial strain; culture of only one endometrial swab yielded more than nine colonies. When a speculum approach was employed, three of 12 swab specimens from 12 mares yielded between one and three colonies. The stage of cycle had no effect on the extent of contamination, but the proportion of positive cultures was significantly smaller when swabs were taken via a vaginal speculum approach, compared to a manual transvaginal approach. Complete preclusion of vulvovestibular contamination of endometrial swab specimens was not achieved; however, fewer than ten colonies can be expected even in mares in which the vulvovestibular area has been thoroughly contaminated with a broth culture, provided that the perineal area is adequately cleaned prior to swabbing.     

The Effect of Manual Removal of Placenta Immediately after Foaling on Subsequent Fertility Parameters in the Mare

Retained placenta is considered to be a common problem in postpartum mares. The incidence varies from 6% to 54% depending on the breed, with higher incidence in heavy draught mares than in light-weight mares. Retained placenta has been linked to lower postpartum oxytocin concentration, impaired uterine involution, and dystocia. The objective of this study was to assess the effect of early manual removal of placenta immediately postpartum on subsequent fertility parameters (development of free intrauterine fluid, inflammatory status of endometrium, and on pregnancy rates) and to compare them with mares with spontaneous expulsion of placenta. A total of 29 mares, mainly Irish draught, were closely monitored during foaling by closed circuit television and allocated to two groups: (1) mares that expelled the placenta spontaneously within 3 hours of foaling; and (2) mares that were cleansed manually immediately after foal delivery. All mares were examined and scanned 5 and 9 days postpartum, and free intrauterine fluid was recorded; endometrial swabs were taken 9 days postpartum for endometrial cytology and culture. None of the fertility parameters analyzed showed statistical difference between groups 1 and 2. Therefore, it can be concluded that early manual removal of placenta has no detrimental effects on subsequent fertility of mares and, therefore, can be recommended when a veterinarian attends a foaling.     

Evaluation of brush cytology in the diagnosis of chronic intranasal disease in cats

Brush cytology was used as a diagnostic aid in 85 cats affected with chronic intranasal disease. Fifty-three of these cases, sampled over a five-year period, were included in this study, while the other cases were excluded due to poor cellularity of the cytological samples (nine cases) or a lack of histological or follow-up data (23 cases). Thirty-six brush samples were classified by cytology as inflammatory. Subsequent histological examination revealed a false negative diagnosis of neoplasia in six cats, two of which had malignant tumours (one adenocarcinoma and one lymphoma), the remaining four having benign tumours (two adenomas and two osteochondromas). Seventeen samples were classified by brush cytology as neoplastic. This was confirmed in 16 of these cases by histology or follow-up (nine epithelial malignant tumours, six lymphomas and one osteosarcoma). In the remaining case, a false positive diagnosis of lymphoma was made. The procedure had an overall 86.8 per cent (46/53) agreement between the diagnosis of inflammatory conditions versus neoplasia, with a sensitivity of 72.7 per cent, a specificity of 96.8 per cent, a predictive value of a positive test of 94.1 per cent and a predictive value of a negative test of 83.3 per cent.     

Quantitative assessment of the risks of reducing the routine swabbing requirements for the detection of Taylorella equigenitalis

The transmission of contagious equine metritis (CEM) on stud farms in Britain, Ireland and other European countries is prevented by following the recommendations in the Horserace Betting Levy Board's Code of Practice on CEM. A quantitative risk assessment was undertaken to estimate the likely impact of removing the recommendation, from the 2002 code, to culture endometrial or cervical swabs microaerophilically for the presence of Taylorella equigenitalis, the causative organism. The scientific literature was reviewed for evidence about the anatomical distribution of T. equigenitalis at different times after infection and it was found that, in chronically infected mares, the organism was detectable in the clitoral swabs of nearly 93 per cent, but in the cervical swabs of only 31 per cent. In contrast, in acutely infected mares, the organism was detectable in the clitoral swabs of nearly 69 per cent, but in the cervical swabs of 84 per cent. By using these results, a quantitative risk assessment was undertaken, assessing the likely effects of removing the recommendation that swabs from the cervix of low-risk mares should be cultured for T. equigenitalis. The results were sensitive to the prevalence of the infection, but when it was low, there appeared to be few benefits in continuing to culture cervical swabs routinely. However, such swabs are vital when the disease is suspected.     

Diagnosis and correction of twin pregnancy in the mare

Reproductive Physiology 1. Twin pregnancies result in high rates of abortion, stillbirth, and neonatal mortality. 2. Twins develop subsequent to multiple ovulations. Multiple ovulations are related to breed, parity, and mare history. Multiple ovulations are most frequently seen in Thoroughbred and Draft mares. Multiple ovulations are more common in barren and perhaps maiden mares than in lactating mares, and they are more common in certain individual mares. 3. Equine embryos are motile in the uterus from the time of first detection (Days 9 to 10) until fixation (Day 16). They are frequently located in the uterine body on Days 9 and 10. 4. Twin embryos have a pattern of motility and fixation similar to that of single embryos, and fixation is more frequently unilateral than bilateral (70 per cent versus 30 per cent, respectively). 5. Mares have an efficient natural embryo-reduction mechanism to eliminate excess (greater than 1) embryos resulting from multiple ovulations. Natural embryo reduction is more successful in unilateral than bilateral twin pregnancies (89 per cent versus 11 per cent successful reduction, respectively). 6. After the establishment of endometrial cups (Days 35 to 40), mares that are aborted will frequently not cycle for several months. Management of Twin Pregnancy 1. Breed all mares regardless of the number of preovulatory follicles. Withholding mares with preovulatory follicles from breeding does not decrease the incidence of twin pregnancy, but it decreases the overall pregnancy rate and results in a loss of breeding time. 2. Check all mares for twins, regardless of the number of detected ovulations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Equine endometrial cytology

The simplicity of collection of material for cytologic preparations belies the complexity of smear interpretation beyond recognition of neutrophils. Knowledge concerning cancer cytology moves rapidly, for cancer is a progressive, often fatal disease so that tissue for comparison and confirmation of interpretation often becomes available. This is not true for cytologic study of the equine endometrium. Lesions detected by means of cytology smears may be transient and regress, offering little information concerning their etiology or consequences. They may be focal and missed in the corresponding biopsy. Such experiences should lead to the abandonment of equine endometrial cytology, but have only strengthened our interest and enthusiasm for this technique in the study of the uterus. We have found it to be useful in a limited number of clinical circumstances in which other techniques have failed. This, plus the growing number of supportive clinicians using cytopathology service and the large number of mares with fertility problems, leads us to believe that further investigation of equine endometrial cytology may prove to be even more helpful as a clinical tool.     

Comparison of cytologic and histologic evaluations of the conjunctiva in the normal equine eye

OBJECTIVE: To describe the cells observed in conjunctival brush cytology (CBC) from normal horses and compare these findings with conjunctival structural histology so as to understand which cells are recovered from CBC. METHODS: This study was divided into three parts. (1) Conjunctival brush smears were collected from 20 healthy horses on both eyes and a differential count on 300 cells was carried out on May Grünwald-Giemsa (MGG) smears. (2) A similar protocol was used for whole eyes from five horses obtained rapidly after death from a slaughterhouse. The eyes were then assessed for conjunctival histology. (3) Cytobrush smears were collected from five healthy horses. Smears were examined after MGG or periodic acid Schiff (PAS) staining. RESULTS: The differential cell count showed a majority of deep and intermediate epithelial cells with very few superficial and goblet cells in both eyes. A stratified columnar to cuboidal epithelium was observed on nearly the whole surface of the conjunctiva. A stratified squamous type was observed at the palpebral and bulbar edges. Areas with highest mucus cell indices were found from the nasal to the temporal edge of the equine inferior conjunctiva in the upper palpebral segment near the fornix and in a part of the nasal fornix. In MGG smears no mucus cells were identified; however, they were numerous in PAS smears (22.6% +/- 11) and were mostly cylindrical cells (42.5% +/- 14.4 PAS positive). CONCLUSIONS: Cytobrush smears in the healthy horse are characterized by a majority of polyhedral and cylindrical cells and a few squamous cells. The cylindrical cells may be mucous cells and probably originate from the main stratified columnar to cuboidal epithelium.     

Efficiency of uterine fluid cytology in the diagnosis of subclinical endometritis in the water buffalo (Bubalus bubalis)

This study compared endometrial cytology vis‐a‐vis uterine fluid cytology for assessment of uterine health in clinically normal and subclinical endometritis (SE)‐affected buffaloes. Uterine fluid samples and endometrial samples were collected from the buffaloes (n = 38) at oestrus using blue sheath and cytobrush, respectively. The smears were stained with Field stain for 3 minutes, and a minimum of 400 cells were counted in each smear for determination of the percentage of polymorphonuclear (PMN) leucocyte. The incidence of subclinical endometritis, based on the cytobrush cytology, was 23.08%. The correlation between cytobrush cytology with uterine fluid cytology was positive and significant (r = .37; p = .02). The ratio of PMN leucocyte in cytobrush cytology to uterine fluid cytology was 1:2.4. ROC analysis revealed that the threshold value of 6.16% PMN leucocyte in uterine fluid cytology showed a diagnostic sensitivity and specificity of 100% in differentiating normal from SE‐affected buffaloes. In conclusion, collection of uterine fluid was easier compared to collection of endometrial samples using cytobrush and the percentage of PMN leucocyte in uterine fluid cytology can be used as a tool for diagnosis of subclinical endometritis in buffaloes.     

Development and Comparison of Sampling Techniques for a Broad Range,Semiquantitative Polymerase Chain Reaction Assay for Detection of Bacterial DNA in the Equine Uterus

Microbial culture from a double-guarded culture swab is commonly used to diagnose infectious endometritis. The objective of this study was to develop a quantitative polymerase chain reaction (qPCR) assay to detect a broad range of bacteria from equine uterine samples. Twenty-seven mares with a clinical history of endometritis had a double-guarded culture swab collected for analysis by qPCR and microbial cultures. An additional 12 mares had a uterine biopsy sample collected for qPCR analysis, microbial culture, and histopathology. Subsequently, a double-guarded culture swab for microbial culture and a cytology brush sample were also collected. The qPCR assay detected bacterial DNA in nine of 27 mares from a double-guarded swab and six of 12 mares from an endometrial biopsy. Positive microbial growth was detected in nine of 27 mares and four of 12 mares from a double-guarded culture swab. Bacterial DNA was detected in two of 27 mares and two of 12 mares without subsequent microbial growth. The simple presence of an organism's DNA allows for detection by nonculture-based systems, both live and dead organisms can be identified. In conclusion, the qPCR assay was determined to be a sensitive diagnostic technique for identifying pathogens associated with infectious endometritis. The primary application of the qPCR assay is detection of potential pathogenic bacteria in the uterus of a mare suspected of having infectious endometritis when a traditional microbial culture is negative. Further work is warranted to determine if mares positive for bacterial DNA and negative for microbial culture are affected clinically.     

Serological surveillance of equine viral arteritis in the United Kingdom since the outbreak in 1993

Serological analysis of blood samples submitted to the Animal Health Trust showed that during 1995, 185 of 9203 unvaccinated horses (2.0 per cent) tested positive for antibodies to equine arteritis virus (EAV), and that during 1996, 46 of 8851 unvaccinated horses (0.52 per cent) tested positive. During both years thoroughbreds were the predominant breed tested and only a small proportion of these (<0.3 per cent), consisting predominantly of imported mares, were seropositive. In contrast, among standardbred horses, from which samples were actively solicited in 1995, 84 of 454 (18.5 per cent) were seropositive. Among standardbreds there was a difference in prevalence between types of horses, with 3.7 per cent of racing horses, 25 per cent of non-racing horses and 41 per cent of stallions testing seropositive. Investigations of seropositive stallions identified during 1994 and 1995 demonstrated that clinically inapparent equine viral arteritis (EVA) had occurred previously in the UK. Of 50 seropositive stallions, nearly half were standardbreds and nearly all had been imported from either North America or the European Union. Whether 34 seropositive stallions were shedding virus in their semen was established either by test mating, by the serology of the covered mares, or by investigation by MAFF following the introduction of the Equine Viral Arteritis Order 1995. Nine of the stallions (26.5 per cent) were identified as presumptive shedders of EAV in semen and among specific breeds, viral shedding was identified in six of 15 (40 per cent) standardbreds and three of nine (33 per cent) warmbloods. In contrast with the outbreak of EVA in the UK in 1993, no signs of disease typical of EAV infection were reported during these investigations, even in mares test mated to stallions shedding the virus.