Identification and characterization of the primary infectious agents associated with ovine ulcerative balanoposthitis and vulvovaginitis in South Africa

Abstract

Ulcerative balanoposthitis and vulvovaginitis is a disease characterized by erosion and ulceration of the glans penis and muco-cutaneous junction of the vulval lips of sheep. The disease was first recognized in South Africa in 1979 in the Calvinia district, Northern Cape province, and its distribution has since extended to all major Dorper sheep farming areas of the country with serious economic consequences. It is now a major concern for Dorper sheep breeders and farmers due to the fact that the disease has a detrimental effect on conception and subsequent lambing percentages. The aetiology and epidemiology of the disease are not well established. During this study the microbial flora in the genital tract of both clinically healthy and infected sheep were compared. The aim was to isolate and identify the pathogenic microorganism/s that contribute to the disease in sheep and to determine the in vitro antimicrobial susceptibility of the isolates. Flocks of sheep in the Northern Cape province showing clinical signs of ulcerative balanoposthitis and vulvovaginitis were examined. The microbiological flora of 116 clinically unaffected sheep and 104 affected sheep from 15 different farms, and with characteristic ulcerative lesions were examined. Swabs from rams and ewes were collected aseptically, and put into cryovials consisting of transport medium for bacterial, mycoplasmal and viral maintenance. Swabs for Chlamydophila species antigen detection were placed into tubes without transport medium. All specimens were processed for virus isolation in cell culture, Chlamydophila antigen detection with ELISA, and bacteria and mycoplasmas were isolated on standard culture media. The latter were further identified with biochemical tests and indirect immunofluorescence antibody (IFA) test. The IFA test was found to be useful when contamination with other microorganisms was prevalent, especially in genital tract specimens. This procedure had reduced the necessity for sub-culturing and cloning. The IFAT was found to be effective for the identification of Mycoplasma spp. growing on primary growth media in mixed cultures. The technique also helped to confirm the presence of mycoplasmas that did not produce typical colonies, and it was possible to identify mycoplasma colonies overgrown by bacterial contaminants. Bacteriological examination of materials from affected and unaffected ewes and rams resulted in the isolation of a sizeable number of Arcanobacterium pyogenes. It was isolated from 44.2 % affected sheep and 17.2 % healthy ones. This isolation difference was statistically significant (p<0.01). Seventy four per cent of the isolates came from severe clinical cases. There were no significant differences in isolations of Corynebacterium species and streptococcus species between normal and clinically affected sheep. The mollicutes isolated from the genito-urinary tract of the sampled sheep included Mycoplasma mycoides mycoides large colony, Mycoplasma species Group 7, Mycoplasma capricolum, Mycoplasma capri, Mycoplasma bovigenitalium, Mycoplasma agalactiae, Mycoplasma arginini, and unidentified Mycoplasma spp. and Acholepalsma laidlawii and Ureaplasma species. Mycoplasma was isolated from 49.3 % of 116 clinically normal sheep and 78.2 % of 104 affected sheep. There were significant differences in rates of isolation among clinical groups (p<0.05). Of all the mycoplasma isolates, Mycoplasma mycoides mycoides LC was isolated from 61.5 % of clinically diseased sheep while 6.0 % of the isolates were from apparently healthy animals (p<0.05). There was a significant association between the degree of the severity of the lesion and the rate of isolation of M mm LC (p<0.05). The extent of the isolation of M mm LC suggests a causal relationship with ulcerative balanitis and vulvitis in sheep in South Africa. The present findings, together with those of Trichard et al, (1993), showed that M mm LC is the major pathogenic mycoplasma incriminated in ulcerative balanitis and vulvitis in sheep in South Africa. However, results also point towards other pathogens such as A. pyogenes playing a role in the pathogenesis of the disease and predisposing the genital tract to infection with mycoplasma. A number of other identified and unidentified strains of mycolasma were isolated from both clinically affected and healthy sheep. However, in genital tract infection is uncertain. Virus isolation efforts in cell culture and Chlamydophila antigen capture ELISA yielded negative results in affected rams and ewes, and healthy sheep. Current clinical observations during this project have shown that the typical ulceration appears to be confined to the glans penis and lips of the vulva and no ulceration has been observed on the shaft of the penis and vaginal vestibule. In uncomplicated cases inflammation of the prepuce and vaginal vestibule is not a regular feature of the disease. Therefore, the name ulcerative balanitis and vulvitis most accurately describe the clinical signs of the disease in South Africa. Age-related susceptibility to the disease was observed. Young sheep are 2.5 times more likely to have the lesion than adult sheep (p<0.05). It was also observed that male sheep acquire severe lesions more often than female sheep. Since, infection in both male and female sheep can spread by coitus, it may valuable to attempt detection of infected animals and follow a strict isolation policy. In addition, further studies to elucidate predisposing factors related to management and environment are required. The in vitro activities of enrofloxacin, florfenicol, oxytetracycline and spiramycin were determined against field isolates of MmmLC by means of the broth microdilution technique. The minimum inhibitory concentrations (MIC) of these antimicrobial drugs were determined for a representative number of 10 isolates and one type strain. The susceptibility of A. pyogenes to enrofloxacin, oxytetracycline and tilmicosin was determined by means of agar disk diffusion test. The MICs of enrofloxacin, florfenicol, oxytetracycline and spiramycin were within the ranges of 0.125-0.5, 1.0-2.0, 2.0-4.0 and 4.0-8.0 µg mℓ-1, respectively. This study has shown that resistance of MmmLC against enrofloxacin, florfenicol, oxytetracycline and spiramycin was negligible. All tested field strains of A. pyogenes were susceptible to enrofloxacin, oxytetracycline and tilmicosin with mean inhibition zones of 30.6, 42.3&35.8 mm, respectively. Although, there is a lack of data on in vivo efficacy and in vitro MIC breakpoints of these antimicrobial drugs for MmmLC, the MIC results indicate that these 4 classes of antimicrobial drugs should be effective in the treatment of ulcerative balanitis and vulvitis in sheep in South Africa.