GRANULAR VULVOVAGINITIS ( GVV ) IN SHEEP EXPERIMENT ALLY INDUCED WITH MYCOPLASMA OVINE / CAPRINE SEROGROUP 11

Kumar D., P. P. Gupta, J. S. Rana, H. S. Banga: Granular Vulvovaginitis (GVV) in Sheep Experimentally Induced with Mycopiasma Ovine/Caprine Serogroup 11. Acta vet. Brno, 61, 1992: 241-249. To have insight into the pathogenicity of Mycoplasma ovine/caprine serogroup 11 for female genital system of sheep, 8 apparently healthy lambs were taken. Two ml of M. ovine/caprine serogroup 11 (2-D) culture containing 6 x 108 CFU (colony forming units) ml-l was inoculated into lightly scarified vulvovaginal canal of 7 lambs, while 2 ml of sterile mycoplasma broth was similarly inoculated in one lamb, which served as control. All the infected lambs exhibited swelling of vulva along with mucus discharge from next day post-inoculation (DPI), and this persisted up to 10-12 DPI. The experiment was of 70 d duration during which one lamb was sacrificed at every 10 d interval. The control lamb was also sacrificed on 70th d. Grossly, in the vulva and vagina, congestion, oedema and fine nodules were discernible by 10 DPI. Microscopically, all the infected lambs developed lymphocytic vulvitis, vaginitis, cervicitis, and endometritis, which aggravated with the increase in DPI. M. ovine/caprine serogroup 11 could be re-isolated from vaginal swabs of all the infected lambs up to 70th DPI. The findings reported here indicate that M. ovine/ /caprine serogroup 11 is pathogenic for female genital tract of sheep as it causes granular vulvovaginitis (GVV), and it is the first description of the pathologic features of GVV in sheep induced by this organism. Vulva, vagina, cervix, uterus, lymphoid follicles, GVV, sheep Mycoplasma ovine/caprine serogroup 11 was first isolated from an outbreak of granular vulvovaginitis (GVV) in sheep in Australia (Cottew et aI. 1974). Subsequently, this organism was isolated from spontaneous cases of GVV in sheep in India (Tiwana 1982). Despite its association with spontaneous cases of vulvovaginitis in sheep, its pathogenicity to female genital tract and its role in producing GVV has not been proven experimentally. Therefore, the present work was undertaken to assess its pathogenicity and to study the sequential pathological changes in. the female genital tract of sheep infected with this organism. Materials and Methods Experimental animals Eight female lambs aged 6-8 months were kept under observation for 1 week before starting the experiment. During this period, no mycoplasma organisms or any grossly visible vulvar/vaginal lesions were detected and all the animals were adjudged healthy.

Mycoplasma ovine/caprine serogroup 11 was first isolated from an outbreak of granular vulvovaginitis (GVV) in sheep in Australia (Cottew et aI. 1974).Subsequently, this organism was isolated from spontaneous cases of GVV in sheep in India (Tiwana 1982).Despite its association with spontaneous cases of vulvovaginitis in sheep, its pathogenicity to female genital tract and its role in producing GVV has not been proven experimentally.Therefore, the present work was undertaken to assess its pathogenicity and to study the sequential pathological changes in. the female genital tract of sheep infected with this organism.

Experimental animals
Eight female lambs aged 6-8 months were kept under observation for 1 week before starting the experiment.During this period, no mycoplasma organisms or any grossly visible vulvar/vaginal lesions were detected and all the animals were adjudged healthy.

Infectious agents
Forty eight hour old culture of M. ovine/caprine serogroup 11 (2-D) at 3rd passage level, containing 6 x lOG colony forming units (CFU) per ml was used for inoculation. :242 .Mode of infection Two ml of this culture was inoculated into the vaginal canal of 7 lambs which was lightly scarified using sterilized dental brush, while 2 ml of sterilized mycoplasma broth was inoculated into the •vaginal canal of a control lamb.
Experimental design "The experiment was continued for 70 d during which daily rectal temperature was rcc)rded as well as, all the lambs were observed for any gross change in the appearance of vulva and abnormal discharge, if any.Haematologica1 values of all lambs were estimated on 3rd, 6th and 10th day :post-inoculation (DPI) and subsequently, on every 10th d up to 70th DPI.The control animal •was also killed on 70th d of the experiment.The.genital tract and other organs of all the lambs •were examined grossly and microscopically.For microscopic examination, 5 I'm thick paraffin •sections were cut and stained with haematoxylin and eosin.The vaginal swabs were taken and --cultured on mycoplasma medium for Mycoplasma isolation by the method described by Banerjee •et al. (1979).

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Clinically, the appetite of all the lambs remained unaffected during the experiment.However, their body temperature showed a mild rise of 1_20 F up to 3-4 DPI.From 2nd DPI, the vulvar lips of all the infected lambs were slightly 'swollen and mild mucus discharge was observed from their vulva up to 10-12 DPI.Haematological analysis of the infected animals revealed a marked increase in the average total leucocyte count (TLC) from the pre-inoculation basal value of 6.34 X 10 3 /cmm to 9.01 X 10 3 /cmm and 9.74 X 103/cmm of blood on 3rdand 20th DPI, respectively.Thereafter, TLC started declining gradually but remained higher than the control (6.10 X 10 3 /cmm) as well as the pre-inoculation basal values (6.34 X 10 3 /cmm) till the end of the experiment.The differential leucocyte -count (DLC) showed a progressive neutrophilia on 3rd DPI followed by a gradual • lymphocytosis which peaked at 20th DPI.Other haematological components did not show any significant change.M. ovine/caprine serogroup 11 was re-isolated from the vaginal swabs of all the infected lambs up to 70th DPI.
Grossly, the mucosa of vulva and vagina of the lamb killed on lOth DPI was .congested,thickened and rough due to scattered fine nodules raising the mucosa.The vulvovaginal mucosa of the lambs killed on 20th and 30th DPI showed pin .head-sizedtranslucent nodules arranged in a linear fashion giving it a corrugated .appearance.The animals necropsied subsequently, showed similar but more severe changes in their vulva and vagina than those seen at earlier days.There was marked increase in the number and size of the nodules.Seventy days after infection, the GVV became very severe.The vaginal mucosa was completely -occupied by relatively large sized translucent nodules which were raising the mucosa.
Microscopically, all the infected lambs showed lymphocytic vulvitis, vaginitis, • •cervicitis, endometritis and oophoritis but their extent and severity increased with increasing period after infection.The sections of vulva, vagina and cervix -of the lamb killed on lOth DPI showed diffuse lymphocytic infiltration and lymphoid nodules in the sub-epithelial region of the mucosa (Figs. 1 & 2) as well •as in the tunica muscularis.At places, the epithelium over the lymphoid nodules was exfoliated.There was also perivascular plasma cells and lymphocytic cuffing in the sub-mucosa as well as in the tunica serosa of these organs.The superficial epithelial cells lining the mucous membrane showed hydropic degeneration.Mild lymphocytic infiltration was also noticed below the endometrial epithelium, .around the endometrial glands and also in the ovarian stroma.In the lamb killed

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• on 20th DPI, the microscopic changes in the vulva, vagina, cervix, uterus and ovary were similar but more pronounced than those seen on 10th DPI.The perivascular plasma cells and lymphocytic cuffing (Fig. 3) as well as hydropic degeneration in the mucosal epithelium of the vulva and vagina were more severe than those seen on 10th DPI.On 30th DPI, the cervix showed diffuse lymphocytic and plasma cells infiltration below the epitheliwn lining its lumen and in its muscle coat.The muscle coat of cervix also revealed intense perivascular lymphocytic cuffing (Fig. 4).The lymphoid nodules in the lamina propria of vulva and vagina increased in size and number (Fig. 5).The uterus and ovary showed almost similar changes as seen on 20th DPI.In the lambs killed on 40th and 50th DPI, the vulva and vagina revealed large lymphoid follicles in their lamina propria (Figure 6).At this stage, the cervix also showed formation of small lymphoid nodules besides diffuse lymphocytic infiltration below the epitheliwn lining the cervical canal (Fig. 7).The uterine changes were similar to those described earlier but the ovarian stroma also showed small lymphoid aggregates at a few places.On 60th DPI, the vulva and vagina showed increased nwnber of very large lymphoid follicles in the lamina propria (Fig. 8) and also in the tunica muscularis.At this stage, cervix also revealed large lymphoid follicles below the epitheliwn lining its lwnen.The uterus and ovary showed almost similar histopathological changes as seen earlier.The genitalia of the lamb killed on 70th DPI showed almost similar changes as seen on 60th DPI.The genitalia of the control animal killed on 70th d of the experiment did not show any gross or microscopic pathological change.Also no pathological change was detected in other organs of the control as well as the infected lambs.

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The clinical observations in the present study are in accordance with those reported by other workers in spontaneous cases of GVV in sheep due to ureaplasmas, Mycoplasma sp. and Acholeplasma laidlawii (Doig and Ruhnke 1977;Mc Caughey and Ball 1985) and in goats due to M. ovine!caprine serogroup 11 and A. oculi (Tiwana and Singh 1982).The findings of the present study are also in conformity with experimental GVV in sheep due to ureaplasmas (Doig and Ruhnke 1977;McCaughey and Ball 1985).The haematological finding of neutrophilic leucocytosis in the initial stages is in accordance with finding of Hartman et al. (1964) during experimental intrauterine inoculation of M. bovis in heifers.Neutrophilia occurs as a result of systemic response to tissue injury and acute inflammation due to mycoplasma invasion.This systemic response is mediated by interleukin-l (IL-l) which is released by macrophages following exposure to the stimulus of invading mycoplasma and also due to damaged or inflamed tissue.IL-l is chemotactic for neutrophils and acts directly on bone marrow to stimulate the release of neutrophils into the circulation, causing neutrophilia (Tizard 1987).After 3rd DPI, gradual increase in blood lymphocytes might be due to the fact that after completing their short life span, the neutrophils degenerate and release chemotactic factors which attract large number of lymphocytes from bone marrow.
The gross and microscopic lesions in the vulva, vagina, cervix and uterus were almost similar but different in severity to those observed by other workers in -experimentally induced GVV in sheep due to ureaplasmas (Doig and Ruhnke • 1977), and in goats due to M. agalactiae (Singh et al. 1975), A. laidlawii (Gupta et al. 1990) and A. oculi (Sharma et al. 1991).However, in the present study, lymphocytic infiltration was also seen in the ovarian stroma.
The hydropic degeneration of the epithelial cells lining the mucosa of vulva and vagina, observed in this study, indicates that M. ovine/caprine serogroup 11 causes damage to the epithelial cells.Mycoplasmas have special affinity for secretory epithelial surfaces, where they get intimately attached to sialic acid receptors and the receptors other than sialic acid present on host cells, thereby causing damage to host cells by various mechanisms (Razin 1978).
The infiltration of large number of lymphocytes and plasma cells in the epithelial, sub-epithelial, muscular and serosal layers and also around the blood vessels in the genital tract of the infected lambs indicates that strong cell-mediated respon-• ses are directed against the invading M. ovine/caprine serogroup 11 organisms.
M. ovine/caprine serogroup 11 was re-isolated from the vaginal swabs of the infected lambs and not from the blood, indicating that local infection with this organism remains confined to the genital tract and does not cause systemic reaction.Barile (1979) also stated that most Mycoplasma pathogens were not highly invasive, confined themselves toe pithelial surfaces and produced localized infections.
The observations made in the present study lead to the conclusion that M. "ovine/caprine serogroup 11 is pathogenic for female genital tract of sheep and it produces granular vulvovaginitis, lymphocytic cervicitis, endometritis and ,oophoritis.