Comparative pathogenicity of trichomonas vaginalis isolated from symptomatic & asymptomatic cases.R Bhatt, L Deodhar, D Pandit, R Bhise, DK Chatterjee
Department of Microbiology, L.T.M. Medical College, Sion, Mumbai.
Pathogenicity of 19 isolates of T.vaginalis obtained from vaginal specimens were studied in the murine model by intraperitoneal route. Sixteen isolates were recovered from the females with various clinical conditions and 3 isolates were from normal healthy females. Pathogenicity level of these isolates were studied by inoculating 5 mice per isolates through intraperitoneal route and the animals were sacrificed on tenth day post-inoculation. In general, all the isolates recovered produced infection in mice. On comparison with the reference strain obtained from Hoechst India Ltd., seven isolates recovered from symptomatic cases and one strain from healthy females produced severe infection in mice. Though variation in pathogenicity level was observed among the isolates, a definite correlation between clinical picture in natural host and pathogenicity in mice was not observed.
Keywords: Animal, Biological Assay, Disease Models, Animal, Female, Human, Mass Screening, Mice, Trichomonas Vaginitis, classification,epidemiology,parasitology,Trichomonas vaginalis, isolation &purification,pathogenicity,
Trichomonas vaginalis is a cosmopolitan parasite that infests male and female genitourinary tracts. In most man, the infection is asymptomatic and are generally considered to be the carriers of this vector, whereas in women, wide range of clinical picture ranging from acute to chronic or asymptomatic is observed.
The pathogenicity of trichomonad species can be assessed in various experimental animals, but mice have most frequently been used. Various routes - intraperitoneal, intramuscular, subcutaneous, intravaginal and intrapleural route of inoculation in mice has been studied to assess the pathogenicity of various trichomonad strains.
Strain to strain differences and its correlation with clinical condition has been reported by several investigations using murine model,. The purpose of this study was to investigate the relative pathogenicity of strains of T. vaginalis recovered from symptomatic and asymptomatic cases.
High vaginal swabs were collected from 500 female patients attending the gynaecologic Out Patient Department, with the history of vaginal discharge, sterility and females in different gestational period, represented the study group. 100 healthy females attending family planning clinic with no genital complaints, represented control group.
Modified AC medium supplemented with sterile 10% inactivated horse serum, L-cysteine hydrochloride (0.12%), amphotericin B (25 µg/ml) & penicillin G (1000 µ/ml) was used for isolation and maintenance of T. vaginalis.
Out of total 19 isolates studied for pathogenicity in animal model, 16 isolates at random were recovered from study group and 3 isolates were from control group. 5 isolates were from the patients having severe vaginitis, 9 isolates from patients with moderate vaginitis and 5 isolates without any clinical signs of infection.
Kasauli strain of female albino mice weighing 16-18 gms were used as the animal model. A known standard reference strain of T. vaginalis obtained from Hoechst India Ltd. was used throughout for comparative studies.
Pathogenicity of the 19 isolates of t. vaginalis
T. vaginalis strains in an exponential growth phase were used for this study. The cultures were centrifuged at 1500 rpm for 10 min and the cells in the pellet were counted in hemocytometer. The parasites were suspended in the fresh AC medium to achieve a cell count of 2x107 org/ml. 5 mice were injected per isolate of T. vaginalis and 5 mice injected with sterile AC medium represented the control set. The animals were inoculated intraperitoneally with 0.5 ml containing approximately 1x107 orgs and observed for a period of 10 days. The mice that died before 10th day post infection were autopsied, whereas the remaining animals were sacrificed on 10th day post infection.
The following parameters were recorded for each animal:
Microscopic observation: For the presence of motile trichomonads in the infected peritoneal fluid and in the purulent discharge from the infected lesions.
Cultural observation: A drop of infected peritoneal fluid was inoculated into fresh AC medium for the recovery of T. vaginalis.
Histopathology of the infected tissues using hematoxylin eosin method.
Out of 500 patients screened for the presence of T. vaginalis, 85 cases showed the presence of T. vaginalis. Of these 85 isolates, 61 isolates were recovered from the patients with vaginal discharge, 18 isolates from females with different gestational period and 6 isolates from patients with the history of sterility. In control group, T. vaginalis was recovered in 3 cases. Difference in the isolation rate in study and control group was highly significant (p<0.001).
16 strains from study group and 3 strains from control group were tested for pathogenicity through intraperitoneal route of inoculation. In general, all the isolates recovered from both the groups were found to be pathogenic for mice though the severity of infection varied from strain to strain.
The accumulation of peritoneal fluid was observed as bulging at the site of inoculation. The gross pathological picture observed in animals on autopsy was the presence of varying volume of ascitic fluid (0.5 ml to 2.5 ml) and in some cases, infection on various organs in contact with peritoneal fluid. The ascitic fluid recovered was yellowish-grey in colour which was typically fibrinous in nature and coagulated rapidly.
Few strains recovered from study group showed mortality in mice. 40% mortality (2 mice out of 5 inoculated) before or on 10th day post inoculation in isolate number – 144, 160, 181, 187 and 36A whereas strain no. 200 & 23S showed 20% mortality (death in 1 mice out of 5 inoculated). 57.1% (4 out of 7) of strains showing mortality were from patients with severe vaginitis, whereas 42.8% (3 out of 7) strains showing mortality were recovered from patients with moderate vaginitis see [table]
Death in 2 out of 5 mice was observed in one of the isolate recovered from control group, whereas reference strain failed to show any mortality in mice, though on autopsy showed inflammation of liver and stomach adhesion with excessive volume of ascitic fluid.
Several strains recovered from study group showed acute peritonitis with liver damage and frequent liver adhesion to the stomach with purulent lesion on it. Inflammation of intestine was also observed in mice inoculated with strain no.187 & 23S. The purulent discharge from the infected organs revealed plenty of trichomonads along with leucocytes.
The histopathologic section of liver showed vascular congestion, polymorphonuclear (PMN) infiltration, inflammation and surface dissemination. Histopathologic section of stomach revealed macrophages with necrosis, PMN infiltration and hyperemia whereas histopathologic section of infected intestine revealed PMN infiltration and inflammation.
Mice have been used as an experimental animals for more than half a century. Various routes of inoculation – intraperitoneal, intravaginal, subcutaneous, intramuscular has been established to assess the pathogenicity of trichomonas infection with varying degree of success.
In the intraperitoneal mouse assay, the pathogenicity of a given trichomonad strain was estimated by evaluation of the various pathologic manifestations such as volume of ascitic fluid, death of experimental animal or infection of the organs in contact with the ascitic fluid.
43.75% isolates of T.vaginalis recovered from study group whereas 33.3% strain recovered from control group showed mortality in mice. Failure of reference strain to show mortality in mice may be due to the prolonged maintainance of the strain in cell free medium as reported by Lindgen RD & Ivey MH (1964). In contrast the clinical strains were inoculated within 2 months of isolation.
57.14% isolates from patients with severe vaginitis and 42.85% isolates from patients with moderate vaginitis produced mortality in mice. 2 isolates recovered from severe vaginitis failed to show mortality. Similar findings are reported by Reardon et al (1961) & Ivey & Hall (1964). These authors also showed variation in virulence through intraperitoneal route, but failed to correlate with clinical condition in human being. Virtually the effect which these parasites have depend upon the inherent pathogenicity of these strains as well as on various factors.
Authors with to thank Hoehcst India Ltd. for providing the standard strain and animals required for the experiment purpose.
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