Comparative pathogenicity of Trichomonas vaginalis isolated from symptomatic and asymptomatic cases.R Bhatt, D Pandit, L Deodhar, R Bhise, DK Chatterjee
Dept of Microbiology, LTM Medical College, Sion, Bombay, Maharashtra.
Pathogenicity of 19 isolates of Trichomonas vaginalis obtained from vaginal specimens were studied in the murine model by intraperitoneal inoculation. 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 isolate through intraperitoneal route and the animals were sacrificed on the 10th 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 the pathogenicity level was observed among the isolates, a definite correlation between the clinical picture in the natural host and pathogenicity in mice was not observed.
Keywords: Animal, Biological Assay, Case-Control Studies, Disease Models, Animal, Female, Human, Mass Screening, Mice, Severity of Illness Index, Trichomonas Vaginitis, classification,epidemiology,parasitology,prevention &control,Trichomonas vaginalis, isolation &purification,pathogenicity,
Trichomonas vaginalis is a cosmopolitan parasite that infests male and female genitourinary tracts. In most men the infection being asymptomatic are generally considered to be the carriers of this vector, whereas in women, a wide range of clinical pictures ranging from acute to chronic or asyrriptornatic 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 their correlation with the clinical condition has been reported by several investigators using murine models,. The purpose of this study was to investigate the relative pathogenicity of strains of Tmaginalis recovered from symptomatic and asymptomatic cases.
High vaginal swabs were collected in the study group, which consisted of 500 female patients attending the gynaecologic out-patient department, with a history of vaginal discharge, sterility and in different gestational periods. One hundred healthy females attending the family planning clinic with no genital complaints represented the control group.
Modified AC medium supplemented with sterile 10% inactivated horse serum, 1-cysteine hydrochloride (0.12%), amphotericin B (25 ug/ml) and penicillin G (1000 ?/ml) was used for isolation and maintenance of T. vaginalis.
Out of a total 19 isolates studied for pathogenicity in the animal model, 16 isolates at random were recovered from the study group and 3 from the control group. Five isolates were from the patients having severe vaginitis, 9 from patients with a moderate vaginitis and five from patients with no 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 Evaginalis obtained from Hoechst India Ltd was used throughout for comparative studies.
Pathogenicity of the 19 isolates of T. vaginalis: Tmaginalis 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 a hemocytometer. The parasites were suspended in the fresh AC medium to achieve a cell count of 2 x 10 org/ml. Five mice were injected per isolate of Tmaginals and 5 mice injected with sterile AC medium represented the control set. The animals were inoculated intraperitoneally with 0.5 mi containing approximately 1 x 10 organisms 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 the 10th day post-infection.
The following parameters were recorded for each animal:
1. Microscopic observation: For the presence of motile trichomonads in the infected peritoneal fluid and in the purulent discharge form the infected lesions.
2. Cultural observation: A drop of infected peritoneal fluid was inoculated into fresh AC medium for the recovery of Tmaginalis.
3. Histopathology of the infected tissues using hematoxylin eosin stain.
Out of 500 patients screened for the presence of Tmaginalis, 85 showed the presence of Tmaginalis. Of these 85 isolates, 61 were recovered from the patients with vaginal discharge, 18 from females with different gestational periods and 6 from patients with a history of sterility. In the control group, Tmaginalis was recovered in 3 cases. The difference in the isolation rates in study and control groups was highly significant. (P < 0.001)
Sixteen strains from the study group and 3 strains from the 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 a bulging at the site of inoculation. The gross pathological picture observed in animals on autopsy was the presence of varying volumes of ascitic fluid (0.5ml to 2.5ml) 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 the study group showed mortality in mice. A 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 and 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 [Table - 1].
Death in 2 out of 5 mice was observed in one of the isolates recovered from the control group, whereas reference strain failed to show any mortality in mice, though on autopsy inflammation of liver and stomach adhesion with excessive volume of ascitic fluid was noted.
Several strains recovered from the 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 and 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 the 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 experimental animals for more than half a century. Various routes of inoculation - intraperitoneal, intravaginal, subcutaneous, intramuscular have 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 the study group whereas 33.3% strain recovered from the control group showed mortality in mice. Failure of the reference strain to show mortality in mice may be due to the prolonged maintenance of the strain in cell free med iUM4. 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 and lvey and Hall. These authors also showed variation in virulence through intraperitoneal route, but failed to correlate with the clinical condition in human being. Virtually the effect which these parasites have depends upon the inherent pathogenicity of these strains as well as on various factors.
Authors wish to thank Hoechst India Ltd. for providing the standard strain and animals required for the experiments.
[Table - 1]