RESEARCH ARTICLE PUJVol. 5, No. 2, 2012ISSN: 0258-3216

Personal non-commercial use only. PUJ copyright © 2011. All rights reserved PUJ; 2012, 5(2): 175-188

Biological and Biochemical Studies for Characterization of Some Egyptian Trichomonas vaginalis IsolatesLaila M. Boulos1, Mona M. El-Temsahy1, Safia M. Aly1, El Sayed I. El- Agamy2, Eglal I. Amer1

Departments of Parasitology1 and Dairy Science2, Faculties of Medicine1 and Agriculture2, Alexandria University, Alexandria, Egypt

Received: May, 2012 Accepted: September, 2012

ABSTRACT Background: Trichomoniasis has important medical, social and economical implications regarding its serious potentially associated complications, with the possibility of HIV acquisition and transmission. T. vaginalis is a very complex organism. Studying the variation in some biological and biochemical properties of the parasite can be used for characterization of the parasites.Objective: For the characterization of T. vaginalis infecting Egyptian female patients, the growth kinetics of 20 isolates, their pathogenicity, metronidazole susceptibility and electrophoretic protein patterns, were correlated with the recorded clinical manifestations associated with these isolates. Methodology: Positive samples for T. vaginalis were cultured on modified Diamond`s medium. For growth pattern study, trophozoites were counted for each isolate every 24 hours for seven days. The pathogenicity assay was performed using intra-peritoneal inoculation in mice. The isolates susceptibility to different concentrations of metronidazole was recorded by determining the minimal lethal concentration (MLC) and trophozoites viability. The biochemical variability of the studied isolates was performed using 2-dimensional electrophoresis. Results: A broad experimental variability was recorded among the 20 T. vaginalis isolates. There was a clear relationship between 3 isolates obtained from patients with severe vaginitis and the different parameters studied. These isolates had the highest number (20-25 organisms/HPF) in wet mount of vaginal discharge, and the shortest generation time (6:34-7:31 hours); they were also highly pathogenic to mice. Only one isolate (no. 5) proved to be metronidazole resistant. The use of the first dimensional native-polyacrylamide gel electrophoresis (Native-PAGE) demonstrated the presence of some differences. The isolates were classified into two groups according to their proteins net charge. All samples in each group were considered as one isolate. However, when the 2-dimensional electrophoresis (SDS-PAGE) was applied, five different groups could be identified according to proteins molecular weights. Conclusion: There is a broad experimental variability among the studied Egyptian T. vaginalis isolates regarding growth kinetics, metronidazole drug susceptibility, degree of pathogenicity, as well as the electrophoretic protein patterns.

Keywords: Trichomonas vaginalis, Metronidazole, Pathogenicity, Electrophoresis.Corresponding author: Eglal I. Amer, ehabaman2003@yahoo.com

INTRODUCTION

Trichomoniasis is the most common curable non-viral

sexually transmitted disease, and has important medical, social and economical implications(1,2). The disease presents a wide variety of clinical patterns in women ranging from the asymptomatic carrier state to flagrant vaginitis, with one third of asymptomatic infected

patients becoming symptomatic within six months. Serious complications are potentially associated with this disease, such as peri-natal complications, male and female genitourinary tract infections, and HIV acquisition and transmission(1,3).

Despite being a readily diagnosed and treated sexually transmitted disease, trichomoniasis is not a reportable

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infection and its control has received relatively little

emphasis from public health sexually transmitted diseases control programs(4).

The biological variability of T. vaginalis involves experimental pathogenicity and drug susceptibility. Both can be used for characterization of the parasite isolates. The pathogenicity has been assessed in various experimental animals, using various routes of inoculation such as intra-peritoneal, intramuscular, subcutaneous, intravaginal and intra-pleural(5,6). Concerning chemotherapy, metronidazole was found to be highly effective with an excellent cure rate. However, clinically resistant cases were reported(7,8).

Biological characteristics of T. vaginalis were assessed by various techniques such as bioassays(9), serotyping with antibody(10), the generation of electrophoretic analysis(11), isoenzyme profiles(12) and DNA-based techniques(13). The electrophoretic separation of proteins has become one of the main methods for fractionation and characterization of all types of proteins at both the analytical and preparative levels. Two dimensional polyacrylamide gel electrophoresis (2D-PAGE) is now specifically applied for the separation of proteins, including the first dimension according to their net charge, followed by separation in the second dimension according to their molecular weights(14).

Using the variation of some biological and biochemical properties for characterization of the parasites would be of great benefit for effective epidemiological assessment. Hence, the aim of this work was to study the growth pattern, pathogenicity, drug susceptibility and protein composition of different T. vaginalis isolates in correlation with the associated clinical manifestations.

METERIAL AND METHODS

Type of the study: Descriptive analytical study.

A total of 400 female patients in the child bearing period (18-45 years) attending Obstetric and Gynecology outpatient clinics at different hospitals in Alexandria were examined for T. vaginalis infection. During examination, clinical information including symptoms, signs and history of associated systemic diseases were recorded. Vaginal secretion of each patient was collected from the posterior fornix on sterile cotton tipped swabs. Each swab was then immersed in a test tube containing 2 ml sterile physiological saline. The specimens were transported to the laboratory within three hours after collection for wet mount examination(15). Positive samples were cultured in 10 ml of the prepared modified Diamond`s medium consisting of serum, peptone, yeast extract,

maltose, cysteine and ascorbic acid, supplemented with antibiotics (1000 unit of sodium penicillin G, 0.15 mg of streptomycin sulphate and 2 µg of the antifungal amphotericin B) per 1 ml culture medim(16).

Growth pattern study: Three culture tubes for each isolate, containing 10 ml medium/tube, were inoculated with 105 trophozoites (initial inoculum =104/ml). The culture medium of each tube was then dispersed in two sterile screw capped tubes (5 ml volume). The resultant six tubes of each isolate were incubated at 37°C. Trophozoites in 20 µl from each tube were counted using a hemocytometer chamber. The average counted number was multiplied by 104 to calculate the total number of trichomonads per ml. This procedure was repeated for each isolate every 24 hours for seven days(17). The length of the log phase was determined as the time required by the organism after inoculation to reach the maximum growth. Generation time (GT) defined as the time interval for one division was calculated as follows: GT=T Log2/(Log N–Log No), where: T= time elapsed in hours between initial inoculum and second counts, N= second counts, and No= initial inoculum (104/ml). The number of divisions was calculated as T/GT, and the division rate was calculated as 1/GT(17).

Pathogenicity assay: This was performed using intra-peritoneal inoculation of laboratory bred experimental mice(18). A group of ten female albino mice were injected with 107 trichononads of each isolate, and observed for a period of ten days. The following parameters were recorded: a) mortality rate; death before or at the 10th day post infection (pi), b) peritoneal fluid examination by direct microscopy followed by culture, and c) gross and histopathological examination of different organs (peritoneum, spleen, pancreas, stomach, diaphragmatic and visceral surfaces of liver), using hematoxoylin and eosin stain (H&E). The pathogenicity index (PI) of 100 points was calculated for each isolate according to the detected histopathological findings(18). Control group including ten female albino mice were injected intra-peritoneally with parasite-free sterile culture media. All mice were sacrificed ten days pi.

Drug susceptibility: This was assessed using metronidazole (Flagyl, Alexandria Pharm Ind.), in concentrations of 2, 5, 12, 25, 50 and 100 µl/ml. Each one of six culture tubes was inoculated with 104 trophozoites, with a final volume of 2 ml per tube. The different concentrations were added to each isolate and incubated at 37°C under aerobic conditions for 48 hours. Six control tubes (drug-free) for each isolate were incubated simultaneously under the same conditions(19). The drug susceptibility of the different isolates was recorded by

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determining the minimal lethal concentration (MLC), defined as the lowest drug concentration at which no motile organisms were observed. The viability was assessed by re-culture of the inocula previously exposed to the drug on drug-free media, followed by Fluorescein diacetate- Propidium iodide (FDA-PI) staining system(20,21).

Electrophoretic study: Serial subcultures of the parasite were done on modified Diamond`s culture, until no microscopical bacterial contamination was evident. Culture tubes of each isolate were centrifuged repeatedly at 500 × g for ten minutes at room temperature. The resultant pellet of each isolate was suspended in 3 ml PBS, and the parasites were counted and the optimal count for the electrophoretic study was adjusted to 5×107/ml. The pellet was subjected to sonication for two minutes followed by centrifugation at 100 × g for 30 minutes at room temperature. The supernatant containing the proteins was used for gel electrophoresis. An appropriate volume of each sample (containing an equal amount of protein) was mixed with an equal volume of sample buffer(22,23). Electrophoresis was done using 2-dimensional gel electrophoresis. The first dimension electrophoresis, Native-PAGE, was done as follows: prepared proteins were separated on 10% polyacrylamide gel electrophoresis in the absence of SDS and B-mercaptothanol, using the discontinuous buffer system(24). The analysis provided information about the number of separated peptide bands, their migration position, as well as the intensity of each band (band%). The SDS- PAGE was carried out using

reducing agents and the discontinuous buffer system using BIO-RAD mini protein II gel apparatus(25). In this technique, the separation of proteins depends mainly on their molecular weight and the peptide band appears as a spot. Molecular weights of proteins were determined by computerized analysis of SDS-PAGE gels using Gel-Pro documentation software.

Statistical analysis: Statistical analysis of the data was carried out using SPSS version 11.5 for windows. The studied parameters were compared between different T. vaginalis isolates using Student (Unpaired-sample) “t” test and differences were considered significant if the P values were less than 0.001. All values were expressed as means and standard deviation S.D.

Ethical considerations: All animal studies were approved by the ethics committee of Faculty of Medicine, Alexandria University, Egypt. All animal experiments complied with national regulations for animal experimentation. Informed consent was obtained from the patients before taking samples.

RESULTS

Out of 400 samples, 20 were positive for T. vaginalis by wet mount examination and culture on modified Diamond`s medium. Eleven correlated with the clinical manifestations of symptomatic patients (Table 1). Nine were from asymptomatic cases. The latter were ladies attending the Gynecological and family planning clinics either complaining of infertility or asking for the appropriate contraceptive method.

Table (1): The recorded clinical information for each isolate from symptomatic cases.

Isolate Symptoms Vaginitis

1 •Yellowish discharge • Mal odor Moderate2 •Discharge • Mal odor •Urine retension Mild

3 •Dysuria • Discharge •Abdominal colic Moderate•Burning sensation

4 •Dysuria • Discharge Mild

5 •Itching • Mal odor •Whitish discharge Severe

6 •Creamy discharge Mild7 •Itching • Yellowish discharge •Back pain Severe

8 •Dysparunia • Discharge Mild

9 •Itching • Back pain • Discharge Severe10 •Dysporania • Discharge Moderate

11 •Psychic patient • Bad odor Mild

Macroscopically: Mild vaginitis: Slight redness; Moderate vaginitis: Moderate hyperemia, Severe vaginitis: Severe hyperemia.

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Severe clinical manifestations correlated with high intensity of infection, i.e. patients with severe vaginitis had 20-25 organisms/HPF (isolates 5, 7 and 9). Those with moderate vaginitis showed 7-10 organisms/HPF (isolates 1, 3 and 10). In asymptomatic cases (isolates from 12 to 20) and those having mild vaginitis (isolates 2, 4, 6, 8 and 11), only 1-2 organisms/smear were observed.

The growth pattern of T. vaginalis trophozoites in culture started by a logarithmic growth phase (log phase) in which the organisms divided repeatedly till each isolate reached its peak number of parasites. This was followed by gradual decrease in their number until they reached the death phase where each isolate reached zero count (Table 2 and Figure 1). As regards the log phase, 9 isolates (4, 5, 7, 8, 9, 13, 14, 15 and 20) had the shortest log phase of 48 h. Isolates 6 and 11 had the longest log phase of 96

h, while the remaining 9 isolates had a log phase of 72 h (data not shown). Comparison of the peak number of isolates with shortest log phase, after 48 h, showed that the greatest yield was observed in isolate 5 (157.8±3.4), and the least was in isolate 11 (9.0±1.6).

Table (3) shows the comparison between division rate, number of divisions and generation time among T. vaginalis isolates. As regards the division rate, it was observed that isolate 5 had the highest division rate (0.152±0.001) while isolate 11 had the lowest one (0.051±0.002). Concerning the number of divisions, a statistically significant difference was recorded between isolate 5 and all other isolates (P< 0.001). The generation time was found to be highly variable among the studied isolates. The fastest growth was recorded in isolate 5 (6.34 h) and the slowest was in isolate 11 (19.33 h).

Table (2): Growth pattern of the T. vaginalis isolates (mean ± SD/ml X 104)

Isolates 24 h 48 h 72 h 96 h 120 h 144 h 168 hMean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD

1 6±1.5 69.6±3.3 129.2±3.1 46.8±2.6 5.6±3.1 0.0±0.0 0.0±0.0

2 11.1±3.3 31.6±2.8 74.5±3.9 12.3±2.6 2.33±2.0 0.0±0.0 0.0±0.0

3 3.8±0.7 39.6±3.0 61.0±3.7 18.6±3.9 6.1±3.9 1.6±1.8 0.0±0.0

4 5±2.8 60.1±3.9 27.3±3.5 3.5±2.4 0.0±0.0 0.0±0.0 0.0±0.0

5 15±3.2 157.8±3.4 44.8±3.9 9.6±1.2 0.0±0.0 0.0±0.0 0.0±0.0

6 2±1 9.0±2.0 25.0±3.6 47.3±2.6 17.0±2.3 6.8±3.1 1.0±0.89

7 8.1±2.7 119.8±3.5 43.6±1.2 4.3±3.7 0.0±0.0 0.0±0.0 0.0±0.0

8 7.8±2.9 29.8±3.0 16.1±1.4 6.1±3.8 1.6±2.0 0.0±0.0 0.0±0.0

9 8.1±2.4 83.6±3.1 33.0±4.0 7.5±3.8 0.0±0.0 0.0±0.0 0.0±0.0

10 5.8±1.4 57.0±3.5 93.3±4.0 34.0±3.7 29.1±3.0 12.1±2.6 2.1±1.8

11 2.6±1.2 9.0±1.6 14.6±3.0 30.3±3.2 19.8±2.3 4.5±4.0 1.0±0.8

12 2.6±2.0 16.5±3.7 35.3±3.7 11.3±4.0 4.0±1.7 1.5±1.8 0.0±0.0

13 2.6±2.0 77.3±2.3 55.3±3.5 26.5±3.7 7.5±2.7 1.8±1.9 0.0±0.0

14 5.5±2.0 45.6±3.6 26.3±3.6 4.8±3.0 1.8±1.8 0.0±0.0 0.0±0.0

15 7.5±2.6 59.8±3.6 28.1±1.9 12.1±3.6 4.1±3.0 0.6±1.0 0.0±0.0

16 8.8±3.3 16.6±3.0 40.0±1.4 17.6±3.9 8.0±1.5 3.6±1.2 0.6±0.8

17 3.3±0.5 47.8±3.2 98.0±2.5 34.5±2.9 13.5±2.2 5.6±1.0 0.0±0.0

18 3.0±2.1 25.3±3.2 98.8±3.7 45.8±3.5 13.8±33.0 2.8±3.3 0.3±0.8

19 5.1±0.7 16.1±3.4 49.0±2.8 16.0±4.0 2.5±1.7 0.0±0.0 0.0±0.0

20 7.5±2.6 104±3.7 51.1±3.0 8.1±2.9 0.8±0.9 0.0±0.0 0.0±0.0

F 13.446 925.501 578.625 121.487 6.423 21.111 5.952P <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001

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Table (3): Comparison between division rate, number of divisions and generation times among T. vaginalis isolates.

IsolatesDivision rate

(divisions/hour) Mean ± SD

Number of divisions (During log phase)

Mean ± SD

Generation time(Hour:minute)

Mean ± SD

1 0.097 ± 0.000 7.013 ± 0.036 10:16 ± 0:32 0.086 ± 0.001 6.217 ± 0.076 11:35 ± 0:93 0.082 ± 0.001 5.928 ± 0.088 12:9 ± 0:114 0.123 ± 0.002 5.908 ± 0.097 8:8 ± 0:85 0.152 ± 0.001 7.302 ± 0.031 6:34 ± 0:26 0.058 ± 0.001 5.563 ± 0.081 17:16 ± 0:157 0.144 ± 0.001 6.904 ± 0.043 6:57 ± 0:3

8 0.102 ± 0.003 4.892 ± 0.143 9:49 ± 0:17

9 0.133 ± 0.001 6.386 ± 0.054 7:31 ± 0:4

10 0.091 ± 0.001 6.543 ± 0.063 11:0 ± 0:6

11 0.051 ± 0.002 4.916 ± 0.155 19:33 ± 0:37

12 0.071 ± 0.002 5.136 ± 0.154 14:2 ± 0:2513 0.131 ± 0.001 6.272 ± 0.044 7:39 ± 0:314 0.115 ± 0.002 5.509 ± 0.113 8:43 ± 0:11

15 0.123 ± 0.002 5.901 ± 0.087 8:8 ± 0:7

16 0.074 ± 0.001 5.321 ± 0.051 13:32 ± 0:8

17 0.092 ± 0.001 6.614 ± 0.037 10:53 ± 0:4

18 0.092 ± 0.001 6.626 ± 0.054 10:52 ± 0:5

19 0.078 ± 0.001 5.613 ± 0.086 12:50 ± 0:12

20 0.140 ± 0.001 6.711 ± 0.051 7:9 ± 0:3

F 3511.7900 396.2895 1570.5900P <0.001 <0.001 <0.001

P significant ≤ 0.05 As regards pathogenicity, all isolates studied (20) were found to be pathogenic to mice, however the severity of infection varied from one isolate to another (Table 4). The highest PI was recorded in isolate 5 (76.6±12.49), and the lowest in isolate 17 (10.6±1.64). There was a highly statistically significant difference between isolate 5 and all other isolates (P<0.001). The mortality rate was 60% by the 3rd day pi in isolate 5, 30% by the 5th day pi in isolate 7, and 20% by the 8th day pi in two isolates (9 and 11). Only one isolate (16) out of those recovered from asymptomatic cases caused mortality in mice, where mortality rate of 20% was recorded by the 8th day pi (data not shown).

At autopsy, the gross pathological changes observed in mice were mainly ascitis and splenomegaly. Visceral and diaphragmatic surfaces of the liver showed no pathological changes. Ascitis of varying degrees was noticed as bulging at the inoculation site except in isolates 3, 13 and 17 that gave the lowest PI score (Table 4). The ascitic fluid was turbid, yellowish in color and its volume ranged from 0.1-1.5 ml. Its microscopic examination

showed motile T. vaginalis and leukocytes. Re-culture of the infected ascitic fluid was positive for T. vaginalis. Another gross lesion was the marked splenomegaly (2.2 x 0.6 cm) observed in isolate 5, as compared to the rest of the isolates and controls (0.9 x 0.3 cm) (data not shown).

Histopathological examination of H&E stained sections of the studied organs showed different pathological changes with variable degrees of damage. The liver and spleen were the organs mostly affected. Liver sections showed cellular hydropic degeneration, Kupffer cell hyperplasia, nuclear polymorphism with appearance of giant cells, portal lymphocytic infiltrate and marked proliferation and dilatation of blood vessels (Figures 2a-b). The examined spleen sections showed enlarged follicles with prominent germinal centers and hemorrhage in the red pulp with multiple prominent giant cells (Figures 2c-d). The peritoneum showed congestion, necrosis and non-specific inflammatory cell infiltration with the appearance of foamy histiocytes (Figure 2e). The pancreas showed congested blood vessels within

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Table (4): Pathogenecity index (PI) of the T. vaginalis isolates studied.Mortality Ascites Peritoneum Spleen Pancreas Stomach VS liver DS liver

IN % Score (0-5) (0-10) (0-5) (0-5) (0-5) (0-10) (0-10) PI

1 0 0 2.0.±0.47 1.9±0.56 2.0±0.81 2.0±0.0 2.0±0.0 3.6±0.69 5.4±0.96 18.9±1.562 0 0 2.0±0.81 2.3±0.82 1.9±0.73 2.0±0.0 1.3±0.48 2.4±0.84 3.0±0.0 14.9±0.89

3 0 0 0.0±0.0 1.8±0.78 2.3±0.67 0.0±0.0 1.0±0.0 3.0±0.0 3.0±0.94 11.1±1.32

4 0 0 3.3±0.67 4.0±0.0 1.3±0.48 1.6±1.17 2.0±0.66 4.5±0.52 4.5±0.52 21.2±1.675 60 40 5.0±0.0 7.4±0.51 4.0±0.0 3.0±0.0 2.0±0.66 6.8±0.63 8.4±0.69 76.6±12.496 0 0 0.5±0.52 3.0±0.00 2.0±0.0 0.0±0.0 0.0±0.0 4.0±0.0 3.9±1.1 13.4±1.777 30 30 5.0±0.0 4.9±0.56 3.3±0.67 2.1±1.19 3.0±0.0 5.2±0.42 6.4±1.34 59.9±9.2

8 0 0 3.0±0.0 1.6±0.96 1.7±0.48 0.9±0.73 1.2±0.42 4.0±0.0 4.8±0.91 17.2±1.649 20 15 5.0±0.0 6.0±0.0 3.1±1.10 3.0±0.0 2.0±0.0 5.6±0.51 6.5±0.7 46.2±4.0610 0 0 2.0±0.0 4.2±0.91 3.0±0.00 1.0±0.0 1.0±0.0 5.0±1.05 8.0±0.0 24.2±2.6311 20 15 2.0±0.47 4.0±0.0 2.3±0.67 0.0±0.0 0.5±0.52 5.0±0.66 7.0±0.0 35.8±4.85

12 0 0 3.7±0.48 4.2±1.13 4.0±0.0 0.0±0.0 0.8±0.63 4.0±0.0 5.0±0.0 21.7±2.07

13 0 0 0.0±0.0 2.0±0.00 2.2±0.78 1.4±0.51 1.0±0.0 2.0±0.0 3.9±0.87 12.5±1.28

14 0 0 2.0±0.0 1.5±0.70 2.8±1.03 0.0±0.0 2.0±0.0 2.2±0.78 3.0±0.0 13.5±1.14

15 0 0 2.0±0.0 2.0±0.00 2.0±0.0 1.1±0.73 1.0±0.0 2.3±0.82 3.0±0.0 13.4±0.93

16 20 15 1.4±0.69 3.0±0.81 2.2±0.78 0.0±0.0 1.8±0.42 2.0±0.0 3.9±0.87 29.3±4.7217 0 0 0.0±0.0 2.0±0.00 1.4±0.51 0.0±0.0 0.0±0.0 2.9±0.99 4.3±0.48 10.6±1.64

18 0 0 1.6±0.51 3.0±0.94 2.0±0.00 1.0±0.0 0.0±0.0 3.0±0.0 3.0±0.0 13.6±1.28

19 0 0 3.0±0.0 3.0±0.0 1.5±0.52 1.6±0.54 1.0±0.0 1.5±0.52 2.1±1.28 13.7±1.0

20 0 0 3.6±0.84 3.1±1.10 3.0±0.0 2.0±0.0 1.4±0.51 4.0±0.0 5.4±0.51 22.5±1.67F 146.5010P <0.001

IN: Isolate number; VS liver: Visceral surface of liver; DS liver: Diaphragmatic surface of liver.

the lobules, and lymphocytic infiltration with formation of lymphoid follicles. Stomach sections revealed focal erosion, and inflammatory infiltrate with mild nuclear polymorphism or atypia (Figure 2f). Mice injected

intra-peritoneally with parasite free sterile culture media showed no gross pathological lesions. Examination of H&E stained sections of their studied organs revealed absence of any histopathological changes.

Drug susceptibility results are presented in table (5). Isolate 5 was the only one that showed resistance, its MLC was 50µg/ml after 48 h incubation. All other isolates were susceptible with different MLC. The effect of the drug was irreversible, as susceptible treated isolates did not grow after re-culture. Using the FDA-PI staining system, viable trophozoites fluoresced intensely green, while dead ones fluoresced bright orange. Occasionally some organisms appeared black, and then they were seen to convert to green fluorescence during examination.

Using the first dimensional Native-PAGE, the protein pattern of all isolates showed some differences including the number of separated peptide bands on the gel, band migration positions, as well as the intensity of each band (band %) (Figure 3). According to these differences, the 20 isolates could be classified into two different groups.

Group Ι included 5 isolates (2, 4, 5, 14 and 20), while Group ΙΙ included the remaining isolates. Nine peptide bands were detected in all members of group Ι (Table 6); in group ΙI, 5 bands (Table 7) were different from those of group I. The difference in migration positions of peptide bands indicates that they are structurally different, since the faster the migration, the higher the charge density. It was also evident that the peptide bands had different band intensities.

Using the second dimensional SDS-PAGE (Table 8 and Figure 4), the protein patterns of isolates were compared. In group I, the 5 isolates showed three peptide spots on the gel with molecular weights 127, 149 and 152 kDa, indicating that they were identical. In group II, there were four distinguished pattern categories. The first pattern category (group ΙΙa) was present in isolates 6, 7, 9, 11 and 13 in which three peptide spots were noticed

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Table (6): Total laboratory analysis of Native - PAGE (Group I).

Band Isolates (Band%) No. Pos RF 2 4 5 14 201 14 0.051 5.29 6.22 5.73 4.56 3.422 30 0.110 3.33 5.05 5.80 5.25 3.563 36 0.132 4.14 6.77 7.81 5.23 3.684 52 0.191 3.18 4.12 5.85 8.44 8.205 70 0.257 3.92 3.38 5.20 4.62 4.826 102 0.375 9.90 14.01 9.61 9.31 10.857 114 0.419 10.48 10.45 11.83 10.38 12.118 244 0.897 38.85 28.17 29.20 31.21 30.859 259 0.952 20.91 20.80 20.00 21.00 22.01

Table (7): Total laboratory analysis of Native - PAGE (Group II).

Band Isolates (Band%)No. Pos RF 1 3 6 8 9 10 11 12 13 15 16 17 18 19

1 16 0.06 3.9 9.2 8.4 4.5 4.1 5.09 5.93 6.1 5.3 4.5 5.3 7.1 5.1 6.52 118 0.313 27 23.5 28.3 28.3 21.7 22.4 22.5 29.4 21.4 19.1 29.6 26.9 28.5 25.6

3 152 0.559 4.3 5.8 4.3 5.8 5.5 5.0 3.1 5.0 4.4 4.3 3.3 4.4 5.0 6.2

4 193 0.710 17.3 15.6 15.2 12.1 15.4 17.8 13.3 20 17 14.1 11.3 9.9 17.5 19.15 248 0.904 47.5 45.8 43.8 49.3 53.1 49.7 55.2 39.6 51.9 58 57.5 51.8 44.0 42.6

Pos: Position, RF: Relative frequencyTable (8): Total laboratory analysis of SDS-PAGE.

Isolate category Isolate number Number of peptide spots MW of peptide spots in kDa

First group (I) 2, 4, 5, 14, 20 3 127, 149, 152

Second group (II)IIa 6, 7, 9 , 11, 13 3 149, 152, 164IIb 1, 3, 10 5 145, 149, 150.4, 152, 157IIc 12, 15, 16 4 149, 150.6, 151.2, 152IId 8, 17, 18, 19 6 52, 93, 102, 109, 149, 152

* MW: Molecular weight

Pos: Position, RF: Relative frequency

on the gel with molecular weights estimated at 149, 152 and 164 kDa. The second category (group ΙΙb) included isolates 1, 3 and 10 in which five peptide spots were revealed with molecular weights 145, 149, 150.4,152 and 157 kDa. In the third category (group ΙΙc) which involved isolates 12, 15 and

16, four peptide spots were noticed with molecular weights 149, 150.6, 151.2 and 152 kDa. The fourth category (group ΙΙd) included isolates 8, 17, 18 and 19, in which six peptide spots were found, with molecular weights 52, 93, 102, 109, 149 and 152 kDa.

Isolate MLC after 24 hrs (µg/ml)

MLC after 48 hrs (µg/ml) Isolate MLC after 24 hrs

(µg/ml)MLC after 48 hrs

(µg/ml)1 25 5 11 12 22 5 2 12 25 53 50 12 13 50 124 12 2 14 5 25 100 50 15 12 56 12 5 16 5 27 50 25 17 25 128 5 2 18 5 29 12 5 19 25 1210 12 2 20 5 2

Table (5): Susceptibility of T. vaginalis clinical isolates to metronidazole

*MLC: Minimal lethal concentration

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Figure (1): Growth kinetics of the studied Trichomonas vaginalis isolates.a) Isolates 1-10.b) Isolates 11-20

Figure (2): Histopathological study of different organs.Liver section showing (a) hydropic degeneration and ballooning of hepatic cells (arrows) (H & E x400), (b) lymphocytic infiltration of portal tract (arrow) (H & E x400). Spleen section showing (c) enlarged follicles with prominent germinal center and haemorrhage in red pulp (arrows) (H & E x100), (d) multiple prominent giant cells (H & E x400). (e) Peritoneal section showing congestion, necrosis (arrow), and non-specific inflammatory cell infiltration (short arrow) (H & E x100). (f) Stomach section showing mild focal erosion, inflammatory infiltrate with mild nuclear atypia (arrow) (H & E x400).

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Figure (3): The first dimensional Native-PAGE of T. vaginalis isolates.

Figure (4): Peptide mapping by second dimensional SDS-PAGE of the different isolated groups. Protein spots pointed at by arrows in (a) Group I. (b) Group IIa. (c) Group IIb. (D) Group IIc. (e) Group IId.

1 2 3 4 5 6 7 8 9 10

11 12 13 14 15 16 17 18 19 20

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DISCUSSION

Effective epidemiological assessment of sexually-transmitted diseases such as trichomoniasis is achieved with tools that identify the causative agents and measure modes of transmission, prevalence, pathogenicity, virulence and drug resistance(26). In this study, a number of important biological and biochemical parameters were used. Preliminarily, we recorded a direct relationship between the severity of infection in the natural host and the number of organisms per high power field in the vaginal washouts; a fact formerly documented(27).

When we compared the growth kinetics of the 20 T. vaginalis isolates, using an initial inoculum of 104/ml, the log phase was between 48 and 96 h. Similar findings were reported(17), while Kulda et al.(28) using a higher initial inoculum of 7×105 reported that the fast growing strains of T. vaginalis reached maximum growth after 32 h, and 48-52 h for the slowly growing ones. The longer duration at which the maximum growth was reached in our study may be due to the smaller initial inoculum used. In confirmation, Abd El Ghaffar et al.(29) reported that when the starting inoculum was smaller, the peak growth was delayed. In our study as well as those of other researchers(17,30), there was a highly significant difference among all isolates after 24 to 168 h. Comparison of the peak number of organisms reached by the different isolates revealed that after 48 h, the highest yield was (157.81 × 104/ml) and the lowest was (9.0 × 104/ml).

While similar findings were reported in one study(17), results of other studies varied(31,32) probably due to the different growth characteristics of the isolates, as well as the effect of temperature of incubation (35°C), where lower temperature allows degradation of the nutrients in the medium hence more organisms develop and multiply(32). Variations could also be attributed to the different types of media used. Therefore, employment of this biological parameter for differentiation between isolates is only relevant within a particular research study.

Apparently, the parasite generation is indirectly related with its division rate. The fast growing isolates have the higher division rate. Our records of the generation time (GT), which varied between six h and 34 min for the fastest growing isolate and 19 h and 33 min for the slowest growing one, was similar to that obtained by other authors(17,28,33). However, Soliman and Aufy(34) categorized their isolates into slow, medium and fast growing with a GT of more than 11 h, between six and 11 h, and less than six hours respectively.

The pathogenicity of T. vaginalis, assessed by intra-peritoneal inoculation of experimental mice, presented

various manifestations such as mortality of mice, ascitis and pathological changes in various organs. All our clinical isolates produced infection in mice with a wide variability in the PI ranging from 10.6 to 76.6, which agreed with results obtained in other studies(6,35). We observed also that all isolates recovered from patients with severe vaginitis were highly pathogenic to mice (PI > 45). Similarly Tras and Koigas(36) found that strains with higher virulence in mice were mainly from patients with acute and subacute trichomoniasis. Several other studies, that also showed variations in virulence after intra-peritoneal inoculation in mice, did not correlate their findings with the clinical condition in the human host(6,19, 37). Five out of the 20 isolates in our study caused relatively severe peritonitis and death before the 10th day pi. These findings could be explained by the fact that the severity of an experimental infection reflects not only the inherent virulence of the inoculated isolate, but also the response of the individual animal, and the different inoculation routes used in mice(28,33,38). In confirmation, with the natural intra-vaginal route for inoculation in rats no histopathological and immunohistochemical changes

were recorded for symptomatic and asymptomatic isolates(38). The inherent virulence of Trichomonas appears to be related to CP30 (cysteine proteinase of 30 kDa) responsible for cyto-adherence to vaginal epithelial cells. A recent report(39) indicated that CP30, plays an important role in pathogenesis of trichomoniasis and proved that it was intensively higher in isolates from symptomatic as compared to asymptomatic women, indicating higher expression in the former. This probably applies to isolate (5) which was most pathogenic, as its PI was 76.6. Similar results were reported by several authors who found that the more resistant isolates were the highly pathogenic ones(15,35). In the present work, only isolate (5) was found to be resistant at MLC 50 µg/ml.

According to Narcisi and Secor(7), in an aerobic 48 hours assay Trichomonas isolates with MLC of 25 µg/ml and less were considered susceptible to metronidazole, while those with MLC of 50 µg/ml and greater were considered resistant. Variation in susceptibility was also reported in other studies(19,40,41). Investigation into metronidazole resistance in trichomoniasis showed that the microaerobic minimal inhibitory concentration (MIC) of a metronidazole susceptible isolate was around 3.2 µM while that of a clinically, highly metronidazole resistant isolate was 50-100 µM(42). Another study(43) indicated that clinical resistance to metronidazole did not necessitate the development of in vitro resistance, as only 115 out of 175 T. vaginalis isolates from women who had failed at least 2 courses of standard metronidazole

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therapy demonstrated metronidazole resistance in vitro. It was suggested that certain enzyme activities as thioreoloxim reductase and flavin reductase play a role in metronidazole resistance(44). We assessed trophozoites viability by FDA staining. Viable cells fluoresced intensely green, while dead cells stained bright orange with Propidium iodide with no definite internal structures. Organisms that appeared black in color, changed to green fluorescence when stored for some time, and were therefore considered viable. These cells probably did not intercalate Propidium iodide but metabolized the FDA overtime, as previously maintained by the observation that the estimation of cell viability can be done up to one week after staining with the FDA- PI stain(21).

Using two dimensional gel electrophoresis, we identified five different isolates, depending on the number of separated peptides and their corresponding molecular weights. The latter ranged from 52 to 164 kDa. Peptides with 149 and 152 kDa were common among all isolates, while each group had unique, specific peptide(s). Several authors observed an extensive commonality among T. vaginalis isolates, using either one dimensional SDS-PAGE or isoelectric focusing combined with SDS-PAGE(17,45-47). The heterogeneity among T. vaginalis protein composition regarding the number of peptides could be attributed to the use of different molecular techniques. This suggestion is supported by Gao and Tam(48) who analyzed soluble antigens of one T. vaginalis isolate by different techniques (SDS-PAGE, immunoblotting and two dimensional gel electrophoresis). They found a total of 26 distinct protein bands; nine were main bands, eight with molecular weights 15-62 kDa, and one with molecular weight 97 kDa. By immunoblotting, the specific anti-T. vaginalis antibodies raised in rabbit recognized 24 proteins with eight main bands. The researchers’ two-dimensional gel electrophoresis revealed up to 43 individual trichomonad spots, among which nine were the main ones. The molecular weights of these spots ranged from 27 to 100 kDa. Using two- dimensional gel electrophoresis, De Jesus et al.(49) identified proteins in a fresh clinical isolate of T. vaginalis that were classified into sixteen groups. In our study there was no definite relationship between the electrophoretic patterns of T. vaginalis isolates and their associated clinical picture in the natural host since groups (I), (IIa), and (IId) included both isolates from symptomatic and asymptomatic patients. Whereas group (IIb) included only isolates from symptomatic patients with moderate vaginitis, and group (IIc) included only asymptomatic isolates. Similarly, other studies(50,51) did not find any concordance between

random amplified polymorphic DNA (RAPD) and the clinical picture. On the other hand, other studies(40,52) reported that RAPD data correlated with the clinical picture, as patterns of asymptomatic isolates had a distinct form different from that of symptomatic isolates. While Cuervo et al.(11) reported that of the 29 protein spots differentially expressed between the isolates, 19 were over-expressed in the isolate exhibiting high virulence phenotype.

In the present study, there was no relation between drug susceptibility and protein patterns among T. vaginalis isolates, as the only resistant isolate (no. 5) was found to be present among other susceptible ones in group I. This finding is supported by Dunne et al.(53), who reported that there was no obvious difference between the genotypes of metronidazole-susceptible and resistant reproductive lines. On the other hand, El-Okbi et al.(40) reported that the PCR pattern of metronidazole-resistant isolates was different from that of sensitive ones. Others studies that employed RAPD technique showed a correlation between genetic relatedness of strains and similarity in their susceptibility to metronidazole in vitro(50-52). While authors using restriction fragment length polymorphism (RFLP) analysis reported that there were no relations between T. vaginalis infection and metronidazole susceptibility and RFLP subtypes(54).

In the present work, a clear relationship among the isolates obtained from patients with severe vaginitis was observed. These isolates had the highest number of organisms / HPF in wet mount (20-25 /HPF), and were all categorized as fast growing isolates as they had a short generation time. They were also highly pathogenic to mice (PI > 45) and caused 20%-60% mortality. The only metronidazole resistant isolate was one of them. This relationship was not found and could not be applied among isolates obtained from patients with mild or moderate vaginitis as well as those obtained from asymptomatic patients. This indicates that clinical assessment is more or less subjective and that other host factors such as vaginal pH, hormonal level and resident vaginal flora may alter the host parasite relationship. Furthermore follow up of these cases is a must, since the organisms could change their behaviour in the host converting an asymptomatic case to a symptomatic one within six months.

Author contribution: LM Boulos, MM El-Temsahy designed the research. EI El-Agamy performed the electrophoretic study. EI Amer performed the experiment, literature search and wrote the manuscript. All authors revised the manuscript.

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دراسة الخواص ألبيولوجيه والكيميائية الحيوية لبعض فصائل المشعرة المهبلية المصرية المعزولة

ليلي موريس بولس1، مني محمد التمساحي1، صفيه محمود علي1،السيد ابراهيم العجمي2، اجالل ابراهيم عامر1

قسم الطفيليات الطبية، كليه الطب1، وقسم األلبان، كليه الزراعة، جامعة االسكندرية2

المقدمة: المشعرة المهبلية هي طفيل وحيد الخلية واسع االنتشار، وهى المسببة لمرض التريكو مونيازيس. هذا المرض له أهمية اجتماعية وطبية واقتصادية، وقد تنتج عنه مشاكل أثناء فتره الحمل وكذلك زيادة خطر العدوى بفيروس نقص المناعة. إن دراسة االختالف فى النشاط الحيوي الكيماوي بين العديد من الفصائل المعزولة لهذا الطفيل لها منفعة عظيمة فى التقدير الوبائي للمرضالمتسبب عنه، ومن ثم، تم دراسة التركيب البروتينى وعالقته ببعض الخواص الحيوية مثل القدرة على إحداث المرض وتأثره بالعقار.بين العالقة ودراسة المعزولة، المصرية المهبلية المشعرة فصائل من لعدد الكيماوى الحيوى النشاط دراسة البحث: من الهدف

المعايير السابق ذكرها واألعراض المرضية المسجلة لهذه الفصائل المعزولة. طرق البحث: يشتمل العديد من المواد الكيميائية والفئران التجريبية، فلدراسة نمط النمو لعشرين فصيلة من المشعرة المهبلية المصرية المعزولة، تم جمع الطفيل الخاص بكل فصيلة، وتم زرعه على مزرعة دايموند المعدلة وكذلك تم دراسة القدرة على إحداث المرض

باستعمال الحقن البروتينى. ثم تمت دراسة أنماط البروتينات الخاصة بالفصائل المعزولة باستخدام الفصل الكهربائى ثنائى األبعاد. نتائج البحث: قد أظهرت هذه الدراسة أن الفصائل المعزولة من مريضات بالتهابات مهبلية شديدة أسرع نمواً ولديهم أقصر وقت لتنشئة الجيل. كانت الفصائل المعزولة من المريضات ذوات االلتهابات المهبلية الشديدة هي األكثر قدرة على إحداث المرض في الفئران واألكثر فناًء لهم (20-60%). عند استخدام البعد األول للفصل الكهربائي، ظهرت بعض االختالفات، تم علي أثرها تصنيف الفصائل المعزولة إلى مجموعتين، وتم اعتبار فصائل كل مجموعة منهم كفصيلة واحدة. ولكن عندما تم تطبيق البعد الثاني للفصل الكهربائي تبين وجود خمس مجموعات مختلفة تتراوح أوزانهم الجزيئية من 52 إلى 164 وحدة جزيئية. وقد وجد أن البيبتيدات ذات

الوزن الجزيئي 149 و152 شائعة بين كل الفصائل المعزولة بينما كانت لكل مجموعة البيبتيدات الخاصة بها.االستنتاجات والخالصة: أسفرت نتائج البحث عن االستنتاجات اآلتية:

لعقار االستجابة النمو، أطوار ناحية من المعزولة المصرية المهبلية المشعرة فصائل بين واسعة تجريبية اختالفات وجود (1) المترونيدازول، درجة القدرة على إحداث المرض وكذلك أنماط البروتين المعزولة بالفصل الكهربائى.

(2) ال تعكس االختالفات التجريبية بين فصائل المشعرة المهبلية المعزولة سلوكها داخل العائل الطبيعي.هناك عالقة واضحة بينالفصائل أكبر عدد من التي تم دراستها حيث كان لهذه المعايير التهاب مهبلي شديد وبين المعزولة من مريضات ذوات الفصائل الطفيل بالفحص المجهرى األولى، ووقت تنشئة جيل قصير. وكذلك كانت األكثر قدرة على إحداث المرض فى الفئران وكانت الفصيلة

الوحيدة المقاومة لعقار المترونيدازول واحده منهم. (3) يعتبر الفصل الكهربائي ثنائي األبعاد أفضل من الفصل الكهربائي ذو البعد الواحد فى فصل وتصنيف خليط من البروتينات،

وكذلك فى تقدير الوزن الجزيئي لمادة غير معلومة.