Correspondence: Aruna Mittal, Institute of Pathology-ICMR, Safdarjang Hospital Campus, New Delhi 110 029, India. Tel.: +91 011 26198 402/05; fax: +91 011 26198 401; e-mail: firstname.lastname@example.org
The regulation of immune response and chlamydial infectious load in the cervix of human females is largely unknown. Infectious load in terms of inclusion-forming units (IFUs) was determined by quantitative cultures in Chlamydia-positive women, in asymptomatic women, women with mucopurulent cervicitis (MPC) and women with fertility disorders (FD). CD4+, CD8+, CD14+ cells, myeloid and plasmacytoid dendritic cells (mDCs and pDCs) in the cervix were quantified by flow cytometry. Cervical cytokines, levels of β-estradiol and C-reactive protein (CRP) in serum and cervical immunoglobulin A antibody to chlamydial major outer membrane protein antigen, chlamydial heat shock protein 60 and 10 antigens were measured by an enzyme-linked immunosorbent assay. In asymptomatic women, chlamydial load showed significant positive correlations with CD4, mDCs, interleukin-12 (IL-12) and IL-2; however, negative correlations were found with CD8 and IL-8 levels. In women with MPC, chlamydial IFUs correlated positively with CD8, pDC number, IL-8, CRP and interferon-γ (IFN-γ). In women with FD, chlamydial load showed a significant positive correlation with the pDC number, IL-10 and estradiol level and a negative correlation with CD4 and IFN-γ. Overall, these results suggest that the interplay between chlamydial infectious load and host immune responses may be the deciding factor for the clinical condition presented during Chlamydia trachomatis infection.
Chlamydia trachomatis is the leading cause of sexually transmitted bacterial infection (Morrison & Caldwell, 2002; Brunham & Rey-Ladino, 2005). In India, a high prevalence rate of genital chlamydial infection has been reported among symptomatic women, out of which up to 30% have fertility-related disorders such as multiple spontaneous abortions (MSA) or infertility (Gupta et al., 1994; George et al., 2003). Chlamydia trachomatis infections are often asymptomatic and if symptomatic, then they can be treated with antibiotics. However, persistent and recurring infections may lead to severe complications (Fraiz & Jones, 1988; Sherman et al., 1990; Stephens et al., 1998), of which, the utmost concerns are fertility disorders (FD) resulting from damage to uterine and tubal epithelium.
Immunity to chlamydial infection involves both humoral and cell-mediated immune responses (Cain & Rank, 1995; Cotter et al., 1995). Studies in both animal and human models have established that both cytokine-producing T cells and antibody-producing B cells (Bailey et al., 1995; Yang et al., 1999; Cohen et al., 2000; Morrison & Caldwell, 2002) are critically involved in the clearance of chlamydial infection and resistance to reinfection. Recruitment of inflammatory and antigen-presenting cells, such as macrophages and dendritic cells (DCs), to the site of infection with the subsequent release of proinflammatory cytokines appears to be crucial for innate resistance to Chlamydia and is thought to be dependent on initial chlamydial load, but the regulation of infectious load by host immune response or vice versa is not known.
Chlamydia trachomatis undergoes a common intracellular biphasic growth cycle (Fields & Hackstadt, 2002), which includes the elementary bodies, the infectious form and the reticulate bodies. All Chlamydia species can accomplish their entire biosynthesis, replication and differentiation within the cytoplasmic vacuole known as inclusion. The inclusion-forming unit counts (IFUs) in culture can be regarded as a surrogate for infectivity or transmissibility and can help in showing the regulation of chlamydial replication by host factors.
Previous studies have shown the association of chlamydial IFUs with patient characteristics such as age, sex, infecting serovar and race (Workowaski et al., 1992; Arno et al., 1994; Eckert et al., 2000; Geisler et al., 2001), and another study by Brunham et al. (1983) had correlated the immune response to chlamydial infection with quantitative recovery of the pathogen from the cervix, but none of these studies have shown regulation of infectious load by different arms of immune response. In view of these facts, this study was designed to determine the association between infectious load and immune correlates to gain a better understanding of infectious load regulation in the female genital tract.
Materials and methods
Study population and patient classification
After obtaining informed written consent, 1421 patients attending the gynecology outpatient department, Safdarjung Hospital, New Delhi, India, were enrolled for the study. One hundred and twenty-seven asymptomatic women attending the family planning department for birth control measures and testing positive for C. trachomatis were also enrolled. The study received approval from the hospital's ethics review committee. The procedures followed for sample collection were in accordance with the ethical standards for human experimentation established by the Declaration of Helsinki of 1975 (revised in 1983).
At recruitment, a detailed clinical questionnaire was administered to each patient to collect information on the reasons for referral, gynecology history, including menstruation, symptoms of genital and urinary tract infection, obstetric and medical histories.
The Chlamydia-positive patients included in the study comprised of (a) Chlamydia-positive fertile asymptomatic women attending a family planning clinic, (b) Chlamydia-positive fertile women with mucopurulent cervicitis (MPC) [thick discharge and inflammation with number of polymorphonuclear leukocytes (PMNLs)>30] and (c) Chlamydia-positive women with FD. Fertile women (asymptomatic women and women with MPC) were those having the last childbirth within the last 4 months to 1 year. Women with fertility-related disorders included those with infertility and MSA. Infertile women were identified as those who lacked recognized conception after 1.5–2 years of regular intercourse without the use of contraception. The infertile group included women with referred diagnostic laparoscopy (Reddy et al., 2004) and women with unknown reasons. Women with MSA (>2) have been described as those having delivery of a previable fetus before the 20th week of gestation.
Patients taking oral contraceptives, having a positive urine pregnancy test, recent antibiotic therapy, a history of recently treated sexually transmitted infection other than Chlamydia and genital tuberculosis were excluded from the study. Further, women with male factor-related infertility, endometriosis or any other factors such as hormonal imbalances, which could be considered as a cause for infertility, were also excluded. Patients with overlapping group characteristics were also excluded to keep the groups well segregated.
Collection of samples
Cervical samples were collected during midcycle (median 13 days, range 9th–15th day of the menstrual cycle) to avoid variations in sex hormone levels. None of the patients had sexual intercourse 3 days or more before collection of the sample.
After cleaning the exocervix, endocervical swabs (HiMedia, Mumbai, India) were collected for the diagnosis of C. trachomatis and other sexually transmitted disease (STD) pathogens. For cell culture, endocervical swabs were collected in 1 mL of SPG (sucrose, 75 g; KH2PO4, 0.52 g; Na2HPO4, 1.22 g; glutamic acid, 0.72 g; water, 1 L; and pH 7.4–7.6) transport media. Cervical washes for determination of cervical cytokines and cervical cells were collected as described (Agrawal et al., 2007). Samples were not collected from patients with a friable cervix to avoid contact bleeding and to ensure collection of cervical lymphocytes only. Two milliliters of nonheparinized blood for separating serum was also collected. Samples were then stored at 4 °C until transported to the laboratory, and were then processed within 1 h. Swabs for cell culture were frozen immediately in dry ice and stored in a −70 °C freezer.
Microbiology and genotyping
The presence of chlamydial infection was determined as mentioned previously (Agrawal et al., 2007). Gram-stained cervical smears were examined for the presence of yeast cells (candidiasis), and quantification of PMNs per high-powered field was carried out in smears to ensure the presence of MPC. Vaginal smears were analyzed for clue cells, for the diagnosis of bacterial vaginosis. Wet mount microscopy was performed for the diagnosis of Trichomonas vaginalis. Neisseria gonorrhoeae, Mycoplasma hominis and Ureaplasma urealyticum were detected by culturing as described previously (Agrawal et al., 2007). To determine the infecting serovar, genotyping was performed as mentioned previously (Singh et al., 2003).
Quantitative cultures were performed as described previously (Brunham et al., 1983), with modifications (Mittal et al., 1993). Cervical specimens in SPG transport media were vortexed and a 0.1-mL portion was inoculated to a McCoy cell monolayer (grown on coverslips) in each of two wells of a 24-well cell culture plate. In addition, two culture wells were inoculated with 0.1 mL each of specimen for passage to three new culture wells each. After incubation for 48 h at 35 °C and 5% CO2, chlamydial inclusions were detected with a genus-specific antichlamydial lipopolysaccharide monoclonal antibody conjugated with fluorescence. The number of inclusions was determined either by counting all inclusions in each well (if <100 IFUs) or by averaging the number of inclusions in each of the three fields and multiplying that value by the number of fields per well. The average number of inclusions per well was multiplied by 10 to yield the number of IFUs mL−1 of transport medium. If no inclusions were seen in any field, the entire coverslip was scanned to assess the titer. If no inclusions were seen on scanning the entire coverslip, but inclusions did appear when the specimen was blindly passed, the specimen was defined as having <10 IFU mL−1.
Cervical cells were isolated from the cytobrush by vigorously rotating it against the sides of the transport tube after incubating the sample with 5 mM dl-dithiothreitol (Sigma, St. Louis, MO) at 37 °C for 15 min (to reduce the mucus component of the sample). The cell suspension obtained was then filtered through a sterile 70-μm nylon cell strainer (BD Biosciences, San Diego) and centrifuged at 300 g for 10 min; the resultant pellet yielded endocervical cells. The viability of the cells was determined by a trypan blue exclusion assay.
Quantification of different cervical immune cells was performed by standard flow-cytometric technology. Cervical cells were stained with fluorescein isothiocyanate (FITC)-conjugated anti-CD4, -CD8 and -CD14 antibodies (Becton Dickinson, San Jose) for 25 min. For identification of DCs, the following monoclonal antibodies were used: FITC-conjugated lineage cocktail LIN-1 (anti-CD3, anti-CD14, anti-CD16, anti-CD19, anti-CD20 and anti-CD56), CD11c and CD123–phycoerythrin, and HLA-DR-peridin chlorophyll protein (BD Biosciences). Preparations were washed with stain buffer containing 0.1% NaN3 and 2% fetal bovine serum. A total of 10 000 events were acquired for identification of T cells and monocytes and 50 000 events for identification of DCs. Appropriate isotype-matched control antibodies were also used to rule out nonspecific fluorescence. For DC identification, background fluorescence and the presence of lymphocytes were minimized by introduction of an acquisition gate on the forward-scatter vs. the side-scatter profile, which included most of the monocytic and DC fractions and gave reliable differentiation of these cells from epithelial cells, lymphocytes and cell debris. Samples were acquired using a fluorescence-activated cell sorting calibur cytometer and analyzed using cellquest software (Becton Dickinson). DCs were identified as a lineage FITC cocktail-negative and HLA-DR-positive population.
Quantification of cytokines in cervical washes
Quantification of interleukin-1β (IL-1β), IL-2, IL-6, IL-8, IL-10, IL-12 and interferon-γ (IFN-γ) was performed using commercially available enzyme-linked immunosorbent assay (ELISA) kits (eBiosciences, San Diego, CA), according to the manufacturer's instructions. Absorbance was read at 450 nm with a reference absorbance of 650 nm. A log–log standard curve was generated, and unknowns were interpolated. The sensitivities of the cytokine kits were 1 pg mL−1.
Sera of patients and controls were assayed for immunoglobulin G (IgG) antibodies to C. trachomatis surface components using a commercially available ELISA kit (Ridascreen, AG, Germany) according to the manufacturer's instructions. Results were obtained as the mean absorbance of duplicated samples at A450 nm. An OD>1.1 was considered as positive.
Cervical washes of patients and controls were assayed for the presence of IgA antibodies to synthetic peptides for cHSP60, 10 and major outer membrane protein (MOMP) antigen as described previously (Agrawal et al., 2007). Known positive and negative controls (cervical washes of known C. trachomatis-positive and -negative women) were always assayed in parallel to test samples.
Levels of β-estradiol in the sera of patients and controls were measured using commercially available ELISA kits (DRG International Inc.) as per the manufacturer's instructions.
The Kruskal–Wallis nonparametric test was used to compare continuous variables among multiple groups. The Mann–Whitney U-test was used for comparing two groups. Categorical variables were compared using the χ2 test. Correlations were tested with the Spearman correlation coefficient. The results were presented with a 95% confidence interval (CI), and a P value <0.05 was considered significant.
Cervical C. trachomatis infection was diagnosed by direct fluorescence assay/PCR in 480 patients. One thirty-four Chlamydia-positive women were excluded as the infecting serovar was the one other than serovar D, and involving many serovars in the study may influence the relationship of chlamydial inclusion-forming unit counts with clinical and immunological manifestations. Of the 346 women left, 18 patients who were coinfected either with Candida spp., T. vaginalis, M. hominis, U. urealyticum or N. gonorrhoeae or those having bacterial vaginosis were excluded from the study. Chlamydia-positive women were divided into three groups: (a) Chlamydia-positive fertile asymptomatic women attending a family planning clinic (n=127), (b) Chlamydia-positive fertile women with MPC (thick discharge and inflammation with number of PMNs>30) (n=86) and (c) Chlamydia-positive women with FD (n=108). None of the patients enrolled as asymptomatic or with MPC had any previous history of chlamydial infection as confirmed by their previous medical reports and the low titer of IgG antibodies against C. trachomatis in the sera of these women. However, women with FD had recurrent chlamydial infections as shown by their previous medical reports and high titers of IgG antibodies against C. trachomatis (Fig. 1). No significant difference in the median ages of the patients groups was observed (27, 26 and 28 years, respectively).
Relationship of the C. trachomatis inclusion-forming unit with clinical manifestations
The number of C. trachomatis IFU mL−1 was transformed to the base 10 logarithm, and results were expressed as <101, 102, 103, 104 or >104, respectively, if <10, 10–99, 100–999, 1000–9999 or >10 000 IFU mL−1 of transport medium were detected. Table 1 shows the distribution of chlamydial inclusion counts in women enrolled in the three groups. Chlamydia-positive fertile women showed significantly higher inclusion counts compared with women with FD, showing lower recovery of Chlamydia from the cervix of these women.
Table 1. Distribution of chlamydial inclusion counts in Chlamydia trachomatis-positive women
Chlamydia-positive asymptomatic women (n=127)
Chlamydia positive with MPC (n=86)
Chlamydia positive with FD (n=108)
Data are expressed as the percentage of women having IFU in a particular range.
IFUs, inclusion-forming units mL−1 of transport media.
Higher number of CD4+ T lymphocytes and CD14+ monocytes per 10 000 events were observed in the cervical mucosa of women with MPC as compared with the other two groups, but the difference was not significant (CD4 – 594 vs. 353 and 468; CD14 – 493 vs. 259 and 250 for women with MPC vs. asymptomatic women and women with FD, respectively). In contrast, the CD8+ T cell population in the cervix was found to be higher in the symptomatic group compared with the asymptomatic group, but the difference was again not significant (348, 385 and 404 for asymptomatic women, women with MPC and women with FD, respectively). A significantly positive correlation was observed between CD4 cell number and chlamydial recovery in all the groups (Table 2). CD8 cells, on the other hand, showed significant a negative correlation with chlamydial IFUs in asymptomatic women, and women with FD, but showed a positive correlation with chlamydial recovery in women with MPC (Table 2). The number of CD14 cells in the cervix showed a significantly positive correlation with chlamydial IFUs only in asymptomatic women and none of the other groups (Table 2).
Table 2. Correlation of inclusion-forming units (IFUs) with various immune factors
Asymptomatic Chlamydia positive (n=127)
Chlamydia positive with MPC (n=86)
Chlamydia positive with FD (n=115)
Data are Spearman's correlation coefficients obtained between various immune parameters and chlamydial IFUs.
The median and range of absolute numbers of mDCs and pDCs are given in Table 3. Asymptomatic women and women with FD have a significantly higher number of mDCs in their cervical samples as compared with women with MPC (P<0.01) (Table 3). The median number of pDCs was higher in both women with MPC and FD as compared with asymptomatic women (P<0.01) (Table 3), but the difference was significant only for women with FD. mDCs in asymptomatic women showed a significant correlation with chlamydial load (Table 2), whereas, in contrast, pDCs/sample positively correlated with chlamydial load in women with MPC and FD (Table 2).
Table 3. Immune factors studied in relation to Chlamydia trachomatis inclusion counts in women
Asymptomatic CT positive
Chlamydia positive with MPC
Chlamydia positive with FD
Data represent median values. Figures in parentheses denote range.
P<0.05 compared with the other groups.
P<0.05 compared with asymptomatic Chlamydia-positive women.
Concentration of cytokines in cervical washes and their correlation with chlamydial recovery
The median levels of IL-1β, IL-2, IL-6, IL-8, IL-10, IL-12 and IFN-γ in cervical washes of Chlamydia-positive women are shown in Table 3. Significantly higher levels of IL-10 and IFN-γ (P<0.05) were detected in women with FD compared with the other groups. IL-1β, IL-6 and IL-8 levels were significantly higher (P<0.05) in both women with MPC and FD compared with asymptomatic women. IL-12 levels were significantly (P<0.05) lower in women with MPC as compared with the other groups. IL-4 levels were below the detection limits in all the samples (Table 3). In asymptomatic women, IL-2, IL-12 and IFN-γ showed a significantly positive correlation, while IL-8 showed a significantly negative correlation with chlamydial load (Table 2). In women with MPC, IL-1β, IL-8, IL-10 and IFN-γ showed a significant correlation with chlamydial IFUs (Table 2); however, chlamydial load in women with FD showed a significantly positive correlation with IL-10 levels and negative correlations with IL-8 and IFN-γ levels (Table 3).
Cervical antibodies to C. trachomatis and heat shock proteins
Significantly high titers of chlamydial MOMP antibodies were detected in fertile women (data not shown) compared with women with FD, who have significantly high titers of cHSP60 and 10 antibodies (data not shown). Chlamydial MOMP IgA antibodies showed a significantly positive correlation with chlamydial load in all the groups. In contrast, cervical IgA antibodies to cHSP60 and cHSP10 showed a significantly negative correlation with chlamydial IFUs in women with FD (Table 2).
Correlation of infectious load with estrogen and C-reactive protein (CRP) levels
The median β-estradiol and CRP levels are given in Table 3. No significant difference in the estradiol levels was observed between different groups. The median CRP levels were found to be significantly higher in women with MPC and FD as compared with asymptomatic women. A significantly positive correlation was observed between chlamydial load and estradiol levels in both women with MPC and FD (Table 2). CRP, on the other hand, showed a significantly positive correlation with infectious load in women with MPC and a negative correlation in women with FD.
This is, to our knowledge, the first extensive study wherein the correlation of various immune factors with quantitative recovery of chlamydial load was assessed. We determined infectious load by the cell culture method, which, besides having the advantage of giving counts of viable infecting bacteria, also has its own disadvantages in that variables, including the technique and method of specimen collection, the rapidity of transport, the temperature and the methodology used for staining and reading plates, can never be held completely constant (Eckert et al., 2000). In view of these facts, we have attempted to utilize a standardized and uniform approach and kept the variables as constant as possible. Because the relationship of serovars with different clinical manifestations has been shown (Morréet al., 2000; Geisler et al., 2003; Gomes et al., 2006), we performed the correlative analysis in this study with one serovar (serovar D) only.
Our results showed that maximum numbers of women with MPC have a higher number of chlamydial IFUs whereas maximum women with FD have lower chlamydial IFUs. A previous study has also reported that in women with cervical mucopus, significantly higher IFU counts were seen (Hobson et al., 1980; Geisler et al., 2001). No trend was seen for chlamydial recovery in asymptomatic women, showing that during asymptomatic infection, chlamydial IFUs can be high or low, but the immune response is controlled such that no symptoms appear; however, in case of MPC, a persistently high chlamydial load may lead to increased secretion of inflammatory cytokines, thus resulting in acute inflammation. In case of women with FD, lower bacterial recovery and persistent chlamydial infection suggest that while the host immune defense may not be able to remove the bacteria from the cells completely there may, however, be an immunological impact on replication leading to lower replication or persistence, which causes continuous secretion of inflammatory cytokines, ultimately leading to disease sequelae such as infertility (Gomes et al., 2006).
As for immune cell population, CD4 cell numbers and chlamydial recovery were found to be significantly associated in all the groups. CD4+ T cells are identified as a population of cells required for the resolution of genital tract infection in experimental models of chlamydial infection (Su & Caldwell, 1995). Our data suggest that an increase in the number of infecting pathogen increases the proliferation of protective CD4+ T cells to ensure a protective immune response. CD8 cells, on the other hand, showed a significantly negative correlation with chlamydial IFUs both in asymptomatic women and in women with FD; however, a positive correlation was seen in women with MPC. This shows that during acute inflammation, the CD8 cell number increases with chlamydial IFUs. Although CD8 cells are not considered as important as CD4 cells in eliciting a protective immune response to chlamydial infection, some studies have shown otherwise (Matyszak & Gaston, 2004; Roan & Starnbach 2006). Our data suggest that CD8 cells may have a role in limiting the number of pathogens by destroying the epithelial cells harboring the bacteria or by curbing inflammation, but the exact mechanism involved is not clear and needs further elucidation.
mDCs in asymptomatic women showed a significant correlation with chlamydial load, which suggests that mDCs may play a more important role in inducing a protective immune response than cytokines or T-helper cells. In contrast, in women in whom chlamydial infection either causes high inflammation (MPC) or may be the cause of sequelae (FD), a significant correlation of chlamydial load with pDCs number was seen, suggesting that pDCs, unlike mDCs, are responsible for either sustaining the inflammatory response or in the persistence of Chlamydia, as shown by our previous study (Agrawal et al., 2008).
As for the cytokines in cervical secretions, significantly higher levels of IL-10 and IFN-γ were observed in women with FD compared with the other groups. IL-10 is not always an inflammatory/inhibitory cytokine; instead, higher levels of IL-10 probably prevent the pathological effect of inflammatory cytokines such as IL-1β, IFN-γ and tumor necrosis factor-α. These results show that higher secretion of IFN-γ for clearance of infection is counteracted by higher secretion of IL-10, resulting in incomplete removal of bacteria, thus causing persistence and disease outcome especially in women with FD. On the other hand, IL-1β, IL-6 and IL-8 levels were significantly higher in both symptomatic women (with MPC and FD) compared with asymptomatic women. The pathogenic roles of IL-1β, IL-8 and IL-6 have been shown by many studies (Li & Liang, 2000; Gerard et al., 2002). IL-12 levels were significantly lower in women with MPC as compared with the other groups, suggesting that during acute inflammation lower levels of IL-12 are secreted. A significantly positive correlation of IL-2, IL-12 and IFN-γ with chlamydial IFUs in asymptomatic women and a negative correlation of IL-8 show that the inflammatory response is kept at low levels, thus leading to an absence of symptoms. In women with MPC, IL-1β, IL-8, IL-10 and IFN-γ showed a significant correlation with chlamydial IFUs, showing that during inflammation protective cytokines are taken over by inflammatory cytokines and as the pathogen load increases, cytokines also increase. In contrast, chlamydial load in women with FD showed a significantly positive correlation with IL-10 levels and negative correlations with IL-8 and IFN-γ levels, showing that higher levels of inflammatory cytokines restrict chlamydial replication, leading to their persistence.
A significantly positive correlation was observed between chlamydial load and estradiol levels in women with MPC, suggesting that higher estradiol levels either help in persistence of chlamydial infection by modulating host immune responses or enhance the virulence of Chlamydia. In a previous study in humans, it has been shown that women are more susceptible to chlamydial infection under β-estradiol influence, because more chlamydial organisms can be isolated during the proliferative part of the cycle (Dimayuga et al., 2005).
Among cervical antibodies, chlamydial MOMP IgA antibodies showed a significantly positive correlation with chlamydial load both in asymptomatic women and in women with FD. In contrast, cervical IgA antibodies to cHSP60 and cHSP10 showed a significantly negative correlation to chlamydial IFUs in women with MPC, showing that as the chlamydial IFUs increase, the titers of MOMP IgA increase; however, in women with persistent chlamydial infection as in women with FD, as the recovery of Chlamydia from the cervix decreases, the titers of chlamydial heat shock protein 60 and 10 increase, showing that during recurrent infection more of chlamydial stress proteins are exposed. These data confirm our observation that women with FD have persistent infection with the bacteria expressing stress proteins.
In conclusion, we have evaluated the association of quantitative C. trachomatis cultures with various immune factors and clinical conditions. Overall, our results show that in women in whom infection occurs without any symptom, chlamydial IFUs correlated with protective immune response factors (CD4 T cells, CD14 cells, IL-2, IL-12, IFN-γ and anti-chlamydial MOMP antibodies). On the other hand, in women with no fertility complication, but high inflammation (women with MPC), inflammatory factors increased with the chlamydial load (IL-1β, IL-8 and IFN-γ). In contrast to these groups, in women with FD, it was observed that heightened responses of both pro- and anti-inflammatory factors stop chlamydial replication, causing it to persist. Thus, infectious load could prove to be an important marker for the type of host immune response and in turn could facilitate pathogenesis research.
We thank Mrs Madhu Badhwar, Mrs Asha Rani and Mrs Rosamma Thomas for providing technical assistance. This study was supported by an Indo-US grant (BT/IN/USCRHR/AM/2002) from the Department of Biotechnology, Government of India. University Grants Commission is acknowledged for providing assistance to T.A. in the form of a fellowship.