Shigatoxin producing Escherichia coli (STEC) have emerged as important pathogen of zoonotic importance. STECs are found to be associated with outbreaks and sporadic cases of diarrhoea, haemorrhagic colitis (HC), haemolytic uremic syndrome (HUS), acute renal failure and thrombotic thrombocytopenia (TTP) (Beutin et al., 2000). Several outbreaks as well as sporadic cases of these diseases have been reported from many countries including South-East Asia (Caprioli et al., 1994; Bonnet et al., 1998; WHO, 1998). Animals like cattle, sheep, goat may harbour STEC (Wallace et al., 1997) and act as source of human infection. Direct transmission of these pathogens from cattle to human has also been reported (Rice et al., 1996). The present study was undertaken to know the extent of occurrence of STEC among human and animals around Bareilly area of Uttar Pradesh, India.

Human stools from diarrhoeal cases and urine samples from urinary tract infection (UTI) cases were collected from Human Hospital at Indian Veterinary Research Institute, Izatnagar, District Hospital, Bareilly and Primary Health Centres. Faecal samples from diarrhoeal and apparently healthy cattle and buffaloes and from other animals were collected from Veterinary Clinics, Slaughter houses of Bareilly and Rampur Districts, IVRI Dairy Farm and Private Dairy Farms. The samples were collected aseptically in McCarteny bottles containing buffered peptone water and carried to the laboratory under chilled conditions.

Samples collected from human and animal sources were inoculated into Mc-Conkey broth and incubated for 12 h at 37°C. After enrichment inoculum from each tube was streaked onto McConkey solid agar plates and Sorbitol Mc-Conkey agar containing methyl umbelliferyl glucoronidase (SMAC-MUG) and further incubated at 37°C for 24 h. EMB plates were observed for greenish metallic sheen exhibiting colonies. SMAC-MUG plates were observed for non-sorbitol fermenter colonies and fluorescence under UV lamp for presumptive detection of O157 serotype. Cultural and biochemical characterization were performed as per Cruickshank (1969); Edwards and Ewing (1972) and Ratnam et al. (1988). The E. coli isolates were sent to Central Research Institute, Kasauli for serogrouping.

Each serotype (serogroup) was subjected to verotoxicity assay as per method of Konowalchuk et al. (1977) with minor modifications. The isolates were inoculated into the tubes containing 5 ml tryptic soy broth (TSB) and incubated at 37°C for 6 h and reinoculated into 20 ml TSB broth in a low speed shaking water bath at 37°C for 24 h. The cultures were centrifuged at 15000 g for 30 min in a refrigerated centrifuge (Remi). Cell free culture filtrates were obtained after filtration through 0.22 mm pore sized millipore membrane filters. The filtrates were checked for sterillity after streaking onto Brain Heart Infusion agar medium. Vero cell cytotoxicity assay (VCA) was performed in 96 well microtiter plates. Twenty microliter of cell free culture supernatants of each serogroup were inoculated in triplicates into the wells containing vero cell lines maintained in (GMEM) with 10% new born calf serum (NBCS). Cell free culture supernates of Shigella dysenteriae type-1 and one non Shigatoxin producing E. coli were also inoculated as positive and negative controls, respectively. Tissue culture plates were kept under 5% CO₂ tension at 37°C for four days and examined daily at 6 h intervals under inverted microscope to observe cytopathic effects (CPE).

Out of total 527 samples of human diarrhoeal stools, urine from UTI cases, faecal samples from cattle, buffaloes (diarrhoeal and asymptomatic) and from poultry, highest prevalence was reported among human diarrhoeal stools (25.4%), followed by cattle (7.5%) and buffalo (7.6%) and lowest occurrence was reported from poultry intestinal contents (5.2%). The serotyping of E. coli revealed that serotype O2, O9 and O100 were common to human stools and urine samples from UTI cases. These serotypes were also reported from faeces of apparently healthy cattle and buffaloes but not from diarrhoeal faeces (Table 1).

A total of 41 strains of E. coli belonging to different serogroups viz. O9 each from human diarrhoeal stools and UTI cases and O12, O8 and O3 recovered from cattle, buffaloes and poultry faeces, respectively were tested for VCA. Of these 5 (55.5%), 4 (44.4%), 10 (83.3%), 3 (37.5%) and 2 (66.6%), respectively, showed CPE on vero cell lines and were considered as STEC. Apparently healthy cattle showed higher rate of STEC (83.3%). However no significant difference was found among diarrhoeal cattle and buffaloes (Table 2). The actual prevalence may be more as intermittent shedding pattern of STEC at different farms and areas have been reported (Cobbold and Desmarchelier, 2000).

In densely populated cattle sheds the STEC harbouring cows have more chances to infect their offsprings. A closer contact of cattle with other animals, in rural areas with human beings, cattle dung, manure and use of cotaminated water can cause STEC infection among human beings. Hence, more surveys in rural areas are needed to detect the extent of STEC occurrence (Michel et al., 1999). Moreover adoption of intensive cattle feed management systems without adequate hygiene, dietary changes, immunosuppression and pH changes in rumen changing the microbial ecology can affect the shedding of STEC. The STEC group recovered in present study from animals have also been reported from human beings and vice-versa. The findings suggest that animals may act as source of infection and natural reservoir to human beings thus having zoonotic importance because these are associated with serious life threatening UTI, HC and HUS in human. Cattle beef should be considered as critical point to ensure food safety to consumers (WHO, 1998).

References