Prevalence of vero toxic Escherichia coli in fecal samples of domestic as well as wild ruminants in Mathura districts and Kanpur zoo

Aim: The present study was planned to reveal the prevalence of verocytotoxigenic Escherichia coli (VTEC) in fecal samples of domestic and wild ruminants in Mathura district and Kanpur zoo. Materials and Methods: A total of 240 fecal samples comprising 60 each of cattle, buffalo, sheep and deer from Mathura districts and Kanpur zoo were screened for the presence of E. coli and VTEC genes positive by polymerase chain reaction (PCR). Result: Out of 240 fecal samples, 212 E. coli strains were obtained. All the E. coli isolates were screened by PCR to detect virulence genes stx1, stx2, eaeA and hlyA. Of these, 25 isolates were identified as VTEC. The prevalence of VTEC in cattle, buffalo, sheep and deer was found 13.4% (8/60), 13.4% (8/60), 6.67% (4/60) and 8.33% (5/60), respectively. Conclusion: stx1, stx2, eaeA and hlyA genes were prevalent in VTEC isolates from feces of cattle, buffalo, sheep and deer population of Mathura districts and Kanpur zoo. The presence of VTEC isolates in this region may pose a threat to public health.


Introduction
Diarrhea is one of the most common multifactorial diseases of man and animals mainly caused by Escherichia coli [1]. E. coli is the most common observed gastrointestinal flora of animals and environmental contaminant considered as important foodborne pathogen causing serious complications in man and animals [2][3][4][5].
Verocytotoxigenic E. coli (VTEC) was the first identified as a distinct group of E. coli named as VTEC, which had the ability to produce toxins with profound and irreversible effect on vero cells. VTEC is also termed as shiga-like toxin producing E. coli (SLTEC) or shiga toxin producing E. coli or STEC. Acronym STEC is derived from the fact that the toxins are shiga like that is similar to those produced by Shigella dysenteriae Type 1 [6].
The enterohemorragic E. coli belong to the VTEC. VTEC always do not induce clinical signs and are not enterohemorrhagic until addition virulence factor are present like enterohemolysin and adherence factors (intimin). The adherence factor(s) enables the organism to attach to and colonize intestinal mucosal cells [7]. Among VTEC, serotype O157:H7 has been closely associated with the sporadic and clinical outbreaks of hemorrhagic colitis (HC), hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP) in human beings [8][9][10]. Healthy domestic ruminants are recognised as the main natural reservoir of STEC and large game animal may be healthy carriers of STEC [11,12].
Keeping in view the importance of this organism, the present study was planned to reveal the prevalence of VTEC in fecal samples of domestic and wild ruminants in Mathura district and Kanpur zoo.

Ethical approval
This work does not require ethical approval because we have collected fecal samples of animals after defecation.

Sampling and isolation of E. coli
A total of 240 samples of feces (180 domestic ruminants 60 wild ruminants) were collected from Mathura district and Kanpur zoo. The samples were collected aseptically in UV sterile polythene bags Available at www.veterinaryworld.org/Vol.9/January-2016/13.pdf (Fisher Scientific, UK) and immediately transported to the laboratory under chilled conditions for microbiological analysis. For primary isolation of E. coli (VTEC), 10 g of fecal sample were enriched in 90 ml modified trypticase soya broth (mTSB) (Himedia, Mumbai) containing acriflavine (10 mg/ml) to reduce the growth of Gram-positive organism. The method used for collection of materials, and isolation and identification techniques were performed as suggested by the World organization for Animal Health [13]. These samples were incubated at 37°C for 6 h. MacConkey's agar was used as differential media while eosin methylene blue agar (Hi-Media, Mumbai) was used as selective media. Suspected E. coli strains were subjected to morphological, cultural and biochemical characterization as per standard methods [14]. A statistical analysis was done as per the standard method [15].

Molecular characterization
Multiplex polymerase chain reaction (pcr) was used for detection of virulent genes (stx 1 , stx 2 , eaeA, and hlyA) of VTEC. All the E. coli isolates were subjected to genomic DNA isolation. The bacterial growth in mTSB broth (HiMedia, Mumbai) was centrifuged at 3000 rpm for 15 min to make the pellet of bacterial cells. These cells were washed twice with phosphate-buffered saline (pH 7.4) to remove any impurity of broth media. Bacterial DNA was extracted by using DNA extraction kit (Genei, Bangalore) as per the manufacturer's protocol. For the PCR reaction, PCR Master Mix solution (Genei, Bangalore) was used. DNA amplification targeted to virulent genes (stx 1 , stx 2 , eaeA and hlyA) of VTEC was performed using primers on 3 μl of DNA sample in 25 μl reaction mixture [16]. After an initial denaturation step at 95°C for 4 min, 30 amplification cycles were performed, each consisting of 2 min at 94°C, 2 min at 65°C, and 1.5 min at 72°C and followed by a final extension step at 72°C for 2.5 min. After the amplification, amplicons were separated in 1.5% gel in tris acetate EDTA (TAE) buffer at 60 volt for 80 min, stained with 0.5% ethidium bromide solution and visualized under ultraviolet light.

Results and Discussion
Out of 240 fecal samples, a total of 212 E. coli strains were obtained (Table-1). All the strains of E. coli were screened to detect the presence of VTEC genes using PCR. An overall prevalence of VTEC in ruminants (both wild and domestic) was found to be 10.42% (25/240). The highest prevalence of VTEC was reported in cattle and buffalo 13.4% (8/60) in each followed by sheep 6.67% (4/60) and deer 08.33% (5/60). In cattle, 2 VTEC were found to be positive for stx 1 gene (180 bp) and 6 VTEC for stx 1 and stx 2 (180 bp and 255 bp). In buffalo, 3 VTEC were found positive for stx 1 gene and 5 was positive for stx 1 and stx 2 . In sheep, out of 4 VTEC, only one VTEC was having stx 1 gene and 3 VTEC isolates having stx 1 and stx 2 both. In wild ruminants (deer), out of 5 VTEC, only one was found to be positive for stx 1 gene, one isolates was found to be positive for stx 2 with hlyA (534 bp), one stx 1 with eaeA (384 bp) and two VTEC have stx 2 , eaeA and hlyA genes. Two E. coli strains were found having eaeA with hlyA genes, i.e., lacking stx gene and they may be enteropathogenic.
In the previous study, the prevalence of VTEC in sheep, cattle and buffalo were reported as 4.81% [17], 7.4% [18] and 8.9% [19], respectively. However, the prevalence of VTEC in higher level was reported by previous workers as the prevalence of VTEC was 16.66% [20], 18% [21] and 18.47% [22]. In contrast, investigations have shown a higher detection rate of 46% [23] in fecal samples of cattle and buffalo. Lower isolation rate, i.e. 9% [24]. Isolation rate as low as 1.0% has also been reported [25]. In deer previously reported, the prevalence of VTEC 9.3% [26], 16.2% [27] and16.5% [28], which likely similar as present finding. These hazardous strains of E. coli have been given immense attention due to their involvement in serious illnesses like HC (bloody diarrhea, HC), HUS and TTP in human [29][30][31]. The low infective dose, unusual acid tolerance and close association with ruminants have made VTEC a serious global zoonotic problem of great public health significance. VTEC can be present in the intestinal tract of a wide range of domestic and wild animals and ruminants (sheep, goats, cattle, buffalo and deer) [32][33][34][35][36], especially cattle and buffalo are considered as a major reservoir for VTEC [37,38].

Conclusion
The presence of VTEC in feces causes fecal contamination of water, and also contaminates other food sources, thus depicts a dangerous picture regarding human and animal health safety because water is essential for the survival of every living being. Constant monitoring and surveillances program to keep a record of the prevalence from time to time is needed, and proper hygienic measure may reduce the chance of infection.

Authors' Contributions
UJ, BB and AKV designed and planned this research work. RPM collected the samples and executed the isolation and biochemical work. UJ monitored the isolation, biochemical characterization. RPM and UJ was involved in the molecular characterization experiment. Manuscript was drafted and revised by AK, BB & RPM under the guidance of UJ. All authors read and approved the final manuscript.