RESEARCH Detection of Escherichia coli from the udder of the dairy farm buffaloes in Phagwara region, Punjab, India

Aim: To know the presence of Escherichia coli on the udder skin of the dairy farm buffaloes in the Phagwara region, Punjab, India. Materials and Methods: A total of 135 swabbed samples were collected randomly from the udder of buffaloes in ten dairy farms over the period of three months from August to October 2011 without concern to their breed with the prior approval of the farm owners. The sterilized cotton swabs were examined by Gram's staining for the morphology of the culture, culture characteristics was confirmed by growth on different media and by preforming the different biochemical tests like Indole production, Voges- Proskauer test, Urease Production, Nitrate Reduction, Methyl red and Presumptive test. Results: Out of 135 samples were examined, 23(17.03%) were positive for E. coli. Most Probable Number (MPN) results confirmed the one possibility of the bacteria from the contaminated water. Conclusion: The results of the present study suggest that E. coli isolates are present on the udder skin of the dairy farm buffaloes in the Phagwara region, pose a serious threat to the animal as well as consumer health. Thus, more hygienic preventive measures are required to inhibit the bacterial growth, so as to improve the health of the animals as well as the wholesomeness of the milk.


Introduction
mastitis may be due to poor hygienic conditions or intensive use of antimicrobials targeted against Gram Escherichia coli is one of the most intensively positives for mastitis control [5].Mastitis, studied living species.Escherichia coli is a normal inflammation of udder, results in Rs 7,165 crore part of the microbiota of the lower gastrointestinal annual losses to the dairy industry in India and Rs 503 tract of mammals, including humans, and usually exist crore in Punjab [6].The percentage distribution of as a harmless commensal.However, there also exist Gram-negative bacteria causing clinical mastitis is many pathogenic strains of E. coli that can cause a herd dependent, but studies in the United States and variety of diseases in both humans and animals [1].
Europe consistently report that approximately 40% of The mastitis which is caused by E. coli is commonly clinical cases are the result of Gram-negative bacteria called Environmental mastitis [2].This pathogen [7][8][9][10].infects the udder generally through the ducts In terms of milk contamination, the quality of papillaris, which is the only opening of the udder to the milk is determined by aspects of composition and outside world.In the buffalo species, mastitis is the hygiene.Due to its complex biochemical composition most costly disease even though buffaloes have been traditionally considered less susceptible to mastitis and high water activity milk serve as an excellent than cattle [3].
culture medium for the growth and multiplication of However, in comparison with cattle, buffaloes many kinds of microorganisms [11].Among all have some characteristics that may contribute to microorganisms E. coli is frequently contaminating organism and is reliable indicator of fecal pollution greater risk to mastitis such as more pendulous udder generally in insanitary conditions of milk [12].Two and longer teats [4].Higher incidence of E. coli cases of hemolytic uraemic syndrome have been Methylene Blue Agar (CDH Pvt.LTD.) and incubated reported which provide evidence that raw milk may be at 37°C for 24 hrs.The plates were observed for the a vehicle of transmission of E. coli O157: H7, both growth of E. coli.A single Colony was picked and sub affected person consumed raw milk [13].Recovery of cultured on Eosin Methylene Blue Agar for the E. coli from food is an indicative of possible presence purification of the isolate.Gram's staining technique of enteropathogenic and/or toxigenic micro-organism was used for the study of the morphology of the which could constitute a public health hazard.Enteroisolates.The culture characteristics were confirmed by pathogenic E. coli (EEC) can cause severe diarrhea inoculating the pure colonies on Endo DEV Agar and vomiting in infants and young children [14].
(Titan Biotech LTD.), Nutrient Agar (CDH Pvt.LTD), In raw milk samples 9, 20%, isolates of the E. Gelatin Agar (Titan Biotech LTD.) and Nutrient Broth coli were detected, whereas in case of mastitis infected (Titan Biotech LTD. Biochemical tests were buffaloes raw milk 30.12% isolates of the E. coli were performed to confirm the E. coli using Catalase test, detected [12,15,16].The detection of E. coli from the Simmons Citrate Agar (Titan Biotech LTD.), Starch udder indicates a possible contamination of the Agar (Titan Biotech LTD.), Indole production, Vogessurroundings like fecal matter in the soil, Proskauer test, Urease Production, Nitrate Reduction, contaminated milking equipment's, water uses for the Methyl red and Presumptive test [17,18].washing of the udder during milking.So by keeping

Results
these views in mind a study was carried out to detect the E. coli from the udder of the dairy farm buffaloes in The presence of the E. coli on the skin of the udder the Phagwara region, Punjab, India.
was confirmed by the growth pattern on different media and by the biochemical tests whose result were

Materials and Methods
given in the tables 1 and 2 respectively.

Collection of sample: A total of 135 swabbed
The results of the Most Probable Number (MPN) samples were collected randomly from the udder of showed in table-3, which shows the level of contamibuffaloes in ten dairy farms over the period of three nation in per hundred milliliters of water sample months from August to October 2011 without concern collected from the dairy farms [19].The water was to their breed with the prior approval of the farm used for drinking and washing of buffaloes udder, owners.The entire swab samples were collected as per cleaning the utensils (used for the storage of milk) was the guidelines of the International Animal Ethics same, hence there is always probability of contami-Committee.Animals were not used in this experiment nation of E. coli, was very high, which was already in anyway either experimental animals or control.The there in the water, which ultimately reached the skin of sterilized cotton swabs were swabbed onto the overall the udder and utensils through water.This activity outer skin surface area of the udder.Simultaneously brought the bacteria directly into the milk and made it 100ml water sample were also collected for the same unfit for human consumption.The results of the present dairy farms to check the MPN level (most probable study were summarized in the table-4.According to number).The samples were collected in the sterile these results the 23 samples out of 135 showed the sampling bottles that were kept in a cold box and were presence of E. coli (17.03%), although the percentage immediately shifted to the laboratory of Microbiology, was less, but normal flora of the udder restricts the Department of Biotechnology, Lovely Professional growth of E. coli on the udder skin [20].University, India.

Isolation and identification of the Escherichia coli: Each sample was inoculated on the Eosin
Escherichia coli is significant from the point of www.veterinaryworld.orgVeterinary World, Vol.5 No.9 September 2012 523  Biochemical reactions of Escherichia coli +ve view of public as well as animal health, as they have animals kept as individual holding at backyards been responsible for diarrheal disease in human and followed by small holdings in periurban area (42%) environmental mastitis disease in buffaloes [21,22].and the lowest at organized farms with reasonable There are about twelve strains associated with the good management conditions (32%) [28].bovine mastitis and five pathotypes are recognized for While environmental mastitis cannot be totally diarrheal in man [21,23].As the E. coli do not normally eliminated from a herd, the incidence can be held to a live on the skin or in the udder but which enter the teat minimum.The key elements in the control of mastitis canal when the buffaloes comes in contact with a include: sound husbandry practices and sanitation contaminated environment, contagious mastitis is [33].The result of the present study showed that E. transmitted from buffalo-cow to cow by pathogen for coli still present on the udder skin pose a serious threat which the udder is the primary reservoir; it tends to be to the animal as well as consumer health.Thus, more sub-clinical in nature [2].Subclinical mastitis was hygienic preventive measures are required to inhibit found more important in India (varying from 10-50% the bacterial growth, so as to improve the health of the in cows and 5-20% in buffaloes) than clinical mastitis animals as well as the wholesomeness of the milk.
(1-10%) [24].Common contagious pathogens have Training and guidance programs should be started in been reported to infect 7 to 40% of all buffaloes-cow order to develop awareness among farmers emphasizing [25].Frequency of contagious pathogens among mastitis the need for hygienic practice at farm level, as well as cases is greater [26].farm environment should be improved in order to In Faisalabad, 200 milk samples were collected decrease the breeding ground for pathogenic bacteria.from mastitis quarters of buffaloes in which the rate of Wash hands with soap and water, wash teats and udder contamination of the E. coli was (1.40%), whereas in sanitizing solution, thoroughly dry teats and udder 2400 milk samples were collected for screening of the with individual towels, dip teats in an effective sub-clinical mastitis form the six hundred lactating germicidal teat dip reduced the number of pathogenic diary buffaloes from the four districts (Lahore, bacteria from the udder skin [34].Sialkot, Narowal and Okara) in Pakistan, in which the percentage of E. coli was (16.18%) [27,28]

Table - 1
. Culture Characteristics of Escherichia coli on different media

. In Author's contribution Jharkhand
, India, (8.95%) isolates of the E. coli were Rajdeep Palaha carried out the field study (Sample detected from the raw milk of clinical cases of the Collection), Participated in scientific discussion and bovine mastitis [29].Whereas 43.8% cases of E. coli sample analysis.Narendra Chaudhary participated in mastitis were detected in dairy farm buffaloes in the sample analysis, scientific discussion.Harsh Alexandria Desert road, Egypt, as the organism were Kumar participated in the design of the study and present in high level in the water tank as they act as coordination, scientific discussion and wrote the final primary reservoir for this environmental pathogen [30, draft.All authors read and approved the final manuscript.31].Environmental factors such as poor hygiene, poor husbandry, and poor milking technique results the Table-3.MPN Results of the Water Samples

Table - 4
. Occurrence of Escherichia coli growth obtained from the udder