Profile of Antimicrobial Susceptibility from Cattles’s Milk Isolates Suffering from Mastitis in District Lahore

Mastitis is highly prevalent infection in cattle causing cost-effective loss in dairy milk production. Escherichia coli is the most frequently isolated bacteria causing mastitis worldwide. The current study was performed to investigate the mastitis prevalence and effect of different antibiotics against pathogens causing it. In sum, 216 milk samples were collected randomly including 108 each from both cows and buffaloes. These samples were subjected to Surf Field Mastitis Test for prevalence of mastitis that found 18.50% and 23.14% in buffaloes and cows respectively. Mastitis was more prevalent both in buffaloes and cows at the age of 9-10 years that was reported in 35% and 32% respectively. The affected buffaloes showed 65% samples containg watery fluid having blood, pus and mucus whereas 36% samples of affected cows had mucus and watery fluid. Pus and blood content were found to be 12% and 16% respectively. Sub-clinical mastitis was prevalent as 56% and 55% in cows and buffaloes respectively while acute mastitis as 4% and 5% in similar fashion. In both groups Right-for part of udder was frequently affected than any other. Among commonly used four antibiotics, Norfloxacine was observed the most effective antibiotic to control mastitis as compared to Gentamycin, Penicillin and ciprofloxacin. The benefit of this study is to diagnose mastitis and use to isolate pathogens for further processing like antibiotic sensitivity and resistance. This study will help veterinarians in better selection of antibiotic to control mastitis.


INTRODUCTION
Livestock is playing an important role in the development of Pakistan just like other countries and facilitating the development and consumption of different items like meat, milk and various house products. There are 28.4 million buffaloes and 25.5 million cattle involved in the production of 1174.4 tons beef and 31294.4 tons milk respectively (Economic Survey of Pakistan 2005. Mastitis is a common disease in livestock which make the milking animals useless (Sánchez-Macías et al., 2020). Mastitis is the redness, swelling, pain or hardness and heat in the udder of the animals (Zahid, 2004., Jamali et al., 2018. Field survey of Pakistan indicated that mastitis is one of the major diseases of dairy animals in livestock (Hussain et al., 2005). Mastitis causes the inflammation of the teat involving one/ two/three or full four quarters (Mc Dowell et al., 1995).
Worldwide, there is a great loss nearly of 35 billion dollars. Annual loss due to mastitis is about more than 2 billion dollars in United State. About 3 million animals linked to dairy farms show visible symptoms of mastitis (acute) whereas 3 lac die from endotoxins shock (Kivaria and Noordhuizen, 2007). In Punjab, Pakistan, the total annual loss caused by mastitis is about 240 million rupees. The incidence and existence of pathogens connected to diseases (mastitis) differ from herd to herd, time to time and even place to place (Joshi and Gokhale, 2006). Mastitis from livestock and dairy animals cannot totally be eliminated but its effect can be reduces with the help of preventive practices against mastitis (Khan and Khan, 2006). Mastitis present in milking animals is of various types such as clinical and subclinical (Hillerton, 1999). It is more prevalent in cattle than buffaloes (Thepa and Kaphle, 2002). Mastitis causing agents include certain species algae, fungi, bacteria and some viral entities (Thomas et al., 1997). Among all mastitis causing strain of bacteria staphylococcus is one with broad geographic distribution throughout the world (Annemuller et al., 1999).
Pathogens which cause mastitis are mostly found in fecal matter and soil as well (Rodostits et al., 2000). The time of teat canal to remain open after milking is about 1-2 hours that facilitates the entrance of mastitis causing bacteria in to the teat canal (Jones, 2006). Under stress condition animal secretes adrenaline that causes constriction of blood vessels leading to hindrance the supply of oxygen to the mastitis suffering tissue (Rodostits et al., 2000). In animals suffering with mastitis cortisol level decreased while serum calcium, inorganic phosphorus and total protein also decreased (Zaki et al., 2008). Milk proteins such as transferrin, immunoglobulin and serum albumin pass into the milk as the result of vascular system permeability (Khan and Khan, 2006). Few antibiotics such as norfloxacin and gentamycine are more effective while pathogens are resistant against penicillin (Zahid, 2004).
Present study will help to enhance the dairy products with easy identification and control of mastitis at local level. The main objective of the designed study was to find the mastitis prevalence along the effect of various selected antibiotics on mastitis causing agents in buffaloes and cows in District Lahore.

MATERIALS AND METHODS
Milk samples from 216 lactating animals (108 cows and 108 buffaloes) were randomly collected and brought to the Microbiology Laboratory of GCU Lahore. Animals were grouped depending upon the age, part of udder infected, appearance of milk and condition of the disease. Prevalence of mastitis in animals was determined by Surf Field Mastitis Test (SFMT) (Muhammad et al., 1995). Buffaloes and cows were compared for prevalence of mastitis. Four different types of antibiotics i.e., penicillin, gentamycine, norfloxacine and ciprofloxacin discs were used to check which antibiotic is more effective to control mastitis in District Lahore.

Antimicrobial Susceptibility Test
Antimicrobial susceptibility testing was performed on Mueller-Hinton agar by the Kirby-Bauer disk diffusion method (Clinical and Laboratory Standards Institute, 2012). The 4 commercial antibiotic disks used in the experiment included Norfloxacin (10mg), Ciprofloxacin (5mg), Penicillin (10mg), Gentamycin (10mg). The test results were then recorded as susceptible or resistant based on the zone diameter (Clinical and Laboratory Standards Institute, 2013) The data were analyzed with percentage, one and two tail t-tests by using SPSS version 22.0 (SPSS, Chicago, IL, USA). The differences among buffalo and cow at different age groups were found.
As the quality and quantity of milk changes, milk composition and color also changes. Depending upon stage of disease sometimes water, sometimes mucus and sometimes blood appeared in mastitic milk (Mustafa et al., 2007).

Figure 4: Comparison for various stages of mastitis both in buffaloes and cows.
Over all prevalence of mastitis was more in right fore teat of udder both in buffaloes and cows. Left hind teat, left fore teat, right hind teat and right fore teat in buffaloes were 4(20%), 2(10%), 5(25%) and 9(45%) whereas in cows were 1(4%), 9(36%), 12(48%) and 3(12%) effected respectively (Fig 5). According to previous results of various studies it is presented that right fore part is more susceptible for infection because usually it is used first during milking and microbes get entrance in this part of udder (Khan & Muhammad., 2005)

Figure 5: Comparison regarding quarter wise mastitis prevalence both in cows and buffaloes.
Data revealed that among different antibiotics available in market, Norfloxacine was highly sensitive, Ciprofloxacin was sensitive, Gentamycine was mild sensitive and Penicillin was ineffective against control of mastitis both in buffaloes and cows (Table 3). Pathogens are more susceptible against Ciprofloxacin, and Gentamycine whereas resistant against penicillin (Shabbir et al., 2009), because time by time penicillin is used as a medicine to control the infection of mastitis. The resistance against penicillin in mastitis causing agents may be due B-lactamase production that is used to breakdown ring of antibiotic called as B-lectam making antibiotic ineffective against microbial pathogens (Farooq et al., 2008).  Both in buffaloes and cows Right fore part of udder was frequently affected than any other part. Among commonly used four antibiotics, pathogenic bacteria were highly resistant against Penicillin while Norfloxacine was observed the most effective antibiotic to control mastitis as compared to Gentamycin and ciprofloxacin. Pathogens are more susceptible against Ciprofloxacin, and Gentamycin whereas resistant against penicillin, because frequent use of penicillin as a medicine to control the infection of mastitis. The resistance against penicillin in mastitis may be due to Beta-lactamase production which breakdown beta-lectam ring of antibiotic as making antibiotic ineffective against microbial pathogens.

CONCLUSION
The right fore teat canal both in buffalo and cows at the later age is more susceptible to mastitis causing agents those are more sensitive to Norfloxacine then other used antibiotics in District Lahore.