In vitro Antimicrobial Susceptibility Profile of SubClinical Mastitis Isolates from Dairy Goats

| Sub-clinical mastitis is a major problem that causes huge economic loss due to occurrence of antimicrobial resistant organisms. The present study was designed to observe the antimicrobial susceptibility pattern of sub-clinical mastitis bacterial isolates in dairy goats. Bacterial species (n=9) caused sub-clinical mastitis in dairy goats isolated from milk samples were subjected towards the disc diffusion method for antimicrobial sensitivity against twenty six different antimicrobial agents. The results of antimicrobial susceptibility testing revealed that Staphylococcus aureus and Micrococcus luteus were recorded highly sensitive to oxytetracycline with 100% susceptibility, whereas Bacillus cereus was observed highly susceptible (100%) to amoxicillin/clavulanic acid, cephalexin and norfloxacin. Citrobacter species were observed 100% susceptible to ceftriaxone. Likewise, 100% susceptibility of Escherichia coli was detected to ceftriaxone and chloramphenicol; Micrococcus luteus to oxytetracycline; Proteus vulgaris to amikacin, erythromycin and sulphamethoxazole/trimethoprim; Bacillus subtilis to norfloxacin; Staphylococcus epidermidis to doxycycline; and Streptococcus agalactiae to doxycycline and amoxicillin/clavulanic acid. These results suggested that bacterial species caused caprine sub-clinical mastitis could be treated through antibiotics. Moreover, enrofloxacin and doxycycline were observed the most effective antimicrobial agents against the sub-clinical mastitis pathogens.


INTRODUCTION
M astitis is the inflammation of mammary gland of the dairy animals causes huge economic losses till date.Mammary gland affected by bacterial infection have great influence on dairy goats' milk production in terms of decreased production and increased somatic cell count (Boscos et al., 1996).Mastitis is caused by a wide range of microorganism and is characterized by poor quality milk and pathological changes in mammary glands (Radostits et al., 2008).For treatment and prevention of mastitis, indiscriminate use of antimicrobial agents in dairy animals is a serious problem that leads to appearance of resistant bacterial agents of zoonotic importance (Piddock, 1996).It has been suggested that antimicrobial susceptibility testing should be carried out prior to the use of antimicrobials for mastitis treatment.is cheap, wholesome, easily digestible and nutritious.Due to colossal position of dairy goat milk, it is important to supply disease free and healthful milk to the users.Globally dairy goats with mastitis, like dairy cattle with mastitis is a disease of significance importance.In Pakistan losses due to mastitis may be higher due to lack of proper reports on mastitis and poor disease prevention and managements.According to Samiullah et al. (2000) in Khyber Pakhtunkhwa (Pakistan), a massive damage to livestock by culling large number of sick animals is due to mastitis.Bad hygienic environments and poor managements, lack of advanced farming practices like dipping of teat prior and post to milking are some main aspects of this disease development in goats.The prevalence of sub-clinical mastitis in dairy goats ranges from 5 -30% (Contreras et al., 2007).The sub-clinical mastitis is characterized by having no visible signs either in the udder or in the milk.

Journal of Animal
Keeping in view the above facts, the present study was therefore designed to determine the in vitro susceptibility of bacterial isolates of sub-clinical mastitis of dairy goats against the common antimicrobial agents.Furthermore, it would be helpful in formulation of effective treatment plans against the bacteriological sub-clinical mastitis of goats.

bacterial isolates
A total of nine bacterial organisms were isolated from sub-clinical mastitis milk samples (n= 200) of goats that were collected from surroundings of Tandojam.The samples were processed according to standard bacteriological procedures to recover the bacterial pathogens (Pirzada et al., 2016).The bacterial isolates included were Staphylococcus aureus, Staphylococcus epidermidis, Citrobacter species, Escherichia coli, Micrococcus luteus, Proteus vulgaris, Bacillus subtilis, Streptococcus agalactiae and Bacillus cereus.

Staphylococcus aureus isolates
The bacterial species Staphylococcus aureus was found highly sensitive (100%) to oxytetracycline while the quite effective antimicrobials against the organism were erythromycin, enrofloxacin, gentamycin and kanamycin, and their efficacy was recorded as 63.33, 73.33, 63.33 and 50% respectively.However, the efficacy of other drugs tested against the species is given in Table 1.Gentilini et al. (2000) isolated a total of 206 strains of Staphylococcus aureus from bovine clinical and sub-clinical mastitis in Argentina and tested for their in vitro susceptibility to several antimicrobial agents.Minimum Inhibitory Concentrations (MIC) that inhibited 90% of the strains tested per millilitres were 1.5, 0.5, 0.75, 1.50, 0.75, 1.0 and 0.125 for penicillin, oxacillin, cephalotin, gentamycin, erythromycin, clindamycin, and ampicillin-sulbactam, respectively.The resistance against the drugs was detected in 83 (40.3%), 24 (11.6%),16 (7.7%)and 7 (3.4%)Staphylococcus aureus isolates for penicillin, erythromycin, pirlimycin and gentamycin.Sol et al. (1997) and Moroni et al. (2006) carried-out a study on the antimicrobial susceptibility against 68 Staphylococcus aureus isolates collected from milk of cows affected by sub-clinical mastitis.The antimicrobial agents tested were the ß-lactamase penicillin G, amoxicillin, ampicillin, cloxacillin, amoxicillin + clavulanate, cephalonium, and cefoperazone; and other drugs including lincomycin, oxytetracycline, doxycycline, and kanamycin.MIC recorded only certain ß-lactamase-resistant penicillin (specifically cloxacillin) or penicillin combinations (amoxicillin + clavulanate) effective against Staphylococcus aureus, whereas the other ß-lactam derivatives and drugs from other pharmacological groups were found either moderately effective or ineffective.Thus, ß-lactamase-resistant penicillin's are considered to be the antimicrobial agents of choice for the treatment of bovine mastitis resulting from infection by Staphylococcus aureus.

antiMicrobial suscePtibility ProFile oF Bacillus cereus isolates
The results regarding the efficacy of antimicrobials against Bacillus cereus isolates are summarized in the Table 1.Bacillus cereus was observed as highly sensitive to cephaloxin and amoxicillin/clavulanic acid and their efficacy against tested organism was recorded as 100%.Dogruer et al. ( 2010) car-ried-out a study to determine the efficacy of three treatment protocols for Bacillus cercus during lactation in Damascus goats.For this purpose, intramammarian ampicillin dicloxacillin, intramuscular amoxicillin + clavulanic acid and the combination of intramammarian ampicillin + dicloxacillin and intramuscular amoxicillin+ clavulanic acid were used in goats with subclinical mastitis during lactation.The microbiological treatment rates in the intramammarian ampicillin + dicloxacillin, intramuscular amoxicillin + clavulanic acid and the combination of intramammarian ampicillin + dicloxacillin and intramuscular amoxicillin+ clavulanic acid groups in the 7th and 21st days after the treatment were 62.5% and 92.5%, 62.5% and 70%, 87.5% and 87.5%, respectively.The intramammarian and intramuscular com-

Journal of Animal Health and Production
July 2016 | Volume 4 | Issue 3 | Page 75 bination group was found statistically higher effective rate (P< 0.05) than other two groups on day 7th.On day 21st intramammarian and combination groups were found to have statistically better treatment rates (P< 0.05) than that of intramuscular group.The study concluded that the goat subclinical mastitis could be treated, successfully during lactation.While the intramammarian ampicillin + dicloxacillin treatment had the best treatment rates, meanwhile the combination of intramuscular amoxicillin + clavulanic acid was also become successful.Whereas intramuscular amoxicillin + clavulanic acid was observed least effective as intramammarian therapy.

antiMicrobial suscePtibility ProFile oF Citrobacter sPecies
The Citrobacter species showed 100% susceptibility to ceftriaxone while the second most highly effective drugs noted sulphamethoxazole/Trimethoprim and their effects against Citrobacter species are measured as 80% (Table 1).
From the present study, one could conclude that Citrobacter species had exhibited its resistance to majority of the drugs but was highly susceptible to ceftriaxone while quite susceptible to amikacin.Papadopoulos et al. (1997) examined antimicrobial sensitivity against Citrobacter freundii.On testing the sensitivity, the organism was found highly sensitive to ceftriaxone, ampicillin, amoxicillin plus, clavulanic acid, cephalosporin and co-trimoxyzole.Whereas, Baloch et al. ( 2011) reported that Citrobacter species showed 100% susceptibility to amikacin while the second most highly effective drugs noted were ofloxacin, ampicillin and kanamycin (10μg) and their effects against Citrobacter species were measured as 94.73, 84.21 and 52.63% respectively.
Citrobacter species had exhibited its resistance to majority of the drugs but was highly susceptible to amikacin, while quite susceptible to ofloxacin, ampicillin and cefixime.

Escherichia coli isolates
The antimicrobial susceptibility against Escherichia coli recovered from the cases of sub-clinical mastitis in goats was carried-out and results are summarized in Table 1.Escherichia coli was observed highly sensitive to ceftriaxone and chloramphenicol and showed 100% sensitivity while second most highly effective drug demonstrated during investigation was amoxicillin/clavulanic acid (73.33% sensitivity).Whereas the organism was also found quite sensitive to doxycycline and its sensitivity was measured 60%.Khan and Rind (2001) found Escherichia coli resistant to tetracycline, ampicillin and sulphamethoxazole-trimethoprim and it was recorded susceptible to gentamycin, kanamycin, neomycin and chloramphenicol drugs.A similar report was given by Khan et al. ( 2016), who reported that Escherichia coli recovered from table eggs were complete resistant to oxytetracycline, while fully susceptible to second-generation fluoroquinolones including enrofloxacin and ciprofloxacin.These findings are in consistent to our present results for E.coli isolates of sub-clinical mastitis.

Micrococcus luteus isolates
During present survey, oxytetracycline was found 100% effective drug while Norfloxacin were measured the second most highly active (89.65%)drug against Micrococcus luteus species.Baloch et al. (2011) reported that rifampicin was found 100% effective drug while ampicillin, erythromycin (10 units), furazolidone, gentamycin, penicillin G (5units) and enrofloxacin were measured the second most highly active drugs against Micrococcus luteus and their action recorded were 88.23, 88.23, 88.23, 82.35, 82.35 and 76.47% respectively.The drugs found highly active against the species during present survey are in line to the susceptibility of the organism to antimicrobials recorded by the above author.

antiMicrobial suscePtibility ProFile oF Proteus vulgaris sPecies
Proteus vulgaris was found highly sensitive (100%) to amikacin and sulphamethoxazole/trimethoprim, colstin sulphate and erythromycin drugs, and the second most highly active drug (90%) was observed as enrofloxacin.The efficacy of the other drugs tested against the Proteus vulgaris is also given in the same table (Table 1).Baloch et al. (2011) reported that Proteus vulgaris was found highly sensitive to enrofloxacin, this drug showed 100% efficacy against Proteus vulgaris and the second most highly active drugs determined were chloramphenicol, amikacin and enrofloxacin and showed 93.33, 80.0 and 86.66% action against the species respectively.The species Proteus vulgaris was found quite sensitive to gentamycin, doxycycline, kanamycin, norfloxacin, flumequin and neomycin and their efficacy was recorded as 66. 66, 73.33, 66.66, 53.33, 66.66  The CNS isolates tested were found more frequently resistant to ampicillin (36.0%) and kanamycin (6.7%).The results about antimicrobial action against the species recorded in this survey were not in agreement to the findings regarding the action of the antimicrobials against the species observed by the above workers.We recorded some higher action of the antimicrobial against the species while they recorded higher resistance against the antimicrobials.

Streptococcus agalactiae sPecies
On conducting the antimicrobial sensitivity test against the bacterial species during present investigation recovered from sub-clinical mastitis of goats, Streptococcus aglactiae was found highly susceptible to doxycycline and amoxicillin/clavulanic acid (100%) while quite sensitive to erythromycin (15 units), neomycin (10 ug) and amikacin (50%).
The results regarding the efficacy of other antibiotics are given in Table 1.Hameed et al. (2008) carried-out an investigation on lactating cattle and buffaloes in Tehsil Burewala, district Vehari, Pakistan.The bacterial isolates were found sensitive in descending order to enrofloxacin, chloramphenicol, gentamycin, oxytetracycline, doxycycline and amoxicillin.

antiMicrobial suscePtibility ProFile oF Bacillus subtilis sPecies
Bacillus subtilis isolates of sub-clinical mastitis showed 100% sensitivity to neomycin (Table 1).Weber et al. ( 2002) conduct a study to demonstrate the antimicrobial susceptibility of Bacillus subtilis to 18 antimicrobials.Microdilution MIC susceptibility test showed that all Bacillus subtilis strains were susceptible to imipenem, vancomycin, chloramphenicol, gentamicin and ciprofloxacin while non-Bacillus subtilis strains were found most susceptible to neomycin, imipenem, vancomycin, LY146032 and ciprofloxacin.Furthermore, a Disk susceptibility test was conducted to record the susceptibility; the bacterial species was found susceptible to penicillin, semisynthetic penicillins, or cephalosporins with the exception of mezlocillin.Therefore, the present results are somehow in the line to the results about drug susceptibility of the species observed by above workers.
Staphylococcus aureus are known to cause a number of diseases in farm animals including non-ruminants.Our previous study have reported the multidrug resistant Staphylococcus aureus as a causative agent of septic arthritis in poultry birds Journal of Animal Health and Production July 2016 | Volume 4 | Issue 3 | Page 74

Table 1 :
The antimicrobial sensitivity (%) of various organisms identified from sub-clinical mastitic milk samples of goats Bacillus cereus(Nazia et al., 2015).A recent study has demonstrated the host-related differences in susceptibility levels of Staphylococcus aureus isolates from different farm animals, thus indicated a host-microbe interaction and probable role of host in development of antimicrobial resistance in Staphylococcus aureus organisms(Habib et al., 2015).

Journal of Animal Health and Production July 2016 | Volume 4 | Issue 3 | Page 76 were
effective or not at all noted in the present survey are presented in Table1.