Jena et al.

California mastitis test (CMT)

milk sample on to blood agar, nutrient agar, MacConkey’s

The California mastitis test was carried out as screening

agar, Sabourads dextrose agar and Eosine Methylene

test for selections of samples for culture following the

Blue agar plates and the plates were incubated “under

method described by Schalm et al . (1971) and Quinn et al .

aerobic conditions” at 37° C for 24 to 48 hours. The

(1994). A squirt of milk, about 2 ml from each quarter was

staining and cellular morphological features of organisms

placed in each of four shallow cups in the CMT paddle.

were ascertained by microscopic examination of Gram

An equal amount of the commercial reagent was added

stained smears. The bacteria isolated were identified on

to each cup. A gentle circular motion was applied to the

the basis of their cultural, morphological and biochemical

mixtures, in a horizontal plane for 15 sec. The reaction

characteristics as per the method of (Cruickshank et al .,

was interpreted according to Schalm et al . (1971), Quinn

1975).

et al . (1994) and David et al . (2005). The results of visible

reactions were classified into 5 scores: (0) = negative, (±)

Antibiotic susceptibility test

= trace, (+1) = weak positive, (+2) = distinct positive, and

All the isolates were subjected to in vitro drug sensitivity

(+3) = strong positive. In this study, CMT score of 1+ and

test as per method described by Bauer et al. (1966). The

above was considered positive for mastitis and of trace

antimicrobials (µg) available commercially in market

and negative (± and 0) together was considered negative

like cloxacillin (10), amoxycillin(10), streptomycin (10),

for subclinical mastitis.

penicillin G (10), and neomycin (30) were tested for their

in vitro effectiveness against various bacterial isolates.

Milk Sampling

In addition, old and new generation antimicrobials (µg)

Quarters that scored negative and trace were assumed

like chloramphenicol (30), lincomycin (10), gentamicin

healthy and the quarters with different positive scores

(10), ciprofloxacin (10), cephalexin (30), enrofloxacin

through any screening tests were assumed infected.

(10), amikacin (30), cefotaxime (30), ceftriaxone (30),

A cow with at least one affected quarter at the time of

pefloxacin (5), kanamycin (30) and ampicillin (10) were

examination was considered positive for subclinical

also tested. The antibiotic discs (Hi-Media ® , Mumbai,

mastitis. Similar criteria were used to characterize a cow

India) were impregnated on the surface of an agar plate

positive for SCM (Sharma et al. , 2007). All the animals

previously inoculated with a standard amount of the

found positive in SCC were selected for further analysis

organism under scrutiny. The plates were incubated

under bacteriological prevalence and antibiotic sensitivity

at a temperature of 37 ° C for duration of 18 - 24 hours.

test. Only one of the infected quarters from each cow was

Subsequently, the plates were examined for the zone

selected for milk sampling except when the cow had four

of inhibition developed around the discs, followed by

severely infected quarters so one more milk sample was

the diameter of the zone of inhibition was measured

collected. Then the teat end of the selected quarter was

in milimeter and compared with the values listed in

swabbed with cotton soaked in 70% ethyl alcohol and 10

standard chart provided by the manufacturer, on the

milliliters of milk was approximately collected into sterile

basis of which the isolates were categorized as resistant

containers. The first 3-4 streams of milk were discarded.

(R), mildly sensitive (Mi), moderately sensitive (Mo) or

The collecting vial was as near horizontal as possible and

highly sensitive (H) to the antimicrobial contained in that

by turning the teat to a near horizontal position, 15 ml of

particular disc (Ranjan et al ., 2010).

milk was collected into the vial. Samples were transported

to the laboratory in a special box with ice at 4°C for

Statistical analysis

bacteriological investigation (Quinn et al ., 1994).

All collected data were entered in Microsoft Office ®

2007 excel sheet and analyzed by SYSTAT ® version 12

Bacteriological culture and identification of

computer package program. In the present study chi-

microorganisms

square test and confidence intervals were calculated.

The samples were subjected to bacteriological study in

the laboratory by inoculating approximately 0.01 ml of

178

Journal of Animal Research: v.5 n.1. April 2015