INTRODUCTION

An inflammation of the mammary gland is commonly referred as Mastitis, which is primarily occurs in response to intra-mammary bacterial infection, but also to intra-mammary mycoplasma, fungal, or algal infections. Mechanical trauma, thermal trauma, and chemical predispose the gland to intra-mammary infection (IMI). Occurrence of mastitis depends on the interaction of host, agent, and environmental factors. Milk is considered as an excellent medium for growing of many microorganisms. Milk can be contaminated with several bacteria during milking process from the milking personnel, utensils used for milking1. Mastitis usually accompanied by physical, chemical and bacteriological changes in milk and pathological changes in glandular tissue. It is the most costly disease of dairy cattle resulting in the reduction in potential production, losses in quality and quantity of milk, losses due to discarded milk, premature culling, veterinary costs, and labor cost.2

Mastitis in cattle can be broadly classified as: clinical mastitis and sub-clinical mastitis Clinical mastitis is easily identifiable as common signs including flakes in milk and swelling of the udder. Sub-clinical mastitis is usually unnoticed but it is highly prevalent than clinical mastitis and decreases the production and can easily advance into clinical mastitis under adverse conditions. So, identification and control of sub-clinical mastitis is very important among farmers. Average reduction in milk yield due to clinical and sub- clinical mastitis was estimated to be 50 and 17.5 %, respectively. 3

Staphylococcal mastitis is one of the commonest infection associated with dairy animals, mostly prevails in sub-clinical form and once left unidentified, usually flares up with clinical mastitis resulting in huge economic losses to the dairy owners. Staphylococcus aureus infections are difficult to control and are well-known to cause subclinical, clinical, and chronic mastitis, while treatment approaches frequently compromised. The bacterium Staphylococcus aureus is an opportunistic pathogen, may cause mild to severe diseases, ranging from superficial wound infections or food poisoning to bacteremia and other systemic infections.4

In recent days, dairy farming is being considered as one of the most effective means for poverty alleviation, particularly in the hilly region where the majorities of the farmers are either resource poor or land less. Due to ever increasing demand and hiking prices of fresh milk, it is becoming a profitable business among the resource poor farmers in the hills. However, several studies have found that clinical mastitis has a detrimental effect on milk yield.

It poses the risk for transmission of major zoonotic diseases like tuberculosis, leptospirosis, brucellosis, streptococcal sore throat and gastroenteritis.5 Mastitis has become a great threat to the productivity of the lactating animals. Mastitis is one of the most common dairy cow diseases as in other developing countries, dairy animals are key for rural livelihoods in Nepal but often suffer from mastitis a production disease causing economic losses to farmers, challenges to the dairy processing industry, and possible health hazards to consumers.6

It is therefore also important to study the sensitivity pattern of different bacteria isolated time to time from mastitis cows in different geographical zones of the country in order to formulae appropriate therapeutic measures with suitable antibiotics.7

Considering the above facts, this research work was undertaken with the objectives of isolation and identification of bacterial population from California Mastitis Test (CMT) positive milk sample of livestock commercial farm and design their antibiogram profile. Identification of mastitis causing pathogens and the results of the antibiotic resistance of the isolated bacteria are important prerequisites for implementation of effective control of cattle mastitis such information is needed not only to treat and control mastitis but also support public health concerns in developing countries. However, epidemiological and bacteriological studies of cattle mastitis have not been fully conducted in Nepal.

METHODOLOGY

A descriptive cross-sectional study was conducted from January to March 2022 in microbiology section of Veterinary Laboratory, Pokhara. A total of 145 milk samples were collected by using aseptic technique. Each donor were requested to fill the Questionnaire form and were collect milk in provided sterile glass bottle with necessary information. Only the CMT positive milk samples were packed in ice-box and transferred to Microbiology laboratory for further processing and analysis. Colony morphology and characteristics were observed. All isolates were tested for antimicrobial susceptibility by modified Kirby-Bauer disc diffusion method.

RESULTS

A total of 145 milk samples from various cattle ranches were gathered, processed, and examined in the Veterinary Laboratory in Pokhara. Of the 74 cows, 34 (23%) were Jersey and 40 (28%) were Holstein. Also, 71 (49%) of the Buffalo were Murrah in total.

Determination of milk sample by California mastitis test (CMT)

The California mastitis test (CMT) was used to detect mastitis in cattle milk samples. CMT was positive in 76 (52.41%) of the samples and negative in 69 (47.58%).

Table 1: Determination of milk sample by California mastitis test (CMT)

Milk Samples

CMT Test Cow Buffalo Total

Jersey Holstein Murrah

Positive 13(8.96%) 20(13.79%) 43(29.65%) 76(52.41%)

Negative 21(14.48%) 20(13.79%) 28(19.31%) 69(47.58%)

Total 34(23.44%) 40(27.58%) 71(48.96%) 145

Growth Pattern of microorganisms in the milk sample

Bacterial growth was seen in Jersey 10 (76.92%) and Holstein 18 (90%). Murrah 40 had the highest growth rate (93.02%).

Table 2: Growth pattern of microorganisms in the milk sample

Sample Variety Growth No growth Total

Cow Milk Jersey 10 (76.92%) 3 (23.07%) 13 (17.10%)

Holstein 18 (90%) 2 (10%) 20 (26.31%)

Buffalo Milk Murrah 40 (93.02%) 3 (6.97%) 43 (56.57%)

Total 68(89.47%) 8(10.52%) 76

Distribution of isolated microorganism from the milk sample

A total growth of 68 microorganisms from isolated bacterial species were Staphylococcus aureus, Staphylococcus epidermidis and Streptococcus spp, where Staphylococcus aureus showed majority growth among isolated bacteria. Out of 51 Staphylococcus aureus, breed of buffalo (Murrah) showed highest isolates which is 28 (54.90%) followed by jersey 9 (17.64%). Similarly, out of 15 Staphylococcus epidermidis, Murrah 11 (73.33%) showed highest isolates whereas Holstein showed lowest 4 (26.66%).

Table 3: Distribution of isolated microorganism from milk sample

Sample Variety Staphylococcus aureus Staphylococcus epidermidis Streptococcus spp Total

Cow milk

Jersey

9 (17.64%) 0 1 (50%) 10

Holstein

14 (27.45%) 4 (26.66%) 0 18

Buffalo Milk

Murrah

28 (54.90%) 11 (73.33%) 1 (50%) 40

Total 51 15 2 68

Antibiotic susceptibility pattern of the isolated organism

Staphylococcus aureus made up 51 of the 68 isolated organisms, followed by Staphylococcus epidermidis (15), and Streptococcus spp.

Among 51 isolated Staphylococcus aureus, Amikacin 46 (90.19%) and Ceftriaxone 28 (54.90%) demonstrated the highest levels of sensitivity in S. aureus. Enrofloxacin 11 (21.26%) and amikacin 1 (1.96%) were both highly resistant to S. aureus.

Similar to the other Staphylococcus epidermidis isolates, S epidermidis exhibited the maximum sensitivity to Amikacin 14 (93.33%), which was followed by ceftriaxone 6 (40%) in the group of 15 isolates. Tetracycline and enrofloxacin both displayed the highest levels of resistance 4 (26.66%).

Last but not least, no Streptococcus spp. resistance was discovered among the 5 Antibiotiscs that were recovered from 2 separate Streptococcus spp.

Table 4: Antibiotic susceptibility pattern of isolated organism

Antibiotics Staphylococcus aureus Staphylococcus epidermidis Streptococcus

S (%) I (%) R (%) S (%) I (%) R (%) S (%) I (%) R (%)

Ciprofloxacin 34(66.66) 10(19.60) 7(13.72) 12(80) 3(20) 0 2(100) 0 0

Ceftriaxone 28(54.90) 19(37.25) 4(7.84) 6(40) 7(46.66) 2(13.33) 2(100) 0 0

Tetracycline 44(86.27) 1(1.96) 6(11.76) 8(53.33) 3(20) 4(26.66) 2(100) 0 0

Enrofloxacin 36(70.58) 4(7.84) 11(21.56) 7(46.66) 4(26.66) 4(26.66) 1(50) 1(50) 0

Amikacin 46(90.19) 4(7.84) 1(1.96) 14(93.33) 1(6.66) 0 2(100) 0 0

Gentamicin 45(88.23) 4(7.84) 2(3.92) 13(86.66) 2(13.33) 0 2(100) 0 0

DISCUSSION

The study included 145 bovine milk samples obtained from several commercial cattle farms in the Pokhara valley, with the goal of isolating mastitis-causing bacteria in the milk samples. Out of 145 cattle milk samples, 76 (52.41%) were CMT positive, while 69 (47.58%) were CMT negative. CMT positive samples were taken to the Pokhara Veterinary Laboratory to be processed and analyzed. Positive growth of an organism was observed in 68 (89.47%) of Positive CMT, while 8 (10.52%) CMT positive did not display their growth pattern on selective media. 68 organisms were isolated from 76 samples. From the CMT positive sample, three different kinds of bacteria were identified. Whereas 51 Staphylococcus aureus, 15 Staphylococcus epidermidis, and 2 Streptococcus spp. were recovered from Murrah 28 (54.90%), Staphylococcus epidermidis were isolated from Murrah 11 (73.33%). The antibiotic susceptibility of the isolated organism was evaluated using the Kirby-Bauer disk diffusion method with four distinct antibiotic groups.

A total of 145 milk samples were collected. There were 74 cows and 71 buffaloes. Besides, cows came in two varieties. They were Jersey 34 and Holstein 40. The livestock distributions are shown in table 4.1. All of the cattle chosen were lactating less than a month ago. Cows are the most numerous among all cattle. Bovine mastitis is a severe concern around the world due to the economic loss in dairy farms, where the prevalence ranges from 48.62% to 86.2% in cows.8

The screening of mastitis can be done by California Mastitis Test (CMT) in cattle farm. CMT positive sample were 76 out of total sample 145. The field wise screening of this test is also called sub-clinical mastitis. The preliminary test done by CMT in positive milk sample were collected for further analysis and processing for causative agent. Our results showed 76 (52.14%) CMT positive sample which was least finding 56.0%.to the study.9This study represents prevalence of the mastitis is higher in buffalo 43 (29.65%), The highest CMT positive sample result was 52.4%, which was similar to the findings. This finding demonstrated that the scenario in north Ethiopia where cow breed rearing is higher in large scale dairy farms. In contrast, 38.07% prevalence is found in buffalo, which is the least similar to our findings. A big number of gram positive group of organisms were identified from the cow milk sample from the dairy farm in the current study. Three types of bacteria, Staphylococcus aureus, Staphylococcus epidermidis, and Streptococcus spp., were isolated for this investigation, which is consistent with the results. From the cattle milk sample, we identified 75% Staphylococcus aureus, which is comparable to the results, who isolated 73.2% of an organism.10 Our findings varied from 65.9 to 97.7%, with a 72.7% similarity percentage to Gram positive bacteria discovered. In a study on mastitis milk, the pathogenic species of Staphylococcus aureus were the ones that were most frequently isolated from the milk sample. According to a study, Staphylococcus aureus prevalence was 4.8%, which is quite low when compared to our findings. Although one study found 45% Staphylococcus aureus in buffalo milk, we found 54.90% Staphylococcus aureus in buffalo (murrah) milk, the lowest of any study. The buffalo had the highest quantity of Staphylococcus aureus (54.90%) compared to the cow, which is opposite and least to the findings.11

In this investigation, 22% Staphylococcus epidermis was isolated from the sample, which is comparable to the findings of 18.20%. The percentage of Staphylococcus epidermidis isolated was 11.5%. However, Staphylococcus epidermidis was identified from cattle milk samples at a rate of 28%. This scenario depicts the dispersion of Staphylococcus epidermidis in several ecological niches, with the distribution influenced by environmental factors and others.12 Mastitis in cattle is connected with climatic conditions; the most number and species of bacteria were recovered in the summer season, with the most prevalent pathogen in mastitis positive milk samples. Similarly, 17% Staphylococcus epidermidis and 5.62% Streptococcus spp were recovered from a buffalo mastitis positive milk sample.13 we isolated the same number of Bactria from the cow and buffalo. Buffalo milk contains 23% Streptococcus spp. It could be attributed to seasonal variations, proper farming methods, and the teat dipping process.

Antibiotics are a miraculous medicinal discovery that can cure bacterial illnesses. Our results showed that S. aureus, S. epidermidis, and Streptococcus spp. had the highest sensitivity to Amikacin (90.19%) and the lowest result (70%). The highest level of resistance to Enrofloxacin was 21.56%, which was reduced to 69%.14 Staphylococcus epidermidis is 93.33% susceptible to amikacin, both Tetracyclines and Enrofloxacin were resistance 26.66% in our work but other study gets 86.25 sensitive towards Enrofloxacin which is opposite as compare to our result. Less resistance means highest sensitivity of antibiotics, similar results of 25% were found.In our study, two Streptococus species were identified, and all of them were 100% sensitive to antibiotics, with the exception of Enrofloxacin 50%, which was 53% identical to the findings.15

51 isolated Staphylococcus aureus were tested for multidrug resistance, and the resistance to three medications was 3.92%, Staphylococcus aureus was primarily resistant to penicillin and its associated medication, but we did not utilize any antibiotics that were penicillin-related. In addition, research should be conducted to detect resistance in Staphylococcus aureus. Our results were lower than 24.50% in the preceding debate.16 Antibiotic resistance, on the other hand, is spreading like wildfire. Policymakers and the DDA (Department of Drug Administration) should conduct surveillance to discover this condition both globally and in Nepal.