Prevalence of subclinical mastitis in buffaloes in Ayodhya district of Uttar Pradesh

Introduction

Mastitis is inflammation of the parenchyma of mammary glands resulting in bacteriological changes in milk, drop in milk production and pathological alteration in glandular tissues (Bansod et al., 2021) [2] . It is the most widespread and economically important disease worldwide (Kovacevic et al., 2021) [9] . Detection of subclinical mastitis (SCM) is important for its management and control. SCM is the most expensive and devastating disease in the dairy sector, causing significant financial losses. The annual combined cattle and buffalo milk production was 187.75 million tonnes during the year 2019 in India, which has increased by 6.5% when compared to 2018, and 394 grams per day was the per capita availability of milk (BAHS, 2019) [1] . Among all agents causing mastitis, Staphylococcus aureus is a major pathogen which can play vital and significant role in mastitis (Lundberg et al., 2014) [12] . According to study conducted by Krishnamoorthy et al. (2021) [10] the bacterial species-wise analysis revealed a higher prevalence of S. aureus (25%) mainly related to subclinical mastitis and S. epidermidis (9%). There are two forms of mastitis: clinical form and subclinical form. The clinical form is characterized by local visible inflammatory changes in milk and udder tissue with or without systemic clinical signs, whereas subclinical form does not manifest clinical signs of mastitis but increased somatic cell counts with the presence of the causative agent. The diagnosis of the subclinical form of mastitis requires cow side test such as California mastitis test (CMT) or various laboratory tests including somatic cell count (SCC) and milk bacteriological culture. Early diagnosis of subclinical form of mastitis is very much essential for successful treatment and control of infection (Lakshmi, 2016) [11] Subclinical mastitis has a negative influence on the performance of dairy cows and buffaloes. It has a significant impact on production, nearly 10-20% decrease in milk production, as it causes an undesirable effect on the milk constituents, which affects its nutritional value and renders it unfit for processing and consumption (Iraguha et al., 2015) [7] . It includes reduced milk production, changes in milk quality and treatment cost. Rathod et al. (2017) [17] estimated the economic losses due to subclinical form of mastitis were approximately in the range of INR 21,677 to INR 88,340 per animal for a lactation period. Mastitis, specifically sub clinical type is well known for development of multiple drug resistance strains and emergence of new pathogens due to indiscriminate use of antimicrobials over long period (Raberg et al., 2007) [15] . Hence it becomes important to diagnose the etiological agent of subclinical mastitis and subsequently the effective antimicrobial to prevent the indiscriminate use of antibiotics. Thus, the present study was designed to determine the prevalence of subclinical mastitis in buffaloes of Ayodhya district.

Materials and Methods

Selection of animals

The area for study was at Ayodhya district of Uttar Pradesh. Milk samples were collected from lactating buffaloes with the history of decreased milk yield but showed no visible physical abnormality in udder and changes in milk. Most of the buffaloes under study were between four to eight years of age. On the basis of stratified random sampling technique three blocks were selected, then two villages from each block and thirty samples were collected from each village (total 180 samples). The physical examination of udder was performed for exclusion of abnormalities of udder and teat (Schalm et al., 1971) [18] .

Clinical examination of udder of buffalo

Thorough examination of udder was done to find any abnormalities in the udder like presence of any lesion, pain, heat and swelling. Milk from each quarter was stripped out for detecting the abnormalities in the milk related to colour and consistency. These examinations were continued until final selection of buffaloes that had sub clinical mastitis (Radostits et al., 2007) [16] .

Collection of milk samples

Collection of milk sample in each case was as follows, washing the udder and teat with clean water and mild antiseptic solution (potassium permanganate), wiping the thoroughly with dry clean cloth, discarding first 2 to 3 milking streams from each quarter and then collecting about 10 ml of milk sample in a sterile container.

Screening of buffaloes for sub clinical mastitis

Tests namely White side test and California mastitis test were used to detect sub clinical mastitis in buffaloes and those samples which showed strong positive reaction were selected for isolation of S. aureus. The CMT was performed to detect the presence of sub clinical infections, procedure described by Quinn et al. (2011) [14] . White side test was performed using standard procedures as described by Schalm et al. (1971) [18] .

Microbiological assay of milk sample

Milk samples collected from buffaloes with subclinical mastitis were incubated in sterile nutrient broth at 37 °C for 24 hr to check growth of bacteria; and then bacteria were streaked on mannitol salt agar (MSA) plate and incubated in incubator, the grown bacterial colonies were stained with Gram's staining. Gram positive bacteria were transferred to nutrient agar slant.

Identification of S. aureus

Each sample of milk was directly inoculated onto mannitol salt agar (MSA) and incubated at 37 °C for 24 hours to identify S. aureus, as described by Talan et al. (1989) [21] . Mannitol fermented colonies from primary cultures were purified by subculture onto nutrient agar slants and incubated at 37 °C for 24-48 hour and stored at 4 °C until further use. Grams stained slides were examined according to Barrow and Feltham, (2003) [3] .

Biochemical characterization of Staphylococcus aureus

For biochemical characterization of bacteria, Methyl Red test, Voges-Proskauer test, catalase test, Slide and Tube coagulase test, Nitrate reduction test and haemolysis on blood agar were performed.

Results and Discussion

The screening of animals for detection of sub clinical mastitis was done by selecting three different blocks with two different villages in each block in Ayodhya district. Total 180 buffaloes from 3 blocks and 6 villages were screened using California Mastitis Test (CMT) and White Side Test (WST). Among the total of 180 buffaloes screened 72 (40%) buffaloes were tested as positive for sub clinical mastitis. Amongst three blocks that were selected Milkipur block had a greater number of positive cases i.e., 28 (46.66%), followed by Harrington Ganj block with 23 (38.33%) positive cases and Khandasa block with least number of cases i.e., 21 (35%). Our findings are in agreement with findings of Hussain et al. (2020) [6] which showed 43.6% prevalence of mastitis. Hussain et al. (2020) [6] reported higher prevalence of subclinical mastitis in buffalos i.e., 79.1%. Biswas et al. (2020) [4] found that the overall prevalence of SCM in buffalo was 20.0% and Sharma et al. (2018) [19] also found that overall prevalence of SCM in buffaloes on animal basis was 23.85% in buffaloes, which were less than the findings of present study. Amongst six villages selected, Nimadi village had a highest number of positive cases i.e., 16 (53.33%), followed by Deogiri 14 (46.67%), Kadanpur 12 (40%), Gadouli 11 (36.67%), Kanji 10 (33.33%) and Reona village with least number of cases i.e., 9 (30%). The prevalence findings of subclinical mastitis in villages Reona and Kanji are in agreement with findings of Sharma et al. (2018) [19] and Srinivasan et al. (2013) [20] which showed prevalence of subclinical mastitis as 33.76%, and 28.33% respectively. Out of total 180 animals' samples screened for subclinical mastitis, 59 (32.77%) samples were tested as positive for S. aureus. Among the samples that were positive for mastitis i.e., 72, the number of samples that were positive for S. aureus was 59 (81.94%). Amongst three blocks that were selected Khandasa and Milkipur both had 20 (33.33%) samples positive for S. aureus and Harrington Ganj block had 19 (31.67%) samples positive for S. aureus. According to Chhabra et al. (2020) [5] prevalence of S. aureus was 36.46% which is in accordance with the present study. Study report of Krishnamoorthy et al. (2021) [10] also suggests that prevalence of S. aureus was 28%. Amongst six villages selected Nimadi village had a highest number of positive cases for S. aureus i.e., 12 (40%), followed by Deogiri and Gadouli both with 11 (36.67%), Kanji with 9 (30%) and then Kadanpur and Reona villages with least number of cases i.e., 8 (26.67%). Maalik et al.

(2019) [13] suggested that 25% of the samples tested were positive for S. aureus. The findings of present study are lesser than findings of Hussain et al. (2020) [6] who reported that the frequency of Staphylococcus aureus mastitis was 65% and also lesser than the 45% reported by Khan et al.

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