Evaluation of Surf Field Mastitis Test (SFMT) For the Detection of Subclinical Mastitis in Dairy Cows

Mastitis is inflammation of the udder characterized by elevated somatic cells and changes in physical, chemical and microbial quality in milk (Waseem et al., 2020). High producing dairy animals suffer from mastitis leading to economic losses to the farmers. Broadly, mastitis is categorized as clinical and subclinical mastitis; of these, the subclinical mastitis (SCM) occurs in higher frequency and eventually leads to clinical mastitis (Mdegela et al., 2009). Prevalence of cow-wise and quarter-wise subclinical mastitis has been estimated to be 46.35% (95% CI 39.38; 53.46) and 23.25% (95% CI 18.15; 29.27), respectively among dairy cows in India (Bangar et al., 2015). Diagnosis of clinical mastitis is based on examination of udder for physical and structural changes, milk for increased markers (SCC, enzymes, etc) and detection of mastitogens (Perreten, 2013; Kotb, 2014). However, detection of SCM is more difficult as changes in udder and milk are non obvious; this requires indirect detection techniques such as California Mastitis Test (CMT), electrical conductivity, enzyme (e.g. LDH, NAGase, etc) measurements (Sharma et al., 2018). A diagnostic test to be ideal for the detection of SCM in cows must be sensitive, specific, rapid, repeatable and economic. Isolation and identification of mastitis causing agents is still considered as gold standard for the diagnosis of mastitis (El-Sayed et al., 2017; Karabasanavar et al., 2019). Nevertheless, it cannot be applied for routine diagnosis of SCM at the field levels. Hence, various detection tests are in vogue for the field level detection of SCM. Therefore, present study was undertaken to evaluate cow-side tests for their application at field level for the detection of SCM.

Sample collection and analysis
 
Milk samples were collected from individual quarters from crossbred (Holstein Friesian or Jersey) lactating dairy cows (n=72) of Harave village of Chamarajanagar district (Karnataka state, India) from June 2018 to June 2019. Samples were brought to laboratory (Department of VPE, Veterinary College, Hassan) under chilled conditions and screened for mastitis using California mastitis test (CMT) as described by Schalm and Noorlander (1957) and CMT was scored using the grading procedure of Schneider and Jasper (1964). Somatic cell count (SCC) was performed in accordance with Sharma and Rajani (1969). Surf field mastitis test (SFMT) test was performed in accordance with Muhammad et al., (2010). Briefly, 3% household detergent Surf Excel(R) (Unilever Ltd.) was prepared in water and shaken for a minute followed by testing with equal volumes of milk and detergent solutions. After thorough mixing by swirling, appearance of floccules or gel of varying degrees was read in accordance with Muhammad et al., (1995).
       
Milk samples were collected from individual quarters of 18 crossbred (Holstein Friesian and Jersey) dairy cows and screened for subclinical mastitis (SCM) using CMT, SCC and SFMT. Cows testing positive for CMT (scores 1 to 3) and SCC (count >2 Lakh cells per millilitre) were clinically examined for mastitis such as cardinal signs of udder inflammation, deviations in cow’s health and gross changes in milk; cows affected with only subclinical mastitis (SCM) i.e. without any obvious signs of mastitis were identified as SCM positive cases.
 
Statistical analysis
 
Descriptive statistics were calculated using MS-Excel program and online statistical tool (Wessa, 2017). Diagnostic tests were evaluated using standard reference (Ausubel et al., 1997; Gardner and Greiner, 1999) and agreements between tests was evaluated based on kappa value in accordance with Sachs (1984) and Viera and Garret, (2005) i.e. <0.1 (no); 0.1-0.4 (weak) i.e. 0.1-0.2 (sight) and 0.21-0.40 (fair); 0.41-0.60 (clear/moderate); 0.61-0.80 (strong/substantial) and 0.81-0.99 (almost perfect/nearly complete) agreement or concordance.

Elevated somatic cell count (SCC) during the process of intramammary infection in cows is a cardinal sign of mastitis. Somatic cells comprise of mostly of leucocytes and epithelial cells in milk, whose number increase significantly during mastitis consequent to invasion of pathogens in the udder (Schukken et al., 2003; Green et al., 2004). Normal somatic cell counts range from 10,000 to 100,000 cells/mL in the milk obtained from uninfected cows (Hillerton, 1999); during intra-mammary infections SCC raises above 2,00,000 cells/mL (Kandeel et al., 2018). However, in case of subclinical mastitis, cow’s udder even though infected, the signs of mastitis such as physico-chemical changes in the milk or udder changes are non-obvious (Sinha et al., 2018). This requires indirect methods such as measurement of cells, electrical conductivity, enzyme estimations, biomarker analysis, etc (Hillerton 1999). Even though validated rapid farm level cell counters are available for screening of SCM in organized dairies, cost of consumables and instrumentation at times hinder their application at the field level. To suit cow-side SCM detection, CMT has emerged as the most preferred test at the field level (Malinowski et al., 2008; Kandeel et al., 2018). The CMT is cow-side on farm semi-quantitative SCM screening test that also works on the principle of chemical reaction with milk somatic cells.
       
After mixing with the anionic surfactant in CMT, somatic cells breakdown leading to the release of DNA and pH dependent ionic interaction results in surfactant-DNA binding, it is evidenced by the gel formation (Nageswararao et al., 1969; Whyte et al., 2005). Similar effect occurs when Surf Excel(R) is added to the milk containing somatic cells in the SFMT. Nevertheless, diagnostic gel formation to detect SCM is achieved by cheaper and widely available household detergent instead of CMT reagent (Muhammad et al., 2010).
       
The CMT can be performed by mixing milk with equal volume of CMT reagent and looking for changes in the milk and grading it as T, 1, 2 and 3 scores so as to approximately indicate somatic cell count. The CMT screening of SCM takes a minute time and has been one of the most widely used tests worldwide over six decades. Nevertheless, the CMT reagent is required for testing; which is a major limiting factor for screening cows under small holder rural setups. Interestingly, household detergent Surf Excel® performs same function as like that of CMT reagent. The results of CMT and SFMT are compared with SCC and presented in Table 1.      
 

       
In the present study, CMT and SFMT showed equal proximity to SCC as measured by kappa concordance and correlation coefficient. In a similar study, Badiuzzaman et al., (2015) evaluated different SCM diagnosis tests and compared them with cultural isolation (gold standard); wherein, SCC showed highest sensitivity and specific of 86.6% and 97.8% followed by CMT (80.08%, 69.40%), white side test (61.71%, 63.38%)  and SFMT (60.54%, 60.66%), respectively. Further, CMT showed higher analytical accuracy and also positive and negative predictivity compared to SFMT. Iqbal et al., (2006) also compared a battery of tests and concluded strong similarity of SFMT to CMT; and suggested SFMT for field use due to its low cost, easy availability and readily acceptance by the stakeholders.
       
Although, CMT has been recognized as popular cow-side test, owing to its diagnostic performance; SFMT also deserves its merit for the detection of SCM under small holder rural conditions. Cost-wise, testing SCM by SFMT incurs just one hundredth of the expenditure of CMT reagent; 12 g Surf ExcelTM (M/s. Hindustan Unilever Ltd.) sachet in the grocery shop costs just Rs. 2/- and as many as 20 cows can be tested with 3% surf solution (12 g in 400 ml water). Surf Excel detergent is widely available in rural groceries; dairy farmers can be easily trained to prepare 3% surf in water; after mixing equal volume of 3% surf with milk, gel formation can be easily observed so as to indicate the SCM.
       
Kumari et al., (2019) studied low cost interventions for SCM and found SFMT as an alternative to costly tests such as SCC, electrical conductivity, etc. Comparative evaluation of field level SCM detection tests made in this study indicated SFMT as one of the simplest, economical and reliable cow-side screening test that do not require laboratory setup, equipment and testing can be performed rapidly thereby aiding farmers to opt for further control strategies (Sargeant et al., 2001).

Mastitis continues to be the major economic burden of dairy farms worldwide and infections affect about 30% of dairy cattle (Hillerton and berry 2005; Halasa et al., 2007); timely interventions suiting local conditions must be discovered to tackle SCM. Results of the present study indicated practical applications of SFMT at the field level owing to easy the availability of the test material. Keeping in view the economic significance of SCM to the dairy sector, SFMT requires popularization among dairy farmers for screening cows for SCM so that suitable interventions could be initiated in-time thereby protecting farmers from economic losses associated with mastitis and also ensure supply of safe milk to the public.

This study was funded by World Bank sponsored Sujala Watershed Project-Component III, Government of Karnataka. Authors duly acknowledge Director of Research, KVAFSU, Bidar, Nodal Officer of the project and Dean, Veterinary College, Hassan.