Md. Humayun Kabir
Bangladesh Livestock Research Institute, Savar, Dhaka, Bangladesh.
Md. Ershaduzzaman
Bangladesh Livestock Research Institute, Savar, Dhaka, Bangladesh.
K. H. M. Nazmul Hussain Nazir
Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh, Bangladesh
Mohammad Sirajul Islam
Bangladesh Livestock Research Institute, Savar, Dhaka, Bangladesh.
Razia Khatun
Bangladesh Livestock Research Institute, Savar, Dhaka, Bangladesh.
Md. Shahjahan Ali Sarker
Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh, Bangladesh
Md. Abu Yousuf
Bangladesh Livestock Research Institute, Savar, Dhaka, Bangladesh.
Yousuf Ali
Bangladesh Livestock Research Institute, Savar, Dhaka, Bangladesh.
Nathu Ram Sarkar
Bangladesh Livestock Research Institute, Savar, Dhaka, Bangladesh.
Md. Giasuddin
Bangladesh Livestock Research Institute, Savar, Dhaka, Bangladesh.
Accuracy, BMT, CMT, Efficacy, Mastitis, Validity
Animal Health and Management
Milk, Performance evaluation
Ethical statement: The milk samples from the animals were collected by field veterinarians by following the international standard considering animal welfare and ethics. Development of BLRI mastitis test (BMT) kit: After a series of trials, BMT was developed at the Bangladesh Livestock Research Institute Regional Station, Sirajganj. Composition of the BMT: sodium carbonate (1%), sodium lauryl ethyl sulphate (0.7%), and bromocresol purple (0.01%). Selection of study area, duration, and study animal: The present study was conducted at Shahjadpur, Sirajganj, Sathia, Pabna, and Mymensingh during July 2017–June 2018. A total of 400 quarters milk samples from 100 apparently healthy crossbred dairy cows were collected. The milk samples were subjected to the screening of SCM by using the newly developed BMT. In addition, 150 milk samples were collected and subjected to somatic cell count (SCC) by direct microscopic count (DMC) and DCC (De Laval cell counter®) to validate the results of BMT and California Mastitis Test (CMT). Sample collection: In this research work, a total of 550 bovine milk samples were collected during morning time. Before the collection of milk, the udder including teat and tips of teat were hygienically washed with water and soaked with 70% alcohol. The milk samples (15 ml from each quarter) were collected in pre-labeled screw-capped vials. CMT and BMT were done at the field prior to milk sample collection. The milk samples were kept in an icebox and transported to the Laboratory of Animal Health, Bangladesh Livestock Research Institute Regional Station, Baghabari, Sirajganj, where maximum tests were performed. As replicas, the samples were kept at 4ºC in a refrigerator for further laboratory investigations at the Department of Microbiology and Hygiene, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh, and Department of Medicine, Faculty of Veterinary Medicine, Chittagong Veterinary and Animal Sciences University, Chittagong. California mastitis test (CMT) and BLRI mastitis test (BMT): A total of 400 quarter milk samples from 100 crossbred dairy cows were subjected to BMT to justify its efficacy to validate as an individual test kit as compared to CMT in detecting SCM based on SCC through DMC. CMT was performed with a commercial CMT kit (Immucell California Mastitis Test Kit, Portland) and the results were scored according to the manufacturer’s instructions. Another study with 150 milk samples, the SCCs were determined by DMC and DCC (De Laval cell counter®) categorized by CMT and BMT scores including average result were performed. The milk samples were mixed properly for homogenization of cream. A drop (0.01 ml/10 μl) of milk was spread evenly over an area of 12 cm on a microscopic glass slide and was air-dried. Then, the milk fat from the slide was removed. For this, the glass slides were dipped in Xylene for 1–2 min and dried again. The dried slide was immersed in 95% ethanol for 2–5 min. Staining with Broadhurst-Paley stain for at least 5 sec was done if necessary. The leukocytes present in 10 microscopic fields were counted as per the method described by Schalm et al. [18]. The following criteria were used in making the cell count: Within a field count all nucleated somatic cells including those at the periphery with more than 50% of the cell body in view. Free nuclei representing more than 50% of the nuclear material are counted. A cytoplasmic mass without a nucleus and small cell fragments with little nuclear material are not counted. Animals were considered as positive for mastitis when CMT and BMT score was ≥1+ and SCC value was ≥2 × 105/ ml of milk (threshold value). The following diagnostic test characteristics were determined using the milk somatic count result as a gold standard control. Accuracy = TP + TN/TP + FP + TN + FN × 100, Sensitivity = TP/TP + FN × 100, Specificity = TN/TN + FP × 100, PPV = TP/TP + FP × 100, NPV = TN/TN + FN × 100, where: TP = True Positive, FP = False Positive, TN = True Negative, FN = False Negative. Sensitivity, specificity, and accuracy test: We used CMT as a gold standard. Number of positive and negative quarters in CMT and BMT was recorded and sensitivity, specificity, accuracy, and predictive value were calculated, as per the method described by Greiner et al. Statistical analysis: Data analysis was done using STATA version 12.1 (STATA Corp., College Station, TX). The percentage accuracy of the tests and sensitivity, specificity, and the predictive values of the CMT and BMT results, compared to SCC were calculated using standard two-by-two contingency tables. Data were also analyzed by Chi-square test to observe the significant influence of CMT and BMT.
J Adv Vet Anim Res. 2019 Sep; 6(3): 425–430.
Journal