Development of ELISA against milk haptoglobin for diagnosis of subclinical mastitis in goats

Abstract

The study described the development of a haptoglobin-based diagnostic tool for mastitis in Ettawa crossbreed goats. Fifty eight milk samples were collected from a flock of goats in Yogyakarta, Central Java, Indonesia. All samples were tested for mastitis using the California Mastitis Test (CMT), Somatic Cell Count (SCC), and Polymerase Chain Reaction (PCR) to identify Staphylococcus aureus , Streptococcus uberis and Streptococcus agalactiae . The presence of haptoglobin mRNA and proteins in the milk somatic cells was detected using Sanger sequencing and SDS-PAGE, respectively. Milk haptoglobin levels were subsequently estimated using an indirect enzyme-linked immunosorbent assay (ELISA) developed in this study. The Receiver Operating Characteristic (ROC) analysis was performed to compare the sensitivity and specificity of CMT, SCC, and the ELISA using the PCR as the reference standard. Kappa test was used to determine the agreement between the three imperfect tests. Results indicated that somatic cells of goat milk expressed a haptoglobin mRNA with a size of 174 bp and two haptoglobin proteins with molecular weights of 18 kDa and 32 kDa. The PCR test showed that 81% of samples were diagnosed positive for mastitis. At a specificity level of 50%, the ROC indicated that the ELISA was more sensitive compared to SCC or CMT (consecutively, 96%, 94%, and 92%). Kappa values between haptoglobin ELISA and CMT or SCC were high (0.84 and 0.81, respectively). This study indicates that somatic cells of goat milk were capable of synthesizing and secreting haptoglobin. Milk haptoglobin can be a potential target for an early detection of mastitis in goats.

Development of antibodies against recombinant staphylococcal enterotoxin B from food poisoning cases

“…This method is very sensitive and can be used to detect low concentrations of antibodies [17]. The ELISA is a fundamental tool for detecting toxins such as SEB [18]. Based on the principles of antibody interactions, ELISA allows for easy visualization of results and can be completed without the use of additional radioactive materials.…”

Food safety and quality. Scientific proceedings

“…agalactiae, Str. dysgalactiae (Windria et al 2021). Intramamárnu infekciu v chove malých prežúvavcov môžu taktiež spôsobiť baktérie ako Bacillus spp., Listeria spp., Corynebacterium spp., Pseudomonas spp., Mycoplasma spp., Enterococcus spp., Mannheimia haemolytica, Clostridium perfringens a Escherichia coli (Granado et al, 2014;Bogdanovičová et al, 2015).…”

The Relationship between Mastitis and Antimicrobial Peptide S100A7 Expression in Dairy Goats

“…The milk somatic cell count determination was used with a direct microscopic somatic cell count (DMSCC). The SCC was counted according to the Diff-Quik Stain manual (Solarbio, G1540, Beijing, China) according to previous researches [40,41]. Briefly, a mixed sample, a pipette of 10 µL of the sample, was placed in the center of a glass slide; the tip of the pipette gently drew a circle and averagely coasted it into an area of 1 cm 2 , fixed formaldehyde for 5 min, immersed in Diff-QuickIDyeing for 5 min and immersion in Diff-QuickIIdyeing for 5 min, 95% alcohol decolored for 5 s, 100% alcohol decolored for 30 s, transparency with xylene for 1 min and sealing, and observed under a microscope.…”