Phenotypic and Molecular Characterization of Bovine Mastitis Milk Origin Bacteria and Linkage of Intramammary Infection With Milk Quality

Abstract

Mastitis is a multi-etiological complex disease of dairy cows and negatively affects the quality and quantity of milk. Milk is a nutritious food for human being; milk quality is negatively affected by intramammary infection of dairy cows. A total of 300 milk samples were collected from mastitis dairy cows irrespective of parity and stage of lactation, 235 (78.33%) samples were culturally positive and yielded 1,100 bacterial isolates. Staphylococcus aureus was found to be the prime etiological agent involved in the mastitis of dairy cows, followed by Escherichia coli and other environmental pathogens. On the molecular characterization of isolates obtained from the milk culture, various toxic genes such as nuc, seb, hla, stx1, stx2, hly, and Sagl were found on different isolated bacteria. Milk somatic cell counts (SCC) were found to be directly related to the severity of mastitis. On drawing the SCC correlation with milk components, it was found that SCC had a significant negative correlation with fat, lactose, solid not fat (SNF), and ash. It was concluded that mastitis-affected milk contains numerous pathogenic bacteria, toxins, and reduced milk quality, which is unfit for human consumption.

Keywords: bacterial isolates; mastitis; milk quality; somatic cell count; toxic genes.

Figure 1

Isolation and cultural characteristics of bacterial isolates from bovine mastitis milk. (a) Staphylococcus aureus on nutrient agar showing mucoid colonies; (b) growth of S. aureus on mannitol salt agar showing yellow colonies (4,5,6) whereas S. epidermidis show pink colonies (1,2,3) (c) S. aureus on blood agar showing βhemolysis; (d) growth of E. coli on MacConkey agar (lactose fermenting, pink colonies); (e) growth of E. coli on EMB agar (typical green metallic sheen) sample no. 2, 4, 5, 6; (f) E. coli showing typical green metallic sheen on EMB agar; (g) Pseudomonas on nutrient agar showing blue-green colony; (h) Pseudomonas on MacConkey agar showing mucoid colony; (i) Klebsiella on EMB agar showing mucoid colony.

Figure 2

Characterization of bacterial isolates by biochemical tests. (a) Catalase test: Effervescence seen on the addition of S. aureus to 3% H₂O₂; (b) tube coagulase test: coagulation observed for S. aureus; (c) oxidase test—development of purple color on disc within 10 s of adding the organism; (d) halo zone observed for S. aureus on DNase agar.

Figure 3

Gram's staining of bacterial isolates. (a) Depicts the Gram negative rods (E. coli) and (b) depicts the Gram positive cocci (bunches) S. aureus.

Figure 4

Amplified PCR products of S. aureus and S. agalactiae genes isolated from mastitis milk samples. (a) PCR product of nuc gene at 270 bp. Lane M: 100 bp ladder; Lane 1: positive control; Lanes 2, 3, 4, 7, and 8 are nuc gene positive; Lane 5, 6: are nuc gene negative; Lane 9: negative control; (b) PCR product of seb gene at 643 bp. M: 100 bp ladder; Lane 1: positive control; Lanes 2, 4, 5, 6, and 8 are seb gene positive; Lane 3, and 7: are seb gene negative; Lane 9: negative control; (c) PCR product of hla gene at 535 bp. M: 100 bp ladder; Lane 1: positive control; Lanes 2, 5, 7, and 8 are hla gene positive; Lane 3, 4, and 6: are hla gene negative; Lane 9: negative control; (d) Lane M: 100 bp ladder; Lane 1: positive control; Lanes 2, 3, 4, 6, 7, and 8: are S agl gene positive; Lane 5: is S agl gene negative; Lane 9: negative control.

Figure 5

Amplified PCR products of E. coli gene isolated from mastitis milk samples. (a) PCR product of E. coli gene at 232 bp. Lane M: 100 bp ladder; Lane 1: negative control; Lanes 2: positive control; Lane 3, 4, 5, 7, 8, and 9: are E. coli gene positive; Lane 6: is E. coli gene negative; (b) PCR product of stx1gene at 180 bp. M: 100 bp ladder; Lane 1: positive control; Lanes 2, 3, 4, 6, 7, and 8: are stx1 gene positive; Lane 5: is stx1gene negative; Lane 9: negative control; (c) PCR product of stx2 gene at 255 bp. M: 100 bp ladder; Lane 8: positive control; Lanes 2, 3: are stx2 gene positive; Lane 1, 4, 6, 7, and 5: are stx2 gene negative; Lane 9: negative control; (d) PCR product of hly gene at 180 bp. M: 100 bp ladder; Lane 1: positive control; Lanes 2, 5, 7, 8: are hly gene positive; Lane 3, 4, 6: are hly gene negative; Lane 9: negative control.