Detection and characterization of pathogenic Pseudomonas aeruginosa from bovine subclinical mastitis in West Bengal, India

Abstract

Aim: Subclinical mastitis in bovines is mainly responsible for the huge economic loss of the dairy farmers, of which Pseudomonas aeruginosa is one of the causative agents. The study was aimed at a screening of suspected milk samples from different cattle farms of West Bengal for detection and confirmation of P. aeruginosa strains followed by their characterization.

Materials and methods: Around 422 milk samples were screened from different dairy farms primarily by on-spot bromothymol blue (BTB) test and then in the lab by somatic cell counts (SCC) to finally consider 352 samples for detection of P. aeruginosa. Selective isolation and confirmation of the isolates were done using selective media, viz., cetrimide and Pseudomonas agar followed by confirmation by fluorescent technique. Molecular characterization of the strains was done by polymerase chain reaction for the presence of toxA (enterotoxin A, 352 bp) and exoS (exoenzyme S, 504 bp) genes.

Results: Approximately, 371 (87.9%) samples were positive in on-spot BTB test among which 352 (94.8%) samples revealed high SCC values (more than 3 lakh cells/ml) showing infection when screened. Among these, 23 (6.5%) samples yielded typical Pseudomonas sp. isolates out of which only 19 (5.4%) isolates were confirmed to be P. aeruginosa which showed characteristic blue-green fluorescence due to the presence of pigment pyoverdin under ultraviolet light. Out of these 19 isolates, 11 isolates were positive for toxA, 6 isolates for exoS, and 2 for both these pathogenic genes.

Conclusion: Approximately, 5.4% cases of bovine subclinical mastitis infections in South Bengal were associated with P. aeruginosa which possess pathogenic genes such as toxA (63.2%) and exoS (36.8%).

Keywords: Pseudomonas aeruginosa; bovines; characterization; exoS; subclinical mastitis; toxA.

Figure-1

Pink colored bacilli of Pseudomonas aeruginosa isolates in Gram’s staining.

Figure-2

Blue-green fluorescence under ultraviolet light of Pseudomonas aeruginosa isolates.

Figure-3

Detection of toxA gene (352 bp) in Pseudomonas aeruginosa isolates by polymerase chain reaction. Lane 2-4, 6-9: Test samples, 1: Positive control, 5: Negative control.

Figure-4

Detection of exoS gene (504 bp) in Pseudomonas aeruginosa isolates by polymerase chain reaction. Lane 3-4, 6-8: Test samples, 2: Positive control, 5: Negative control.