Real-time polymerase chain reaction for detection and enumeration of <i>Staphylococcus aureus</i> and <i>Streptococccus agalactiae</i> using different milk samplings

Dibbern, Aline Gerato; Gonçalves, Juliano Leonel; Alves, Bruna Gomes; Barreiro, Juliana Regina; Anderson, Kevin; Santos, Marcos Veiga dos

doi:10.37496/rbz5220210086

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Animal production systems and agribusiness • R. Bras. Zootec. 52 • 2023 • https://doi.org/10.37496/rbz5220210086 copy

Real-time polymerase chain reaction for detection and enumeration of Staphylococcus aureus and Streptococccus agalactiae using different milk samplings

Authorship SCIMAGO INSTITUTIONS RANKINGS

ABSTRACT

The objective of the present study was to evaluate the qPCR for detection and enumeration of Staphylococcus aureus and Streptococcus agalactiae using different milk samplings in comparison to the conventional microbiology. Four dairy herds with a history of subclinical mastitis caused by S. aureus and S. agalactiae were selected. Sampling approach included milk samples from bulk tank (BT), cow level (composite samples, CO), and mammary quarter level (MQ) from 785 lactating cows. Three consecutive monthly milk samplings were carried out, totaling 3347 MQ milk samples, 912 CO, and 12 from BT. All collected milk samples were subjected to conventional microbiology and qPCR for detection and enumeration of S. aureus and S. agalactiae. The qPCR showed 71.5% of diagnostic sensitivity for S. aureus isolated from MQ milk samples, 71.8% for CO, and 50% for BT milk samples compared with conventional microbiology methodology. Taken together, the diagnostic sensitivity for S. agalactiae isolated from MQ milk samples was 90.2, 87.7 for CO, and 90.9% for BT milk samples. In general, the qPCR methodology enabled the detection of S. aureus and S. agalactiae, regardless of the type of milk sampling. The direct use of milk samples to estimate the counting of S. aureus by qPCR demonstrated lower sensitivity than the counting of S. agalactiae, which can be explained by the pathogen infection dynamics and differences in milk sample type.

bovine mastitis; identification; molecular microbiology; qPCR

Gene	Primer	Bp	Tm (℃)
SAG¹	F: 5′-TTTGGTGTTTACACTAGACTG-3′ R: 5′-TGTGTTAATTACTCTTATGCG-3′	120 bp	77-78
SAU²	F: 5’-CCTGAAGCAAGTGCATTTACGA-3’ R: 5’-CTTTAGCCAAGCCTTGACGAACT-3’	166 bp	75-76

Sample type	S. aureus		S. agalactiae		Total
Sample type	MC	qPCR	MC	qPCR	Total
Composite	76 (8.3%)	90 (9.9%)	58 (6.3%)	150 (16.4%)	912
Mammary quarters	134 (4.0%)	98 (2.9%)	161 (4.8%)	147 (4.4%)	3347

S. aureus					S. agalactiae
		Microbiological culture					Microbiological culture
	MQ¹	Presence	Absence	Total		MQ²	Presence	Absence	Total
qPCR	Presence	93	0	93	qPCR	Presence	138	0	138
qPCR	Absence	37	0	37	qPCR	Absence	15	0	15
	Total	130	0	130		Total	153	0	153
		Microbiological culture					Microbiological culture
	CO¹	Presence	Absence	Total		CO²	Presence	Absence	Total
qPCR	Presence	56	16	72	qPCR	Presence	50	65	115
qPCR	Absence	22	20	42	qPCR	Absence	7	10	17
	Total	78	36	114		Total	57	75	132
		Microbiological culture					Microbiological culture
	BT¹	Presence	Absence	Total		BT²	Presence	Absence	Total
qPCR	Presence	6	0	6	qPCR	Presence	10	2	12
qPCR	Absence	6	0	6	qPCR	Absence	1	0	1
	Total	12	0	12		Total	11	2	13

Pathogen	Count equivalence				Identification agreement
Pathogen	N	CI 95%	SEM	P-value	ҡ	CI (95%)	SEM
S. aureus	5	−0.5191 to 0.5207	0.1872	0.9968	0.71	−1.00 to 0.79	0.052
S. agalactiae	4	−0.6077 to 0.6113	0.1915	0.9931	0.64	−1.00 to 0.75	0.066

Sample type	Pathogen	ҡ	CI (95%)	SEM
Bulk tank	S. aureus	0.00	−1.00 to 0.00	0.000
Bulk tank	S. agalactiae	0.28	−1.00 to 0.29	0.005
Composite	S. aureus	0.00	−1.00 to 0.00	0.000
Composite	S. agalactiae	0.29	−1.00 to 0.32	0.017
Mammary quarters	S. aureus	0.00	−1.00 to −1.00	0.000
Mammary quarters	S. agalactiae	0.29	−1.00 to 0.36	0.043

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E-mail: rbz@sbz.org.br

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[1] *Corresponding author: mveiga@usp.br