Abstract
BackgroundEnterococcus hirae is considered a part of the normal intestinal biota of several domestic animals, including poultry. However, this species is also associated with infective endocarditis in chickens, a disease that leads to unexpected deaths and serious economical losses. Enterococcus hirae is identified predominantly with the use of conventional bacteriological methods, biochemical tests and PCR. Rapid, sensitive and specific methods for detecting E. hirae in clinical samples are required in poultry production. The aim of this study was to use the Loop-Mediated Isothermal Amplification (LAMP) for the identification and quantification of E. hirae in heart samples from broiler chickens.ResultsThe specificity of the LAMP method was confirmed for 7 enterococcal strains and 3 non-enterococcal strains. E. hirae was detected in all of the 22 analyzed clinical bacterial isolates and in all of the 9 heart samples. Three sets of primers supported the detection of E. hirae with high sensitivity and specificity within one hour. The highest detection rate of a LAMP product was approximately 7 min for an E. hirae strain and 12 min for a positive heart sample. The detection limit for the E. hirae ATCC 10541 standard was 1.3 × 102 CFU (43.4 fg) or 13.8 copies of the E. hirae genome equivalent per reaction. The reaction was 10-fold more sensitive than conventional species-specific PCR. The LAMP assay supported the determination of the E. hirae load in chicken hearts with endocarditis in field cases. The average number of E. hirae cells in hearts was 5.19 × 107 CFU/g of tissue, and the average number of E. hirae genome equivalents in hearts was 5.51× 106 copies/g of tissue. Bacterial counts were significantly higher in the LAMP assay than in the standard plate count.ConclusionsThe LAMP assay is a useful diagnostic tool and an effective alternative to conventional methods for the detection of this enterococcal species. The sodA-based LAMP assay supported direct identification of E. hirae from pure cultures and heart samples without previous bacterial cultivation. This is the first study to apply the LAMP method for the purpose of diagnosing E. hirae-associated endocarditis in poultry.
Highlights
Enterococcus hirae is considered a part of the normal intestinal biota of several domestic animals, including poultry
The first amplified products of the sodA gene fragment from the reference strain of E. hirae were detected within 8 min with loop primers (Cq 16.13; Melting temperature (Tm) 86.65 °C), and within 18 min (Cq 35.55; Tm 86.65 °C) without loop primers (Fig. 3)
The nucleotide sequence of the LoopMediated Isothermal Amplification (LAMP) product of the control strain was highly similar (99%) to E. hirae, and it was deposited in GenBank (MG581167)
Summary
Enterococcus hirae is considered a part of the normal intestinal biota of several domestic animals, including poultry. This species is associated with infective endocarditis in chickens, a disease that leads to unexpected deaths and serious economical losses. Sensitive and specific methods for detecting E. hirae in clinical samples are required in poultry production. E hirae was first described as a new species in 1985 by Farrow and Collins in strains that had been previously referred to as Enterococcus faecium. Enterococcus hirae is considered a part of normal intestinal biota and an opportunistic pathogen in birds and mammals [2,3,4]. In chickens from tropical regions, E. hirae was detected in cecal and cloacal swabs only in birds older than 8 weeks [6]
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