The application of the loop-mediated isothermal amplification (LAMP) method for diagnosing Enterococcus hirae-associated endocarditis outbreaks in chickens

Abstract

Background: Enterococcus 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.

Results: The 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.

Conclusions: The 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.

Keywords: Broiler chickens; CFU; Endocarditis; Enterococcus hirae; LAMP; qPCR.

Fig. 1

The chicken heart with severe lesions (arrows) on the right atrioventricular valve (asterisk) associated with E. hirae infection

Fig. 2

Location and sequences of primers used in the LAMP assays. The positions of the LAMP primers are shown relative to the sodA gene fragment of Enterococcus hirae (accession no. CP003504.1). Right and left arrows indicate sense and complementary sequences. Green boxes: indicate the outer primers F3 and B3 (product size 248 bp); Blue boxes: indicate forward inner primer FIP (F1c + F2); Grey boxes: indicate backward inner primer BIP (B1c + B2); Yellow boxes: indicate Loop primers LF and LB. Red font indicates the location and sequence of LAMP product (139 bp). Blue font indicates the location of species-specific primers used in standard PCR (product size 187 bp)

Fig. 3

LAMP amplification graph and dissociation curves for E. hirae ATCC 10541 generated by running the assays with two and three sets of primers. Assay with the loop primers resulted in Cq value of 16.13, and melting peak at Tm 86.65 °C, whilst the one without the loop primers Cq value of 35.55, and melting peak at Tm 86.65 °C

Fig. 4

Amplification graph (a) and agarose gel (1.5% w/v) electrophoresis of LAMP products (b) amplified from genomic DNA of E. hirae, other Enterococcus and non-Enterococcus strains. Lane 1: Neg-negative control, Lane 2: Pos-positive control, E. hirae (ladder-like band pattern), Lane 3: M-100-bp DNA ladder (Thermo Fisher Scientific Inc., USA), Lane 4–13: E. casseliflavus, E. cecorum, E. durans, E. faecalis, E. faecium, E. gallinarum, E. raffinosus, Escherichia coli, Staphylococcus aureus, Rimerella anatipestifer

Fig. 5

Agarose gel electrophoresis after (A) LAMP, and (B) PCR assay with F3/B3 LAMP primers and (C) species-specific primers using 10-fold dilutions of DNA E. hirae ATCC 10541 as a sensitivity indicator. a) Neg-Negative control, M-100-bp DNA ladder, Lane 3–11: LAMP assay products using serial dilutions b) Neg-Negative control, M-50-bp DNA ladder (SM0373,Thermo Fisher Scientific Inc., USA), Lane 3–11: PCR results using serial dilutions and F3/B3 LAMP primers (product length 248 bp) c) Neg-Negative control, M-50-bp DNA ladder, Lane 3–11: PCR results using serial dilutions and species-specific primers (product length 187 bp)

Fig. 6

Results of LAMP assay for E. hirae load in affected chicken heart samples (log of CFU/μl of DNA template). Results for hearts were visualized on standard curve, which was generated from a dilution series of genomic DNA E. hirae ATCC 10541 by plotting the quantification cycle values (Cq) against the log of the bacterial quantity (log₁₀ CFU equivalent per μl of DNA template). Linear equation: y = − 3.319x + 41.23. R² = 0,99. Amplification factor = 2.00

Fig. 7

Melting curves generated after LAMP assay using (a) DNA of bacterial strains isolated from heart tissue samples and (b) DNA isolated directly from affected heart tissue samples of broiler chickens. Agarose gel electrophoresis of the LAMP products amplified from DNA of (c) bacterial strains isolated from heart tissue and (d) heart tissue samples of broiler chickens. Pos – positive control (E. hirae ATCC 10541), NTC - Non Template Control, Neg-Negative control, M-100-bp DNA ladder (SM0323, Thermo Fisher Scientific Inc., USA), c) Lane 4–19: bacterial isolates 1–16. d) Lane 4–12: 9 heart samples

Fig. 8

Agarose gel electrophoresis showing the PCR products generated using DNA of bacterial isolates and (a) F3/B3 LAMP primers or (b) species-specific primers, and showing the PCR products generated using DNA isolated directly from the heart tissue samples and (c) F3/B3 LAMP primers or (d) species-specific primers. Neg-Negative control, Pos-Positive control, M-50-bp or 100 bp DNA ladder (Thermo Fisher Scientific Inc., USA), a), b) Lane 3–11: 22 bacterial isolates. c), d) Lane 4–12: 9 heart tissue samples