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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 × 10 2  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 × 10 7  CFU/g of tissue, and the average number of  E. hirae  genome equivalents in hearts was 5.51× 10 6  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  sod A-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.

bmc microbiology

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