A PCR-based strategy for simple and rapid detection of Lactobacillus spp. in content from Duodenum, Jejunum, Ileum and Cecum of broiler chickens

The gastrointestinal microflora is known to affect the health and performance of broilers. Prevention of the intestinal epithelium from pathogenic bacteria invasion by Lactobacillus spp. strains and performance improvement of broilers has been reported. This microflora has been analyzed by the use of bacteriological culture methods. These approaches have problems such as low sensitivities, inability to detect non-cultivable bacteria and unknown species, time-consuming aspects and low levels of reproducibility. The goal of this study was to develop a molecular method allowing rapid detection of the Lactobacillus spp. strains at gastrointestinal segments and different ages. Thirty-six Commercial broilers were raised and at the ages 4, 14 and 30 days, eight birds were randomly sacrificed. Contents of duodenum, jejunum, ileum and cecum were prepared and their DNA extracted. The specific detection of the Lactobacillus spp. were based on PCR amplification of the 16S rRNA gene using oligonucleotide primers LAA-f and LAA-r which PCR product size was 286 bp. For total intestine segments, a total of 77 samples were positive for naturally occurring Lactobacillus spp. by PCR method. This value represented an incidence of 80.21% with respect to the total gut samples. Furthermore, 65.62, 90.62 and 84.37% samples were positive for naturally occurring Lactobacillus spp. by PCR method at 4, 14 and 30 days respectively. Meanwhile anterior segments exhibited nearly higher levels of Lactobacillus spp. compared with the posterior segments. The method presented in this manuscript was developed for routine, careful and rapid detection of a large number of samples on a daily basis. (author)