Detection and Identification of Lactobacillus Species in Crops of Broilers of Different Ages by Using PCR-Denaturing Gradient Gel Electrophoresis and Amplified Ribosomal DNA Restriction Analysis

Abstract

The microflora of the crop was investigated throughout the broiler production period (0 to 42 days) using PCR combined with denaturing gradient gel electrophoresis (PCR-DGGE) and selective bacteriological culture of lactobacilli followed by amplified ribosomal DNA restriction analysis (ARDRA). The birds were raised under conditions similar to those used in commercial broiler production. Lactobacilli predominated and attained populations of 10 8 to 10 9 CFU per gram of crop contents. Many of the lactobacilli present in the crop (61.9% of isolates) belonged to species of the Lactobacillus acidophilus group and could not be differentiated by PCR-DGGE. A rapid and simple ARDRA method was developed to distinguish between the members of the L. acidophilus group. HaeIII-ARDRA was used for preliminary identification of isolates in the L. acidophilus group and to identify Lactobacillus reuteri and Lactobacillus salivarius. MseI-ARDRA generated unique patterns for all species of the L. acidophilus group, identifying Lactobacillus crispatus, Lactobacillus johnsonii, and Lactobacillus gallinarum among crop isolates. The results of our study provide comprehensive knowledge of the Lactobacillus microflora in the crops of birds of different ages using nucleic acid-based methods of detection and identification based on current taxonomic criteria.The digestive tracts of mammals and birds are home to a diverse collection of bacterial species, collectively referred to as the gut microflora (28). From gnotobiotic animal studies, the microflora is known to influence the biochemistry, immunology, physiology, and nonspecific resistance to intestinal infection of the host (9). The impact of the gut microflora on the nutritional status of farm animals is of particular interest, especially where intensive farming practices are used (4).The crop, ileum, cecum, and colon of poultry are known to harbor bacterial populations (16,27). Recent reports have investigated the composition of the ileal (13) and cecal (35) microflora using bacteriological culture and culture-independent methods. Lactobacilli are numerous in the ileum of broilers, whereas the cecal microflora is dominated by obligately anaerobic bacteria and bacteria yet to be cultivated. From the results of culture-based studies, it has been determined that the microflora of the crop has a simple composition and is dominated by lactobacilli (16,27). Colonization of the surface of the stratified, squamous epithelium of the crop by lactobacilli has been reported by Fuller (6) and Morishita et al. (18). Lactobacillus salivarius, Lactobacillus fermentum or Lactobacillus reuteri, and Lactobacillus acidophilus were the species most commonly detected (16,27). These studies were conducted prior to the reclassification of L. acidophilus, which has been divided into two DNA homology groups containing six related species (5,11,15). DNA homology group A consists of L. acidophilus (A1), Lactobacillus crispatus (A2), Lactobacillus amylovorus (A3), and Lactobacillus gallinarum (A4); DNA homology gr...

Identification of lactic acid bacteria with bio-preservative potential isolated from contaminated avian blood obtained at the slaughterhouse

“…Our results are in agreement with those of other studies where ARDRA is accurate enough to identify species level (Guan et al 2003, Soto et al 2010. However, the bacteria most frequently detected in our experiment were L. salivarius and E. faecalis in contrast with earlier studies where the strains most frequently isolated from the chicken GIT microbiota were Lactobacillus crispatus, Lactobacillus gallinarum, Lactobacillus johnsonii, and Lactobacillus reuteri (Abbas et al 2007, Bjerrum et al 2006, Guan et al 2003. Bacterial diversity and the composition of the microbiota are related to age, rearing environment, production system and diet of chickens (Knarreborg et al 2002, Lu et al 2003.…”

The role of the crop in poultry production

“…Lactobacilli dominate the bacterial community of the crop, but coliforms, streptococci and bifidobacteria have also been found (Abbas Hilmi et al, 2007;Fuller, 1973;Fuller and Brooker, 1974;Fuller and Turvey, 1971;Guan et al, 2003;Peinado et al, 2013;Petr and Rada, 2001). At least some lactobacilli are capable of binding to the crop epithelium to form biofilm layers that are relatively uniquely found on non-secretory stratified squamous epithelium (Edelman et al, 2002;Fuller and Turvey, 1971;Lebeer et al, 2011).…”

Detection, Characterization, and In Vitro and In Vivo Expression of Genes Encoding S-Proteins in Lactobacillus gallinarum Strains Isolated from Chicken Crops

“…Both of the strains adhered to the crop epithelium, consistent with published data predicting that S-proteins of lactobacilli are adhesins. The results of this study provide a basis for the investigation of gene duplication and sequence variation as mechanisms by which bacterial strains of the same species can share the same habitat.Lactobacilli are commonly detected in gut samples collected from animal species, especially those from rodents, pigs (reviewed in reference 35), and chickens (13,15,20,24,45). In the chicken gut, lactobacilli are present in the crop (15), the ileum (20, 24), and the ceca (45).…”

“…Lactobacilli are commonly detected in gut samples collected from animal species, especially those from rodents, pigs (reviewed in reference 35), and chickens (13,15,20,24,45). In the chicken gut, lactobacilli are present in the crop (15), the ileum (20, 24), and the ceca (45).…”