PRJNA169064
Chicken Fecal Samples Targeted Locus
Antibiotics are used widely in animal feeds to treat animal disease or as growth promoter in developing countries, especially in some feedlot with much higher density of animals, thus may increase the antibiotic resistant bacteria and change the fecal microbiota in the animal. Feces from 8 chicken’s feces from a small farm house with total 8 chicken raised and scarcely using antibiotic and 10 chicken’s feces from in a huge feedlot with high chicken density and frequently using antibiotic were collected. The fecal microbiota from the feces were separately analyzed by pyro-sequencing 16S rRNA V3. The results suggested that the fecal microbiota of small farm-house chicken is more diverse than that in huge feedlot chicken. The mean Shannon diversity index for the farm-house-chicken samples was 5.321 (SD 0.590), significantly higher than that for the huge feedlot-chicken samples 4.398 (SD 0.440) (P=0.008). Furthermore, 69 aerobic-cultivatable-tetracycline-resistant strains were randomly isolated from the chicken’s feces in small farm house and 65 strains from those in a huge feedlot farm, the kind of species of huge feedlot resources is more diverse than that in small farm house. The study concluded that microbiota of farm house chicken feces was more diverse than that in huge feedlot. To the contrary, the aerobic-cultivatable-tetracycline-resistant bacteria was more diverse in huge feedlot. The results suggested that attention should be paid to the changes of microbiota and antibiotic resistant bacteria in the chicken raised in huge feedlot m, as well as their risks in influencing human health.  To ensure that there were enough 16S rDNA V3 hypervariable region PCR products to harvest, two-step PCR strategy was used. The PCR enrichment of the 16S rDNA V3 hypervariable region was performed with forward primer 5’-barcode-TACGGGAGGCAGCAG-3’ and reverse primer 5’-barcode-ATTACCGCGGCTGCTGG-3’. The 5’ terminal of every primer contained an 8-base-oligonucleotide tag (before the hyphen), while the sequence after the hyphen was able to pare the sequences of the V3 end region. The detail refer to (42). After the PCR reaction, the electrophoresis was immediately performed to isolate the enriched V3 region DNA fragments from the reaction mixture with loading buffer and SybGreen. All of the products were approximately 200 bp in length and were harvested using a gel extraction kit (Gel Extraction Kit, OMEGA Bio-Tek, USA) following the manufacture’s instructions. All of the enriched DNA were mixed to establish the library (50 ng/sample).    The 8 chicken recruit from a small farm house in Licheng town, Li Yan city , JIANG SU province, China, there are total 8 chicken raised in the farm house. The chicken were uptaked with cetotetrine hydrochloride one time through adding into water 13 days ago before sampling because they were ill, the age of chicken is 37 days, the average weight of the chicken is 125 g, the diets of chicken are rice with skin, green vegetable Chinese cabbage and corn-meal-mush. The 10 chicken we enrolled from a huge feedlot ( they have 10,000 chicken) in Nandu town, Li Yan city, JIANG SU province, the chicken’s age is 35 days, average weight of chicken is 7450 g. on the age of 10th day, the chicken were inoculated with Newcastle disease attenuated vaccine by adding into water, on the age of 15th day , they were inoculated with influenza vaccine, before sampling, they were using penicillin in past month, 1-2U/kg adding into water, extends over 3 days; and using amplidox extends over 5 days by add into feed (feed/amplidox=200 g/g), the feed were fed bought from Nantong CHIA TAI Co.,LTD.