Recipient Organization
UNIV OF ARKANSAS
(N/A)
PINE BLUFF,AR 71601
Performing Department
Department of Agriculture
Non Technical Summary
Though there has been more focus on the use of probiotics in large ruminants and their impact on the gut and fecal microbiota (microbiome) and the known foodborne pathogens, there has been much less attention on the small ruminants and their commensal microbiota and its role in spreading foodborne infections and multi-drug resistance (MDR) pathogens. A few recent studies started to focus on the prevalence of antimicrobial resitance (AMR) genes in the commensal microbiota. Hence, the gut microbiota shows greater rate of horizontal gene transfer (HGT) than that of bacteria in other environments. For better understanding and finding ways to combat the spread of MDR and to improve the food safety measures in small ruminants (goats) farms, we need to have more studies focusing on the probiotics in goat and their effect on the gut and fecal microbiome.
Animal Health Component
60%
Research Effort Categories
Basic
20%
Applied
60%
Developmental
20%
Goals / Objectives
The overall objective of this proposal is to study the effect of using the probiotics (live microbes) for food animals to enhance the microbiome (microbial diversity) in gut and fecal matter to force out the pathogenic microbes which are the potential source of microbial contamination.The aim of the current study: To investigate the changes in the microbiome composition after exposure to probiotics treatment, and AMR transfer dynamics. To achieve the study aims, several approaches will be used including the 16S rRNA gene sequencing to determine bacterial taxonomy of gut microbiota populations; shotgun metagenomics analyses to identify the diversity of genes present within the microbiota, such as AMR genes and those associated with HGT; and meta-transcriptomic analyses for profiling of community-wide gene expression (RNA-seq) to evaluate factors that impact to the development and spread of AMR.
Project Methods
Study design: A finishing goat experiment will be conducted in two phases during the summer 2018 and winter 2019. The experimental design will be a randomized complete block design with sampling group and initial body weight (BW). Animal experiments will be conducted according to the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC). The animal studies protocol will be submitted for approval by the Institutional Animal Care and Use Committee (IACUC), at the University of Arkansas at Pine Bluff (UAPB). Treatments will be assigned randomly to pens, consists of a product with (L. acidophilus strain), a 2nd treatment group consists of a product with (Bifidobacteria strain) and 3rd with no supplementation (control) group. Treatments will be given every day of the experiment. Fecal samples will be collected from the rectum of each animal every 15 days starting on day 0 and extending through the duration of the finishing period.First experiment, 12 male goats (buckling) age around 5-6 months with an average initial BW of 50 lb will be procured. Initial BW will be the average of BW taken for two consecutive days. Following the initial weigh day, buckling will be assigned randomly within pens using a random number generator to 4 pens (one head per pen)/ each group. Pens will be assigned randomly to either control or treated. On the second day of the initial weigh period (day 0), buckling will be fed the finishing diet with or without the probiotic products (control). Buckling will be sampled every 2-weeks during 10 weeks experimental period, resulting in one pretreatment sampling and five separate test period samplings. The pretreatment sample will be collected on day 0, whereas the five test-period samplings will be collected on days 15, 30, 45, 60, and 75.In the second experiment, goats will be sampled every 21 days, resulting in one pretreatment sampling and six separate test period samplings. The pretreatment sample will be collected on day 0, whereas the five test-period samplings will be collected on days 21, 42, 63, 84, and 105.In each year of the study, commercial probiotic products will be used and will be applied to the feed truck mixing box and fed per animal daily according to the manufacturers recommendations. In both years, buckling will be fed once daily.Separate feed equipment and utensils will be used to feed the control and treated diets to eliminate the chance of cross contamination.Performance measures collected included average daily gain, dry matter intake measured on a daily basis, and feed efficiency.Samples collection:In both years, fecal samples will be collected directly from the rectum of each animal as they will be restrained in a small ruminants processing chute. Each fecal sample will be placed in an individually labeled specimen cup. All samples will be placed in a cooler with ice packs and immediately transported to the laboratory. A blinded design experiment will be used as the laboratory personnel will be blinded to treatments and field investigators will be blinded to test results until the study is completed.Microbial analysis:Fecal samples will be analyzed for presence of Salmonella species and E. coli O157:H7, using procedures previously described (Melendez et al. 2010; Smith et al. 2005), respectively. Samples will be diluted in peptone water and dilutions of sample supernatant will be spiral plated on selective media (TBX, Difco) which will allow for the simultaneous determination of coliforms, E. coli (generic) and Salmonella. Isolates that collectively are sorbitol non-fermenting, lactose-fermenting, negative for r-glucuronidase activity, and positive for the 0157 antigen will be tested in a five-primer pair multiplex PCR assay. The multiplex PCR detected genes for E. coli O157: H7 (fliCh 7 ), Shiga toxins 1 (stx1) and 2 ( stx2 ), and intimin (eae O157 ) (Potter et al. 2004). Detection of genes for O157: H7, and at least one other target genes will be considered for confirmation. Salmonella characterization will be performed following our previously published study (Sanad et al. 2016).Rumenal juice samples:For rumenal juice samples collection, a stomach tube sampling method will be used. A flexible PVC tube (2 mm of wall thickness and 6 mm of internal diameter with about 20 holes of 3 mm diameter in the 12 cm-probe head will be warmed-up using hot water and inserted to the rumen via the mouth and esophagus. Rumen samples (50 ml) will be collected using an electric vacuum pump. Before being strained through a nylon membrane. A 20 cm long handle sampling scoop will be used to collect rumen contents samples through the cannula from different parts of the dorsal sac in the rumen. An average of three samples will be taken, composited, aliquoted (20 ml). For each animal, samples will be first collected via stomach tube before morning feeding. The pH will be measured using a pH-meter. All these samples will be immediately frozen and stored at−80?C until subsequent molecular analyses (Ramos-Morales et al. 2014).Microbial community (Microbiome) analysisDNA Extraction:The DNA will be isolated from 200 mg of frozen rumen fluid and fecal samples following the methods previously described by Khafipour et al. (2009) using the ZR fecal DNA kit (D6010; Zymo Research Corp., Orang, CA, USA) that included a bead-beating step for mechanical disruption of microbial cells.b.Illumina sequencingMicrobial community structure of the samples will be assessed using Illumina sequencing of 16S rRNA genes isolated from the gut microbiota. The 16S rRNA genes will be amplified using specific primers that target the V3-V4 hypervariable regions as described before (Derakhshani et al. 2016), libraries prepared the purified products sequenced with the Miseq platform (Illumina, San Diego, CA) using V3 chemistry with 300 bp paired-end reads. Sequences from the 16S rRNA amplicon data will be analysed using the QIIME pipelines.c.Shotgun metagenome sequencing and analysisThe metagenome will be fragmented, tagged and quantified according to the Nextera® XT Sample preparation guide (Illumina, USA). Sequencing will be done on the MiSeq platform using V3 chemistry and 150 bp paired-end reads. Metagenome data mapping and assembly will be performed on Geneious following the recommended criteria.Reference genomes for assembly and annotation will be downloaded from the NCBI database.Statistical analysis:The effect of the probiotics products will be tested by modeling the probability of detecting E. coli O157:H7 from feces using the logit link function in a binomial distribution multivariable generalized estimation equation model. A first-order autoregressive correlation structure will be defined to account for repeated measures of animals over time, sampling blocks, and year. The data from our experiments will be analyzed using STATA statistical software (StataCorp LLC, College Station, Texas, USA). P values of <0.05 will be considered significant for our findings. Also, we will use CLC Main Workbench 7.8 (Qiagen Bioinformatics) for sequence data analysis.