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Veterinary World Jul 2018A critical prerequisite for studying rumen microbial community by high throughput molecular biology methods is good quality community DNA. Current methods of extraction...
BACKGROUND AND AIM
A critical prerequisite for studying rumen microbial community by high throughput molecular biology methods is good quality community DNA. Current methods of extraction use kits designed for samples from the different origin for rumen. This puts stress on the development of a relevant manual method for DNA extraction. The objective of this study was to modify the existing methods of community DNA extraction and thereby systematic comparison of their efficiency based on DNA yield, purity, 16S rRNA gene sequencing, and identification to determine the optimal DNA extraction methods whose DNA products reflect targeted bacterial communities special to rumen.
MATERIALS AND METHODS
Enzymatic method, Chemical method, Enzymatic + Chemical method, and Enzymatic + Chemical + Physical method were modified toward evaluation of community DNA extraction from solid, squeezed, and liquid fractions of goat rumen digesta. Each method was assessed critically for nucleic acid yield and its quality. The methods resulting in high nucleic acid yield, optimal purity ratios with intact band on agarose gel electrophoresis were optimized further. Optimized methods were studied using standard polymerase chain reaction (PCR) with universal bacterial primers and 16S rRNA primers of targeted rumen bacteria. Methods denoting the presence of targeted rumen bacteria were assessed further with 16S rRNA gene sequencing and identification studies. It led toward methods efficacy estimation for molecular biology applications. Effect of rumen sample preservation on community DNA extraction was also studied. Their mean standard deviation values were calculated to understand sampling criticality.
RESULTS
Modified Chemical method (Cetrimonium bromide) and Enzymatic+Chemical+Physical (ECP) method (Lysozyme-Cetrimonium bromide-Sodium Dodecyl Sulfate-freeze-thaw) could extract 835 ng/µl and 161 ng/µl community DNA from 1.5 g solid and 2 ml squeezed rumen digesta with purity ratios of 1.8 (A/A) and 2.3 (A/A) respectively. Comparative analysis showed the better efficiency of ECP method and chemical method toward freshly squeezed rumen digesta and solid rumen digesta. However, sample preservation at -80°C for 1.5 months drastically affected the yield and purity ratios of community DNA. New protocol revealed targeted microbial community having Gram-positive as well as Gram-negative bacteria such as , , , and .
CONCLUSION
To date, this is the first report of modified methods wherein least chemicals and steps lead toward PCR and 16S rRNA gene sequencing quality community DNA extraction from goat rumen digesta. Detection of targeted rumen bacteria in solid and squeezed rumen digesta proves their strongest association with rumen fiber mat. It also marks the presence of distinct microbial communities in solid and squeezed rumen fractions that in turn differs the performance of each different method employed and yield of nucleic acid obtained. It also leaves a possibility of the presence of complex microbial consortia in squeezed rumen digesta whose DNA extraction methods need more attention. Finally, manual protocols of community DNA extraction may vary in different ruminant which suggests undertaking rigorous research in their establishment.
PubMed: 30147271
DOI: 10.14202/vetworld.2018.990-1000 -
Avian Diseases Jun 2018Streptococcal bacterial species represent common inhabitants of the intestinal tract of animals and humans with a potential for opportunistic infections. Streptococcosis...
Streptococcal bacterial species represent common inhabitants of the intestinal tract of animals and humans with a potential for opportunistic infections. Streptococcosis has been identified in turkey poults ( Meleagris gallopavo), ducklings and goslings (Anatidae), broiler chickens, semimature-adult chickens ( Gallus gallus domesticus), and young and adult pigeons (Columbidae). However, the exact underlying factors that lead to bacterial invasion of the blood stream and tissue colonization have not been completely elucidated. The electronic database of the California Animal Health and Food Safety laboratory (Fresno, Tulare, and Turlock branches) was searched for necropsy cases in which streptococcosis was diagnosed in different avian species between January 2000 and August 2017. A total of 95 cases, involving both commercial operations and noncommercial premises, were analyzed. Streptococcus spp., Streptococcus bovis, and Streptococcus gallolyticus were identified from multiple organs, with macroscopic or histopathologic lesions (or both) indicative of septicemia in 23 (24%), 40 (42%), and 30 (32%) cases, respectively. Streptococcus pluranimalium and Streptococcus lutetiensis were also isolated from one (1%) and two (2%) cases, respectively. Turkey poults, broiler chickens, and ducklings were the most-commonly affected species with streptococcosis. Splenitis and hepatitis were the most-common lesions observed and these were the organs with the highest isolation rate. An overview of the clinical and pathologic presentation, and possible predisposing conditions associated with this bacterial infection, is provided.
Topics: Animals; Bird Diseases; California; Chickens; Columbidae; Ducks; Poultry Diseases; Streptococcal Infections; Streptococcus; Turkeys
PubMed: 29944398
DOI: 10.1637/11765-103117-Reg.1 -
Frontiers in Microbiology 2018The complex (SBSEC) comprises several species inhabiting the animal and human gastrointestinal tract (GIT). They match the pathobiont description, are potential... (Review)
Review
The complex (SBSEC) comprises several species inhabiting the animal and human gastrointestinal tract (GIT). They match the pathobiont description, are potential zoonotic agents and technological organisms in fermented foods. SBSEC members are associated with multiple diseases in humans and animals including ruminal acidosis, infective endocarditis (IE) and colorectal cancer (CRC). Therefore, this review aims to re-evaluate adhesion and colonization abilities of SBSEC members of animal, human and food origin paired with genomic and functional host-microbe interaction data on their road from colonization to infection. SBSEC seem to be a marginal population during GIT symbiosis that can proliferate as opportunistic pathogens. Risk factors for human colonization are considered living in rural areas and animal-feces contact. Niche adaptation plays a pivotal role where subsp () retained the ability to proliferate in various environments. Other SBSEC members have undergone genome reduction and niche-specific gene gain to yield important commensal, pathobiont and technological species. Selective colonization of CRC tissue is suggested for , possibly related to increased adhesion to cancerous cell types featuring enhanced collagen IV accessibility. can colonize, proliferate and may shape the tumor microenvironment to their benefit by tumor promotion upon initial neoplasia development. Bacteria cell surface structures including lipotheichoic acids, capsular polysaccharides and pilus loci (, and govern adhesion. Only human blood-derived contain complete pilus loci and other disease-associated surface proteins. Rumen or feces-derived and other SBSEC members lack or harbor mutated pili. Pili also contribute to binding to fibrinogen upon invasion and translocation of cells from the GIT into the blood system, subsequent immune evasion, human contact system activation and collagen-I-binding on damaged heart valves. Only carrying complete pilus loci seem to have highest IE potential in humans with significant links between bacteremia/IE and underlying diseases including CRC. Other SBSEC host-microbe combinations might rely on currently unknown mechanisms. Comparative genome data of blood, commensal and food isolates are limited but required to elucidate the role of pili and other virulence factors, understand pathogenicity mechanisms, host specificity and estimate health risks for animals, humans and food alike.
PubMed: 29692760
DOI: 10.3389/fmicb.2018.00603 -
Journal of Basic Microbiology May 2016The presence of the toxic amino acid mimosine in Leucaena leucocephala restricts its use as a protein source for ruminants. Rumen bacteria degrade mimosine to 3,4- and...
The presence of the toxic amino acid mimosine in Leucaena leucocephala restricts its use as a protein source for ruminants. Rumen bacteria degrade mimosine to 3,4- and 2,3-dihydroxypyridine (DHP), which remain toxic. Synergistes jonesii is believed to be the main bacterium responsible for degradation of these toxic compounds but other bacteria may also be involved. In this study, a commercial inoculum provided by the Queensland's Department of Agriculture, Fisheries, and Forestry was screened for isolation and characterization of mimosine, 3,4- and 2,3-DHP degrading bacterial strains. A new medium for screening of 2,3-DHP degrading bacteria was developed. Molecular and biochemical approaches used in this study revealed four bacterial isolates - Streptococcus lutetiensis, Clostridium butyricum, Lactobacillus vitulinus, and Butyrivibrio fibrisolvens - to be able to completely degrade mimosine within 7 days of incubation. It was also observed that C. butyricum and L. vitulinus were able to partially degrade 2,3-DHP within 12 days of incubation, while S. lutetiensis, was able to fully degrade both 3,4 and 2,3 DHP. Collectively, we concluded that S. jonesii is not the sole bacterium responsible for detoxification of Leucaena. Comprehensive screening of rumen fluid of cattle grazing on Leucaena pastures is needed to identify additional mimosine-detoxifying bacteria and contribute to development of more effective inoculums to be used by farmers against Leucaena toxicity.
Topics: Animals; Bacteria; Cattle; Fabaceae; Inactivation, Metabolic; Mimosine; Pyridines; Rumen
PubMed: 26773324
DOI: 10.1002/jobm.201500590