Manreetpal Singh Brar

Gene-centric metagenomics analysis of feline intestinal microbiome using 454 junior pyrosequencing

Abstract The feline gastrointestinal microbiota have direct influence on feline health and also human health as a reservoir for potential zoonotic pathogens and antibiotic resistant bacterial strains. In order to describe the feline gastrointestinal microbial diversity, fecal samples from cats have been characterized using both culture-dependent and culture-independent methods. However, data correlating total microbial composition and their functions are lacking. Present descriptive study evaluated both phylogenetic and metabolic diversity of the feline intestinal microbiota using GS Junior titanium shotgun pyrosequencing. A total of 152,494 pyrosequencing reads (5405 assembled contigs) were generated and classified into both phylogenetic and metabolic profiles of the feline intestinal microbiota. The Bacteroides/Chlorobi group was the most predominant bacterial phylum comprising ~68% of total classified diversity, followed by Firmicutes (~13%) and Proteobacteria (~6%) respectively. Archaea, fungi and viruses made up the minor communities in the overall microbial diversity. Interestingly, this study also identified a range of potential enteric zoonotic pathogens (0.02–1.25%) and genes involved in antimicrobial resistance (0.02–0.7%) in feline fecal materials. Based on clustering among nine gastrointestinal metagenomes from five different monogastric hosts (dog, human, mice, cat and chicken), the cat metagenome clustered closely together with chicken in both phylogenetic and metabolic level (>80%). Future studies are required to provide deeper understandings on both intrinsic and extrinsic effects such as impact of age, genetics and dietary interventions on the composition of the feline gastrointestinal microbiome.

Recombination Is Associated with an Outbreak of Novel Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Viruses in China

ABSTRACT In 2009 to 2010, there was a marked increase in the number of infections with highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) in China. Through phylogenetic analysis, we show that viruses from this outbreak originated from a single recombination event, illustrating the potential importance of this process for disease emergence.

Microbial community in microbial fuel cell (MFC) medium and effluent enriched with purple photosynthetic bacterium ( Rhodopseudomonas sp. )

High power densities have been obtained from MFC reactors having a purple color characteristic of Rhodopseudomonas. We investigated the microbial community structure and population in developed purple MFC medium (DPMM) and MFC effluent (DPME) using 16S rRNA pyrosequencing. In DPMM, dominant bacteria were Comamonas (44.6%), Rhodopseudomonas (19.5%) and Pseudomonas (17.2%). The bacterial community of DPME mainly consisted of bacteria related to Rhodopseudomonas (72.2%). Hydrogen oxidizing bacteria were identified in both purple-colored samples: Hydrogenophaga and Sphaerochaeta in the DPMM, and Arcobacter, unclassified Ignavibacteriaceae, Acinetobacter, Desulfovibrio and Wolinella in the DPME. The methanogenic community of both purple-colored samples was dominated by hydrogenotrophic methanogens including Methanobacterium, Methanobrevibacter and Methanocorpusculum with significantly lower numbers of Methanosarcina. These results suggeste that hydrogen is actively produced by Rhodopseudomonas that leads to the dominance of hydrogen consuming microorganisms in both purple-colored samples. The syntrophic relationship between Rhodopseudomonas and hydrogenotrophic microbes might be important for producing high power density in the acetate-fed MFC under light conditions.

Genomic Evolution of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Isolates Revealed by Deep Sequencing

Most studies on PRRSV evolution have been limited to a particular region of the viral genome. A thorough genome-wide understanding of the impact of different mechanisms on shaping PRRSV genetic diversity is still lacking. To this end, deep sequencing was used to obtain genomic sequences of a diverse set of 16 isolates from a region of Hong Kong with a complex PRRSV epidemiological record. Genome assemblies and phylogenetic typing indicated the co-circulation of strains of both genotypes (type 1and type 2) with varying Nsp2 deletion patterns and distinct evolutionary lineages (“High Fever”-like and local endemic type). Recombination analyses revealed genomic breakpoints in structural and non-structural regions of genomes of both genotypes with evidence of many recombination events originating from common ancestors. Additionally, the high fold of coverage per nucleotide allowed the characterization of minor variants arising from the quasispecies of each strain. Overall, 0.56–2.83% of sites were found to be polymorphic with respect to cognate consensus genomes. The distribution of minor variants across each genome was not uniform indicating the influence of selective forces. Proportion of variants capable of causing an amino acid change in their respective codons ranged between 25–67% with many predicted to be non-deleterious. Low frequency deletion variants were also detected providing one possible mechanism for their sudden emergence as cited in previous reports.

The spread of type 2 porcine reproductive and respiratory syndrome virus (prrsv) in North America: A phylogeographic approach

The emergence and spread of Type 2 Porcine Reproductive and Respiratory Syndrome virus (Type 2 PRRSV) in North America is heavily influenced by the multiple site production system used in the hog industry. However, it is unclear how anthropogenic factors such has this have shaped the current spatial distribution of PRRSV genotypes. We employed Bayesian phylogeographic analyses of 7040 ORF5 sequences to reveal the recent geographical spread of Type 2 PRRSV in North America. The directions and intensities in our inferred virus traffic network closely mirror the hog transportation. Most notably, we reveal multiple viral introductions from Canada into the United States causing a major shift in virus genetic composition in the Midwest USA that went unnoticed by the regular surveillance and field epidemiological studies. Overall, these findings provide important insights into the dynamics of Type 2 PRRSV evolution and spread that will facilitate programs for control and prevention.

Genetic dissection of complete genomes of Type 2 PRRS viruses isolated in Denmark over a period of 15 years

Type 2 Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) was first detected in Europe in 1996 co-incident with the introduction of a live attenuated vaccine. Since then, only limited ORF5 and ORF7 sequences of Type 2 PRRS viruses have been reported throughout Europe. In the present study, the genetic and antigenic diversity of 11 complete genomes and 49 ORF5 and 55 ORF7 nucleotide sequences obtained from 57 viruses in Denmark from 2003 to 2012 were examined. The genetic identity of the 11 complete genomes to the vaccine strain (Ingelvac PRRS MLV) ranged between 93.6 and 99.6% while the 49 ORF5 sequences examined were 94.0-99.8% identical to the vaccine strain. Among the Danish sequences, the pairwise nucleotide identity was 90.9-100% and 93.0-100.0% for ORF5 and ORF7, respectively. Analysis of the genetic region encoding NSP2 revealed high diversity among the Danish viruses with an 86.6-98.9% range in similarity. Furthermore, several of the sequenced viruses harbored deletions in the NSP2 coding region. Phylogenetic analysis in a global Type 2 PRRSV framework classified all Danish isolates to a single cluster (sub-lineage 5.1) which comprised strains closely-related to the Type 2 prototype isolate VR2332.

Porcine reproductive and respiratory syndrome virus in Ontario, Canada 1999 to 2010: Genetic diversity and restriction fragment length polymorphisms

Classification of Ontario porcine reproductive and respiratory syndrome virus (PRRSV) field isolates (n = 505) from 1999 to 2010, based on a global type 2 PRRSV ORF5 phylogenetic framework, revealed genetic diversity comparable to PRRSV in the USA, with sequences assigned to five of nine lineages (1, 2, 5, 8 and 9). Importantly, the tree topology indicated a Canadian ancestry for the highly virulent MN184-related strains that first emerged in 2001 in the USA. Mapping of the RFLP patterns onto the phylogenetic tree revealed numerous examples of different RFLP patterns located within the same phylogenetic cluster. Statistical analysis showed occurrences where similar RFLP patterns masked diverse genetic distances and instances of close genetic proximity with divergent RFLP patterns. Collectively, extensive genetic diversity prevails in type 2 PRRSV in one region of the North American swine industry, and it is not described adequately by RFLP typing, which might have value in differentiating strains at the local farm level.

Triterpenoid herbal saponins enhance beneficial bacteria, decrease sulfate-reducing bacteria, modulate inflammatory intestinal microenvironment and exert cancer preventive effects in Apc Min/+ mice

Saponins derived from medicinal plants have raised considerable interest for their preventive roles in various diseases. Here, we investigated the impacts of triterpenoid saponins isolated from Gynostemma pentaphyllum (GpS) on gut microbiome, mucosal environment, and the preventive effect on tumor growth. Six-week old ApcMin/+ mice and their wild-type littermates were fed either with vehicle or GpS daily for the duration of 8 weeks. The fecal microbiome was analyzed by enterobacterial repetitive intergenic consensus (ERIC)-PCR and 16S rRNA gene pyrosequencing. Study showed that GpS treatment significantly reduced the number of intestinal polyps in a preventive mode. More importantly, GpS feeding strikingly reduced the sulfate-reducing bacteria lineage, which are known to produce hydrogen sulfide and contribute to damage the intestinal epithelium or even promote cancer progression. Meanwhile, GpS also boosted the beneficial microbes. In the gut barrier of the ApcMin/+ mice, GpS treatment increased Paneth and goblet cells, up-regulated E-cadherin and down-regulated N-cadherin. In addition, GpS decreased the pro-oncogenic β-catenin, p-Src and the p-STAT3. Furthermore, GpS might also improve the inflamed gut epithelium of the ApcMin/+ mice by upregulating the anti-inflammatory cytokine IL-4, while downregulating pro-inflammatory cytokines TNF-β, IL-1β and IL-18. Intriguingly, GpS markedly stimulated M2 and suppressed M1 macrophage markers, indicating that GpS altered mucosal cytokine profile in favor of the M1 to M2 macrophages switching, facilitating intestinal tissue repair. In conclusion, GpS might reverse the hosts inflammatory phenotype by increasing beneficial bacteria, decreasing sulfate-reducing bacteria, and alleviating intestinal inflammatory gut environment, which might contribute to its cancer preventive effects.

Tumor Grafting Induces Changes of Gut Microbiota in Athymic Nude Mice in the Presence and Absence of Medicinal Gynostemma Saponins

Recent findings have revealed that gut microbiota plays a substantial role in modulating diseases such as autism, rheumatoid arthritis, allergies, and cancer that occur at sites distant to the gut. Athymic nude mice have been employed for tumorigenic research for decades; however, the relationships between the gut microbiome and host’s response in drug treatment to the grafted tumors have not been explored. In this study, we analyzed the fecal microbiome of nonxenograft and xenograft nude mice treated with phytosaponins from a popular medicinal plant, Gynostemma pentaphyllum (Gp). Analysis of enterobacterial repetitive intergenic consensus (ERIC)-PCR data showed that the microbiota profile of xenograft mice departed from that of the nonxenograft mice. After ten days of treatment with Gp saponins (GpS), the microbiota of the treated mice was closer to the microbiota at Day 0 before the implantation of the tumor. Data obtained from 16S pyrosequencing of fecal samples reiterates the differences in microbiome between the nonxenograft and xenograft mice. GpS markedly increased the relative abundance of Clostridium cocleatum and Bacteroides acidifaciens, for which the beneficial effects on the host have been well documented. This study, for the first time, characterizes the properties of gut microbiome in nude mice responding to tumor implant and drug treatment. We also demonstrate that dietary saponins such as GpS can potentially regulate the gut microbial ecosystem by increasing the number of symbionts. Interestingly, this regulation of the gut ecosystem might, at least in part, be responsible for or contribute to the anticancer effect of GpS.