DETERMINATION OF SPECIFIC ENTEROPATHOGEN PRESENCE IN CAPTIVE CHEETAHS (ACINONYX JUBATUS) FED VARIOUS DIETS USING FLUORESCENCE IN SITU HYBRIDIZATION.

Chronic enteropathies pose an important difficulty in the captive management of cheetahs (Acinonyx jubatus) because of suspected multifactorial pathogenesis and the complex nature of enteric microbiota dynamics. Enterobacteriaceae, Campylobacter spp., Clostridium perfringens, Helicobacter spp., and Salmonella spp. are enteropathogens of interest because of their zoonotic potential and suspected contribution to enteropathies. This study aimed to determine the presence of these enteropathogens of interest in fecal samples from cheetahs (N = 48) fed different diets from three different institutions and to investigate the associations between diet, fecal score, and specific enteropathogen presence. Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes were used to visualize and quantify putative enteropathogens in each sample concurrent with selective culturing for Salmonella and Clostridium perfringens. From FISH counts, carcass-fed animals had greater numbers of Enterobacteriaceae compared with animals fed low-fat dog food, although this trend was not statistically significant (P = 0.088). Furthermore, no significant associations were found between fecal score and bacterial load. Abundance of Campylobacter spp., Clostridium perfringens, or Helicobacter spp. as measured by FISH were not correlated with diet or fecal score. On the basis of these data, in agreement with published literature, it is concluded that these microbes may be commensals in the cheetah gastrointestinal tract and do not appear to be a primary cause of abnormal fecal scores.

Phenotypic Characterization and Draft Genome Sequence Analyses of Two Novel Endospore-Forming Sporosarcina spp. Isolated from Canada Goose (Branta canadensis) Feces.

In an attempt to isolate new probiotic bacteria, two Gram-variable, spore-forming, rod-shaped aerobic bacteria designated as strain A4 and A15 were isolated from the feces of Canada geese (Branta canadensis). Strain A4 was able to grow in high salt levels and exhibited lipase activity, while A15 did not propagate under these conditions. Both were positive for starch hydrolysis, and they inhibited the growth of Staphylococcus aureus. The strains of the 16S rRNA sequence shared only 94% similarity to previously identified Sporosarcina spp. The ANI (78.08%) and AAI (82.35%) between the two strains were less than the species threshold. Searches for the most similar genomes using the Mash/Minhash algorithm showed the nearest genome to strain A4 and A15 as Sporosarcina sp. P13 (distance of 21%) and S. newyorkensis (distance of 17%), respectively. Sporosarcina spp. strains A4 and A15 contain urease genes, and a fibronectin-binding protein gene indicates that these bacteria may bind to eukaryotic cells in host gastrointestinal tracts. Phenotypic and phylogenetic data, along with low dDDH, ANI, and AAI values for strains A4 and A15, indicate these bacteria are two novel isolates of the Sporosarcina genus: Sporosarcina sp. A4 sp. nov., type strain as Sporosarcina cascadiensis and Sporosarcina sp. A15 sp. nov., type strain Sporosarcina obsidiansis.

Cecal Microbial Hydrogen Cycling Potential Is Linked to Feed Efficiency Phenotypes in Chickens.

In chickens, early life exposure to environmental microbes has long-lasting impacts on gastrointestinal (GI) microbiome development and host health and growth, via mechanisms that remain uncharacterized. In this study, we demonstrated that administrating a fecal microbiome transplant (FMT) from adults to day-of-hatch chicks results in significantly higher body mass of birds and decreased residual feed intake (RFI), implying enhanced feed efficiency, at 6 weeks of age. To assess the potential mechanisms through which FMT affects adult bird phenotype, we combined 16 S rRNA gene amplification, metagenomic, and comparative genomic approaches to survey the composition and predicted activities of the resident microbiome of various GI tract segments. Early life FMT exposure had a long-lasting significant effect on the microbial community composition and function of the ceca but not on other GI segments. Within the ceca of 6-week-old FMT birds, hydrogenotrophic microbial lineages and genes were most differentially enriched. The results suggest that thermodynamic regulation in the cecum, in this case via hydrogenotrophic methanogenic and sulfur-cycling lineages, potentially serving as hydrogen sinks, may enhance fermentative efficiency and dietary energy harvest capacity. Our study provides a specific mechanism of action through which early-life microbiome transplants modulate market-relevant phenotypes in poultry and, thereby, may represent a significant advance toward microbiome-focused sustainable agriculture.

Considerations and best practices in animal science 16S ribosomal RNA gene sequencing microbiome studies.

Microbiome studies in animal science using 16S rRNA gene sequencing have become increasingly common in recent years as sequencing costs continue to fall and bioinformatic tools become more powerful and user-friendly. The combination of molecular biology, microbiology, microbial ecology, computer science, and bioinformatics-in addition to the traditional considerations when conducting an animal science study-makes microbiome studies sometimes intimidating due to the intersection of different fields. The objective of this review is to serve as a jumping-off point for those animal scientists less familiar with 16S rRNA gene sequencing and analyses and to bring up common issues and concerns that arise when planning an animal microbiome study from design through analysis. This review includes an overview of 16S rRNA gene sequencing, its advantages, and its limitations; experimental design considerations such as study design, sample size, sample pooling, and sample locations; wet lab considerations such as field handing, microbial cell lysis, low biomass samples, library preparation, and sequencing controls; and computational considerations such as identification of contamination, accounting for uneven sequencing depth, constructing diversity metrics, assigning taxonomy, differential abundance testing, and, finally, data availability. In addition to general considerations, we highlight some special considerations by species and sample type.

Emerging Pathogenic Gammaproteobacteria Wohlfahrtiimonas chitiniclastica and Ignatzschineria Species in a Turkey Vulture (Cathartes aura).

New World vultures, such as turkey vultures (Cathartes aura), are obligate scavengers with large geographic ranges. In a preliminary characterization of the turkey vulture (TV) gastrointestinal microbiome in Southern California, we identified 2 recently described emerging bacterial pathogens not previously known to be associated with this avian species. High-throughput sequencing of broad-range 16S rRNA gene amplicons revealed sequences from TV cloacal swabs that were related closest to Wohlfahrtiimonas chitiniclastica and Ignatzschineria species, both Gammaproteobacteria considered by the United States Centers for Disease Control and Prevention as emerging zoonotic pathogens. None of these bacterial sequence types have been previously identified from samples obtained from the turkey vulture gastrointestinal microbiome. With the use of bioinformatics workflows previously established by our research group, we designed specific and sensitive polymerase chain reaction primer sets that represent novel diagnostic assays for the genera Wohlfahrtiimonas and Ignatzschineria. These primer sets were validated by Sanger sequence confirmation from complex TV samples. Because the genera Wohlfahrtiimonas and Ignatzschineria are both known to have dipteran hosts, the molecular diagnostic tools we present here should be useful for better understanding the role of flies, vultures, and other scavengers in the ecology and epidemiology of the genera Wohlfahrtiimonas and Ignatzschineria from a One Health perspective.

Data analysis workflow for the detection of canine vector-borne pathogens using 16 S rRNA Next-Generation Sequencing.

BACKGROUND: Vector-borne diseases (VBDs) impact both human and veterinary medicine and pose special public health challenges. The main bacterial vector-borne pathogens (VBPs) of importance in veterinary medicine include Anaplasma spp., Bartonella spp., Ehrlichia spp., and Spotted Fever Group Rickettsia. Taxon-targeted PCR assays are the current gold standard for VBP diagnostics but limitations on the detection of genetically diverse organisms support a novel approach for broader detection of VBPs. We present a methodology for genetic characterization of VBPs using Next-Generation Sequencing (NGS) and computational approaches. A major advantage of NGS is the ability to detect multiple organisms present in the same clinical sample in an unsupervised (i.e. non-targeted) and semi-quantitative way. The Standard Operating Procedure (SOP) presented here combines industry-standard microbiome analysis tools with our ad-hoc bioinformatic scripts to form a complete analysis pipeline accessible to veterinary scientists and freely available for download and use at https://github.com/eltonjrv/microbiome.westernu/tree/SOP . RESULTS: We tested and validated our SOP by mimicking single, double, and triple infections in genomic canine DNA using serial dilutions of plasmids containing the entire 16 S rRNA gene sequence of (A) phagocytophilum, (B) v. berkhoffii, and E. canis. NGS with broad-range 16 S rRNA primers followed by our bioinformatics SOP was capable of detecting these pathogens in biological replicates of different dilutions. These results illustrate the ability of NGS to detect and genetically characterize multi-infections with different amounts of pathogens in a single sample. CONCLUSIONS: Bloodborne microbiomics & metagenomics approaches may help expand the molecular diagnostic toolbox in veterinary and human medicine. In this paper, we present both in vitro and in silico detailed protocols that can be combined into a single workflow that may provide a significant improvement in VBP diagnostics and also facilitate future applications of microbiome research in veterinary medicine.

Assessment of bacterial and fungal populations in urine from clinically healthy dogs using next-generation sequencing.

BACKGROUND: Urine from clinically healthy dogs is not sterile. Characterizing microbial diversity and abundance within this population of dogs is important to define normal reference ranges for healthy urine. OBJECTIVES: To establish composition and relative representation of bacterial and fungal microbiomes in urine of clinically healthy dogs. ANIMALS: Fifty clinically healthy dogs. METHODS: Analytic study. Urine sampling via cystocentesis. Comprehensive evaluation of urine including standard urinalysis, culture and sensitivity, next-generation sequencing (NGS), and bioinformatics to define bacterial and fungal microbiome. RESULTS: Culture did not yield positive results in any samples. Next-generation sequencing of urine established low presence of bacteria, fungi, or both in all samples. Diversity and abundance of bacterial and fungal communities varied between urine samples from different dogs. Struvite crystals were associated with bacterial community structure (P = .07) and there was a positive correlation between struvite crystals and pH. CONCLUSIONS AND CLINICAL IMPORTANCE: The microbiome in urine of clinically healthy dogs has diverse bacterial and fungal species These findings highlight limitations of conventional culture testing and the need for culture-independent molecular diagnostics to detect microorganisms in urine.

Broiler chickens and early life programming: Microbiome transplant-induced cecal community dynamics and phenotypic effects.

The concept of successional trajectories describes how small differences in initial community composition can magnify through time and lead to significant differences in mature communities. For many animals, the types and sources of early-life exposures to microbes have been shown to have significant and long-lasting effects on the community structure and/or function of the microbiome. In modern commercial poultry production, chicks are reared as a single age cohort and do not directly encounter adult birds. This scenario is likely to initiate a trajectory of microbial community development that is significantly different than non-industrial settings where chicks are exposed to a much broader range of environmental and fecal inocula; however, the comparative effects of these two scenarios on microbiome development and function remain largely unknown. In this work, we performed serial transfers of cecal material through multiple generations of birds to first determine if serial transfers exploiting the ceca in vivo, rather than the external environment or artificial incubations, can produce a stable microbial community. Subsequently, we compared microbiome development between chicks receiving this passaged, i.e. host-selected, cecal material orally, versus an environmental inoculum, to test the hypothesis that the first exposure of newly hatched chicks to microbes determines early GI microbiome structure and may have longer-lasting effects on bird health and development. Cecal microbiome dynamics and bird weights were tracked for a two-week period, with half of the birds in each treatment group exposed to a pathogen challenge at 7 days of age. We report that: i) a relatively stable community was derived after a single passage of transplanted cecal material, ii) this cecal inoculum significantly but ephemerally altered community structure relative to the environmental inoculum and PBS controls, and iii) either microbiome transplant administered at day-of-hatch appeared to have some protective effects against pathogen challenge relative to uninoculated controls. Differentially abundant taxa identified across treatment types may inform future studies aimed at identifying strains associated with beneficial phenotypes.

Draft Genome Sequences of Two Clostridium Isolates from the Poultry Gastrointestinal Tract.

Here, we announce the draft genome sequences of two Clostridium strains, C8-1-8 and C2-6-12, isolated from the cecal contents of commercial broiler chickens (in Athens, GA). These strains may represent potentially novel species within the genus Clostridium, and these draft genomes allow further investigation into potential probiotics for poultry.

Draft Genome Sequences of Two Potentially Novel Bacillus Isolates from Backyard and Commercial Chicken Gastrointestinal Tracts.

Here, we present the draft genome sequences of two Bacillus strains, HF117_J1_D and USDA818B3_A, isolated in Pomona, California, from the gastrointestinal (GI) tract of backyard and commercial broiler chickens, respectively. The draft genomes of both strains appear to represent novel species.

Draft Genome Sequences of Two Paenibacillus Isolates from Goose and Chicken Gastrointestinal Material.

Here, we present the draft genome sequences of two Paenibacillus strains, An7 and USDA918EY, isolated from goose feces (Bend, OR, USA) and chicken ceca (Pomona, CA, USA), respectively. These data may assist with analyses of microorganisms associated with free-ranging and commercial avian species.

Characterization of LysBC17, a Lytic Endopeptidase from Bacillus cereus.

Bacillus cereus, a Gram-positive bacterium, is an agent of food poisoning. B. cereus is closely related to Bacillus anthracis, a deadly pathogen for humans, and Bacillus thuringenesis, an insect pathogen. Due to the growing prevalence of antibiotic resistance in bacteria, alternative antimicrobials are needed. One such alternative is peptidoglycan hydrolase enzymes, which can lyse Gram-positive bacteria when exposed externally. A bioinformatic search for bacteriolytic enzymes led to the discovery of a gene encoding an endolysin-like endopeptidase, LysBC17, which was then cloned from the genome of B. cereus strain Bc17. This gene is also present in the B. cereus ATCC 14579 genome. The gene for LysBC17 encodes a protein of 281 amino acids. Recombinant LysBC17 was expressed and purified from E. coli. Optimal lytic activity against B. cereus occurred between pH 7.0 and 8.0, and in the absence of NaCl. The LysBC17 enzyme had lytic activity against strains of B. cereus, B. anthracis, and other Bacillus species.

Microbial-derived products as potential new antimicrobials.

Due to the continuing global concerns involving antibiotic resistance, there is a need for scientific forums to assess advancements in the development of antimicrobials and their alternatives that might reduce development and spread of antibiotic resistance among bacterial pathogens. The objectives of the 2nd International Symposium on Alternatives to Antibiotics were to highlight promising research results and novel technologies that can provide alternatives to antibiotics for use in animal health and production, assess challenges associated with their authorization and commercialization for use, and provide actionable strategies to support their development. The session on microbial-derived products was directed at presenting novel technologies that included exploiting CRISPR-Cas nucleases to produce sequence-specific antimicrobials, probiotics development via fecal microbiome transplants among monogastric production animals such as chickens and mining microbial sources such as bacteria or yeast to identify new antimicrobial compounds. Other research has included continuing development of antimicrobial peptides such as newly discovered bacteriocins as alternatives to antibiotics, use of bacteriophages accompanied by development of unique lytic proteins with specific cell-wall binding domains and novel approaches such as microbial-ecology guided discovery of anti-biofilm compounds discovered in marine environments. The symposium was held at the Headquarters of the World Organisation for Animal Health (OIE) in Paris, France during 12-15 December 2016.

Characterization of two glycosyl hydrolases, putative prophage endolysins, that target Clostridium perfringens.

Clostridium perfringens, a spore-forming anaerobic bacterium, causes food poisoning and gas gangrene in humans and is an agent of necrotizing enteritis in poultry, swine and cattle. Endolysins are peptidoglycan hydrolases from bacteriophage that degrade the bacterial host cell wall causing lysis and thus harbor antimicrobial therapy potential. The genes for the PlyCP10 and PlyCP41 endolysins were found in prophage regions of the genomes from C. perfringens strains Cp10 and Cp41, respectively. The gene for PlyCP10 encodes a protein of 351 amino acids, while the gene for PlyCP41 encodes a protein of 335 amino acids. Both proteins harbor predicted glycosyl hydrolase domains. Recombinant PlyCP10 and PlyCP41 were expressed in E. coli with C-terminal His-tags, purified by nickel chromatography and characterized in vitro. PlyCP10 activity was greatest at pH 6.0, and between 50 and 100 mM NaCl. PlyCP41 activity was greatest between pH 6.5 and 7.0, and at 50 mM NaCl, with retention of activity as high as 600 mM NaCl. PlyCP10 lost most of its activity above 42°C, whereas PlyCP41 survived at 50°C for 30 min and still retained >60% activity. Both enzymes had lytic activity against 75 C. perfringens strains (isolates from poultry, swine and cattle) suggesting therapeutic potential.

The cecal microbiome of commercial broiler chickens varies significantly by season.

Next-generation DNA sequencing is rapidly becoming a powerful tool for food animal management. One valuable use of this technology is to re-examine long-standing observations of performance differences associated with animal husbandry practices to better understand how these differences may be modulated by the gastrointestinal (GI) microbiome. The influences of environmental parameters such as air temperature and relative humidity on broiler chicken performance have commonly been observed, but how the GI microbiome may respond to seasonal environmental changes remains largely unknown. The purposes of this study were therefore to: (1) characterize the cecal microflora of commercial broilers (N = 87) collected at harvest across all 4 seasons, and (2) identify any significant changes of the GI microbiome and specific taxa according to season and Campylobacter status. Finding taxa with significant positive or negative correlations with Campylobacter could be useful by identifying indicator or antagonistic taxa and could also inform inferences regarding the ecological niche of Campylobacter. Whole GI tracts were removed from commercial broilers representing 87 independent flocks between April 2013 and May 2014 in the U.S. state of Georgia. Intact ceca were separated, cultured for Campylobacter and cecal contents were frozen. The cecal microbiome was characterized using barcoded sequencing of 16S rRNA genes on the Illumina MiSeq platform. The composition of the microbiome measured at processing was generally not affected by Campylobacter status but was most significantly affected by season of grow-out. Significantly fewer bacterial genera were found in winter than spring or summer. Bacterial genera with prior evidence for both positive or negative influences on gut health outcomes were significantly less abundant in the fall. Identifying specific members of the GI microbiota that vary according to season may help develop novel interventions to improve husbandry practices and growth performance.

Assessing Cat Flea Microbiomes in Northern and Southern California by 16S rRNA Next-Generation Sequencing.

Flea-borne diseases (FBDs) impact both human and animal health worldwide. Because adult fleas are obligately hematophagous and can harbor potential pathogens, fleas act as ectoparasites of vertebrates, as well as zoonotic disease vectors. Cat fleas (Ctenocephalides felis) are important vectors of two zoonotic bacterial genera listed as priority pathogens by the National Institute of Allergy and Infectious Diseases (NIAID-USA): Bartonella spp. and Rickettsia spp., causative agents of bartonelloses and rickettsioses, respectively. In this study, we introduce the first microbiome analysis of C. felis samples from California, determining the presence and abundance of relevant pathogenic genera by characterizing the cat flea microbiome through 16S rRNA next-generation sequencing (16S-NGS). Samples from both northern (NoCal) and southern (SoCal) California were assessed to expand current knowledge regarding FBDs in the state. We identified Rickettsia and Bartonella, as well as the endosymbiont Wolbachia, as the most abundant genera, followed by less abundant taxa. In comparison to our previous study screening Californian cat fleas for rickettsiae using PCR/digestion/sequencing of the ompB gene, the 16S-NGS approach applied herein showed a 95% level of agreement in detecting Rickettsia spp. There was no overall difference in microbiome diversity between NoCal and SoCal samples. Bacterial taxa identified by 16S-NGS in this study may help to improve epidemiological investigations, pathogen surveillance efforts, and clinical diagnostics of FBDs in California and elsewhere.

Environmental reservoirs of pathogenic mycobacteria across the Ethiopian biogeographical landscape.

The Mycobacterium genus comprises over one-hundred-and-fifty recognised species, the majority of which reside in the environment and many of which can be pathogenic to mammals. Some species of environmental mycobacteria may interfere with BCG vaccination efficacy and in tuberculin test interpretation. Examining biogeographic trends in the distribution of members of the mycobacteria across a number of physicochemical and spatial gradients in soil and water environments across Ethiopia using oligotyping identified differential distributions of pathogenic and significant species. The tuberculosis complex was identified in more than 90% of water samples and taxonomic groups implicated in lower BCG vaccine efficiency were core in both soil and water Mycobacterium communities. A reservoir of Mycobacterium bovis was identified in water, with up to 7.3×102 genome equivalents per ml. Elevation, temperature, habitat and vegetation type were important predictors of both soil and water Mycobacterium communities. These results represent the first step in understanding the potential risk of exposure to environmental mycobacteria that may undermine efforts to reduce disease incidence.

Spatial and Temporal Changes in the Broiler Chicken Cecal and Fecal Microbiomes and Correlations of Bacterial Taxa with Cytokine Gene Expression.

To better understand the ecology of the poultry gastrointestinal (GI) microbiome and its interactions with the host, we compared GI bacterial communities by sample type (fecal or cecal), time (1, 3, and 6 weeks posthatch), and experimental pen (1, 2, 3, or 4), and measured cecal mRNA transcription of the cytokines IL18, IL1ß, and IL6, IL10, and TGF-ß4. The microbiome was characterized by sequencing of 16S rRNA gene amplicons, and cytokine gene expression was measured by a panel of quantitative-PCR assays targeting mRNAs. Significant differences were observed in the microbiome by GI location (fecal versus cecal) and bird age as determined by permutational MANOVA and UniFrac phylogenetic hypothesis tests. At 1-week posthatch, bacterial genera significantly over-represented in fecal versus cecal samples included Gallibacterium and Lactobacillus, while the genus Bacteroides was significantly more abundant in the cecum. By 6-week posthatch, Clostridium and Caloramator (also a Clostridiales) sequence types had increased significantly in the cecum and Lactobacillus remained over-represented in fecal samples. In the ceca, the relative abundance of sequences classified as Clostridium increased by ca. 10-fold each sampling period from 0.1% at 1 week to 1% at 3 week and 18% at 6 week. Increasing community complexity through time were observed in increased taxonomic richness and diversity. IL18 and IL1ß significantly (p < 0.05, pairwise t-tests) increased to maximum mean expression levels 1.5 fold greater at week 3 than 1, while IL6 significantly decreased to 0.8- and 0.5-fold expression at 3- and 6-week posthatch, respectively relative to week 1. Transcription of pro-inflammatory cytokines was generally negatively correlated with the relative abundance of various members of the phylum Firmicutes and positively correlated with Proteobacteria. Correlations of the microbiome with specific cytokine mRNA transcription highlight the importance of the GI microbiome for bird health and productivity and may be a successful high-throughput strategy to identify bacterial taxa with specific immune-modulatory properties.

Genome Sequence of a Urease-Positive Campylobacter lari Strain.

Campylobacter lari is frequently isolated from shore birds and can cause illness in humans. Here, we report the draft whole-genome sequence of a urease-positive strain of C. lari that was isolated in estuarial water on the coast of Delaware, USA.

A Thermophilic Phage Endolysin Fusion to a Clostridium perfringens-Specific Cell Wall Binding Domain Creates an Anti-Clostridium Antimicrobial with Improved Thermostability.

Clostridium perfringens is the third leading cause of human foodborne bacterial disease and is the presumptive etiologic agent of necrotic enteritis among chickens. Treatment of poultry with antibiotics is becoming less acceptable. Endolysin enzymes are potential replacements for antibiotics. Many enzymes are added to animal feed during production and are subjected to high-heat stress during feed processing. To produce a thermostabile endolysin for treating poultry, an E. coli codon-optimized gene was synthesized that fused the N-acetylmuramoyl-L-alanine amidase domain from the endolysin of the thermophilic bacteriophage ɸGVE2 to the cell-wall binding domain (CWB) from the endolysin of the C. perfringens-specific bacteriophage ɸCP26F. The resulting protein, PlyGVE2CpCWB, lysed C. perfringens in liquid and solid cultures. PlyGVE2CpCWB was most active at pH 8, had peak activity at 10 mM NaCl, 40% activity at 150 mM NaCl and was still 16% active at 600 mM NaCl. The protein was able to withstand temperatures up to 50° C and still lyse C. perfringens. Herein, we report the construction and characterization of a thermostable chimeric endolysin that could potentially be utilized as a feed additive to control the bacterium during poultry production.