Porcine fungal mock community analyses: Implications for mycobiome investigations.

Introduction: The gut microbiome is an integral partner in host health and plays a role in immune development, altered nutrition, and pathogen prevention. The mycobiome (fungal microbiome) is considered part of the rare biosphere but is still a critical component in health. Next generation sequencing has improved our understanding of fungi in the gut, but methodological challenges remain. Biases are introduced during DNA isolation, primer design and choice, polymerase selection, sequencing platform selection, and data analyses, as fungal reference databases are often incomplete or contain erroneous sequences. Methods: Here, we compared the accuracy of taxonomic identifications and abundances from mycobiome analyses which vary among three commonly selected target gene regions (18S, ITS1, or ITS2) and the reference database (UNITE - ITS1, ITS2 and SILVA - 18S). We analyze multiple communities including individual fungal isolates, a mixed mock community created from five common fungal isolates found in weanling piglet feces, a purchased commercial fungal mock community, and piglet fecal samples. In addition, we calculated gene copy numbers for the 18S, ITS1, and ITS2 regions of each of the five isolates from the piglet fecal mock community to determine whether copy number affects abundance estimates. Finally, we determined the abundance of taxa from several iterations of our in-house fecal community to assess the effects of community composition on taxon abundance. Results: Overall, no marker-database combination consistently outperformed the others. Internal transcribed space markers were slightly superior to 18S in the identification of species in tested communities, but Lichtheimia corymbifera, a common member of piglet gut communities, was not amplified by ITS1 and ITS2 primers. Thus, ITS based abundance estimates of taxa in piglet mock communities were skewed while 18S marker profiles were more accurate. Kazachstania slooffiae displayed the most stable copy numbers (83-85) while L. corymbifera displayed significant variability (90-144) across gene regions. Discussion: This study underscores the importance of preliminary studies to assess primer combinations and database choice for the mycobiome sample of interest and raises questions regarding the validity of fungal abundance estimates.

Temporal dynamics of the chicken mycobiome.

The microbiome is an integral part of chicken health and can affect immunity, nutrient utilization, and performance. The role of bacterial microbiota members in host health is relatively well established, but less attention has been paid to fungal members of the gastrointestinal tract (GIT) community. However, human studies indicate that fungi play a critical role in health. Here, we described fungal communities, or mycobiomes, in both the lumen and mucosa of the chicken ileum and cecum from hatch through 14 days of age. We also assessed the effects of delayed access to feed immediately post-hatch (PH) on mycobiome composition, as PH feed delay is commonly associated with poor health performance. Chicken mycobiomes in each of the populations were distinct and changed over time. All mycobiomes were dominated by Gibberella, but Aspergillus, Cladosporium, Sarocladium, Meyerozyma, and Penicillium were also abundant. Relative abundances of some taxa differed significantly over time. In the cecal and ileal lumens, Penicillium was present in extremely low quantities or absent during days one and two and then increased over time. Meyerozyma and Wickerhamomyces also increased over time in luminal sites. In contrast, several highly abundant unclassified fungi decreased after days one and two, highlighting the need for improved understanding of fungal gut biology. Mycobiomes from chicks fed during the first 2 days PH versus those not fed during the first 2 days did not significantly differ, except during days one and two. Similarities observed among mycobiomes of fed and unfed chicks at later timepoints suggest that delays in PH feeding do not have long lasting effects on mycobiome composition. Together, these results provide a foundation for future mycobiome studies, and suggest that negative health and production impacts of delayed feeding are not likely related to the development of fungal populations in the GIT.

One-Step Homology Mediated CRISPR-Cas Editing in Zygotes for Generating Genome Edited Cattle.

Selective breeding and genetic modification have been the cornerstone of animal agriculture. However, the current strategy of breeding animals over multiple generations to introgress novel alleles is not practical in addressing global challenges such as climate change, pandemics, and the predicted need to feed a population of 9 billion by 2050. Consequently, genome editing in zygotes to allow for seamless introgression of novel alleles is required, especially in cattle with long generation intervals. We report for the first time the use of CRISPR-Cas genome editors to introduce novel PRNP allelic variants that have been shown to provide resilience towards human prion pandemics. From one round of embryo injections, we have established six pregnancies and birth of seven edited offspring, with two founders showing >90% targeted homology-directed repair modifications. This study lays out the framework for in vitro optimization, unbiased deep-sequencing to identify editing outcomes, and generation of high frequency homology-directed repair-edited calves.

Temporal Dynamics of the Gut Bacteriome and Mycobiome in the Weanling Pig.

Weaning is a period of environmental changes and stress that results in significant alterations to the piglet gut microbiome and is associated with a predisposition to disease, making potential interventions of interest to the swine industry. In other animals, interactions between the bacteriome and mycobiome can result in altered nutrient absorption and susceptibility to disease, but these interactions remain poorly understood in pigs. Recently, we assessed the colonization dynamics of fungi and bacteria in the gastrointestinal tract of piglets at a single time point post-weaning (day 35) and inferred interactions were found between fungal and bacterial members of the porcine gut ecosystem. In this study, we performed a longitudinal assessment of the fecal bacteriome and mycobiome of piglets from birth through the weaning transition. Piglet feces in this study showed a dramatic shift over time in the bacterial and fungal communities, as well as an increase in network connectivity between the two kingdoms. The piglet fecal bacteriome showed a relatively stable and predictable pattern of development from Bacteroidaceae to Prevotellaceae, as seen in other studies, while the mycobiome demonstrated a loss in diversity over time with a post-weaning population dominated by Saccharomycetaceae. The mycobiome demonstrated a more transient community that is likely driven by factors such as diet or environmental exposure rather than an organized pattern of colonization and succession evidenced by fecal sample taxonomic clustering with nursey feed samples post-weaning. Due to the potential tractability of the community, the mycobiome may be a viable candidate for potential microbial interventions that will alter piglet health and growth during the weaning transition.

Yeasts of Burden: Exploring the Mycobiome-Bacteriome of the Piglet GI Tract.

Interactions between the bacteria and fungi in the gut microbiome can result in altered nutrition, pathogenicity of infection, and host development, making them a crucial component in host health. Associations between the mycobiome and bacteriome in the piglet gut, in the context of weaning, remain unknown. Weaning is a time of significant stress, dietary changes, microbial alterations, and a predisposition to infection. The loss of animal health and growth makes potential microbial interventions of interest to the swine industry. Recent studies have demonstrated the diversity and development of the microbiome in the gastrointestinal (GI) tract of piglets during weaning, resulting from the dietary and physiological changes. Despite these advances, the role of the mycobiota in piglet health and its contribution to overall microbiome development remains mostly unknown. In this study we investigated the bacteriome and the mycobiome after weaning in the GI tract organs and feces from 35-day old piglets. Following weaning, the α-diversity and amplicon sequence variants (ASVs) counts of the bacteriome increased, proximally to distally, from the stomach to the feces along the GI tract, while the mycobiome α-diversity and ASV counts were highest in the porcine stomach. ß-diversity analyses show distinct clusters based on organ type in the bacteriome and mycobiome, but dispersion remained relatively constant in the mycobiome between organ/fecal sites. Bacteroidetes, Firmicutes, and Epsilonbacteraeota were the most abundant bacterial phyla present in the GI tract and feces based on mean taxonomic composition with high variation of composition found in the stomach. In the mycobiome, the dominant phyla were Ascomycota and Basidiomycota, and the stomach mycobiome did not demonstrate the same high level of variation observed in the bacteriome. Potential interactions between genera were found in the lower piglet GI bacteriome and mycobiome with positive correlations found between the fungus, Kazachstania, and several bacterial species, including Lactobacillus. Aspergillus demonstrated negative correlations with the short chain fatty acid-producing bacteria Butyricoccus, Subdoligranulum, and Fusicatenibacter. This study demonstrates the distinct colonization dynamics between fungi and bacteria in the GI tract and feces of piglets directly following weaning and the potential interactions of these microbes in the porcine gut ecosystem.

The piglet mycobiome during the weaning transition: a pilot study1.

The importance of the microbiota in the gastrointestinal tract of animals is recognized as a critical player in host health. Recently, the significance of the mycobiome has been recognized, but culture-independent studies are limited, especially in swine. Weaning is a time of stress, dietary changes, and a predisposition to infections, making it a time point of interest to industry. In this pilot study, we sought to assess and characterize the mycobiome in the feces of swine from birth through the critical weaning transition to investigate the mycobiome population and its temporal dynamics in piglet feces. Cultured fecal samples demonstrate a significant increase in fungal burden following weaning that does not differ from adult levels, suggesting stable colonization. Culturable fungi were not found in any environmental samples tested, including water, food, sow milk or colostrum. To determine the fungal diversity present and to address the problem of unculturable fungi, we performed a pilot study utilizing ITS and 16S rRNA focused primers for high-throughput sequencing of fungal and bacterial species, respectively. Bacterial populations increase in diversity over the experimental timeline (days 1 to 35 postbirth), but the fungal populations do not demonstrate the same temporal trend. Following weaning, there is a dynamic shift in the feces to a Saccharomycetaceae-dominated population. The shift in fungal population was because of the dominance of Kazachstania slooffiae, a poorly characterized colonizer of animal gastrointestinal tracts. This study provides insights into the early colonization and subsequent establishment of fungi during the weaning transition in piglets. Future studies will investigate the effect of the mycobiome on piglet growth and health during the weaning transition.