Effects of Eimeria acervulina infection on the luminal and mucosal microbiota of the cecum and ileum in broiler chickens.

Coccidiosis, an intestinal disease caused by Eimeria parasites, is responsible for major losses in the poultry industry by impacting chicken health. The gut microbiota is associated with health factors, such as nutrient exchange and immune system modulation, requiring understanding on the effects of Eimeria infection on the gut microbiota. This study aimed to determine the effects of Eimeria acervulina infection on the luminal and mucosal microbiota of the cecum (CeL and CeM) and ileum (IlL and IlM) at multiple time points (days 3, 5, 7, 10, and 14) post-infection. E. acervulina infection decreased evenness in CeL microbiota at day 10, increased richness in CeM microbiota at day 3 before decreasing richness at day 14, and decreased richness in IlL microbiota from day 3 to 10. CeL, CeM, and IlL microbiota differed between infected and control birds based on beta diversity at varying time points. Infection reduced relative abundance of bacterial taxa and some predicted metabolic pathways known for short-chain fatty acid production in CeL, CeM, and IlL microbiota, but further understanding of metabolic function is required. Despite E. acervulina primarily targeting the duodenum, our findings demonstrate the infection can impact bacterial diversity and abundance in the cecal and ileal microbiota.

Cecal microbiota composition differs under normal and high ambient temperatures in genetically distinct chicken lines.

Modern broilers, selected for high growth rate, are more susceptible to heat stress (HS) as compared to their ancestral jungle fowl (JF). HS affects epithelia barrier integrity, which is associated with gut microbiota. The aim of this study was to determine the effect of HS on the cecal luminal (CeL) and cecal mucosal (CeM) microbiota in JF and three broiler populations: Athens Canadian Random Bred (ACRB), 1995 Random Bred (L1995), and Modern Random Bred (L2015). Broiler chicks were subjected to thermoneutral TN (24 °C) or chronic cyclic HS (8 h/day, 36 °C) condition from day 29 until day 56. HS affected richness in CeL microbiota in a line-dependent manner, decreasing richness in slow-growing JF and ACRB lines, while increasing richness in faster-growing L1995 and L2015. Microbiota were distinct between HS and TN conditions in CeL microbiota of all four lines and in CeM microbiota of L2015. Certain bacterial genera were also affected in a line-dependent manner, with HS tending to increase relative abundance in CeL microbiota of slow-growing lines, while decreases were common in fast-growing lines. Predictive functional analysis suggested a greater impact of HS on metabolic pathways in L2015 compared to other lines.

Physiological effects of in ovo delivery of bioactive substances in broiler chickens.

The poultry industry has improved genetics, nutrition, and management practices, resulting in fast-growing chickens; however, disturbances during embryonic development may affect the entire production cycle and cause irreversible losses to broiler chicken producers. The most crucial time in the chicks' development appears to be the perinatal period, which encompasses the last few days of pre-hatch and the first few days of post-hatch. During this critical period, intestinal development occurs rapidly, and the chicks undergo a metabolic and physiological shift from the utilization of egg nutrients to exogenous feed. However, the nutrient reserve of the egg yolk may not be enough to sustain the late stage of embryonic development and provide energy for the hatching process. In addition, modern hatchery practices cause a delay in access to feed immediately post-hatch, and this can potentially affect the intestinal microbiome, health, development, and growth of the chickens. Development of the in ovo technology allowing for the delivery of bioactive substances into chicken embryos during their development represents a way to accommodate the perinatal period, late embryo development, and post-hatch growth. Many bioactive substances have been delivered through the in ovo technology, including carbohydrates, amino acids, hormones, prebiotics, probiotics and synbiotics, antibodies, immunostimulants, minerals, and microorganisms with a variety of physiological effects. In this review, we focused on the physiological effects of the in ovo delivery of these substances, including their effects on embryo development, gastrointestinal tract function and health, nutrient digestion, immune system development and function, bone development, overall growth performance, muscle development and meat quality, gastrointestinal tract microbiota development, heat stress response, pathogens exclusion, and birds metabolism, as well as transcriptome and proteome. We believe that this method is widely underestimated and underused by the poultry industry.

Effects of Eimeria acervulina infection on the luminal and mucosal microbiota of the duodenum and jejunum in broiler chickens.

The intestinal disease coccidiosis, caused by Eimeria parasites, impacts nutrient absorption in broiler chickens, leading to weight gain depression and major losses in the poultry industry. To develop alternatives to antibiotics for treating infected chickens, the gut microbiota has been researched because of its association with health factors such as nutrient exchange, immune system modulation, digestive system physiology, and pathogen exclusion. The aim of this study was to determine the effect of Eimeria acervulina infection on the luminal and mucosal microbiota of both the duodenum (DuoL and DuoM) and jejunum (JejL and JejM) at multiple time points (days 3, 5, 7, 10, and 14) post-infection. 16S rRNA amplicon sequencing was utilized to characterize the microbiota and analyze differences in alpha and beta diversity between infected (IF) and control (C) birds at each time point. Alpha diversity differed between IF and C birds in DuoM and JejM microbiota. Combined with beta diversity results, DuoM microbiota appeared to be affected by infection in the longer-term, while JejM microbiota were affected in the shorter-term. Relative abundances of bacterial taxa known for short-chain fatty acid (SCFA) production, such as Lachnospiraceae, Subdoligranulum, and Peptostreptococcaceae, tended to be lower in IF birds for all four microbiota. Moreover, predicted functional abundances showed MetaCyc pathways related to SCFA production, especially butyrate, may be influenced by these differences in bacterial relative abundance. Our findings expand understanding of how Eimeria infection affects luminal and mucosal microbiota in the duodenum and jejunum, and further research on metagenomic function may provide insights on the degree of influence duodenal and jejunal bacteria have on chicken health.

Temporal changes of genes associated with intestinal homeostasis in broiler chickens following a single infection with Eimeria acervulina.

Infection with the protozoan parasite Eimeria can cause the economically devastating disease coccidiosis, which is characterized by gross tissue damage and inflammation resulting in blunted villi and altered intestinal homeostasis. Male broiler chickens at 21 d of age were given a single challenge with Eimeria acervulina. Temporal changes in intestinal morphology and gene expression were investigated at 0, 3, 5, 7, 10, and 14 d postinfection (dpi). There were increased crypt depths for chickens infected with E. acervulina starting at 3 dpi and continuing to 14 dpi. At 5 and 7 dpi, infected chickens had decreased Mucin2 (Muc2), and Avian beta defensin (AvBD) 6 mRNA at 5 and 7 dpi and decreased AvBD10 mRNA at 7 dpi compared to uninfected chickens. Liver-enriched antimicrobial peptide 2 (LEAP2) mRNA was decreased at 3, 5, 7, and 14 dpi compared to uninfected chickens. After 7 dpi, there was increased Collagen 3a1 and Notch 1 mRNA compared to uninfected chickens. Marker of proliferation Ki67 mRNA was increased in infected chickens from 3 to 10 dpi. In addition, the presence of E. acervulina was visualized by in situ hybridization (ISH) with an E. acervulina sporozoite surface antigen (Ea-SAG) probe. In E. acervulina infected chickens, Ea-SAG mRNA was only detectable on 5 and 7 dpi by both ISH and qPCR. To further investigate the site of E. acervulina infection, Ea-SAG and Muc2 probes were examined on serial sections. The Muc2 ISH signal was decreased in regions where the Ea-SAG ISH signal was present, suggesting that the decrease in Muc2 by qPCR may be caused by the loss of Muc2 in the localized regions where the E. acervulina had invaded the tissue. Eimeria acervulina appears to manipulate host cells by decreasing their defensive capabilities and thereby allows the infection to propagate freely. Following infection, the intestinal cells upregulate genes that may support regeneration of damaged intestinal tissue.

The Supplementation of FloraMax-B11 Did Not Affect the Bile Acid Neosynthesis and the Enterohepatic Circulation in Broiler Chickens.

Most probiotics possess bile salt hydrolase enzymes and may increase bile acid excretion and negatively affect fat digestion and absorption. Therefore, the study objective was to determine the time course effects of a commercial probiotic (P) FloraMax-B11 (FM) supplementation on bile acid neosynthesis and enterohepatic circulation in broiler chickens. Fertile Ross 708 eggs were incubated under standard commercial conditions. At hatch, chicks (n = 550) were randomly assigned to 5 treatment groups (n = 5 replicates per treatment group) with 22 birds per pen. The 5 treatment groups consisted of: control group (C, normal water from hatch to 35 days of age without supplements); P3, water supplemented with FM for the first 3 days post-hatch followed by normal water until day 35; P10, water supplemented with FM for the first 10 days post-hatch followed by normal water until day 35; P35, water supplemented with FM from hatch to day 35; and AGP, water supplemented with antibiotic growth promoter (AGP) from hatch until day 35. Ileum, liver, and plasma were collected at hatch, days 3, 10, 21, and 35 post-hatch. The relative mRNA expression of genes involved in bile acid synthesis (CYP7A1, CYP8B1, FXR, FGFR4, and FGF19) and transport (ASBT, I-BABP, OSTα, OSTß, and BSEP) as well as ileal deoxycholic acid and plasma cholic acid were determined. There was no FM and AGP interaction for any of the response criteria. No FM or AGP effects were observed (p > 0.05) for any genes, except FGF19, which expression was increased (p < 0.0001) in AGP compared to P35. No FM or AGP effects were observed (p > 0.05) for levels of deoxycholic and cholic acids. However, all the genes, deoxycholic acid, and plasma cholic acid were affected by age (p < 0.0001). In general, the data indicate that FM did not negatively impact bile acid metabolism and enterohepatic circulation, which appeared to be age dependent. However, more research should be conducted to confirm these results and investigate the effects of FM on bile acid metabolism, fat digestion, and intestinal microbiota in broiler chickens.

Delayed access to feed early post-hatch affects the development and maturation of gastrointestinal tract microbiota in broiler chickens.

BACKGROUND: The first two weeks of post-hatch (PH) growth in broilers (meat-type birds) are critical for gut development and microbiota colonization. In the current broiler production system, chicks may not receive feed and water for 24 to 72 h due to variations in hatching time and hatchery management. Post-hatch feed delay affects body weight, feed efficiency, mortality, and gut development. The goal of this study was to investigate changes in the microbiome in broiler chickens early PH and the effect of delayed access to feed on the microbiota. RESULTS: Chicks either received feed and water immediately after hatch or access to feed was delayed for 48 h to mimic commercial hatchery settings (treatment, TRT). Both groups were sampled (n = 6) at -48, 0, 4 h, and 1 (24 h), 2 (48 h), 3 (72 h), 4 (96 h), 6 (144 h), 8 (192 h), 10 (240 h), 12 (288 h) and 14 (336 h) days PH. Ileal (IL) and cecal (CE) epithelial scrapings (mucosal bacteria, M) and digesta (luminal bacteria, L) were collected for microbiota analysis. Microbiota was determined by sequencing the V3-V4 region of bacterial 16S rRNA and analyzed using QIIME2. The microbiota of early ileal and cecal samples were characterized by high abundance of unclassified bacteria. Among four bacterial populations (IL-L, IL-M, CE-L, CE-M), IL-M was the least affected by delayed access to feed early PH. Both alpha and beta diversities were affected by delayed access to feed PH in IL-L, CE-M and CE-L. However, the development effect was more pronounced. In all four bacterial populations, significant changes due to developmental effect (time relative to hatch) was observed in taxonomic composition, with transient changes of bacterial taxa during the first two weeks PH. Delayed access to feed has limited influence on bacterial composition with only a few genera and species affected in all four bacterial populations. Predicted function based on 16S rRNA was also affected by delayed access to feed PH with most changes in metabolic pathway richness observed in IL-L, CE-L and CE-M. CONCLUSIONS: These results show transient changes in chicken microbiota biodiversity during the first two weeks PH and indicate that delayed access to feed affects microbiota development. Proper microbiota development could be an important factor in disease prevention and antibiotic use in broiler chickens. Moreover, significant differences in response to delayed access to feed PH between luminal and mucosal bacterial populations strongly suggests the need for separate analysis of these two populations.

Altered gene expression of selected matrix metalloproteinase system proteins in the broiler chicken gastrointestinal tract during post-hatch development and coccidia infection.

Matrix metalloproteinases (MMPs) are a family of proteases, that can process extracellular matrix (ECM) components and non-ECM molecules. MMPs can also function intracellularly in proteolytic and nonproteolytic functions. The participation of MMPs in the remodeling of the chicken gastrointestinal tract is largely unknown. The aim of the present study was to examine 1) the early neonatal developmental changes and effect of delayed access to feed immediately post-hatch (PH) and 2) the effect of Eimeria infection on mRNA expression of selected MMPs, their tissue inhibitors (TIMPs), and a disintegrin and metalloproteinase (ADAM) metallopeptidase with thrombospondin type 1 motif 8 (ADAMTS8) in the gastrointestinal tract of chicken. Protein localization of MMPs and TIMPs was also carried out in the normal ileal wall at -48, 24, and 336 h relative to hatch using immunofluorescence. In experiment 1, newly hatched Ross 708 chicks received feed and water immediately PH or were subjected to 48 h delayed access to feed. Chickens were sampled at -48, 0, 4, 24, 48, 72, 96, 144, 192, 240, 288, and 336 h PH. Ileum was collected for investigation of gene expression or fixed in paraformaldehyde for immunofluorescence. In experiments 2 and 3, Ross 708 male broilers were infected, at 21 d of age with Eimeria maxima or E. acervulina or sham-infected with water. Intestinal tissues were collected at 7 and 10 d postinfection for gene expression analysis. In general, mRNA expression patterns of all examined genes showed downregulation during the first 2 wk PH and were not affected by delay in feed access. These development-dependent changes in expression and tissue-dependent localization in the ileum of selected MMPs and TIMPs indicate that these molecules participate in the remodeling of chicken intestinal tissues during PH development. Increased expression of MMP-7 and MMP-9 transcripts in the intestine of Eimeria infected birds suggests an important role for these enzymes in the process of tissue remodeling and destruction in pathological conditions. The findings of this study are important for understanding the relationship between the expression of the MMP system and intestinal development, as well its role in gastrointestinal infection and subsequent recovery.

Research Note: Choice of microbiota database affects data analysis and interpretation in chicken cecal microbiota.

The chicken microbiota is often analyzed to address questions about the effects of diet or disease on poultry health. To analyze the microbiota, bioinformatic platforms such as QIIME 2 and mothur are used, which incorporate public taxonomic databases such as Greengenes, the ribosomal database project (RDP), and SILVA to assign taxonomies to bacterial sequences. Many chicken microbiota studies continue to incorporate the Greengenes database, which has not been updated since 2013. To determine whether a choice of database could affect results, this study compared the results of bioinformatic analyses obtained using the Greengenes, RDP, and SILVA databases on a cecal luminal microbiome dataset. The QIIME 2 platform was used to process 16S bacterial sequences and assign taxonomies with Greengenes, RDP, and SILVA. Linear discriminant analysis effect size (LEfSe) was performed, allowing for the comparison of taxonomies considered significantly differentially abundant between the three databases. Some notable differences between databases were observed in results, in particular the ability of SILVA database to classify members of the family Lachnospiraceae into separate genera, while these members remained in one group of unclassified Lachnospiraceae through Greengenes and RDP. LEfSe analyses showed that the SILVA database produced more differentially abundant genera, in large part due to the classification of these separate Lachnospiraceae genera. Additionally, the relative abundance of unclassified Lachnospiraceae in SILVA results was significantly lower than in RDP results. Our results show the choice of taxonomic database can influence the results of a microbiota study at the genus level, potentially affecting the interpretation of the results. The use of the SILVA database is recommended over Greengenes in chicken microbiota studies, as more specific classifications at the genus level may provide more accurate interpretations of changes in the microbiota.

Delay of Feed Post-Hatch Causes Changes in Expression of Immune-Related Genes and Their Correlation with Components of Gut Microbiota, but Does Not Affect Protein Expression.

Because the delay of feed post-hatch (PH) has been associated with negative growth parameters, the aim of the current study was to determine the effect of delayed access to feed in broiler chicks on the expression of immune-related genes and select proteins. In addition, an analysis of the correlation between gene expression and components of the gut microbiota was carried out. Ross 708 eggs were incubated and hatched, and hatchlings were divided into FED and NONFED groups. The NONFED birds did not have access to feed until 48 h PH, while FED birds were given feed immediately PH. The ileum from both groups (n = 6 per group) was sampled at embryonic day 19 (e19) and day 0 (wet chicks), and 4, 24, 48, 72, 96, 144, 192, 240, 288, and 336 h PH. Quantitative PCR (qPCR) was carried out to measure the expression of avian interleukin (IL)-1ß, IL-4, IL-6, IL-8, IL-18, transforming growth factor (TGF-ß), toll-like receptor (TLR)2, TLR4, interferon (IFN)-ß, IFN-γ, and avian ß-defensins (AvBD) I, 2, 3, 5, 6, 7, 8, 9, and 10. Protein expression of IL-10, IL-1ß, IL-8, and IL-18 were measured using ELISAs. A correlation analysis was carried out to determine whether any significant association existed between immune gene expression and components of the ileal luminal and mucosal microbiota. Expression of several immune-related genes (TGF-ß, TLR4, IFN-γ, IL-1ß, IL-4, IL-6, and AvBDs 8 and 9) were significantly affected by the interaction between feed status and age. The effects were transient and occurred between 48 and 96 h PH. The rest of the genes and four proteins were significantly affected by age, with a decrease in expression noted over time. Correlation analysis indicated that stronger correlations exist among gene expression and microbiota in NONFED birds. The data presented here indicates that delay in feed PH can affect genes encoding components of the immune system. Additionally, the correlation analysis between immune gene expression and microbiota components indicates that a delay in feed has a significant effect on the interaction between the immune system and the microbiota.

Ileal microbial composition in genetically distinct chicken lines reared under normal or high ambient temperatures.

BACKGROUND: Heat stress (HS) has negative effects on poultry productivity, health and welfare resulting in economic losses. Broiler chickens are particularly susceptible to HS due to their high metabolic rate and rapid growth. The commensal intestinal bacterial populations have an important physiological role in the host and could ameliorate the negative effect of HS on the host. Thus, the aim of this study was to compare changes in the ileal (IL) microbiota in four different broiler lines during HS. RESULTS: Day-old broiler chicks from Giant Jungle Fowl (JF), Athens Canadian Random Bred (ACRB), 1995 Random Bred (L1995), and Modern Random Bred (L2015) lines were raised under thermoneutral (TN) conditions until day (d) 28. On d 29 birds were subjected to TN (24 °C) or chronic cyclic HS (8 h/d, 36 °C) condition till d 56. On d 56 two birds per pen were euthanized, and IL luminal content (IL-L) and mucosal scrapings (IL-M) were collected for bacterial DNA isolation. Libraries were constructed using V3-V4 16S rRNA primers and sequenced using MiSeq. DNA sequences were analyzed using QIIME2 platform and SILVA 132 database for alpha and beta diversity, and taxonomic composition, respectively. Functional property of microbiota was predicted using the PICRUSt 2 pipeline and illustrated with STAMP software. Shannon index was significantly elevated in IL-M under HS. ß-diversity PCoA plots revealed separation of microbial community of L2015-TN from JF-TN, JF-HS, ACRB-TN, and ACRB-HS in the IL-M. PERMANOVA analysis showed a significant difference between microbial community of L1995-HS compared to ACRB-HS and JF-TN, L1995-TN compared to ACRB-HS and JF-TN, L2015-HS compared to ACRB-HS and ACRB-TN, L2015-HS compared to JF-TN, L2015-TN compared to ACRB-HS and JF-TN, and ACRB-HS compared to JF-TN in the IL-L. The impact of HS on microbial composition of IL-M was more prominent compared to IL-L with 12 and 2 taxa showing significantly different relative abundance, respectively. Furthermore, differences in microbiota due to the genetic line were more prominent in IL-M than IL-L with 18 and 8 taxa showing significantly different relative abundance, respectively. Unlike taxonomy, predicted function of microbiota was not affected by HS. Comparison of L2015 with JF or ACRB showed significant changes in predicted function of microbiota in both, IL-M and IL-L. Differences were most prominent between L2015 and JF; while there was no difference between L2015 and L1995. CONCLUSIONS: These data indicate the genetic line × temperature effect on the diversity and composition of IL microbiota. Moreover, the data showcase the effect of host genetics on the composition of IL microbiota and their predicted function. These data are of critical importance for devising nutritional strategies to maintain GIT microbial balance and alleviate the negative effects of HS on broiler chickens' performance and health.

Effects of Eimeria tenella on Cecal Luminal and Mucosal Microbiota in Broiler Chickens.

The intestinal disease coccidiosis, caused by parasitic Eimeria species, severely impacts poultry production, leading to an estimated $14 billion in annual losses worldwide. As the poultry industry moves away from antibiotics as a treatment for diseases, a better understanding of the microbiota is required to develop other solutions such as probiotics, prebiotics, and nutritional supplements. This study aimed to investigate the effects of Eimeria tenella infection on luminal (cecal contents [CeC]) and mucosal (cecal epithelial scrapings [CeS]) microbial populations in 288 Ross 708 broiler chickens at multiple time points postinfection (PI). By use of 16S rRNA amplicon sequencing, it was revealed that microbial diversity differed in infected (IF) chickens in comparison to the control (C) in both CeC and CeS microbiota at the peak of infection (7 days PI), when simultaneously IF birds saw reduced body weight gain and a higher feed conversion ratio. Infection resulted in a significant differential abundance of some bacterial taxa, including increases in potential secondary pathogens Escherichia coli, Enterococcus, Clostridium, and Proteus and a decrease in the short chain fatty acid-producing family Lachnospiraceae. Predicted metagenomic pathways associated with E. coli, such as those responsible for amino acid biosynthesis, were differentially expressed in IF birds. In conclusion, our results show that E. tenella infection disturbs luminal and mucosal microbiota balance in chickens. Moreover, the luminal microbiota seems to be more susceptible to prolonged imbalance due to IF, whereas the mucosal microbiota appeared to be affected only in the short term, demonstrating the importance of researching both the luminal and mucosal microbiota of the cecum.

Efectos de Eimeria tenella sobre la microbiota luminal y de la mucosa de los ciegos en pollos de engorde. La coccidiosis, una enfermedad intestinal causada por especies parasitarias de Eimeria, afecta gravemente la producción avícola, lo que genera pérdidas anuales estimadas en 14,000 millones de dólares en todo el mundo. A medida que la industria avícola se aleja de los antibióticos como tratamiento para enfermedades, se requiere de un mejor conocimiento de la microbiota para desarrollar otras soluciones como probióticos, prebióticos y suplementos nutricionales. Este estudio tuvo como objetivo investigar los efectos de la infección por Eimeria tenella en las poblaciones microbianas luminales (contenido cecal [CeC]) y de la mucosa (raspados del epitelio cecal [CeS]) en pollos de engorde Ross 708 (288) en diferentes puntos de tiempo después de la infección (PI). Mediante el uso de la secuenciación de amplicones de ARNr 16S, se reveló que la diversidad microbiana difería en los pollos infectados (IF) en comparación con el grupo control (C) tanto en la microbiota del contenido cecal como de la mucosa durante el pico de infección (7 días después de la infección), cuando de manera simultánea las aves infectadas mostraron una reducción en la ganancia de peso corporal reducido y una tasa de conversión alimenticia más alta. La infección resultó en una abundancia diferencial significativa de algunos taxones bacterianos, incluidos aumentos en los patógenos secundarios potenciales como Escherichia coli, Enterococcus, Clostridium y Proteus y una disminución en la familia Lachnospiraceae productora de ácidos grasos de cadena corta. Las vías metagenómicas predichas asociadas con E. coli, como las responsables de la biosíntesis de aminoácidos, se expresaron diferencialmente en las aves infectadas. En conclusión, estos resultados muestran que la infección por E. tenella perturba el equilibrio de la microbiota luminal y de la mucosa en pollos. Además, la microbiota luminal parece ser más susceptible a un desequilibrio prolongado debido a la infección, mientras que la microbiota mucosa parece verse afectada solo a corto plazo, lo que demuestra la importancia de investigar tanto la microbiota luminal como la de la mucosa en el ciego.

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.

Choice of 16S ribosomal RNA primers affects the microbiome analysis in chicken ceca.

We evaluated the effect of applying different sets of 16S rRNA primers on bacterial composition, diversity, and predicted function in chicken ceca. Cecal contents from Ross 708 birds at 1, 3, and 5 weeks of age were collected for DNA isolation. Eight different primer pairs targeting different variable regions of the 16S rRNA gene were employed. DNA sequences were analyzed using open-source platform QIIME2 and the Greengenes database. PICRUSt2 was used to determine the predicted function of bacterial communities. Changes in bacterial relative abundance due to 16S primers were determined by GLMs. The average PCR amplicon size ranged from 315 bp (V3) to 769 bp (V4-V6). Alpha- and beta-diversity, taxonomic composition, and predicted functions were significantly affected by the primer choice. Beta diversity analysis based on Unweighted UniFrac distance matrix showed separation of microbiota with four different clusters of bacterial communities. Based on the alpha- and beta-diversity and taxonomic composition, variable regions V1-V3(1) and (2), and V3-V4 and V3-V5 were in most consensus. Our data strongly suggest that selection of particular sets of the 16S rRNA primers can impact microbiota analysis and interpretation of results in chicken as was shown previously for humans and other animal species.

The effects of tributyrin supplementation on weight gain and intestinal gene expression in broiler chickens during Eimeria maxima-induced coccidiosis.

Butyrate is a feed additive that has been shown to have antibacterial properties and improve gut health in broilers. Here, we examined the performance and gene expression changes in the ileum of tributyrin-supplemented broilers infected with coccidia. Ninety-six, Ross 708 broilers were fed either a control corn-soybean-based diet (-BE) or a diet supplemented with 0.25% (w/w) tributyrin (+BE). Birds were further divided into groups that were inoculated with Eimeria maxima oocysts (EM) or sham-inoculated (C) on day 21 posthatch. At 7 d postinfection (7 d PI), the peak of pathology in E. maxima infection, tributyrin-supplemented birds had significantly improved feed conversion ratios (FCR, P < 0.05) and body weight gain (BWG, P < 0.05) compared with -BE-infected birds, despite both groups having similar feed intake (FI, P > 0.05). However, at 10 d post-infection (10 d PI) no significant effects of feed type or infection were observed. Gene expression in the ileum was examined for insights into possible effects of infection and tributyrin supplementation on genes encoding proteins related to immunity, digestion, and gut barrier integrity. Among immune-related genes examined, IL-1B and LEAP2 were only significantly affected at 7 d PI. Transcription of genes related to digestion (APN, MCT1, FABP2, and MUC2) were primarily influenced by infection at 7 d PI and tributyrin supplementation (FABP2 and MUC2) at 10 d PI. With exception of ZO1, tight junction genes were affected by either infection or feed type at 7 d PI. At 10 d PI, only CLDN1 was not affected by either infection or feed type. Overall tributyrin shows promise as a supplement to improve performance during coccidiosis in broiler chickens; however, its effect on gene expression and mode of action requires further research.

Metagenomic Analysis of 16S Clostridium perfringens Amplicons Corroborates C. perfringens Counts on Select Agar and C. perfringens PCR Analyses of Bacteria in Broiler Farm Litter.

The purpose of this study was twofold-first, to determine whether analysis of bacterial 16S ribosomal RNA (rRNA) in poultry litter corroborated standard Clostridium perfringens counts and PCR assay, and second, to find whether a correlation between 16S rRNA analysis and netB or Tpel toxin PCR intensity with chick mortality existed. At three time points of growout (0, 2, and 4 wk) litter samples were collected from 23 broiler houses representing eight farms during a coccidiosis vaccine control program. DNA extracted from these samples was used for microbiota determination by sequencing the hypervariable V3-V4 region of bacterial 16s rRNA. Obtained sequences were analyzed by QIIME 2 and the Greengenes database for taxonomic composition and relative abundance of C. perfringens in the litter bacterial population. Clostridium perfringens counts on select agar and semiquantitative PCR for C. perfringens were compared with 16S analysis for equivalence testing. Relative abundance of C. perfringens estimated by 16S analysis and semiquantitative PCR for netB and Tpel toxin DNA were analyzed by Pearson linear correlation and statistical equivalence analyses with cumulative chick mortality at 4 and 9 wk growout. When data from all time points were combined, abundance estimates by C. perfringens 16S were statistically equivalent (α = 0.10) to both C. perfringens PCR and C. perfringens counts. Yet, no correlations were observed between any estimate of C. perfringens abundance and cumulative percent chick mortality at 4 or 9 wk growout. However, correlation analyses revealed a significant linear relationship between netB signal at 0 wk (r = 0.55) and 4 wk (r = 0.46) and cumulative mortality at 9 wk growout (P < 0.05). Similarly, abundance of Tpel at 0 and 2 wk showed a linear relationship with cumulative percent mortality at both 4 and 9 wk growout (0.44 ≤ r ≤ 0.54, P < 0.05). No correlations were observed between any other genera or species determined by 16S and cumulative percent chick mortality.

Effects of heat stress on performance, blood chemistry, and hypothalamic and pituitary mRNA expression in broiler chickens.

This study was conducted to evaluate potential hormonal mechanisms associated with the stress response, thermoregulation, and metabolic changes of broiler chickens exposed to high environmental temperature. Nine hundred 1-day-old male broiler chicks (Ross 708) were placed in floor pens and raised to 24 d. At 24 d, chicks were randomly assigned to 1 of 2 treatments, heat stress (HS) or no HS, and allocated into battery cages in 8 batteries (10 birds per cage, 2 cages per battery). On day 31, blood was collected prior to HS and analyzed using an iSTAT analyzer. Half of the batteries were then moved into 2 rooms with an elevated ambient temperature (35°C) for 8 h. The remaining batteries stayed in the thermoneutral rooms with an ambient temperature of 22°C. Beginning at 5 h after the initiation of HS, blood was collected and analyzed using an iSTAT analyzer, birds were euthanized, and hypothalamus and pituitary samples were collected (16 birds per treatment), flash frozen, and stored at -80°C until RNA extraction. Reverse transcription-quantitative PCR was used to compare mRNA levels of key corticotropic and thyrotrophic genes in the hypothalamus and pituitary. Levels of mRNA for each target gene were normalized to PGK1 (pituitary) and GAPDH (hypothalamus) mRNA. Differences were determined using mixed model ANOVA. HS decreased (P < 0.05) feed intake, BW, bicarbonate, potassium, CO2, and triiodothyronine, while it increased mortality, glucose, pH, plasma thyroxine, and corticosterone. Expression of pituitary corticotropin-releasing hormone receptor 1 was downregulated (P < 0.001), while corticotropin-releasing hormone receptor 2 mRNA levels were higher (P = 0.001) in HS birds. HS increased expression of thyroid hormone receptor ß (P = 0.01) (2.8-fold) and thyroid stimulating hormone ß (P = 0.009) (1.4-fold). HS did not affect levels of mRNA of genes evaluated in the hypothalamus. Results showed that HS significantly affected both the thyrotropic and corticotropic axes. Understanding the role and regulation of these pathways during HS will allow researchers to better evaluate management strategies to combat HS.

Effect of delayed feeding post-hatch on expression of tight junction- and gut barrier-related genes in the small intestine of broiler chickens during neonatal development.

The gut not only plays a key role in digestion and absorption of nutrients but also forms a physical barrier and first line of defense between the host and the luminal environment. A functional gut barrier (mucus and epithelial cells with tight junctions [TJ]) is essential for optimal health and efficient production in poultry. In current broiler system, chicks are deprived of food and water up to 72 h due to uneven hatching, hatchery procedures, and transportation. Post-hatch feed delay results in lower BW, higher FCR and mortality, and delayed post-hatch gut development. Little is known about the effects of early neonatal development and delayed feeding immediately post-hatch on gut barrier function in chickens. Therefore, the aim of the present study was to characterize the expression pattern of gut barrier-related and TJ-related genes in the small intestine of broiler chickens during early development and delay in access to feed. Newly hatched chicks received feed and water immediately after hatch or were subjected to 48 h delayed access to feed to mimic commercial hatchery setting and operations. Birds were sampled (n = 6) at -48, 0, 4, 24, 48, 72, 96, 144, 192, 240, 288, and 336 h post-hatch. Jejunum and ileum were collected, cleaned of digesta, and snap-frozen in liquid nitrogen or fixed in paraformaldehyde. The relative mRNA levels of gut barrier- and TJ-related protein genes were measured by quantitative PCR and analyzed by 2-way ANOVA. In both tissues, changes (P < 0.05) in gene expression pattern of gut barrier-related and TJ-related genes were detected due to delayed access to feed post-hatch and/or development. In general, expression of TJ-related genes was downregulated while mRNA levels of gut barrier-related genes were upregulated during development. Histological differences and changes in mucin staining due to age and treatment were observed. These results suggest that delayed access to feed post-hatch may affect TJ structure and/or function and therefore gut barrier function and overall health of the chicken small intestine.

Research Note: Effect of butyric acid glycerol esters on ileal and cecal mucosal and luminal microbiota in chickens challenged with Eimeria maxima.

Coccidiosis is one of the most prevalent diseases seen in the poultry industry leading to excessive economic losses. The aim of this study was to investigate the effect of butyric acid glycerol esters (BE) on the ileal and cecal microbiota in birds challenged with Eimeria maxima (EM). Ross 708 male broilers were fed a diet supplemented with 0 (control) or 0.25% BE from day 1. On day 21, half of the birds were infected with 103 EM oocysts. For determing microbiota, ileal and cecal contents and epithelial scrapings were collected at 7 and 10 D postinfection (PI). Alpha diversity of bacterial communities was mostly affected (P < 0.05) by time PI and EM infection. The richness of luminal bacterial populations in the ileum and ceca was affected (P < 0.05) by addition of BE and by time PI × EM × BE interaction, respectively. In the ileal and cecal luminal and mucosal bacterial communities, permutational multivariate analysis of variance (PERMANOVA, unweighted UniFrac) showed significant (P < 0.05) differences because of time PI and interaction between time PI, EM, and BE. Significant (P < 0.05) differences in taxonomic composition at the family level were observed in microbiota of luminal and mucosal populations of the ileum and ceca owing to time PI, EM, BE, and their interactions. The bacterial community present in the cecal lumen was characterized by the lowest number of differential bacteria, whereas the cecal mucosal community was characterized by the highest number of differentially abundant bacteria. In conclusion, our results show that EM infection and time PI has the biggest impact on microbial diversity in the chicken gut. The presence of BE in the diet had a limited effect on gut microbiota.