Avian ceca are indispensable for hindgut enteric nervous system development.

The enteric nervous system (ENS), which is derived from enteric neural crest cells (ENCCs), represents the neuronal innervation of the intestine. Compromised ENCC migration can lead to Hirschsprung disease, which is characterized by an aganglionic distal bowel. During the craniocaudal migration of ENCCs along the gut, we find that their proliferation is greatest as the ENCC wavefront passes through the ceca, a pair of pouches at the midgut-hindgut junction in avian intestine. Removal of the ceca leads to hindgut aganglionosis, suggesting that they are required for ENS development. Comparative transcriptome profiling of the cecal buds compared with the interceca region shows that the non-canonical Wnt signaling pathway is preferentially expressed within the ceca. Specifically, WNT11 is highly expressed, as confirmed by RNA in situ hybridization, leading us to hypothesize that cecal expression of WNT11 is important for ENCC colonization of the hindgut. Organ cultures using embryonic day 6 avian intestine show that WNT11 inhibits enteric neuronal differentiation. These results reveal an essential role for the ceca during hindgut ENS formation and highlight an important function for non-canonical Wnt signaling in regulating ENCC differentiation.

Disentangling the biotic and abiotic drivers of bird-building collisions in a tropical Asian city with ecological niche modeling.

Bird collisions with buildings are responsible for a large number of bird deaths in cities around the world, yet they remain poorly studied outside North America. We conducted one of the first citywide fine-scale and landscape-scale analyses of bird-building collisions in Asia and used maximum entropy modeling (as commonly applied to species distribution modeling) in a novel way to assess the drivers of bird-building collisions in the tropical city-state of Singapore. We combined 7 years of community science observations with publicly available building and remote sensing data. Drivers of bird-building collisions varied among taxa. Some migratory taxa had a higher relative collision risk that was linked to areas with high building densities and high levels of nocturnal blue light pollution. Nonmigratory taxa had a higher collision risk in areas near forest cover. Projecting our results onto official long-term land-use plans, we predicted that future increases in bird-building collision risk stemmed from increases in blue light pollution and encroachment of buildings into forested areas and identified 6 potential collision hotspots linked to future developments. Our results suggest that bird-building collision mitigation measures need to account for the different drivers of collision for resident and migratory species and show that combining community science and ecological modeling can be a powerful approach for analyzing bird-building collision data.

Modelos de nicho ecológico para esclarecer los causantes bióticos y abióticos de las colisiones entre aves y edificios en una ciudad tropical asiática Resumen Las colisiones entre aves y edificios son causa de un gran número de muertes en todas las ciudades del mundo, y aun así se estudian muy poco fuera de América del Norte. Realizamos uno de los primeros análisis a escala fina y a escala de paisaje en una ciudad asiática y usamos el modelo de entropía máxima (como se aplica con frecuencia a los modelos de distribución de especies) de manera novedosa para analizar los causantes de estas colisiones en Singapur, una ciudad­estado tropical. Combinamos siete años de observaciones de ciencia comunitaria con los datos públicos de teledetección y construcción. Los causantes de las colisiones entre aves y edificios variaron entre taxones. Algunos taxones migratorios tuvieron un riesgo de colisión relativamente más alto relacionado con áreas de alta densidad de edificios y niveles elevados de contaminación lumínica de luz azul nocturna. Los taxones no migratorios tuvieron un riesgo de colisión más elevado en las áreas cercanas a la cobertura forestal. Con la proyección de nuestros resultados sobre los planes oficiales de uso de suelo a largo plazo, pronosticamos que el incremento en el futuro de colisiones entre aves y edificios vendrá del incremento en la contaminación de luz azul y la invasión de edificios en las áreas forestales; también identificamos seis potenciales puntos calientes de colisión relacionados a futuros desarrollos inmobiliarios. Nuestros resultados sugieren que para mitigar estas colisiones se necesita considerar los diferentes causantes de dichas colisiones para las especies migratorias y residentes y también muestran que la combinación de la ciencia comunitaria y los modelos ecológicos puede ser una estrategia poderosa para analizar los datos de colisiones entre aves y edificios.

Influence of an indigenous yeast, CECA, from the Ningxia wine region of China, on the fungal and bacterial dynamics and function during Cabernet Sauvignon wine fermentation.

BACKGROUND: Saccharomyces cerevisiae CECA was a potential indigenous Chinese wine yeast that can produce aroma and flavor in Cabernet Sauvignon wines. High-throughput sequencing combined with metabolite analysis was applied to analyze the effects of CECA inoculation on the native microbial community interaction and metabolism during Cabernet Sauvignon wine fermentation. RESULTS: Fermentations were performed with three different inoculant strategies: spontaneous fermentation without inoculation, inoculation with CECA after grape must sterilization, and direct inoculation of CECA. Results showed that the diversity of bacteria (P = 0.033) is more sensitive to CECA inoculation than fungi (P = 0.563). In addition, CECA inoculation altered the species composition of core microorganisms (relative abundance >1%) and the keystone species (accounting for the top 1% of the most important interactions), as well as of the biomarkers (linear discriminant analysis > 3.0, P < 0.05). Furthermore, the inoculation could change the cluster of metabolites, and these differential metabolite sets were correlated with four fungal taxa of Issatchenkia, Issatchenkia orientalis, Saccharomycetales, Saccharomycetes and two bacterial taxa of Pantoea, Tatumella ptyseos, were significantly correlated. Inoculated fermentation also altered the correlation between dominant microorganisms and aroma compounds, giving Cabernet Sauvignon wines more herbal, floral, fruity, and cheesy aromas. CONCLUSION: Saccharomyces cerevisiae CECA and dimethyl dicarbonate (DMDC) inhibition treatments significantly altered the microbial community structure of Cabernet Sauvignon wines, which in turn affected the microbial-metabolite correlation. These findings will help winemakers to control the microbial dynamics and functions during wine fermentation, and be more widely used in regional typical wine fermentations. © 2024 Society of Chemical Industry.

Cecal Microbiota Development and Physiological Responses of Broilers Following Early Life Microbial Inoculation Using Different Delivery Methods and Microbial Sources.

Broilers in intensive systems may lack commensal microbes that have coevolved with chickens in nature. This study evaluated the effects of microbial inocula and delivery methods applied to day-old chicks on the development of the cecal microbiota. Specifically, chicks were inoculated with cecal contents or microbial cultures, and the efficacies of three delivery methods (oral gavage, spraying inoculum into the bedding, and cohousing) were evaluated. Also, a competitive study evaluated the colonization ability of bacteria sourced from extensive or intensive poultry production systems. The microbiota of inoculated birds presented higher phylogenetic diversity values (PD) and higher relative abundance values of Bacteroidetes, compared with a control. Additionally, a reduction in the ileal villus height/crypt depth ratio and increased cecal IL-6, IL-10, propionate, and valerate concentrations were observed in birds that were inoculated with cecal contents. Across the experiments, the chicks in the control groups presented higher relative abundance values of Escherichia/Shigella than did the inoculated birds. Specific microbes from intensively or extensively raised chickens were able to colonize the ceca, and inocula from intensive production systems promoted higher relative abundance values of Escherichia/Shigella. We concluded that Alistipes, Bacteroides, Barnesiella, Mediterranea, Parabacteroides, Megamonas, and Phascolarctobacterium are effective colonizers of the broiler ceca. In addition, oral gavage, spray, and cohousing can be used as delivery methods for microbial transplantation, as indicated by their effects on the cecal microbiota, intestinal morphology, short-chain fatty acids concentration, and cytokine/chemokine levels. These findings will guide future research on the development of next-generation probiotics that are able to colonize and persist in the chicken intestinal tract after a single exposure. IMPORTANCE The strict biosecurity procedures employed in the poultry industry may inadvertently hinder the transmission of beneficial commensal bacteria that chickens would encounter in natural environments. This research aims at identifying bacteria that can colonize and persist in the chicken gut after a single exposure. We evaluated different microbial inocula that were obtained from healthy adult chicken donors as well as three delivery methods for their effects on microbiota composition and bird physiology. In addition, we conducted a competitive assay to test the colonization abilities of bacteria sourced from intensively versus extensively raised chickens. Our results indicated that some bacteria are consistently increased in birds that are exposed to microbial inoculations. These bacteria can be isolated and employed in future research on the development of next-generation probiotics that contain species that are highly adapted to the chicken gut.

Essential Role of BMP4 Signaling in the Avian Ceca in Colorectal Enteric Nervous System Development.

The enteric nervous system (ENS) is principally derived from vagal neural crest cells that migrate caudally along the entire length of the gastrointestinal tract, giving rise to neurons and glial cells in two ganglionated plexuses. Incomplete migration of enteric neural crest-derived cells (ENCDC) leads to Hirschsprung disease, a congenital disorder characterized by the absence of enteric ganglia along variable lengths of the colorectum. Our previous work strongly supported the essential role of the avian ceca, present at the junction of the midgut and hindgut, in hindgut ENS development, since ablation of the cecal buds led to incomplete ENCDC colonization of the hindgut. In situ hybridization shows bone morphogenetic protein-4 (BMP4) is highly expressed in the cecal mesenchyme, leading us to hypothesize that cecal BMP4 is required for hindgut ENS development. To test this, we modulated BMP4 activity using embryonic intestinal organ culture techniques and retroviral infection. We show that overexpression or inhibition of BMP4 in the ceca disrupts hindgut ENS development, with GDNF playing an important regulatory role. Our results suggest that these two important signaling pathways are required for normal ENCDC migration and enteric ganglion formation in the developing hindgut ENS.

Effects of floor- and net-rearing systems on intestinal growth and microbial diversity in the ceca of ducks.

BACKGROUND: Rearing systems can affect livestock production directly, but whether they have effects on intestinal growth states and ceca microorganisms in ducks is largely unclear. The current study used Nonghua ducks to estimate the effects of rearing systems on the intestines by evaluating differences in intestinal growth indices and cecal microorganisms between ducks in the floor-rearing system (FRS) and net-rearing system (NRS). RESULTS: The values of relative weight (RW), relative length (RL) and RW/RL of the duodenum, jejunum, ileum and ceca in the FRS were significantly higher than those in the NRS during weeks 4, 8 and 13 (p < 0.05). A total of 157 genera were identified from ducks under the two systems, and the dominant microorganisms in both treatments were Firmicutes, Bacteroidetes, Actinobacteria and Proteobacteria at the phylum level. The distribution of microorganisms in the ceca of the two treatments showed significant separation during the three time periods, and the value of the Simpson index in the FRS was significantly higher than that in the NRS at 13 weeks (p < 0.05). Five differential microorganisms and 25 differential metabolic pathways were found in the ceca at week 4, seven differential microorganisms and 25 differential metabolic pathways were found in the ceca at week 8, and four differential microorganisms and two differential metabolic pathways were found in the ceca at week 13. CONCLUSIONS: The rearing system influences duck intestinal development and microorganisms. The FRS group had higher intestinal RL, RW and RW/RL and obviously separated ceca microorganisms compared to those of the NRS group. The differential metabolic pathways of cecal microorganisms decreased with increasing age, and the abundance of translation pathways was higher in the NRS group at week 13, while cofactor and vitamin metabolism were more abundant in the FRS group.

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.

Combined antagonist treatment of glucocorticoid and mineralocorticoid receptor does not affect weight loss of fasting rainbow trout but inhibits a fasting-induced elevation of cortisol secretion.

The gastrointestinal system of fish reacts rapidly to food deprivation. The relative masses of digestive organs and activities of digestive enzymes decrease within days of fasting. This is believed to be an energy-conserving strategy as the metabolic cost of maintaining digestive capacity is high. Cortisol is known for its role in energy mobilization following stress exposure, and prolonged elevated cortisol levels have been shown to reduce growth rates in fish. Fish experiencing chronic cortisol elevations show structural changes to their digestive tissues and overall reductions in relative digestive tissue masses. In fish fasting for prolonged periods, circulating cortisol levels have been reported to be downregulated, upregulated, or unchanged compared to feeding fish. This study aimed to investigate if RU486 and spironolactone, antagonists of the glucocorticoid receptor (GR), and mineralocorticoid receptor (MR), respectively, alone or in combination affect circulating cortisol levels during prolonged starvation. In addition, we tested the effects of blocking GR and MR, on the down-regulation of relative digestive tissue mass during starvation, and its effects on weight loss. Three treatment groups of rainbow trout were intraperitoneally implanted with either GR, MR, or GR and MR blockers. A fourth group was implanted with cortisol, while a fifth group served as a control. All treatment groups were sampled over a course of four weeks of food deprivation and compared against each other and fed control fish at day 0 of the trial. Starvation for 2 weeks and longer significantly increased circulating cortisol levels in all groups except for the group implanted with GR and MR antagonists. Loss of body mass occurred most rapidly during the first week of starvation. Spironolactone treatment resulted in significantly reduced loss of mass during the first week, however, over the following weeks, no differences in mass loss were observed in the groups implanted with blockers, while cortisol-treated fish showed the highest decrease in body mass over time. Relative digestive tissue mass decreased in all groups but apparently, the fasting-induced elevation in plasma cortisol levels did not affect the relative weight loss of digestive tissues as no differences were observed between control fish and GR + MR antagonist treated fish. Very high cortisol levels caused by cortisol treatment however caused a faster decrease in the relative mass of some digestive organs, particularly the stomach.

Design, expression and evaluation of novel chimeric protein constructed from colorectal tumor-associated antigen.

There were 134,000 new diagnosis and 49,000 deaths in 2016 due to colorectal cancer. Similar to most cancers, early diagnosis increases the chance of successful treatment. Detection of tumor-associated antigens or the immune response against such markers is one of the most common methods of diagnosis. In that regard, we aimed to design and express a chimeric protein from the most common tumor-associated antigens in colorectal cancer and assess its ability to detect the immune response in comparison with the parental tumor-associated antigens in patient's sera. Through bioinformatics approaches a chimeric protein from carcinoembryonic antigen (CEA) and carbohydrate antigen 19.9 (CA19-9) was designed and expressed in E. coli (BL21DE3). Proper folding, expression levels and immune reactivity were assessed by western blot, ELISA and immunohistochemistry. Recombinant proteins functionality and immune reactivity were confirmed by ELISA and Western blot. Results showed that recombinant CEA, recombinant CA19.9 and chimeric protein of CEA- CA19.9 have strong reactivity with antibodies in the sera of colorectal cancer patients, whereas no reactivity was seen with the sera of healthy volunteers. Significantly stronger immune reactivity was seen with the chimeric protein than each of the CEA or CA19.9 alone. Overall, it was concluded that the designed recombinant proteins in this study could be used to detect autoantibodies produced against the colorectal tumor-associated antigens. The chimeric CEA-CA19.9 protein shows a stronger reactivity with the sera antibodies of colorectal cancer patients that CEA or CA19.9 alone.

Reduced-Particle-Size Wheat Bran Is Efficiently Colonized by a Lactic Acid-Producing Community and Reduces Levels of Enterobacteriaceae in the Cecal Microbiota of Broilers.

In the present study, we investigated whether reducing the particle size of wheat bran affects the colonizing microbial community using batch fermentations with cecal inocula from seven different chickens. We also investigated the effect of in-feed administration of regular wheat bran (WB; 1,690 µm) and wheat bran with reduced particle size (WB280; 280 µm) on the cecal microbial community composition of broilers. During batch fermentation, WB280 was colonized by a lactic acid-producing community (Bifidobacteriaceae and Lactobacillaceae) and by Lachnospiraceae that contain lactic acid-consuming butyric acid-producing species. The relative abundances of the Enterobacteriaceae decreased in the particle-associated communities for both WB and WB280 compared to that of the control. In addition, the community attached to wheat bran was enriched in xylan-degrading bacteria. When administered as a feed additive to broilers, WB280 significantly increased the richness of the cecal microbiota and the abundance of bacteria containing the butyryl-coenzyme A (CoA):acetate CoA-transferase gene, a key gene involved in bacterial butyrate production, while decreasing the abundances of Enterobacteriaceae family members in the ceca. Particle size reduction of wheat bran thus resulted in the colonization of the bran particles by a very specific lactic acid- and butyric acid-producing community and can be used to steer toward beneficial microbial shifts. This can potentially increase the resilience against pathogens and increase animal performance when the reduced-particle-size wheat bran is administered as a feed additive to broilers.IMPORTANCE Prebiotic dietary fibers are known to improve the gastrointestinal health of both humans and animals in many different ways. They can increase the bulking capacity, improve transit times, and, depending on the fiber, even stimulate the growth and activity of resident beneficial bacteria. Wheat bran is a readily available by-product of flour processing and is a highly concentrated source of (in)soluble dietary fiber. The intake of fiber-rich diets has been associated with increased Firmicutes and decreased Proteobacteria numbers. Here, we show that applying only 1% of a relatively simple substrate which was technically modified using relatively simple techniques reduces the concentration of Enterobacteriaceae This could imply that in future intervention studies, one should take the particle size of dietary fibers into account.

Denaturing Gradient Gel Electrophoresis-Polymerase Chain Reaction Comparison of Chitosan Effects on Anaerobic Cultures of Broiler Cecal Bacteria and Salmonella Typhimurium.

Enteropathogen colonization and product contamination are major poultry industry problems. The emergence of antibiotic resistance, and associated risks to human health, is limiting the use of antibiotics as first-line defense against enteropathogens in poultry. The chitin derivative, chitosan, has drawn substantial attention for its bactericidal properties. Different molecular weight (MW) chitosans can have varied effects against different bacteria in monoculture. In the current study, cecal contents from each of three market-age broilers and Salmonella Typhimurium, as indicator enteropathogen, were exposed to in vitro anaerobic culture to three chitosan preparations (0.08%, wt/vol), low (LMW), medium (MMW), and coarse (CMW). Effects of chitosan and the carrier solvent acetic acid, on cecal bacteria and Salmonella, were examined by denaturing gradient gel electrophoresis (DGGE) and Salmonella enumeration. Bacterial profiles for the three cecal contents were shown by DGGE to be very different. Each of the three cecal contents grown in the presence of 0.08% acetic acid was very different from the same contents grown without the chitosan solvent. Culturing cecal contents in the presence of chitosan altered the bacterial DGGE profiles from the control and acetic acid-only cultures. The DGGE chitosan-treated profiles for all three cecal sources were identical to each other regardless of the MW chitosan in the culture medium. Compared with Salmonella in monoculture, Salmonella decreased (p < 0.05) by about 1.5 log CFU/mL when grown in mixed culture with cecal contents. Salmonella monocultures in the presence of 0.08% of the chitosan solvent acetic acid decreased (p < 0.05) counts by almost 3.5 log CFU/mL. Combining acetic acid and cecal contents reduced (p < 0.05) Salmonella by 7 log CFU/mL. Adding the chitosan preparations to the mixtures reduced (p < 0.05) Salmonella by 8 log CFU/mL.

Effect of Vancomycin, Tylosin, and Chlortetracycline on Vancomycin-Resistant Enterococcus faecium Colonization of Broiler Chickens During Grow-Out.

Broiler chickens may serve as reservoirs for human colonization by vancomycin-resistant Enterococcus (VRE). We examined the effects of vancomycin and two commonly used antimicrobial feed additives on VRE colonization in broiler chickens during grow-out. Chicks received unsupplemented feed or feed containing vancomycin, chlortetracycline, or tylosin from day of hatch to grow-out at 6 weeks. At 3 days of age, chicks received by crop gavage 107 colony-forming units (CFUs) of a human or poultry VRE isolate. Cecal contents were monitored weekly for VRE, short-chain fatty acids (SCFAs), and bacterial denaturing gradient gel electrophoresis (DGGE) profile methods. Vancomycin promoted persistent and high-level colonization with human- and poultry-derived VRE to grow-out in comparison with controls, while treatment with chlortetracycline and tylosin did not. Colonization by the poultry isolate in control, chlortetracycline, and tylosin groups persisted throughout the grow-out period with low concentrations present at 6 weeks, whereas the human isolate decreased to an undetectable level by week 6. Vancomycin resulted in significant reductions in cecal acetic acid and butyric acid in comparison with controls, but chlortetracycline and tylosin did not. DGGE profiles contained two main clusters with all vancomycin profiles in a smaller cluster and all other profiles in a larger cluster. These results demonstrate that vancomycin, but not chlortetracycline or tylosin, disrupted the indigenous microbiota and SCFA patterns of broiler chickens and promoted colonization by VRE.

On-farm Campylobacter and Escherichia coli in commercial broiler chickens: Re-used bedding does not influence Campylobacter emergence and levels across sequential farming cycles.

Limitations in quality bedding material have resulted in the growing need to re-use litter during broiler farming in some countries, which can be of concern from a food-safety perspective. The aim of this study was to compare the Campylobacter levels in ceca and litter across three litter treatments under commercial farming conditions. The litter treatments were (a) the use of new litter after each farming cycle; (b) an Australian partial litter re-use practice; and (c) a full litter re-use practice. The study was carried out on two farms over two years (Farm 1, from 2009-2010 and Farm 2, from 2010-2011), across three sheds (35,000 to 40,000 chickens/shed) on each farm, adopting three different litter treatments across six commercial cycles. A random sampling design was adopted to test litter and ceca for Campylobacter and Escherichia coli, prior to commercial first thin-out and final pick-up. Campylobacter levels varied little across litter practices and farming cycles on each farm and were in the range of log 8.0-9.0 CFU/g in ceca and log 4.0-6.0 MPN/g for litter. Similarly the E. coli in ceca were ∼log 7.0 CFU/g. At first thin-out and final pick-up, the statistical analysis for both litter and ceca showed that the three-way interaction (treatments by farms by times) was highly significant (P<0.01), indicating that the patterns of Campylobacter emergence/presence across time vary between the farms, cycles and pickups. The emergence and levels of both organisms were not influenced by litter treatments across the six farming cycles on both farms. Either C. jejuni or C. coli could be the dominant species across litter and ceca, and this phenomenon could not be attributed to specific litter treatments. Irrespective of the litter treatments in place, cycle 2 on Farm 2 remained Campylobacter-free. These outcomes suggest that litter treatments did not directly influence the time of emergence and levels of Campylobacter and E. coli during commercial farming.

The addition of charcoals to broiler diets did not alter the recovery of Salmonella Typhimurium during grow-out.

Two experiments evaluated prebiotics added to feed on the recovery of Salmonella in broilers during grow-out and processing. In Experiment 1, "seeder" chicks were inoculated with Salmonella Typhimurium and placed with penmates. Treatments were: basal control diet, added 0.3% bamboo charcoal, 0.6% bamboo charcoal, or 0.12% Aromabiotic (medium chain fatty acids). The ceca from seeders and penmates were sampled to confirm Salmonella colonization at 3, 4, and 6 wk, and pen litter was sampled weekly. At 3 wk, charcoal fed chicks had significantly lower cecal recovery (37% lower) of Salmonella via direct plating but no differences at wk 4 or 6. At 6 wk, broilers fed Aromabiotic had no recovery of Salmonella from ceca with direct plating and significantly, 18%, lower recovery with enrichment. In Experiment 2, the treatments were: basal control diet, added 0.3% bamboo charcoal, 0.3% activated bamboo charcoal, or 0.3% pine charcoal. At placement, 2 seeders were challenged with Salmonella and commingled with penmates and ceca sampled at 1 and 2 wk, and ceca from 5 penmates/pen at 3 to 6 wk. Weekly, the pH of the crop and duodenum was measured from 1 penmate/pen and the litter surface sampled. At the end of grow-out broilers were processed. Results showed that penmates had colonized at 1 and 2 wk. Cecal Salmonella showed no differences except at 4 wk, when activated bamboo charcoal had a 18% lower recovery of Salmonella (enrichment) compared to the control (88%). Similar to Experiment 1, the recovery of Salmonella from the litter was not significantly different among treatments, however an overall decrease in recovery by 4 wk with direct plating reoccurred. The pH of the duodenum and the crop were not different among treatments. Crop pH (6.0) for all treatments were significantly higher at wk 1 compared to wk 2 to 6. Charcoals had minimal effect on Salmonella recovery in the ceca, but following defeathering, broilers fed charcoals had significantly lower Salmonella recovery from breast skin (charcoals 5+/60 compared to control 8+/20). While the addition of charcoals to broilers feed did not significantly affect Salmonella recovery during production (from litter or ceca samples) there was a lower Salmonella recovery from breast skin following scalding and defeathering.

Effects of dietary fiber on cecal short-chain fatty acid and cecal microbiota of broiler and laying-hen chicks.

This experiment was conducted to evaluate the effects of feeding dietary fiber on cecal short-chain fatty acid (SCFA) concentration and cecal microbiota of broiler and laying-hen chicks. The lower fiber diet was based on corn-soybean meal (SBM) and the higher fiber diet was formulated using corn-SBM-dried distillers grains with solubles (DDGS) and wheat bran to contain 60.0 g/kg of both DDGS and wheat bran from 1 to 12 d and 80.0 g/kg of both DDGS and wheat bran from 13 to 21 d. Diets were formulated to meet or exceed NRC nutrient requirements. Broiler and laying-hen chicks were randomly assigned to the high and low fiber diets with 11 replicates of 8 chicks for each of the 4 treatments. One cecum from 3 chicks was collected from each replicate: one cecum underwent SCFA concentration analysis, one underwent bacterial DNA isolation for terminal restriction fragment length polymorphism (TRFLP), and the third cecum was used for metagenomics analyses. There were interactions between bird line and dietary fiber for acetic acid (P = 0.04) and total SCFA (P = 0.04) concentration. There was higher concentration of acetic acid (P = 0.02) and propionic acid (P < 0.01) in broiler chicks compared to laying-hen chicks. TRFLP analysis showed that cecal microbiota varied due to diet (P = 0.02) and chicken line (P = 0.03). Metagenomics analyses identified differences in the relative abundance of Helicobacter pullorum and Megamonas hypermegale and the genera Enterobacteriaceae, Campylobacter, Faecalibacterium, and Bacteroides in different treatment groups. These results provide insights into the effect of dietary fiber on SCFA concentration and modulation of cecal microbiota in broiler and laying-hen chicks.

Effects of DNA extraction methods on the real time PCR quantification of Campylobacter jejuni, Campylobacter coli, and Campylobacter lari in chicken feces and ceca contents.

Polymerase chain reaction (PCR) method was coupled with a DNA extraction to enumerate Campylobacter spp. from poultry gastrointestinal tract samples. Three experiments were conducted that included: 1) Development of a DNA standard curve related to bacterial DNA primers; 2) Design of a cell/genomic DNA extraction protocol to isolate Campylobacter spp. DNA from complex samples such as poultry feces; and 3) Comparison of PCR quantification to standard plate count methodology. The standard curve using primers for Campylobacter spp. was created for DNA extracted from environmental isolates with a linear range (R2 > 0.95) and with a high specificity for C. coli and C. jejuni recovered from poultry, swine and laboratory isolates. A 2-step extraction process of bacterial DNA from poultry feces was developed in which the cells were first concentrated using a gradient-centrifugation step followed by comparison of 4 DNA extraction methods. Two commercial DNA extraction methods (Zymo Research Quick DNA, and Invitrogen magnetic separation), a traditional phenol-chloroform DNA extraction method using proteinase K to inactivate DNAses, and an in-house isolation method for DNA extraction based on chaotropic salts were used. The middle gradient layer recovered 89% to 98% of the bacteria cells from the sample, with recovery dependent upon the Campylobacter genus. The 4 DNA extractions methods recovered 112 to 302 ug/nL of DNA. Finally, the qPCR and standard plate methods were highly correlated for enumerating Campylobacter spp. in the 2.0 to 8.0-log CFU range. Analyses of the results from this study demonstrate that the combination of the standard curve for Campylobacter spp. DNA primers, the gradient cell concentration method and DNA extraction techniques with qPCR can be used to enumerate Campylobacter spp. from poultry samples with findings similar those of traditional plate count methodology.

The Hatching Time of Broiler Chickens Modifies Not Only the Production Traits but Also the Early Bacteriota Development of the Ceca.

This trial was carried out to find out the effects of the parent flock and hatching time of broiler chickens on the production traits and bacteriota development of animals. Two sets of 730 hatching eggs were collected from two different parent flocks with ages of 25 and 50 weeks. In the hatchery, both groups were divided into two subgroups: those hatched during the first 10 and the subsequent 10 h of the hatching window. A feeding trial was carried out afterwards, using the four treatments in six replicate floor pens and feeding commercial starter, grower, and finisher diets that contained all the nutrients according to the breeder's recommendations. The day-old chickens of the older parent flock and those hatched later were heavier, and this advantage remained until the end of the production period. The different ages and origins of the parent flocks failed to modify the microbiological parameters of the chicken's ceca; however, the hatching time significantly influenced the different bacteriota diversity indices: the late-hatched chickens showed higher Bacteroidetes and lower Firmicutes and Actinobacteria abundances at day 11. These treatments resulted in differences in the main families, Ruminococcaceae, Lactobacillaceae, and Bacteroidaceae. These differences could not be found at day 39.

Effect of sprouted whole pearl millet on growth performance, intestinal development, bacterial count, and blood indices of broiler chickens.

This study investigated the effects of varying levels of sprouted whole grain pearl millet (SPM) on growth performance, intestinal morphology, microbial count, and blood indices of broiler chickens. A maize-soybean meal basal diet was formulated and fed to broiler chickens as starter (0 to 21 d) and finisher (22 to 42 d) diets. The diets comprised of 0%, 25%, 50%, 75%, and 100% of SPM incorporated as whole grain. On 0 d, 180 unsexed broiler chickens were allocated to experimental diets in a completely randomized design. Each treatment was replicated three times; each replicate had 12 chicks. All diets were isonitrogenous and isocaloric to meet the nutrient requirements of broiler chickens. Diets and water were provided ad libitum for 42 d. Results showed that the body weight gain (BWG) of broiler chickens on SPM compared favorably with those on the control diet. BWG showed trends in increment (P < 0.10) while FCR showed decreased trends (P < 0.10) with partial inclusion of SPM at 42 d and 0 to 42 d. The drumstick weight showed quadratic effect (P = 0.044) while the wing weight showed linear effect (P = 0.047) to treatment diets at 21 d. The liver weights of broiler chickens showed linear response (P = 0.018) at 21 d and (P = 0.004) at 42 d to SPM inclusion in diets. Sprouted whole PM consistently increased low-density lipoprotein concentration and mean corpuscular hemoglobin concentration (P < 0.05). Length and weight of small intestine and ceca showed decreasing trends on SPM levels in the treatment diets. Digesta pH assessment revealed that pH in the crop was lower (P < 0.05) on partial SPM while pH in proventriculus was reduced (P < 0.05) with inclusion of SPM in treatment diets. Lactobacilli count decreased linearly (P = 0.010) with SPM inclusion. This study suggests that SPM could be used as an alternative source of energy in production of broiler chickens. Therefore, partial replacement of maize with SPM in broiler diet had no negative effect on performance, physiological status, and overall health of broiler chickens.

Sex differences in risk factors for end-stage kidney disease and death in type 2 diabetes: A retrospective cohort study.

BACKGROUND: This study investigated the sex differences in the risk of end-stage kidney disease (ESKD) and mortality, as well as the effect modification of sex on associated factors in patients with type 2 diabetes. METHODS: This multicenter observational cohort study included 4328 patients with type 2 diabetes. Hazard ratios (HRs) with 95% confidence intervals (CIs) of sex for ESKD and death were estimated using Cox proportional regression with adjustment for baseline covariates. For assessing risk modification, HRs and incidence rates for ESKD and death were compared between sexes across patient characteristics using Cox proportional and Poisson regression models. RESULTS: During a median follow-up of 7 years, 276 patients (70% men) developed ESKD, and 241 patients (68% men) died. Men had higher risks of ESKD (HR 1.34; 95% CI 1.02-1.75; p = .034) and death (HR 1.64; 95% CI 1.24-2.16; p = .001) versus women after adjusting for multiple covariates. Among patients with microalbuminuria, men had a substantially higher risk of ESKD versus women, compared to those with normo- and macroalbuminuria (p for interaction .04). Incidence rates were also increased in men versus women with albuminuria of around 300 mg/g. No differences were detected in the association of sex and death across baseline patient subgroups. CONCLUSIONS: In type 2 diabetes, men had an increased risk of ESKD and death versus women. Moderately increased albuminuria was strongly associated with sex difference in developing ESKD.

Application of a Commercial Salmonella Real-Time PCR Assay for the Detection and Quantitation of Salmonella enterica in Poultry Ceca.

ABSTRACT: Foodborne salmonellosis is commonly associated with poultry and poultry products, necessitating continued development of pre- and postharvest food safety interventions and risk management strategies. Evaluation of technologies and strategies is limited by availability of cost-effective, rapid laboratory methods. The objective of this study was to evaluate a commercial qualitative PCR assay and its novel quantitative application to detect and enumerate Salmonella in poultry ceca as an analytical matrix. Ceca were collected at harvest, the contents were homogenized, and paired samples were evaluated with buffered peptone water (BPW) and BAX MP + Supplement (MPS) preenrichment broths followed by PCR screening with a BAX System Q7 PCR and by culture isolation. Additional ceca were inoculated with Salmonella to develop a standard curve for the BAX System SalQuant quantitative PCR application (QA), and estimates were obtained by the QA and most-probable-number (MPN) methods. For preenrichment media, PCR outcomes were equivalent to those of culture isolation for detecting Salmonella in ceca with 95.65 and 87.88% sensitivity and 82.00 and 100.00% specificity (P = 0.074) for BPW and MPS, respectively. However, at the sample level, BPW performed significantly worse (47.92%) than did MPS (68.75%) for overall isolation of Salmonella (P < 0.0001). After standard curve development, the mean QA estimates obtained for the inoculated samples were 1.14 (95% confidence interval [CI]: 0.62 to 1.66), 1.79 (1.50 to 2.08), 2.91 (2.65 to 3.17), and 3.76 (3.26 to 4.25) log CFU/mL for each targeted inoculation of 1.0, 2.0, 3.0, and 4.0 log CFU/mL, respectively, and were within or comparable to the 95% CI values of paired MPN estimates. These data support the use of MPS for the detection and isolation of Salmonella enterica from poultry ceca when screening with PCR and indicate that QA may be useful as an alternative tool to estimate Salmonella loads in poultry ceca, which may support preharvest food safety interventions.