The effect of gut microbiota-derived carnosine on mucosal integrity and immunity in broiler chickens challenged with Eimeria maxima.

In the first study, an in vitro culture system was developed to investigate the effects of carnosine on macrophage proinflammatory cytokine response using an established chicken macrophage cell line (CMC), gut integrity using a chicken intestinal epithelial cell line (IEC), muscle differentiation in quail muscle cells (QMCs) and primary chicken embryonic muscle cells (PMCs), and direct anti-parasitic effect against Eimeria maxima sporozoites. Cells to be tested were seeded in 24-well plates and treated with carnosine at 4 different concentrations (0.1, 1.0, and 10.0 µg). After 18 h of incubation, cells were harvested to measure gene expression of proinflammatory cytokines in CMC, tight junction (TJ) proteins in IECs, and muscle cell growth markers in QMCs and PMCs. In vivo trials were conducted to investigate the effect of dietary carnosine on disease parameters in broiler chickens challenged with E. maxima. One hundred and twenty male broiler chickens (0-day-old) were allocated into 4 treatment groups: 1) basal diet without infection (NC), 2) basal diet with E. maxima infection (PC), 3) carnosine at 10.0 mg/kg feed with PC (HCS), and 4) carnosine at 1.0 mg/kg feed with PC (LCS). All groups except NC were orally infected with E. maxima on d 14. Jejunal samples were collected for lesion scoring and jejunum gut tissues were used for transcriptomic analysis of cytokines and TJ proteins. In vitro, carnosine treatment significantly decreased IL-1ß gene expression in CMC following LPS stimulation. In vivo feeding studies showed that dietary carnosine increased BW and ADG of chickens in E. maxima-infected groups and reduced the jejunal lesion score and fecal oocyst shedding in HCS group. Jejunal IL-1ß, IL-8, and IFN-γ expression were suppressed in the HCS group compared to PC. The expression levels of claudin-1 and occludin in IECs were also increased in HCS following carnosine treatment. In conclusion, these findings highlight the beneficial effects of dietary carnosine supplementation on intestinal immune responses and gut barrier function in broiler chickens exposed to E. maxima infection.

Effects of dosing non-toxigenic Clostridia on the bacterial populations and immunological responses in the intestinal tract of lactating dairy cows.

Understanding the effects of dosing non-toxigenic Clostridia to cows is rare and has received little attention so far. In the present study, a total of eight lactating dairy cows were divided in two groups: control (n = 4) or Clostridia challenged (oral supplementation of five diverse strains of Paraclostridium bifermentans, n = 4). Bacterial communities were analyzed by qPCR and next-generation sequencing (NGS) in the buccal mucosa as well as digesta and mucosal samples of the gastrointestinal (GI) tract from rumen to rectum (10 compartments), as well as fecal samples. Transcriptomic analysis of barrier and immune-related gene expression was performed on rumen, jejunum, and liver samples. We observed increased microbial populations with the Clostridial challenge in the buccal tissues and the proximal GI tract (forestomach), correlating with Clostridial loads in the feed. Otherwise, there were no significant differences in microbial populations (p > 0.05) throughout the distal part of the GI tract. The NGS approach, however, revealed that the Clostridial challenge changed the relative abundance of gut and fecal microbiota. In particular, in the challenge group, no Bifidobacterium was observed in the mucosa-associated microbiota and abundance of Pseudomonadota increased in the feces. These results indicated potential adverse effects of Clostridia to cow health. In general, immune responses to the Clostridial challenge were weak. However, transcriptional analysis revealed the down-regulation of junction adhesion molecule encoding gene (-1.44 of log2 fold-change), which might impact intestinal permeability.

Gut Microbiota-Derived Indole-3-Carboxylate Influences Mucosal Integrity and Immunity Through the Activation of the Aryl Hydrocarbon Receptors and Nutrient Transporters in Broiler Chickens Challenged With Eimeria maxima.

Two studies were conducted to evaluate the effects of indole-3-carboxylate (ICOOH) as a postbiotic on maintaining intestinal homeostasis against avian coccidiosis. In the first study, an in vitro culture system was used to investigate the effects of ICOOH on the proinflammatory cytokine response of chicken macrophage cells (CMCs), gut integrity of chicken intestinal epithelial cells (IECs), differentiation of quail muscle cells (QMCs), and primary chicken embryonic muscle cells (PMCs) and anti-parasitic effect against Eimeria maxima. Cells to be tested were seeded in the 24-well plates and treated with ICOOH at concentrations of 0.1, 1.0, and 10.0 µg. CMCs were first stimulated by lipopolysaccharide (LPS) to induce an innate immune response, and QMCs and PMCs were treated with 0.5% and 2% fetal bovine serum, respectively, before they were treated with ICOOH. After 18 h of incubation, cells were harvested, and RT-PCR was performed to measure gene expression of proinflammatory cytokines of CMCs, tight junction (TJ) proteins of IECs, and muscle cell growth markers of QMCs and PMCs. In the second study, in vivo trials were carried out to study the effect of dietary ICOOH on disease parameters in broiler chickens infected with E. maxima. One hundred twenty male broiler chickens (0-day-old) were allocated into the following four treatment groups: 1) basal diet without infection (CON), 2) basal diet with E. maxima (NC), 3) ICOOH at 10.0 mg/kg feed with E. maxima (HI), and 4) ICOOH at 1.0 mg/kg feed with E. maxima (LO). Body weights (BWs) were measured on 0, 7, 14, 20, and 22 days. All groups except the CON chickens were orally infected with E. maxima on day 14. Jejunal samples were collected for lesion score and the transcriptomic analysis of cytokines and TJ proteins. In vitro, ICOOH increased the expression of TJ proteins in IECs and decreased IL-1ß and IL-8 transcripts in the LPS-stimulated CMCs. In vivo, chickens on the HI diet showed reduced jejunal IL-1ß, IFN-γ, and IL-10 expression and increased expression of genes activated by aryl hydrocarbon receptors and nutrient transporters in E. maxima-infected chickens. In conclusion, these results demonstrate the beneficial effects of dietary ICOOH on intestinal immune responses and barrier integrity in broiler chickens challenged with E. maxima. Furthermore, the present finding supports the notion to use microbial metabolites as novel feed additives to enhance resilience in animal agriculture.

Comparative Genomics of Clostridium perfringens Reveals Patterns of Host-Associated Phylogenetic Clades and Virulence Factors.

Clostridium perfringens is an opportunistic pathogenic bacterium that infects both animals and humans. Clostridium perfringens genomes encode a diverse array of toxins and virulence proteins, which continues to expand as more genomes are sequenced. In this study, the genomes of 44 C. perfringens strains isolated from intestinal sections of diseased cattle and from broiler chickens from diseased and healthy flocks were sequenced. These newly assembled genomes were compared to 141 publicly available C. perfringens genome assemblies, by aligning known toxin and virulence protein sequences in the assemblies using BLASTp. The genes for alpha toxin, collagenase, a sialidase (nanH), and alpha-clostripain were present in at least 99% of assemblies analyzed. In contrast, beta toxin, epsilon toxin, iota toxin, and binary enterotoxin of toxinotypes B, C, D, and E were present in less than 5% of assemblies analyzed. Additional sequence variants of beta2 toxin were detected, some of which were missing the leader or signal peptide sequences and therefore likely not secreted. Some pore-forming toxins involved in intestinal diseases were host-associated, the netB gene was only found in avian isolates, while netE, netF, and netG were only present in canine and equine isolates. Alveolysin was positively associated with canine and equine strains and only present in a single monophyletic clade. Strains from ruminant were not associated with known virulence factors and, except for the food poisoning associated clade, were present across the phylogenetic diversity identified to date for C. perfringens. Many C. perfringens strains associated with food poisoning lacked the genes for hyaluronidases and sialidases, important for attaching to and digesting complex carbohydrates found in animal tissues. Overall, the diversity of virulence factors in C. perfringens makes these species capable of causing disease in a wide variety of hosts and niches.

Effects of Dietary Maltol on Innate Immunity, Gut Health, and Growth Performance of Broiler Chickens Challenged With Eimeria maxima.

Two studies were conducted to evaluate the effects of maltol as a postbiotic on innate immunity, gut health, and enteric infection. In the first study, an in vitro culture system was used to evaluate the effects of maltol on the innate immune response of chicken macrophage cells (CMC), gut integrity of chicken intestinal epithelial cells (IEC), anti-parasitic activity against Eimeria maxima, and differentiation of quail muscle cells (QMC) and primary chicken embryonic muscle cells (PMC). All cells seeded in the 24-well plates were treated with maltol at concentrations of 0.1, 1.0, and 10.0 µg. CMC and IEC were stimulated by lipopolysaccharide to induce an innate immune response, and QMC and PMC were treated with 0.5 and 2% fetal bovine serum, respectively. After 18 h of incubation, pro-inflammatory cytokines, tight junction proteins (TJPs), and muscle cell growth markers were measured. In the second study, the dietary effect of maltol was evaluated on disease parameters in broiler chickens infected with E. maxima. Eighty male 1-day-old broiler chickens were allocated into the following four treatment groups: (1) Control group without infection, (2) Basal diet with E. maxima, (3) High maltol (HI; 10.0 mg /kg feed) with E. maxima, and (4) Low maltol (LO; 1.0 mg/kg feed) with E. maxima. Body weights (BW) were measured on days 0, 7, 14, 20, and 22. All chickens except the CON group were orally infected with 104 E. maxima per chicken on day 14. Jejunum samples were collected for gut lesion scoring, and the gene expression of cytokines and TJPs. Data was analyzed using PROC MIXED in SAS. In vitro, maltol not only increased TJPs in IEC and cytokines in the LPS-stimulated CMC but also showed direct cytotoxicity against sporozoites of E. maxima. In vivo, the HI group improved the BW, reduced the gut lesion scores and fecal oocyst shedding, and decreased jejunal TNFSF15 and IL-1ß expression in E. maxima-infected chickens. In conclusion, these results demonstrate the beneficial effects of dietary maltol in the enhancement of growth performance, gut health, and coccidiosis resistance and the applicability of maltol as a postbiotic for the replacement of antibiotic growth promoters in commercial poultry production.

Forming 4-Methylcatechol as the Dominant Bioavailable Metabolite of Intraruminal Rutin Inhibits p-Cresol Production in Dairy Cows.

Rutin, a natural flavonol glycoside, elicits its diverse health-promoting effects from the bioactivities of quercetin, its aglycone. While widely distributed in the vegetables and fruits of human diet, rutin is either absent or inadequate in common animal feed ingredients. Rutin has been supplemented to dairy cows for performance enhancement, but its metabolic fate in vivo has not been determined. In this study, plasma, urine, and rumen fluid samples were collected before and after the intraruminal dosing of 100 mg/kg rutin to 4 Holsteins, and then characterized by both targeted and untargeted liquid chromatography-mass spectrometry (LC-MS)-based metabolomic analysis. In plasma and urine, 4-methylcatechol sulfate was identified as the most abundant metabolite of rutin, instead of quercetin and its flavonol metabolites, and its concentration was inversely correlated with the concentration of p-cresol sulfate. In rumen fluid, the formation of 3,4-dihydroxyphenylacetic acid (DHPAA) and 4-methylcatechol after rapid degradation of rutin and quercetin concurred with the decrease of p-cresol and the increase of its precursor, 4-hydroxyphenylacetic acid. Overall, the formation of 4-methylcatechol, a bioactive microbial metabolite, as the dominant bioavailable metabolite of rutin and quercetin, could contribute to their beneficial bioactivities in dairy cows, while the decrease of p-cresol, a microbial metabolite with negative biological and sensory properties, from the competitive inhibition between microbial metabolism of rutin and tyrosine, has the potential to reduce environmental impact of dairy operations and improve the health of dairy cattle.

Dietary Supplementation With Bacillus subtilis Direct-Fed Microbials Alters Chicken Intestinal Metabolite Levels.

Direct-fed microbials (DFMs) are dietary supplements containing live microorganisms which confer a performance and health benefit to the host, but the mechanisms are unclear. Here, a metabolomics approach was used to identify changes in intestinal metabolite levels in chickens fed an unsupplemented diet or a diet supplemented with B. subtilis strain 1781 or strain 747. Body weight gains of chickens fed the B. subtilis-supplemented diets were increased up to 5.6% in the B. subtilis 1781 group and 7.6% in the B. subtilis 747 group compared with chickens fed the unsupplemented diet. Compared with unsupplemented controls, the levels of 83 metabolites were altered (p < 0.05) (25 increased, 58 decreased) in chickens given the B. subtilis 1781-supplemented diet, while 50 were altered (p < 0.05) (12 increased, 38 decreased) with the B. subtilis 747-supplemented diet. Twenty-two metabolites were altered (p < 0.05) (18 increased, 4 decreased) in the B. subtilis 1781 vs. B. subtilis 747 groups. A random forest analysis of the B. subtilis 1781 vs. control groups gave a predictive accuracy of 87.5%, while that of the B. subtilis 747 vs. control groups was 62.5%. A random forest analysis of the B. subtilis 1781 vs. B. subtilis 747 groups gave a predictive accuracy of 75.0%. Changes in the levels of these intestinal biochemicals provided a distinctive biochemical signature unique to each B. subtilis-supplemented group, and were characterized by alterations in the levels of dipeptides (alanylleucine, glutaminylleucine, phenylalanylalanine, valylglutamine), nucleosides (N1-methyladenosine, N6-methyladenosine, guanine, 2-deoxyguanosine), fatty acids (sebacate, valerylglycine, linoleoylcholine), and carbohydrates (fructose). These results provide the foundation for future studies to identify biochemicals that might be used to improve poultry growth performance in the absence of antibiotic growth promoters.

Quantification of Propionibacterium acidipropionici P169 bacteria in environmental samples by use of strain-specific primers derived by suppressive subtractive hybridization.

A quantitative PCR (qPCR) assay targeting a gene identified by suppressive subtractive hybridization (SSH) was developed to detect Propionibacterium acidipropionici P169, with a threshold of 10(4) CFU/U of dairy feed or rumen fluid. The report is the first using a molecular marker generated by SSH to quantify a bacterial strain in environmental samples.

An outbreak of gangrenous dermatitis in commercial broiler chickens.

The present report describes an outbreak of gangrenous dermatitis (GD) infection in a commercial poultry farm in Delaware involving 34-day-old broiler chickens. In addition to obvious clinical signs, some GD-affected broilers also showed severe fibrino-necrotic enteritis and large numbers of Gram-positive rods in the necrotic tissue. Histopathological findings included haemorrhage, degeneration and necrosis of parenchymatous cells, especially of skin, muscle, and intestine. Immunofluorescence staining revealed Clostridium-like bacilli in the skin and the intestine. Both Clostridium perfringens and Clostridium septicum genomic sequences were identified by polymerase chain reaction in bacterial cultures isolated from the skin, muscle, and intestine, and in the frozen tissues from the GD-affected birds. Serological analysis demonstrated that both affected and clinically healthy birds from the same house had high serum antibody titres against C. perfringens, C. septicum, Eimeria, chick anaemia virus, and infectious bursal disease virus. These results are discussed in the context of the relationship between the different Clostridium spp. and the pathogenesis of GD.

Prevalence and diversity of toxigenic Clostridium perfringens and Clostridium difficile among swine herds in the midwest.

Clostridium perfringens and Clostridium difficile are associated with scours in the neonatal piglet and are an economic concern in swine production. The objective of this study was to characterize the prevalence and diversity of C. perfringens and C. difficile isolates obtained from scouring neonatal piglets in a large integrated production system, as well as in smaller independently owned regional farms. Rectal swabs were collected from 333 pigs at 11 sites in an integrated swine production system and from an additional 180 pigs at 16 regional farms located throughout the Midwest. C. perfringens was isolated from 89.8% of the pigs swabbed at the integrated sites, and C. difficile was isolated from 57.7% of these pigs. Of the pigs from the regional farms sampled, 95.6% were positive for isolation of C. perfringens and 27.2% were positive for C. difficile. Toxigenic isolates were typed using random amplified polymorphic DNA (RAPD) PCR, and were placed in four dendrograms for C. perfringens and C. difficile populations isolated from the integrated sites and regional farms. Diversity indices showed that there was greater diversity in C. difficile populations and in populations isolated from the regional farms. A subset of isolates from the C. difficile dendrograms were further toxinotyped by amplification of the pathogenicity locus and subsequent digestion by HincII, AccI, and EcoRI. Of the 45 isolates typed, 44 were determined to be toxinotype V. The results of this study illustrate the diversity of C. perfringens and C. difficile isolates and the prevalence of these pathogens in swine production sites.

MLST analysis reveals a highly conserved core genome among poultry isolates of Clostridium septicum.

Clostridium septicum is a highly virulent, anaerobic bacterium capable of establishing necrotizing tissue infections and forming heat resistant endospores. Disease is primarily facilitated by secretion of numerous toxic products including a lethal pore-forming cytolysin. Spontaneously occurring clostridial myonecrosis involving C. septicum has recently reemerged as a concern for many poultry producers. However, despite its increasing prevalence, the epidemiology of infection and population structure of C. septicum remains largely unknown. In this study a multilocus sequence typing (MLST) approach was utilized to examine evolutionary relationships within a diverse collection of C. septicum isolates recovered from poultry flocks experiencing episodes of gangrenous dermatitis. The 109 isolates examined represented 42 turkey flocks and 24 different flocks of broiler chickens as well as C. septicum type strain, ATCC 12464. Isolates were recovered predominantly from gangrenous lesions although isolates from livers, gastrointestinal tracts, spleens and blood were included. The loci analyzed were csa, the major lethal toxin produced by C. septicum, and the housekeeping genes gyrA, groEL, dnaK, recA, tpi, ddl, colA and glpK. These loci were included in part because of their previous use in MLST analysis of Clostridium perfringens and Clostridium difficile. Results indicated a high level of conservation present within these housekeeping gene fragments when compared to what has been previously reported for the aforementioned clostridia. Of the 5352 bp of sequence data examined for each isolate, 99.7% (5335/5352) was absolutely conserved among the 109 isolates. Only one of the ten unique sequence types, or allelic profiles, identified among the isolates was recovered from both turkeys and broiler chickens suggesting some host species preference. Phylogenetic analyses identified two unique clusters, or clonal complexes, among these poultry isolates which may have important epidemiological implications for poultry producers in the United States. This work indicates a predominantly clonal population structure for C. septicum although some evidence of recombination was also observed.

Effects of feeding two levels of propionibacteria to dairy cows on plasma hormones and metabolites.

To determine the effect of feeding propionibacteria on metabolic indicators during lactation, multiparous and primiparous Holstein cows were fed one of three dietary treatments in a 2 x 3 factorial design from 2 weeks prepartum to 30 weeks post partum: (1) Control (primiparous n=5, multiparous n=8) fed a total mixed ration (TMR); (2) high-dose group (primiparous n=6, multiparous n=5) fed TMR plus 6 x 10 (11) cfu/head daily (high-dose P169) of propionibacterium strain P169; or (3) low-dose group (primiparous n=8, multiparous n=6) fed TMR plus 6 x 10(10) cfu/head daily (low-dose P169) of P169. Blood samples were collected weekly and analysed for plasma concentrations of glucose, insulin, insulin-like growth factor-I (IGF-I), leptin, nonesterified fatty acids (NEFA) and cholesterol. Between weeks 25 and 30, all groups received bovine somatotropin (bST) every 2 weeks. Low-dose P169 multiparous cows had lower (P<0.05) plasma insulin and glucose concentrations than high-dose P169 multiparous cows, whereas high-dose P169 primiparous cows had lower glucose but greater insulin concentartions than low-dose P169 primiparous cows (P<0.05). Plasma insulinratioglucose molar ratios were 13-18% lower (P<0.05) in low-dose P169 cows than in control or high-dose P169 cows. Plasma IGF-I, NEFA and leptin levels did not differ among diet groups between weeks 1 and 25. Low-dose P169 multiparous cows had 25% greater plasma cholesterol levels than high-dose P169 and control multiparous cows, but cholesterol levels in primiparous cows did not differ. During bST treatment, high-dose P169 multiparous cows and low-dose P169 primiparous cows had lower IGF-I levels than their respective controls and, regardless of parity, high-dose P169 cows had greater NEFA than control cows. Although supplemental feeding of P169 altered plasma hormones and metabolites, the particular effects were dependent on dose of P169 and parity of cows.