Changes in gene expression in the intestinal mucus of broilers with woody breast myopathy.

Previous work has shown that dietary treatments affect woody breast (WB) incidence differently, which indicates that gut conditions such as gut barrier function, inflammation, and oxidative stress are likely related to WB. In this study, dietary supplementation with antibiotics (bacitracin) or probiotics (Bacillus subtilis) was investigated for their effects on the expression of transcripts related to gut barrier function, inflammation, and oxidative stress in the mucus lining of the jejunum from broilers with or without WB. A split-plot experimental design was used in this study. The dietary treatments served as the main plot factor and the breast muscle condition was the subplot factor. On d 41, jejunum mucus was collected from 1 bird from each of 3 replicate pens in each 3 dietary treatment groups that exhibited WB and an additional bird that contained a normal breast (3 biological replicates/treatment/phenotype; 3 × 3 × 2, total N = 18). Total RNA was extracted using a commercial RNA extraction kit. The expression levels of CLDN1, MUC6, TLR2A, TLR2B, TLR4, IFN-γ, IL-1ß, IL-8L1, IL-10, NOS2, and SOD were determined using 2-step RT-qPCR analysis. The gene expression difference in ΔCt values was determined after normalizing with the chicken 18S rRNA gene. When the significant differences occurred between treatments, the relative fold change was calculated using the ΔΔCt method and the significance level was calculated. The PROC GLM procedure of SAS 9.4 was used, and the level of significance was set at P ≤ 0.05. There were no significant interactive effects between diet and the breast muscle condition on the expression of any of the genes tested. However, birds with WB exhibited higher MUC6 (P < 0.0001) gene expression levels than birds with normal breast muscles. In addition, the expression of SOD decreased in birds that were fed the antibiotic diet when compared to birds that were fed the probiotic diet (P = 0.014). In conclusion, WB identified in broilers tested in the current study is attributed to increased expression of mucin, indicating a correlation between WB incidence and gel-forming mucin secretion and pathogen signaling.

In silico prediction and expression analysis of vaccine candidate genes of Campylobacter jejuni.

Campylobacter jejuni (C. jejuni) is the most common food-borne pathogen that causes human gastroenteritis in the United States. Consumption of contaminated poultry products is considered as the major source of human Campylobacter infection. An effective vaccine would be a promising alternative to antibiotic supplements to curb C. jejuni colonization in poultry gastrointestinal (GI) tract. However, the genetic diversity among the C. jejuni isolates makes vaccine production more challenging. Despite many attempts, an effective Campylobacter vaccine is not yet available. This study aimed to identify suitable candidates to develop a subunit vaccine against C. jejuni, which could reduce colonization in the GI tract of the poultry. In the current study, 4 C. jejuni strains were isolated from retail chicken meat and poultry litter samples and their genomes were sequenced utilizing next-generation sequencing technology. The genomic sequences of C. jejuni strains were screened to identify potential antigens utilizing the reverse vaccinology approach. In silico genome analysis predicted 3 conserved potential vaccine candidates (phospholipase A [PldA], TonB dependent vitamin B12 transporter [BtuB], and cytolethal distending toxin subunit B [CdtB]) suitable for the development of a vaccine. Furthermore, the expression of predicted genes during host-pathogen interaction was analyzed by an infection study using an avian macrophage-like immortalized cell line (HD11). The HD11 was infected with C. jejuni strains, and the RT-qPCR assay was performed to determine the expression of the predicted genes. The expression difference was analyzed using ΔΔCt methods. The results indicate that all 3 predicted genes, PldA, BtuB, and CdtB, were upregulated in 4 tested C. jejuni strains irrespective of their sources of isolation. In conclusion, in silico prediction and gene expression analysis during host-pathogen interactions identified 3 potential vaccine candidates for C. jejuni.

Effects of dietary bacitracin or Bacillus subtilis on the woody breast myopathy-associated gut microbiome of Eimeria spp. challenged and unchallenged broilers.

Study suggested that dysbiosis of the gut microbiota may affect the etiology of woody breast (WB). In the current study, the cecal microbiota and WB in chickens fed three different diets were investigated. A total of 504 male chicks were used in a randomized complete block design with a 3 (Diet) × 2 (Challenge) factorial arrangement of treatments with 6 replicates per treatment, 6 treatments per block, and 14 birds per treatment. The experimental diets were a control diet (corn-soybean meal basal diet), an antibiotic diet (basal diet + 6.075 mg bacitracin/kg feed), and a probiotic diet (basal diet + 2.2 × 108 CFU Bacillus subtilis PB6/kg feed). On d 14, birds that were assigned to the challenge treatment received a 20 × live cocci vaccine. On d 41, breast muscle hardness in live birds was palpated and grouped into normal (NB) and WB phenotypes. Cecal contents were collected and their bacterial compositions were analyzed and compared. The genomic DNA of the cecal contents was extracted and the V3 and V4 regions of 16S rRNA gene were amplified and sequenced via an Illumina MiSeq platform. There were no differences (P > 0.05) in Shannon and Chao 1 indexes between the challenges, diets, and phenotypes (NB vs. WB). However, there was a difference (P = 0.001) in the beta diversity of the samples between the challenged and nonchallenged groups. Relative bacterial abundance differed (false discovery rate, FDR < 0.05) between the challenge treatments, but there were no significant differences (FDR > 0.05) among the three diets or two phenotypes. Predicted energy metabolism, nucleotide metabolism, and amino acid and coenzyme biosynthesis activities only differed (q-value < 0.05) between challenged and nonchallenged groups. The cocci challenge altered the gut microbial composition on Butyricicoccus pullicaecorum, Sporobacter termitidis, and Subdoligranulum variabile, but the dietary antibiotic and probiotic treatments did not impact gut microbial composition. No strong association was found between WB myopathy and gut microbial composition in this study.

Effects of in ovo probiotic administration on the incidence of avian pathogenic Escherichia coli in broilers and an evaluation on its virulence and antimicrobial resistance properties.

Avian pathogenic Escherichia coli (APEC) causes colibacillosis in poultry, which has been traditionally controlled by the prophylactic in-feed supplementation of antibiotics. However, antibiotics are being removed from poultry diets owing to the emergence of multidrug-resistant (MDR) bacteria. Therefore, alternatives to control APEC are required. This study aimed to evaluate the effects of in ovo inoculation of probiotics on the incidence of APEC in broilers and evaluate the virulence and antimicrobial resistance properties of the APEC isolates. On embryonic day 18, 4 in ovo treatments (T) were applied: T1 (Marek's vaccine [MV]), T2 (MV and Lactobacillus animalis), T3 (MV and Lactobacillus reuteri), and T4 (MV and Lactobacillus rhamnosus). A total of 180 male broilers per treatment were randomly placed in 10 pens. The heart, liver, spleen, and yolk sac were collected on day 0, 14, 28, and 42. Presumptive E. coli isolates were confirmed by real-time PCR. The positive isolates were screened for the APEC-related genes (iroN, ompT, hlyF, iss, and iutA), and E. coli isolates containing one or more of these genes were identified as APEC-like strains. A total of 144 APEC-like isolates were isolated from 548 organ samples. No differences (P > 0.05) among treatments were observed for the incidence of APEC-like strains in all organs when averaged over sampling days. However, when averaged over treatments, the incidence in the heart, liver, and yolk sac was different among sampling days; a significant increase was observed in these organs on day 14 compared with day 0. Twenty-five antimicrobial resistance genes were evaluated for all APEC-like isolates, and 92.4% of the isolates carried at least one antimicrobial resistance gene. Thirty-seven isolates were then selected for antimicrobial susceptibility testing; MDR strains accounted for 37.8% of the isolates. In conclusion, the in ovo inoculation of a single probiotic strain did not confer protection against APEC strains in broilers. The high prevalence of MDR isolates indicates that further research on antibiotic alternatives is required to prevent APEC infections in broilers.

Effects of coccidial vaccination and dietary antimicrobial alternatives on the growth performance, internal organ development, and intestinal morphology of Eimeria-challenged male broilers.

Effects of the coccidial vaccination and dietary antimicrobial alternatives on growth performance, internal organ development, and intestinal morphology of male broilers subjected to an Eimeria challenge were determined. A total of 1,120 one d-old Ross × Ross 708 male broilers were randomly distributed to 80 floor pens (10 treatments, 8 replication pens/treatment, and 14 chicks/pen). A 2 × 5 factorial arrangement of treatments was used to determine the main and interaction effects of the coccidial vaccination (vaccinated or non-vaccinated) and the dietary additive [1) corn and soybean-meal basal diet, 2) basal diet + antimicrobials (bacitracin and salinomycin), 3) basal diet + probiotics (3 Bacillus subtilis strains), 4) basal diet + prebiotics (mannan-oligosaccharides and ß-glucans), and 5) basal diet + probiotics + prebiotics]. To mimic the Eimeria challenge, all chicks were gavaged with a 20x dose of a different coccidial vaccine (live Eimeria oocysts) at Day 14. The coccidial vaccination decreased Day 0-14 and 29-42 BW gain (BWG) and subsequently decreased Day 0-56 BWG. Broilers fed diets with antimicrobials exhibited the lowest feed conversion ratio (FCR) during the periods of Day 0-14 and 15-28, the shallowest ileal crypt depth on Day 28, and the lowest relative duodenum weight on Day 28 and 42. The Pre+Pro diets helped the broilers to reach a lower overall FCR than did the Pro alone diets and helped the broilers reach a FCR similar to that of the Anti diets. However, broilers fed diets supplemented with prebiotics and probiotics exhibited the deepest intestinal crypt depth on Day 28. There was no interaction between coccidial vaccination and dietary additive on growth performance or any carcass yield. In conclusion, antimicrobial additives might reduce the intestinal size of broilers; whereas prebiotic and B. subtilis-based probiotic additives might promote the growth of several digestive organs. Prebiotics can be safely used with B. subtilis-probiotics in broiler feed without compromising feed conversion ability.

Effects of Bacillus subtilis and zinc on the growth performance, internal organ development, and intestinal morphology of male broilers with or without subclinical coccidia challenge.

Effects of antibiotic (bacitracin), anticoccidial (narasin), and alternative (Bacillus subtilis and zinc) feed additives on growth performance, internal organ development, and intestinal morphology of commercial broilers with or without subclinical coccidia challenge were determined. A total of 1,344 1-day-old male Ross × Ross 708 broilers were randomly distributed into 12 treatments (6 diets × 2 challenge treatments, 8 replication pens/treatment) in 96 floor pens. The 6 dietary treatments were as follows: a control diet (corn and soybean-meal basal diet), a probiotic diet (basal diet + Bacillus subtilis), a zinc diet (basal diet + 100 ppm zinc), a probiotic and zinc combined diet, an anticoccidial diet (basal diet + narasin), and a practical diet (basal diet + narasin + bacitracin). On day 21, each chick in the challenge treatment was gavaged with a 10× dose of a commercial vaccine containing live Eimeria oocytes, whereas each chick in the non-challenge treatment was gavaged with equivalent distilled water. The subclinical coccidia challenge increased the relative weights of pancreas and decreased the ileal crypt depth of broilers at 26 d of age, increased feed conversion ratios from day 15 to 28 and 29 to 40, and increased the relative weights of duodenum and bursa on day 54. As compared to other diets, anticoccidial and practical diets increased BW gain and decreased feed conversion ratio from day 15 to 28, and increased the day 40 carcass weights. As compared to control diets, probiotic diets decreased BW gain and increased the mortality from day 15 to 28; however, probiotic diets did not affect the overall growth performance from day 0 to 54 or carcass yield on day 54. Growth measurements during periods of day 29 to 40 and day 41 to 54 were not affected by any feed additive. From this study, a subclinical coccidia challenge enlarged specific internal organs and compromised the feed conversion ability of broilers. Dietary Bacillus subtilis did not affect overall growth rate or carcass yield of broilers under subclinical coccidia challenge.