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Subclinical necrotic enteritis (NE) is an enteric disease that inflicts significant economic losses in the poultry industry, primarily by reducing performance in commercial flocks but without significant mortality. This study evaluated the effects of a variety of phytogenic blends on broilers’ performance and carcass composition during an induced NE challenge. In this study, 1120 day (d)-old male broilers were allocated to four treatments groups (14 replicate floor pens, 20 birds/pen): the control (CONT) group, fed a basal corn-soybean diet, and three phytogenic blend dietary additives (PHYTO1, PHYTO2, and PHYTO3) added to the basal diet at 150, 250, and 500 mg/kg feed, respectively. Subclinical NE was induced by spraying a concentrated coccidiosis vaccine onto the feed and litter 24 h post-placement. On day 8, two birds/pen were necropsied for NE lesions. On days 8, 14, 28, and 42, the average daily gain (ADG), feed intake (ADFI), and feed conversion ratio (FCR) were calculated. On day 42, two birds/pen were euthanized to assess carcass composition using dual-energy X-ray absorptiometry (DXA). Statistical analyses for all data were performed using the ANOVA procedure (JMP, Pro 16) and significance (p ≤ 0.05) between treatments was determined by the LSD test. There was no effect of treatment on NE lesions. PHYTO1, PHYTO2, and PHYTO3 significantly improved FCR from days 9 to 14, 0 to 14, and 0 to 42 and resulted in greater ADG from days 9 to 14, 29 to 42 and cumulatively on days 0 to 42. Carcass composition data revealed a numerically higher lean-to-fat ratio in the PHYTO groups compared to the CONT group. These results indicate that the dietary supplementation of phytogenic blends could alleviate the adverse effects of NE challenge on broilers’ performance and carcass composition.

Impaired intestinal integrity in broilers reduces performance and health, highlighting the importance of accurately measuring intestinal permeability (IP) to maintain gut health. The objective of this study was to evaluate the efficiency of iohexol as an IP marker in broilers challenged with Eimeria maxima, Clostridium perfringens, or both during both peak challenge (day [d] 21) and recovery (d 28) periods. One-day-old male Ross 708 birds (n = 56) were distributed into 4 treatment groups: NC (no-challenge control); EM (challenged with 5,000 E. maxima sporulated oocysts/bird on d 15); CP (challenged with 1.0 × 108 CFUs/bird of C. perfringens on d 19 and d 20); and EM + CP (challenged by co-infection of E. maxima and C. perfringens as described). On d 21 and d 28, each bird received an iohexol dose of 64.7 mg/kg body weight via oral gavage. One hour later, blood samples were collected from 14 birds (12 in EM) per group on d 21 and from 7 birds (6 in EM) on d 28. For lesion scoring and ileum collection, 7 birds per group (6 birds in EM) were sampled on each d 21 and d 28. Birds in the EM and EM + CP groups had lower body weight gain (BWG) compared to the NC and CP groups on d 19–21 (P ≤ 0.05). These birds also exhibited significantly greater lesion scores and markedly higher serum iohexol levels on d 21 (P ≤ 0.05). However, no significant differences in serum iohexol levels were observed among treatment groups following recovery on d 28. Moreover, significant differentials were observed in the mRNA abundance of key tight junction proteins (CLDN1, CLDN2, and ZO3), pro-inflammatory cytokines (IL-1β, IFNγ, and IL-22), and gut health markers (GLP2, OLFM4, and MUC2) in the EM and EM + CP groups compared to the NC and CP groups on d 21. In conclusion, this study demonstrates that iohexol is an effective marker for assessing IP in broilers under different enteric challenge conditions.

Necrotic enteritis (NE), an enteric disease caused by Clostridium perfringens, results in damage to the intestinal epithelial lining disrupting its function, nutrient absorption, and utilization. This study evaluated the effects of in ovo and post-hatch applications of a Saccharomyces cerevisiae-based postbiotic on performance and nutrient transporter genes of broilers during a NE challenge. At embryonic d 18, Ross 708 fertile eggs were injected with 0.2 mL of either water or postbiotic. A total of 288 male hatchlings were assigned to one of the following treatment groups: 1) NC (in ovo water injection, no challenge); 2) PIW (postbiotic in ovo and in drinking water, no challenge); 3) NC+ (NC with NE challenge); and 4) PIW+ (PIW with NE challenge). On d 14, all birds in the NE-challenged groups were orally gavaged with 3,000 Eimeria maxima sporulated oocysts followed by two doses of ∼1×108 CFU/mL/bird of C. perfringens on d 19 and d 20. Hatchability, weekly performance, intestinal lesion scores, and mRNA abundance of nutrient transporters in the jejunum and ileum were assessed. Data were analyzed by 2-way ANOVA in JMP and significance between treatments identified by LSD test (P ≤ 0.05). A significant postbiotic treatment and NE challenge interaction was observed in performance during d 21-28 with a greater ADG in PIW compared to NC and PIW+. Lesion scores in the jejunum and ileum were significantly reduced in PIW+ compared to NC+. On d 7, mRNA abundance of SGLT1 was significantly greater in PIW compared to the NC group. On d 14, birds in PIW had greater levels of GLUT2 and EAAT3 than NC group. No significant interaction effects were observed on d 21. PIW+ had significantly greater EAAT3 mRNA levels compared to PIW in jejunum and PIW and NC+ in ileum on d 28. In conclusion, in ovo and water supplementation of this postbiotic presents a potential to improve the performance, ameliorate pathology detriments associated with NE, and positively regulate the mRNA levels of key nutrient transporters during NE challenge.

Caused by the Gram-positive bacteria Clostridium perfringens, necrotic enteritis (NE) is an enteric disease with significant economic implications in broiler production. This study employed an experimental NE model involving co-infection with Eimeria maxima and C. perfringens to assess whether sulfate polysaccharides extracted from marine macroalgae could mitigate the adverse effects of NE in broilers. A total of 600 day (d)-old Ross 708 male broilers were randomly assigned to one of four treatment groups: NC (negative control, fed a corn-soybean meal diet); PC (positive control, NC + 15 ppm Avilamycin and 125 ppm Amprolium); AGS (Algimun® Standard, NC + Algimun added at 0.1 % of the diet); and AGH (Algimun High, NC + Algimun added at 0.2 % of the diet). Average daily feed intake and weight gain were calculated and adjusted for daily mortality on d 14, 21, 28, and 42. On d 21, four birds/pen were examined for intestinal NE lesions. On d 14, 21, and 42, serum and jejunum samples from one bird/pen were collected to measure the concentration of the biomarker calprotectin and mRNA abundance of cytokines and tight junction proteins. Data were analyzed using JMP and significance between treatments identified by LSD (P ≤ 0.05). NE lesion scores on d 21, and mortality and FCR during d 0 to 42 were lower in PC, AGS, and AGH compared to NC birds. AGS birds had the lowest levels for serum calprotectin on d 21 while PC, AGS, and AGH had lower levels than the NC group on d 42. On d 14 and d 42, mRNA abundance of CLDN1, 3, ZO1, IL1β, IFNγ, IL10, and IL12B was greater in AGS and AGH birds compared to NC. Also, AGH had a greater abundance of TNFα on d 14 and d 42 compared to NC. Further, mRNA abundance of CLDN3, ZO1, 2, OCLDN, IL1β, IL10, IL12B, IFNγ, and ANXA1 was greater in PC on d 21 compared to NC and AGS. Based on these findings, dietary supplementation of this macroalgae-derived sulfated polysaccharides yielded a similar response to an antibiotic growth promoter presenting potential as an alternative additive.

ABSTRACT: The effects of traditional and on-farm hatching systems on broiler performance and health under a subclinical necrotic enteritis (NE) challenge were evaluated in this study. A 2×2 factorial study explored the effects of place of hatch (on-farm hatched [OFH] vs. hatchery hatched [HH]) and NE challenge (nonchallenged vs. challenged) on broilers. Cobb 500 eggs (∼E19) were acquired from a commercial hatchery; 840 eggs were placed in pens on clean shavings in prewarmed floor pens and allowed to hatch out, while 927 eggs were placed in a hatcher set under standard practices. On day (d) of hatch, all chicks were weighed and randomly distributed to 4 treatments (8 replicate pens each and 30 birds/pen). The OFH birds were placed immediately after sorting while HH birds were placed back in the hatcher overnight to simulate commercial hatchery procedures. After placing HH birds, feed and litter in the challenge group pens were sprayed with a live oocyst coccidia vaccine as a predisposing factor to NE. The small intestines of 3 male chicks per pen were scored for NE lesions (n = 24) on d 8 (peak NE challenge) and jejunal samples were collected from 1 bird per pen for RNA extraction and qPCR on d 8 and d 14. Data were analyzed using JMP Pro17 and significance between treatments was identified by LSD (P ≤ 0.05). Regardless of the hatching system, the subclinical NE challenge caused a significant reduction in average daily gain (ADG) and average daily feed intake (ADFI), and increased feed conversion ratio (FCR) until d 28 (P ≤ 0.05). Moreover, OFH birds exhibited significantly better growth (P ≤ 0.05) through d 28 but had similar performance to HH birds by d 42. There were no significant differences in NE lesion scores between HH and OFH groups. In conclusion, OFH system resulted in better broiler performance compared to HH system under both no-challenge and challenge conditions during the starter and grower periods. This practice may hold potential for further exploration by the industry as an alternative to traditional hatching, aiming to improve the welfare and productivity of broilers.

ABSTRACT: In this comparative study, the differential responses of heritage (ACRB; Athens Canadian Random Bred) and modern (Cobb) broilers to a necrotic enteritis (NE) challenge were evaluated. The design was a 2×2 factorial with breed (ACRB and Cobb) and challenge (non-challenged and NE-challenged) as main factors. On day (d) of hatch, 96 male chicks (48 ACRB and 48 Cobb) were allocated to 4 experimental groups with 8 replicate cages and 3 birds/cage. On d 14, birds in the NE-challenged groups were orally gavaged with 3,000 Eimeria maxima sporulated oocysts followed by 2 doses of ∼1×108 CFU of Clostridium perfringens on d 19 and 20. On d 21, 2 birds/cage were necropsied to score NE lesions, and spleen and cecal tonsils (CT) samples were collected from 1 bird/cage for assessing mRNA abundance. Challenged ACRB birds exhibited reduced growth performance and relative growth performance compared to challenged Cobb birds. There was no significant interaction between breed and challenge during the challenge period (d 14–21) for mortality. However, there was a challenge main effect (P ≤ 0.05) on mortality as manifested by greater NE-associated mortality compared to non-challenged birds. No significant breed × challenge interaction or breed main effect on lesion scores were observed in the duodenum, jejunum, and ileum. NE-challenged Cobb birds exhibited greater mRNA abundance of IL-18, TNFα, TLR1.2, TLR2.1, CCR5, CCR6, CCL20, and AvBD1 in CT compared to NE challenged ACRB birds. There was a significant breed × challenge interaction effect on mRNA abundance of IL-10, AvBD13, NK-Lysin, and LEAP2 in the spleen. Moreover, a main effect of breed was observed in IL-1β, IL-18, TNFα, TLR2.1, CCR5, CCL20, and NK-Lysin where ACRB birds had higher mRNA abundance than Cobb birds (P ≤ 0.05). The observed differences in performance, pathology, and mRNA abundance between ACRB and Cobb broilers during the NE challenge highlight the distinct immune response profiles of heritage and modern breeds, emphasizing the need for breed-specific nutritional, managerial, and genetic selection programs for modulating immune responses during enteric disease challenges.

ABSTRACT: Transfer of Salmonella to internal organs of broilers over a 35 d grow-out period was evaluated. A total of 360 one-day old chicks were placed in 18 floor pens of 3 groups with 6 replicate pens each. On d 0, broilers were orally challenged with a cocktail of Salmonella (equal population of marked serovars; nalidixic acid-resistant S. Typhimurium, rifampicin-resistant S. Infantis, and kanamycin-resistant S. Reading) to have 3 groups: L (low; ∼2 log CFU/bird); M (medium; ∼5 log CFU/bird); and H (High; ∼8 log CFU/bird). On d 2, 7 and 35, 4 birds/pen were euthanized and ceca, liver, and spleen samples were collected aseptically. Gizzard samples (4/pen) were collected on d 35. The concentration of Salmonella in liver and spleen were transformed to binary outcomes (positive and negative) and fitted in glm function of R using cecal Salmonella concentrations (log CFU/g) and inoculation doses (L, M, and H) as inputs. On d 2, H group showed greater (P ≤ 0.05) cecal colonization of all 3 serovars compared to L and M groups. However, M group showed greater (P ≤ 0.05) colonization of all 3 serovars in the liver and spleen compared to L group. Salmonella colonization increased linearly in the ceca and quadratically in the liver and spleen with increasing challenge dose (P ≤ 0.05). On d 35, L group had greater (P ≤ 0.05) S. Infantis colonization in the ceca and liver compared to M and H groups (P ≤ 0.05). Moreover, within each group on d 35, the concentration of S. Reading was greater than those of S. Typhimurium and S. Infantis for all 3 doses in the ceca and high dose in the liver and gizzard (P ≤ 0.05). Salmonella colonization diminished in the ceca, liver, and spleen during grow-out from d 0 to d 35 (P ≤ 0.05). On d 35, birds challenged with different doses of Salmonella cocktail showed a similar total Salmonella spp. population in the ceca (ca. 3.14 log CFU/g), liver (ca. 0.54 log CFU/g), spleen (ca. 0.31 log CFU/g), and gizzard (ca. 0.42 log CFU/g). Estimates from the fitted logistic model showed that one log CFU/g increase in cecal Salmonella concentration will result in an increase in relative risk of liver and spleen being Salmonella-positive by 4.02 and 3.40 times (P ≤ 0.01), respectively. Broilers from H or M group had a lower risk (28 and 23%) of being Salmonella-positive in the liver compared to the L group when the cecal Salmonella concentration is the same (P ≤ 0.05). Oral challenge of broilers with Salmonella spp. with various doses resulted in linear or quadratic increases in Salmonella colonization in the internal organs during early age and these populations decreased during grow-out (d 35). This research can provide guidance on practices to effectively mitigate the risk of Salmonella from chicken parts and enhance public health.

ABSTRACT: Increased use of genetically modified (GM) plants in the food and feed industry has raised several concerns about the presence of unwanted genes in the food chain and potential associated health risks. In recent years, several studies have compared the nutrient contents of GM crops to conventional counterparts, and some have also tracked the fate of novel DNA fragments and proteins in the gastrointestinal (GIT) and their presence in several tissues. This study was conducted to investigate the fate of transgenic PHP19340A DNA fragment containing gm-fad2-1 (Soybean Event DP-3Ø5423-1) gene in digestive tract contents, blood, internal organs, and muscle tissues. The effects of feeding DP-3Ø5423-1 full-fat soybean meal (FFSBM) to broiler chickens on immune response and blood profiles were also evaluated on d 35. Day-old Ross 308 birds (n = 480) were randomly allocated to 24 floor pens in a 2 × 2 factorial arrangement with diet and gender as main factors. Birds were fed diets containing 20% of either DP-3Ø5423-1 or non-GM FFSBM for 35 d. Data were subjected to a 2-way ANOVA using the GLM procedure of JMP (Pro13). Based on PCR analysis, transgenic PHP19340A DNA fragment containing gm-fad2-1 gene was degraded throughout the digestive system to reach undetectable level in the cecal digesta. Moreover, there was no transgenic gene translocation to blood, organs, or muscle tissue. Feeding DP-3Ø5423-1 FFSBM to broilers had no effect on mRNA abundance of IL-1β, IL-2, IL-6, IL-12B, IL-17A, IFNγ, TNFα, and NF-κB in the spleen or on blood profile. In conclusion, these findings indicate that the examined transgenic fragment in DP-3Ø5423-1 FFSBM progressively degraded in the GIT and did not translocate into blood or tissues. Along with the immune response and blood profile findings, it can be assumed that DP-3Ø5423-1 soybean is safe and unlikely to pose any health risks to broilers or consumers.

ABSTRACT: Several genetically modified (GM) plants have been produced and approved by regulatory agencies worldwide for cultivation and commercialization. Soybean and its by-products are major components of poultry diets and approximately 74% of world production is obtained from GM soybean events. The aim of this study was to evaluate the nutrient composition of DP-3Ø5423-1 extruded full-fat soybean meal (FFSBM) and near isoline non-GM control FFSBM included in broiler diets. Also assessed were their effects on bird performance, body composition, intestinal morphology, tissue fatty acid profile, and mRNA abundance of fatty acid metabolism markers. A total of 480 Ross 308 d of hatch birds were randomly allocated to 24 floor pens in a 2 × 2 factorial arrangement with diet and gender as main factors. Birds were fed diets containing 20% of either DP-3Ø5423-1 or control FFSBM for 35 d. Data were subjected to a 2-way ANOVA using the GLM procedure of JMP (Pro13). No significant interaction (P > 0.05) was observed between treatment groups in terms of performance and carcass composition. Morphological measurements of the jejunum and ileum were not influenced by the SBM treatments. Dietary addition of the DP-3Ø5423-1 FFSBM resulted in higher monounsaturated fatty acid composition of the thigh muscle and abdominal fat. Moreover, dietary treatment had no significant impact on the mRNA abundance of metabolic markers ACCα, FAS, MTTP, SREBP1, PPARα, PPARγ, AMPK-α1, SOD, CAT, and GPx in the liver. In conclusion, our results showed that DP-3Ø5423-1 extruded FFSBM is nutritionally equivalent to non-GM near-isoline counterpart with a comparable genetic background as evidenced by feed analyses except for fatty acid composition. Furthermore, the findings of this study clearly indicate that the examined DP-3Ø5423-1 FFSBM yields similar bird performance as conventional FFSBM.

Pathogen adaptations during host-pathogen co-evolution can cause the host balance between immunity and immunopathology to rapidly shift. However, little is known in natural disease systems about the immunological pathways optimised through the trade-off between immunity and self-damage. The evolutionary interaction between the conjunctival bacterial infection Mycoplasma gallisepticum (MG) and its avian host, the house finch (Haemorhous mexicanus), can provide insights into such adaptations in immune regulation. Here we use experimental infections to reveal immune variation in conjunctival tissue for house finches captured from four distinct populations differing in the length of their co-evolutionary histories with MG and their disease tolerance (defined as disease severity per pathogen load) in controlled infection studies. To differentiate contributions of host versus pathogen evolution, we compared house finch responses to one of two MG isolates: the original VA1994 isolate and a more evolutionarily derived one, VA2013. To identify differential gene expression involved in initiation of the immune response to MG, we performed 3’-end transcriptomic sequencing (QuantSeq) of samples from the infection site, conjunctiva, collected 3-days post-infection. In response to MG, we observed an increase in general pro-inflammatory signalling, as well as T-cell activation and IL17 pathway differentiation, associated with a decrease in the IL12/IL23 pathway signalling. The immune response was stronger in response to the evolutionarily derived MG isolate compared to the original one, consistent with known increases in MG virulence over time. The host populations differed namely in pre-activation immune gene expression, suggesting population-specific adaptations. Compared to other populations, finches from Virginia, which have the longest co-evolutionary history with MG, showed significantly higher expression of anti-inflammatory genes and Th1 mediators. This may explain the evolution of disease tolerance to MG infection in VA birds. We also show a potential modulating role of BCL10, a positive B- and T-cell regulator activating the NFKB signalling. Our results illuminate potential mechanisms of house finch adaptation to MG-induced immunopathology, contributing to understanding of the host evolutionary responses to pathogen-driven shifts in immunity-immunopathology trade-offs.

This study investigated the effects of a synbiotic consisting of inulin, Enterococcus faecium, Pediococcus acidilactici, Bifidobacterium animalis, and Lactobacillus reuteri given orally to day (d)-of-hatch (DOH) broiler chicks at the hatchery and in the feed for a 21 d period. A total of 480 Cobb male broilers were randomly divided into one of four treatments using a 2 × 2 factorial design as follows: (1) control (CTRL) group receiving a gel-only oral application on DOH at the hatchery prior to transport and a non-medicated basal corn/soybean meal starter diet; (2) hatchery synbiotic (HS) receiving an oral gel containing the synbiotic (0.5 mL/bird) at the hatchery and the basal diet; (3) CTRL + dietary synbiotic at 0.5 kg/MT (DS); and (4) HS + dietary synbiotic at 0.5 kg/MT (HSDS). On d 7 and d 21, one bird per pen (eight replicate pens/group) was euthanized, and the ileum was immediately removed for qPCR analysis. Data were subjected to a 2-way ANOVA using GLM procedure (JMP Pro17). A significant diet × hatchery interaction was observed in feed conversion ratio (FCR) from d 14 to d 21 (p = 0.013) where the HS, DS, and HSDS treatments had a significantly lower FCR compared to the CTRL. However, no significant interaction effect was observed for body weight gain (BWG) or FCR during the overall experimental period. No significant interaction was observed in mRNA abundance of the evaluated genes in the ileum on d 7 and d 21. Gel application with the synbiotic significantly reduced sodium-dependent glucose cotransporter 1 (SGLT1) mRNA abundance on d 7 (p = 0.035) in comparison to birds receiving gel alone. Regardless of hatchery application, dietary synbiotic supplementation significantly reduced Toll-like receptor (TLR)2, TLR4, and interleukin (IL)-10 mRNA abundance on d 7 (p = 0.013). In conclusion, these findings showed that hatchery and dietary synbiotic application could have a potential beneficial impact on broiler intestinal immunity by regulating the TLR response, a key element of innate immunity. FCR was improved from d 14 to d 21 after synbiotic application. Future research involving extended grow-out studies with a disease challenge would expand on the implications of an early application of synbiotics.

Probiotics for humans and direct-fed microbials for livestock are increasingly popular dietary ingredients for supporting immunity. The aim of this study was to determine the effects of dietary supplementation of Bacillus subtilis MB40 (MB40) on immunity in piglets challenged with the foodborne pathogen Listeria monocytogenes (LM). Three-week-old piglets (n = 32) were randomly assigned to four groups: (1) basal diet, (2) basal diet with LM challenge, (3) MB40-supplemented diet, and (4) MB40-supplemented diet with LM challenge. Experimental diets were provided throughout a 14-day (d) period. On d8, piglets in groups 2 and 4 were intraperitoneally inoculated with LM at 108 CFU/mL per piglet. Blood samples were collected at d1, d8, and d15 for biochemical and immune response profiling. Animals were euthanized and necropsied at d15 for liver and spleen bacterial counts and intestinal morphological analysis. At d15, LM challenge was associated with increased spleen weight (p = 0.017), greater circulating populations of neutrophils (p = 0.001) and monocytes (p = 0.008), and reduced ileal villus height to crypt depth ratio (p = 0.009), compared to non-challenged controls. MB40 supplementation reduced LM bacterial counts in the liver and spleen by 67% (p < 0.001) and 49% (p < 0.001), respectively, following the LM challenge, compared to the basal diet. MB40 supplementation was also associated with decreased circulating concentrations of monocytes (p = 0.007). Altogether, these data suggest that MB40 supplementation is a safe and well-tolerated approach to enhance immunity during systemic Listeria infection.

Necrotic enteritis (NE) is an intestinal disease that results in poor performance, inefficient nutrient absorption, and has a devastating economic impact on poultry production. This study evaluated the effects of a saponin-based product (Clarity Q, CQ) during an NE challenge. A total of 1200 male chicks were randomly assigned to four dietary treatments (10 pens/treatment; 30 birds/pen): treatment 1 (NC), a non-medicated corn–soybean basal diet; treatment 2 (PC), NC + 50 g/metric ton (MT) of bacitracin methylene disalicylate (BMD); and treatments 3 (CQ15) and 4 (CQ30), NC + 15 and 30 g/MT, respectively. On the day (d) of placement, birds were challenged by a coccidia vaccine to induce NE. On d 8, 14, 28, and 42, performance parameters were measured. On d 8, three birds/pen were necropsied for NE lesions. On d 8 and d 14, jejunum samples from one bird/pen were collected for mRNA abundance of tight junction proteins and nutrient transporter genes. Data were analyzed in JMP (JMP Pro, 16), and significance (p ≤ 0.05) between treatments was identified by Fisher’s least significant difference (LSD) test. Compared to PC and NC, CQ15 had higher average daily gain (ADG), while CQ30 had lower average daily feed intake (ADFI) and feed conversion ratio (FCR). NE lesions in the duodenum were lower in CQ15 compared to all other treatments. On d 8, mRNA abundance of CLDN1, CLDN5, AMPK, PepT2, GLUT2, and EAAT3 were significantly greater in CQ30 (p < 0.05) compared to both PC and NC. On d 14, mRNA abundance of ZO2 and PepT2 was significantly lower in PC when compared to all treatments, while that of ANXA1, JAM3, and GLUT5 was comparable to CQ15. In summary, adding Clarity Q to broiler diets has the potential to alleviate adverse effects caused by this enteric disease by improving performance, reducing intestinal lesions, and positively modulating the mRNA abundance of various tight junction proteins and key nutrient transporters during peak NE infection.

Histomoniasis is caused by the protozoa Histomonas meleagridis (HM) that are laterally transmitted among birds leading to high mortality in commercial flocks. This study tested an HM infection model assessing the lateral transmission of HM in turkey poults raised on floor pens. Day (d)-old female turkey poults (n = 320) were individually wing-tagged and allocated to one of four treatment groups (4 floor pens/group and 20 poults/pen) based on the percentage of poults inoculated with HM: 1) 10% (HM10); 2) 20% (HM20); 3) 30% (HM30); and 4) 40% (HM40). On d 9, seeder poults intracloacally received a 1 mL inoculum/bird containing ∼80,000 histomonads. Poults were individually weighed on d 0, 9, and 25 and feed intake recorded on per pen basis. On d 25, all birds were euthanized by cervical dislocation and ceca and liver were evaluated for HM lesions. Data were analyzed using JMP (Pro16) and significance (P ≤ 0.05) between treatments were determined by LSD test. Mortality was 7.63%, 12.5%, 21.58%, and 20.59%, while transmission rates from inoculated to non-inoculated birds were 62.5%, 57.5%, 92.43%, and 78.75% in HM10, HM20, HM30, and HM40 groups, respectively. Average daily feed intake was proportionally reduced with the increasing number of inoculated poults from HM10 to HM40. Average daily gain was significantly lower in HM30 and HM40 poults compared to those in HM10 and HM20 during the postchallenge period (d 10–25). Therefore, we herein report the successful lateral transmission of HM among turkey poults raised on floor pens. This research model closely resembles commercial field conditions and affords a much-needed platform for conducting relevant basic and applied research on histomoniasis in poultry.

ABSTRACT: High ambient temperature is one of the most common stressors in modern poultry production, resulting in reduced feed intake, weight gain, and increased mortality. This study evaluated the effects of vitamin E (Vit E) and organic selenium (Se) supplementation on performance, body composition, core body temperatures, and mRNA abundance of nutrient transporters in the jejunum of broilers exposed to daily 4-h elevated temperature during d 28 to 35. A total of 640 Cobb male birds were randomly allocated to 32 floor pens in a 2 × 2 factorial arrangement that included ambient temperature (thermoneutral, [TN]; or heat stress, [HS]) and dietary treatments (basal diet or Vit E + Se). Four rooms were used (2 TN and 2 HS) each housing half of the 8 replicate pens per group. Vit E and organic Se were added to the basal diet at the rate of 250 mg/kg and 1 mg/kg diet, respectively. Data were subjected to a 2-way ANOVA using the GLM procedure of JMP (SAS). During the HS period, birds fed the Vit E/Se diet had significantly lower mortality compared to nonsupplemented group (1.92% vs. 7.01%). Moreover, dietary Vit E/Se supplementation had a significant effect on performance by increasing BWG, FI, and European production efficiency factor (EPEF) during the entire experimental period (d 0–35). Dietary Vit E and Se supplementation significantly increased carcass, tissue, lean, and fat weights as well as bone mineral content (BMC) and bone mineral density (BMD) on d 35. Birds fed Vit E/Se supplemented diet had significantly lower (P = 0.010) core body temperature compared to birds fed the basal diet on d 30. Dietary treatment did not influence mRNA abundance of PepT1, SGLT1, or NaPi-IIb on d 28 or d 35. However, HS significantly upregulated levels of PepT1 and NaPi-IIb (P < 0.001) and downregulated that of SGLT1 (P = 0.017) on d 28. In conclusion, dietary Vit E and Se supplementation significantly improved broiler growth performance and carcass composition, and reduced heat-related mortality and core body temperature (on d 30) without influencing the mRNA abundance of intestinal nutrient transporters.

ABSTRACT: This study evaluated the effects of vitamin E (Vit E) and selenium (Se) supplementation on mRNA abundance of antioxidant, immune response, and tight junction genes, as well as taxonomic and functional profiles of ileal microbiota of broilers exposed to daily 4-h elevated temperature during d 28 to 35. A total of 640-day-old Cobb male broiler chicks were randomly allocated to 32 floor pens in a 2 × 2 factorial arrangement that included ambient temperature (thermoneutral [TN] or heat stress [HS]) and dietary treatments (basal diet or Vit E + Se). Vit E and organic Se were added to the basal diet at the rate of 250 mg/kg and 1 mg/kg, respectively. Liver and jejunum tissue samples were taken on d 27 (1 bird/pen), d 28 and d 35 (2 birds/pen) from birds for qPCR analysis. Data were subjected to a 2-way ANOVA using the GLM procedure of JMP. Ileal contents were taken on d 27 and d 35 for microbial profiling. Microbiota data were analyzed in QIIME 2 and significance between treatments identified linear discriminant analysis effect size (LEfSe, P < 0.05). Dietary Vit E/Se significantly downregulated the mRNA levels of HSPs in liver and jejunal tissues of the HS-challenged birds both on d 28 and d 35. Moreover, mRNA abundance of TLR2, TNFα, IFNγ, IL-1β, IL-10, and iNOS in the liver were significantly downregulated in birds fed the Vit E/Se diet on d 35. However, dietary treatment had no significant impact on oxidative stress, immunity, and gut integrity related genes analyzed in jejunal tissues on d 28 and d 35, except downregulation of IFNγ on d 35 (P = 0.052). LEfSe analysis revealed that Lachnospiraceae FE2018 and Ruminococcaceae NK4A214 groups was enriched in the Vit E/Se birds on d 35. Moreover, PICRUSt analysis predicted significant functional differences among the treatment groups. In conclusion, dietary supplementation of Vit E/Se mitigated the negative effects of HS potentially via improving antioxidant status, regulating cytokine responses and modifying ileal microbiota and its function.

Bacterial communities in and on wild hosts are increasingly appreciated for their importance in host health. Through both direct and indirect interactions, bacteria lining vertebrate gut mucosa provide hosts protection against infectious pathogens, sometimes even in distal body regions through immune regulation. In house finches (Haemorhous mexicanus), the bacterial pathogen Mycoplasma gallisepticum (MG) causes conjunctivitis, with ocular inflammation mediated by pro- and anti-inflammatory cytokines and infection triggering MG-specific antibodies. Here, we tested the role of gut bacteria in host responses to MG by using oral antibiotics to perturb bacteria in the gut of captive house finches prior to experimental inoculation with MG. We found no clear support for an impact of gut bacterial disruption on conjunctival pathology, MG load, or plasma antibody levels. However, there was a non-significant trend for birds with intact gut communities to have greater conjunctival pathology, suggesting a possible impact of gut bacteria on pro-inflammatory cytokine stimulation. Using 16S bacterial rRNA amplicon sequencing, we found dramatic differences in cloacal bacterial community composition between captive, wild-caught house finches in our experiment and free-living finches from the same population, with lower bacterial richness and core communities composed of fewer genera in captive finches. We hypothesize that captivity may have affected the strength of results in this experiment, necessitating further study with this consideration. The abundance of anthropogenic impacts on wildlife and their bacterial communities, alongside the emergence and spread of infectious diseases, highlights the importance of studies addressing the role of commensal bacteria in health and disease, and the consequences of gut bacterial shifts on wild hosts.

ABSTRACT: Necrotic enteritis (NE) is a significant enteric disease in commercial poultry with considerable economic effect on profitability manifested by an estimated $6 billion in annual losses to the global industry. NE presents a unique challenge, being a complex enteric disease that often leads to either clinical (acute) or subclinical (chronic) form. The latter typically results in poor performance (reduced feed intake, weight gain and eventually higher feed conversion ratio [FCR]) with low mortality rates, and represents the greatest economic impact on poultry production. The use of antibiotic growth promoters (AGPs) has been an effective tool in protecting birds from enteric diseases by maintaining enteric health and modifying gut microbiota, thus improving broilers’ production efficiency and overall health. The removal of AGPs presented the poultry industry with several challenges, including reduced bird health and immunity as well as questioning the safety of poultry products. Consequently, research on antibiotic alternatives that can support gut health was intensified. Probiotics, prebiotics, essential oils, and organic acids were among various additives that have been tested for their efficacy against NE with some being effective but not to the level of AGPs. The focus of this review is on the relationship between NE pathogenesis, microbiome, and host immune responses, along with references to recent reviews addressing production aspects of NE. With a comprehensive understanding of these dynamic changes, new and programmed strategies could be developed to make use of the current products more effectively or build a stepping stone toward the development of a new generation of supplements.

Necrotic enteritis (NE) caused by Clostridium perfringens is among the most important enteric diseases in poultry production. This study examined the effects of 2 probiotics (Prob) and a synbiotic (Synb) during a naturally occurring NE challenge. On the day of hatch, 1200 Cobb male broilers were randomly allocated to 5 groups (8 pens/treatment, 30 birds/pen) including 1) negative control (NC): corn-soybean meal diet; 2) positive control (PC): NC + 453 g Stafac20/907 kg feed; 3) Prob 1: NC + 453 g Prob 1/907 kg feed; 4) Prob 2: NC + 453 g Prob 2/907 kg feed; and 5) Synb: NC + 453 g Synb/907 kg feed. One day after placement, birds were challenged by a coccidia vaccine to induce NE. Feed intake and body weights were measured on day 8 (NE onset) and end of starter (day 14) and grower (28) periods. On day 8, the small intestines of 3 birds/pen were examined for NE lesions. Ileal mucosal scrapings from one bird/pen were collected on day 8 and day 28 to profile the microbiota using 16S rRNA sequencing. Data were analyzed in JMP or QIIME 2 and significance between treatments identified by LSD or linear discriminant analysis effect size (P

The withdrawal of antibiotic growth promoters from poultry feed has increased the risk of necrotic enteritis (NE) outbreaks. This study examined the effects of a probiotic (PROB) or probiotic/prebiotic/essential oil supplement (PPEO) during a subclinical NE challenge. On day (d) of hatch, 960 male broilers were randomized to four groups (8 pens/treatment, 30 birds/pen) including (1) negative control (NC): corn-soybean meal diet; (2) positive control (PC): NC + 20 g Virginiamycin/ton diet; (3) NC + 227 g PROB/ton diet; and (4) NC + 453 g PPEO/ton diet. One d after placement, birds were challenged by a coccidia vaccine to induce NE. Feed intake and body weights were measured on d 8 (NE onset) and end of each feeding period. On d 8, the small intestines of three birds/pen were examined for NE lesions. Jejunum samples and ileal mucosal scrapings from one bird/pen were respectively collected to measure mRNA abundance (d 8 and d 14) and profile the microbiota (d 8 and d 42). Data were analyzed in JMP or QIIME 2 and significance between treatments identified by LSD (P < 0.05). PROB and PPEO had significantly lower mortality (d 0–14) and NE lesion scores compared to NC. Feed conversion ratio was significantly lower in PC, PROB, and PPEO, while average daily gain was higher in PPEO and PC groups compared to NC from d 0–42. On d 8 and d 14, mRNA abundance of claudin-3 was higher in PPEO compared to NC. On d 14, compared to NC, mRNA abundance of sIgA and PGC-1α in PROB and PPEO were lower and higher, respectively. Compared to NC, PPEO increased mTOR abundance on d 14. On d 8, relative abundance of Clostridium sensu stricto 1, Ruminiclostridium9, Prevotellaceae, Prevotellaceae UCG-014, ASF356, and Muribaculaceae was higher in NC compared to PPEO and PROB, while Lactobacillus was lower in NC. Escherichia-Shigella had higher abundance in PC compared to PPEO and PROB. Collectively, these data indicate that during a subclinical naturally occurring NE, supplementation of PROB or PPEO supports performance and reduces intestinal lesions, potentially through modifying tight junction proteins, gut microbiota, immune responses, and cell metabolism.

Abstract Background A unique clade of the bacterium Mycoplasma gallisepticum (MG), which causes chronic respiratory disease in poultry, has resulted in annual epidemics of conjunctivitis in North American house finches since the 1990s. Currently, few immunological tools have been validated for this songbird species. Interleukin-1β (IL-1β) is a prototypic multifunctional cytokine and can affect almost every cell type during Mycoplasma infection. The overall goal of this study was to develop and validate a direct ELISA assay for house finch IL-1β (HfIL-1β) using a cross-reactive chicken antibody. Methods A direct ELISA approach was used to develop this system using two different coating methods, carbonate and dehydration. In both methods, antigens (recombinant HfIL-1b or house finch plasma) were serially diluted in carbonate-bicarbonate coating buffer and either incubated at 4 °C overnight or at 60 °C on a heating block for 2 hr. To generate the standard curve, rHfIL-1b protein was serially diluted at 0, 3, 6, 9, 12, 15, 18, 21, and 24 ng/mL. Following blocking and washing, anti-chicken IL-1b polyclonal antibody was added, plates were later incubated with detecting antibodies, and reactions developed with tetramethylbenzidine solution. Results A commercially available anti-chicken IL-1β (ChIL-1β) polyclonal antibody (pAb) cross-reacted with house finch plasma IL-1β as well as bacterially expressed recombinant house finch IL-1β (rHfIL-1β) in immunoblotting assays. In a direct ELISA system, rHfIL-1β could not be detected by an anti-ChIL-1β pAb when the antigen was coated with carbonate-bicarbonate buffer at 4°C overnight. However, rHfIL-1β was detected by the anti-ChIL-1β pAb when the antigen was coated using a dehydration method by heat (60°C). Using the developed direct ELISA for HfIL-1β with commercial anti-ChIL-1β pAb, we were able to measure plasma IL-1β levels from house finches. Conclusions Based on high amino acid sequence homology, we hypothesized and demonstrated cross-reactivity of anti-ChIL-1β pAb and HfIL-1β. Then, we developed and validated a direct ELISA system for HfIL-1β using a commercial anti-ChIL-1β pAb by measuring plasma HfIL-1β in house finches.

Poultry coccidiosis is a costly intestinal disease that leads to considerable tissue damage, inefficient nutrient absorption, increased mortality, and predisposition to secondary infections. This study evaluated the effects of a direct feed microbial (DFM) dietary additive on performance, intestinal morphology, and immune response of broilers during a mixed coccidiosis challenge. In total, 840 Cobb500 male broilers were randomly allocated to 3 treatments (7 replicates, 40 birds/pen) including negative control (NC) fed basal diet; positive control (PC) fed basal diet with coccidiosis challenge; and DFM supplemented diet, with coccidiosis challenge. At 15 days of age, all birds except for the NC treatment were orally gavaged with live oocysts of a commercial vaccine. On d 21 (6 days post challenge), 4 birds/pen were randomly selected and euthanized for scoring of coccidia-caused lesions in the duodenum, jejunum, and ceca. Body weight gain (BWG), feed intake (FI), and feed conversion ratio (FCR) were recorded on d 7, 14, 28, and 42. Jejunal and ileal tissue samples were taken for histomorphological assessment from 2 birds/pen on d 21. Ileal samples were also taken for mRNA expression analysis on d 14 and d 21. The DFM birds had significantly greater BWG than PC birds during d 0–21 (P < 0.05). No differences were observed among the treatment groups in terms of FI and FCR. Dietary DFM supplementation significantly reduced lesion scores in the duodenum and jejunum when compared with PC group (P < 0.05). The coccidia challenge significantly reduced (P < 0.05) ileal villus height when compared to the non-challenged group on d 21. Conversely, dietary DFM supplementation alleviated the negative effects of coccidiosis by increasing ileal villus area on d 21 (P < 0.05). The challenged birds had significantly greater expression of IFN-γ and IL-1β in the ileum on d 21. Based on these findings, dietary DFM supplementation may help restore broiler performance during the starter and early grower periods during coccidiosis, likely by maintaining gut integrity via improving intestinal morphology and also by reducing disease severity as manifested by lower lesion scores.

This study investigated the effects of a yeast-derived β-glucan (Auxoferm YGT) supplementation on mRNA expression of immune response genes in the spleen, thymus, and bursa of broiler chickens during a mixed Eimeria infection. Day (d)-old chicks (n = 1440) were fed diets containing 0% or 0.1% YGT. On d 8 post-hatch, half the replicate pens (n = 8) were challenged with a mixed inoculum of E. acervulina, E. maxima, and E. tenella. On d 10 and d 14 post-hatch, the spleen, thymus, and bursa were collected to evaluate mRNA abundance by quantitative real-time PCR. Data were analyzed using PROC GLIMMIX model (2-way interaction) and differences were established by LS-MEANS with significance reported at p ≤ 0.05. In spleen tissues at d 10, expression of interleukin (IL)-10 and inducible nitric oxide synthase (iNOS) were elevated in both 0.1% YGT-fed challenged and non-challenged birds. In thymus tissues at d 14, expression of IL-10, IL-17F, interferon (IFN)-γ, iNOS, and macrophage migration inhibitory factor (MIF) were elevated in challenged birds fed 0.1% YGT. In bursal tissues at d 10 and d 14, expression of IL-10, IFN-γ, iNOS (d 10 only), and MIF were elevated in 0.1% YGT-fed challenged and non-challenged birds. Dietary β-glucan supplementation to chicken diets modulated their immune response to the Eimeria challenge.

The recent emergence of the poultry bacterial pathogen Mycoplasma gallisepticum (MG) in free-living house finches (Haemorhous mexicanus), which causes mycoplasmal conjunctivitis in this passerine bird species, resulted in a rapid coevolutionary arms-race between MG and its novel avian host. Despite extensive research on the ecological and evolutionary dynamics of this host–pathogen system over the past two decades, the immunological responses of house finches to MG infection remain poorly understood. We developed seven new probe-based one-step quantitative reverse transcription polymerase chain reaction assays to investigate mRNA expression of house finch cytokine genes (IL1B, IL6, IL10, IL18, TGFB2, TNFSF15, and CXCLi2, syn. IL8L). These assays were then used to describe cytokine transcription profiles in a panel of 15 house finch tissues collected at three distinct time points during MG infection. Based on initial screening that indicated strong pro-inflammatory cytokine expression during MG infection at the periorbital sites in particular, we selected two key house finch tissues for further characterization: the nictitating membrane, i.e., the internal eyelid in direct contact with MG, and the Harderian gland, the secondary lymphoid tissue responsible for regulation of periorbital immunity. We characterized cytokine responses in these two tissues for 60 house finches experimentally inoculated either with media alone (sham) or one of two MG isolates: the earliest known pathogen isolate from house finches (VA1994) or an evolutionarily more derived isolate collected in 2006 (NC2006), which is known to be more virulent. We show that the more derived and virulent isolate NC2006, relative to VA1994, triggers stronger local inflammatory cytokine signaling, with peak cytokine expression generally occurring 3–6 days following MG inoculation. We also found that the extent of pro-inflammatory interleukin 1 beta signaling was correlated with conjunctival MG loads and the extent of clinical signs of conjunctivitis, the main pathological effect of MG in house finches. These results suggest that the pathogenicity caused by MG infection in house finches is largely mediated by host pro-inflammatory immune responses, with important implications for the dynamics of host–pathogen coevolution.

Necrotic enteritis, caused by Clostridium perfringens, is an enteric disease that leads to poor performance and increased mortality, resulting in significant economic losses in poultry production. This study evaluated the effects of a proprietary prebiotic, probiotic, and plant extract blend on performance of broilers during coccidiosis challenge leading to necrotic enteritis (NE). In total, 744 Cobb500 male broilers were randomly allocated to 3 treatments (8 replicates, 31 birds/pen) including, the negative control (NC) fed a basal diet; the positive control (PC) fed a basal diet with Virginiamycin; and the additive group fed basal diet with a blend of prebiotic, probiotic, and plant extract (BSN). A unique, naturally occurring NE model developed to mimic field conditions was implemented to challenge the birds. This model consists of spraying a concentrated commercial coccidiosis vaccine on litter and feed upon bird placement, which, in conjunction with the presence of C. perfringens spores in the environment, leads to the development of a NE outbreak one week post vaccine application. At the onset of NE on d7, three birds/pen were selected for scoring NE lesions. Body weight gain (BWG), feed intake (FI), and feed conversion ratio (FCR) were recorded on days 7, 14, 28, and 42. Carcass composition was assessed by dual energy X-ray absorptiometry (DXA) analysis on day 42. Dietary supplementation of BSN significantly (p < 0.05) improved FCR during starter and grower periods. Dietary treatments had no effect on NE lesions in the small intestine. DXA analysis revealed slightly higher lean content in BSN birds compared to NC. These results showed that dietary supplementation of the BSN blend significantly improved broilers performance during the early NE challenge phase, as well as in the grower period.

Necrotic enteritis (NE) continues to present major challenges to the poultry industry, and the etiologic agent Clostridium perfringens is the fourth leading cause of bacterially-induced food- borne illnesses in the US. This study was designed to evaluate the effects of a probiotic during naturally occurring NE. On day of hatch, 1080 Cobb 500 male broilers were randomly allocated to three groups (12 replicate pens/treatment, 30 birds/pen) including (1) negative control (NC): corn-soybean meal diet; (2) positive control (PC): NC + 20 mg virginiamycin/kg diet (0.450 kg Stafac®20/ton); and (3) NC + PrimaLac (1.36 and 0.91 kg/ton from 1−21 and 22−42 days, respectively). One day (d) post placement, all birds were challenged by a commercial live oocyst coccidia vaccine as a predisposing factor to NE. Body weight and feed intake were measured at the onset of NE (d 8) and end of each feeding phase. On d 8, small intestines of two birds/pen were examined for NE lesions, and jejunum samples from one bird were collected for mRNA gene expression analysis of tight junction proteins, cytokines, and nutrient transporters. Data were analyzed using the JMP software and significance between treatments identified by LSD (p < 0.05). Compared to NC, supplementation of probiotic reduced d 1−42 mortality; however, PC was the only group with significantly lower mortality. Despite significantly improved feed conversion ratio (FCR) in PC and probiotic groups during d 1−42, average daily gain was only higher in PC (77.69 g/bird) compared with NC (74.99 g/bird). Furthermore, probiotic and PC groups had significantly reduced lesion scores in the duodenum and jejunum compared to NC. Expression of claudin-3 was higher, while expression of zonula occluden-2 tended (p = 0.06) to be higher in probiotic-supplemented birds compared to NC. Moreover, birds fed the probiotic diet had significantly higher expression of IL-10, IL-17, AMPK-α1, and SGLT1 mRNA compared to NC birds. The expression of PepT1 was higher for the probiotic-supplemented group compared to PC. IFN-γ expression was lower in PC compared to NC, while there was no difference between probiotic and NC. There were no differences in gene expression of sIgA, TNF-α, IL-1β, and IL-22 among treatments. Collectively, these data indicate that in a naturally occurring NE model, supplementation of a probiotic helps to improve FCR and reduce lesions, potentially due to the improvements in mRNA expression of tight junctions, cytokines, and nutrient transporters.