Symposium on Gut Health in Production of Food Animals 2021

Chairperson: Mike Kogut / Speaker: Melha Mellata, Iowa State University

Poultry serve as a major reservoir for bacterial Enterobacteriaceae pathogens like Salmonella and Escherichia coli, which are a food safety concern. Although many probiotics are beneficial to poultry productivity, they induce poor host antimicrobial responses against enteric pathogens. We showed that some probiotics can even increase the level of Enterobacteriaceae in the chicken gut by increasing the level of norepinephrine. Furthermore, some bacteria like Salmonella actively promote immunotolerance in the chicken gut, which prevents antibacterial host responses and subsequently results in fecal Salmonella shedding and contamination of poultry products. Thus, novel prophylactics which can stimulate host intestinal responses and overcome these immunotolerant mechanisms to clear intestinal Enterobacteriaceae like Salmonella are needed. Our study demonstrates that prophylactics can alter intestinal immunological responses via neurochemical and metabolic pathways to improve bacterial resistance. These findings provide compelling evidence that targeting the neuroimmunological axis can be an effective strategy to minimize Salmonella persistence in poultry and improve food safety.

Key Words: gut, immunotolerance, prophylactics, neurochemicals, bacterial resistance.

Author: M. Mellata*, Iowa State University, Ames, IA, USA.

Glenn Zhang, Oklahoma State University

The rising concern of antimicrobial resistance highlights a need for effective alternatives to antibiotics for livestock production. Butyrate, forskolin, and lactose are 3 natural products known to induce the synthesis of host defense peptides (HDP), a critical component of innate immunity. In this study, the synergy among butyrate, forskolin, and lactose in enhancing innate host defense, barrier function, and resistance to necrotic enteritis and coccidiosis was investigated. Our results indicated that the 3 compounds synergistically augmented the expressions of multiple HDP and barrier function genes in chicken HD11 macrophages. The compounds also showed an obvious synergy in promoting HDP gene expressions in chicken jejunal explants. Dietary supplementation of a combination of 1 g/kg sodium butyrate, 10 mg/kg forskolin-containing plant extract, and 10 g/kg lactose dramatically improved the survival of chickens from 39% to 94% (P < 0.001) in a co-infection model of necrotic enteritis. Furthermore, the 3 compounds largely reversed growth suppression, significantly alleviated intestinal lesions, and reduced colonization of Clostridium perfringens or Eimeria maxima in chickens with necrotic enteritis and coccidiosis (P < 0.01). Collectively, dietary supplementation of butyrate, forskolin, and lactose is a promising antibiotic alternative approach to disease control and prevention for poultry and possibly other livestock species.

Key Words: antibiotic alternative, host defense peptide, butyrate, necrotic enteritis, coccidiosis.

Authors: Q. Yang and G. Zhang*, Oklahoma State University, Stillwater, OK, USA

Hongyu Xue, Amlan International

Homeostasis of the gut microbial ecosystem is essential for optimal growth performance and disease resistance in postweaning pigs. A220 is a formulated feed additive that features a blend of a proprietary toxin-adsorbing mineral with a select blend of phytogenics shown to have antibacterial properties against a variety of gram-negative and positive bacterial pathogens. This study aimed to assess the effects of A220 on the gut microbiome and further relate this to changes in growth performance and post-weaning diarrhea outcomes in pigs challenged with an enterotoxigenic Escherichia coli (ETEC). At 21 d of age, 36 piglets were weaned and randomly allocated to 1 of 3 groups: control (CON) or A220 supplemented at 0.25 or 0.5%. After a 7-d adaptation, all pigs were orally inoculated with 1010 cfu of F18 ETEC once daily from d 0 to d 2 post-inoculation (PI). Fecal consistency was scored twice daily from d 0–21 PI. Microflora in ileal digesta, ileal mucosa and fecal samples were profiled using 16S rRNA sequencing on d −7, 0, 7, and 21 PI. A220 supplementation at both levels increased feed efficiency from d 14–21 PI and reduced diarrhea frequency during the study (P < 0.05). Compared with CON, 0.5% A220 supplementation increased the relative abundance of Firmicutes, but reduced Bacteroidetes and Proteobacteria in feces on d 7 PI (P < 0.05). Within Firmicutes phylum, A220 at both 0.25% and 0.50% increased the relative abundance of Lactobacillaceae on d 7 PI (P < 0.05). A220 at both levels increased the relative abundance of Prevotellaceae and decreased that of Rikenellaceae in ileal digesta on d 21 PI (P < 0.05). A220 at 0.5% increased the relative abundance of Clostridiaceae and Prevotellaceae, and decreased that of Enterobacteriaceae and Succinivibrionaceae in ileal mucosa on d 21 PI (P < 0.05). A220 supplementation modified gut microbiota in favor of promoting a well-balanced gut microbial ecosystem, which may contribute to enhanced disease resistance and improved growth performance in weanling pigs faced with pathogenic challenges.

Key Words: microbiome, enterotoxigenic Escherichia coli (ETEC), post-weaning, pig.

Authors: H. Xue* 1, D. Wang 1, L. Johnston 1, Y. He 2, C. Jinno 2, Y. Liu 2, and P. Ji 3 / 1 Amlan International, Chicago, IL, USA, 2 Department of Animal Science, University of California, Davis, CA, USA, 3 Department of Nutrition, University of California, Davis, CA, USA.

Jane Leedle, JL Microbiology

Antibiotics and zinc oxide have long been used to prevent or cure post-weaning diarrhea. Now many countries are reducing use or completely banning these substances due to antimicrobial resistance and environmental concerns. Without antimicrobials producers have increased demand for natural solutions. Lactobacilli postbiotics are known to have a strong effect in reducing diarrhea in children. Research suggests this postbiotic prevents pathogen adherence and shifts the gut microbiome toward beneficial flora. Our objective was to evaluate the postbiotic Lactobacillus LB on newly weaned piglet health and performance outcomes compared with Control and Zinc oxide (Zn) in a 42-d E. coli challenge study. Three hundred male pigs 18 to 22 d of age and 2.75 to 7.60 kg BW from a commercial farm were divided into 4 groups each having 15 pens of 5 piglets. The LB treatment was Control feed with 2 kg LB/MT. The Zn treatment was Control with 3000 ppm Zn fed for 21 d and then removed. The 4th treatment was LB+Zn: Control diet with LB and added Zn for the 1st 21 d then Zn was removed. On d 10, all pigs were gavaged with 5 mL of 1.3 × 10e9 cfu E. coli/mL. Response variables were clinical and fecal scores, ADG, and FCR, with data collected on specific study days. Statistical analyses were performed using SAS and a Tukey-Kramer test. Fecal score results showed a strong reduction in diarrhea in the LB group. No watery stool was present in any pen in the LB group, yet it was as high as 89% in Control and 7% in the Zn group. Duration was 5 d post-challenge in LB as opposed to 11 and 7 d for Control and Zn groups, respectively. LB+Zn reduced diarrhea to 5 d as well. The E. coli challenge was severe. Mortality ~40% in the Control group was and 25% in the LB group. At 42 d postweaning, LB piglets had better growth performance. FCR was 13% lower (1.00 vs 1.15; P < 0.01) and ADG was 50% higher (0.21 kg/d vs 0.14 kg/d; P < 0.01) compared with Control. ADG was not different between LB and Zn groups. In summary, the postbiotic Lactobacillus LB significantly reduced postweaning diarrhea and was a good alternative to zinc oxide in piglet production.

Key Words: piglet, postweaning diarrhea, postbiotic.

Authors: P. Tacon 1, S. Della Zassa 2, C. Cull 3, K. Lechtenberg 3, and J. Leedle *4 / 1 ADARE Biome, Houdan, France, 2 Adare Biome, Lawrenceville, NJ, USA, 3 Midwest Veterinary Services Inc., Oakland, NE, USA, 4 JL Microbiology, Hartland, WI, USA.

Luis-Miguel Gomez-Osorio, Alura Inc.

The aim of this work was to determine the in vitro effectiveness of a blend of short-chain fatty acids (SCFA) and medium-chain fatty acids (MCFA) using α-monoglycerides through a minimum inhibitory concentration (MIC) test. Clostridium perfringens type G strains used in this study were previously isolated from field cases of focal duodenal necrosis (FDN) and necrotic enteritis (NE) (Villegas et al., 2020, J. Vet. Diagn. Invest., 32:268–276). Isolated colonies of the tested bacterial species were selected from a 24-h culture on a blood agar plate. MIC tests were done in triplicates using 2 samples of Fractal and 3 strains of Clostridium perfringens. The strains were isolate 1 and isolate 5 (obtained from chickens with FDN) and CP6 (obtained from a broiler chicken with NE). The microbicidal activity of the SCFA and MCFA α-monoglyceride product for CP6 strain is shown in Figures 1, 2 and 3. All isolates had MICs ≤0.08 μg/mL. Furthermore, the MICs for isolate 1 and isolate 5 were 0.04 respectively. For CP6 strain, the MIC was 0.08. The blend of SCFA and MCFA α-monoglycerides demonstrated high antimicrobial activity against different strains of pathogenic Clostridium perfringens isolated from field cases of FDN and NE under an in vitro test.

Key Words: focal duodenal necrosis, necrotic enteritis, α-monoglycerides, short-chain fatty acids, medium-chain fatty acids.

Authors: L.-M. Gomez-Osorio *1, L. Stabler 2, and M. Franca 2 / 1 Alura Inc., Durham, NC, USA, 2 Department of Population Health, Poultry Diagnostic and Research Center, University of Georgia, Athens, GA, USA.

Guillermo Tellez-Isaias, University of Arkansas

Several phytogenics have been evaluated as feed additives in the poultry industry for nutritional purposes. However, phytogenics play an essential role in the prevention of several diseases in poultry due to their antioxidant, anti-inflammatory, antibacterial, antiviral, antifungal, and immunomodulatory properties. Hence, in recent years, our laboratories have studied several phytogenics as feed additives to control salmonellosis and necrotic enteritis, to reduce the severity of aflatoxicosis in broiler chickens, and to control coccidiosis in Leghorn chickens. Curcumin is a bright yellow chemical and the principal curcuminoid of turmeric (Curcuma longa), a member of the ginger family (Zingiberaceae). The aim of this presentation is to summarize the studies that have been published by our laboratories evaluating this remarkable phytochemical alone and in combination with other nutraceuticals: (1) Hernandez et al., 2018, Front. Microbiol. 9:1289; (2) Hernandez et al., 2019, Animals 9:184; (3) Solis et al., 2019, Toxins 11:121; (4) Leyva et al., 2021, J. Anim. Sci. Biotechnol. 12:23; (5) Petrone et al., 2021, Sci. Rep. 11:1–9.

Key Words: curcumin, salmonellosis, coccidiosis, aflatoxicosis, necrotic enteritis.

Authors: G. Tellez-Isaias *1, D. Hernandez-Patlan 2, B. Solis-Cruz 2, V. Petrone-Garcia 2, A. Leyva-Diaz 2, C. Vuong 1, D. Graham 1, C. Selby 1, J. Latorre 1, and B. Hargis 1 / 1 University of Arkansas, Fayetteville, AR, USA, 2 UNAM, Cuautitlan Cuautilan Izcalli, Mexico, Mexico.

Don Ritter, Innovad USA

Compromised intestinal health in modern production has been linked with chronic inflammation and significant economic losses. Although several challenges are artificially introduced in recent poultry models of gut failure/dysbiosis, the ‘too clean’ experimental conditions oppose a major limitation. Here we evaluated (1) a model of chronic gut inflammation under real farming conditions using, as a trigger, high non-starch polysaccharides (without NSPases), and (2) the ability of an in-feed technology (Lumance Innovad: esterified butyrate with plant extracts and essential oils) to alleviate inflammatory responses in broilers. In experiment 1, the high NSP (60% wheat + 5% rye) significantly increased macroscopic morphometric “dysbacteriosis” both at d 21 and d 28 over a standard diet (50% corn + 30% soy) (P < 0.005; 1 bird per pen scored, n = 8 pens/group; n = 30 birds/pen), evaluated according to Teirlynck et al. (2011). In experiment 2, a heatwave elevated the mean temperature to ~32°C for ~12–14 h/d inside the production between d 21 and d 29. Interestingly, although both intestinal and systemic oxidative stress (measured as MDA) in the high NSP diet reduced by ~30% between d 28 and d 35, both inflammatory (IFN-γ) and immune (IgA) responses increased significantly (~33 and 40%, respectively) suggesting a cumulative impact over time. Importantly, Lumance (1 and 2 kg/ton) significantly increased the BW at the end of the life cycle (d 35) over the high-NSP (P= 0.041) and reduced the FCR (P = 0.012) (n = 8 pens/group; n = 30 birds/pen). Also, Lumance reduced, in a dose response manner, both intestinal and systemic MDA at d 28 (P < 0.05), intestinal IFN-γ at d 28 and d 35 (P < 0.05) and numerically both intestinal and systemic IgA at d 28 and d 35. In conclusion, we have successfully established a novel, dietary-induced model of chronic gut inflammation under real farming conditions, which can be seen as the sum of several stress points and evaluated the ability of an in-feed technology (Lumance Innovad) to alleviate its inflammatory responses in broilers.

Key Words: gut health, chronic inflammation model, real farming condition, biomarker, feed additive.

Authors: A. Khadem 1,2, D. Ritter *3 , and C. Gougoulias 1 / 1 Innovad NV/SA, Essen, Belgium, 2 Lab of Nutrition, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium, 3 Innovad USA, Salisbury, MD, USA.

Audrey Duff, The Ohio State University

Heterophil granule component β-d-glucuronidase has been used to assess degranulation activity in cell culture supernatant after bacterial challenge. Adaptation of this assay for site specific degranulation in gastrointestinal (GIT) tissue was evaluated as a potential indicator of localized inflammation in response to Eimeria maxima (EM) infection. Experiments (Exp) 1–3 compared Control (C1, 2, 3) and EM-infected groups (EM1, 2, 3) by t-test (P < 0.05), while Experiment 4 used ANOVA and Dunnett’s post hoc analysis (P < 0.05) to compare C4 to EMlow (5 × 10³ oocysts), -medium (1 × 10⁴ oocysts), and –high (1.5 × 10⁴ oocysts). Intestinal scrapings were collected into RPMI 1% penicillin/streptomycin on ice, gently homogenized, and centrifuged. Supernatants were incubated with RPMI and positive control with serum opsonized zymosan for 1 h at 42°C. Samples were tested in 3 replicates and incubated with 4-methylumbelliferyl-β-d-glucuronide for 4 h at 42°C. Liberated 4-methylumbelliferone (4-MU) was quantified fluorometrically at an excitation/emission wavelength of 360/460 nm. Body weight gain (BWG) was evaluated over the infection period and was significantly suppressed in all EM groups relative to C1–3 (P < 0.05), while only EM-high BWG was lower (P = 0.008) than C4. In Exp 1 and 2, EM1 and EM2 4-MU was significantly lower than C1 and C2 (P = 0.046 and 0.009, respectively), while no differences were observed between C3 and EM3 in Exp 3 (P = 0.351). Exp 4 showed significant decrease in EM-low (P = 0.003) and EM-high (P = 0.003) 4-MU versus C4, but not EM-medium (P = 0.357). All assay positive controls were elevated relative to samples. Suppression of BWG indicates successful EM infection and supports the conclusion of a general observable decease in degranulation in the GIT of EM-challenged broilers. In future iterations, addition of correlation analyses between 4-MU values, BWG, and EM lesion scores on a per bird basis would aid in addressing variability within treatment groups. These results may provide new insight into GIT degranulation events occurring during coccidial infection.

Key Words: degranulation, Eimeria, gastrointestinal (GIT), inflammation, broiler.

Authors: A. Duff*, K. McGovern, M. Trombetta, and L. Bielke, The Ohio State University, Columbus, OH, USA.

D. Ayala, Land O' Lakes

Cellulitis in commercial turkeys has emerged as one of the leading causes of morbidity and mortality in the United States. According to the United States Animal Health Association industry survey, cellulitis ranks among the top 5 health issues for the turkey industry. It also represents a major cause of carcass condemnation at slaughter with significant economic losses for turkey producers. The main bacterial pathogens associated with the disease are Clostridium spp., avian pathogenic Escherichia coli, and Staphylococcus aureus. The aim of this study was to characterize the microbial community of birds with cellulitis and compare it to the microbial profile of healthy birds, to identify potential agents and routes of the infection. A total of 4 sample types including cecum, ileum, skin, and subcutaneous tissue (SBT) from 10 Nicholas turkeys (1) with cellulitis (Cell+ group), (2) without cellulitis (Cell− group), and (3) healthy birds (Control group) were collected between 16 and 18 weeks of age. Samples of Cell+ and Cell− groups were collected at one farm with a history of cellulitis, whereas samples of Control group were collected at a sister farm, with no history of cellulitis. The microbial profile of all samples was characterized by 16S metagenomics. The SBT microbiome of Cell+ samples was dominated by Clostridium sensu stricto with a relative abundance of 65.86% compared with 0.06% and 0.29% in the Cell− and Control groups, respectively. Ileal microbiome of Cell+ group was the second highest in abundance of Clostridium sensu stricto compared with Cell− and Control groups, with relative abundances of 31.27%, 0,09%, and 3.37%, respectively. STB and ileal microbiome from Cell+ group were dissimilar to Cell− and Control groups (P < 0.05). Through bacterial isolation, C. septicum and C. perfringens were isolated from SBT samples elucidating a potential synergistic effect in the development of the disease. Additionally, the high abundance of Clostridium spp. in the ileum and SBT provides insight on the potential translocation of Clostridium spp. from the intestine to subcutaneous breast tissue resulting in cellulitis.

Key Words: cellulitis, Clostridium spp., translocation, gut health.

Authors: D. Ayala*, D. Grum, E. Kimminau, N. Evans, K. Russo, B. Trible, and T. P. Karnezos / Purina Animal Nutrition Center, Gray Summit, MO, USA.

M. Trombetta, The Ohio State University

Necrotic enteritis is an enteric disease primarily caused by overgrowth of Clostridium perfringens (CP) in the small intestine following a variety of predisposing factors. The objective was to determine if a novel probiotic showed anti-clostridial effects, survived pelleting temperatures and harsh environment of the gastrointestinal tract (GIT), and if anti-clostridial effects were retained through the GIT. The probiotic was tested against 8 strains of CP to determine overarching anti-clostridial effect. The probiotic suppressed all 8 strains of CP significantly (P <0.05) when CP inoculated media was overlaid onto a pregrown colony of probiotic and zones of inhibition measured. Next, probiotic efficacy was compared against common antibiotics and commercial probiotics. Two antibiotics, penicillin (0.0625 mg/mL) and metronidazole (0.05 mg/mL), both commercial probiotics, and the experimental probiotic reduced CP growth with the experimental probiotic outperforming both commercial probiotics (P < 0.001) and metronidazole (P = 0.007). The CP strain showed resistance to the third antibiotic, BMD (0.022 mg/mL). A germination and sporulation assay was run to ensure the spores could survive pelleting. A lack of significant change (P= 0.112) in cell recovery was indicated the probiotic’s ability to endure pelleting. A simulation digestive assay was performed mimicking the crop, proventriculus, and intestines to ensure the probiotic could survive digestion. When spores recovered from each section of the GIT were compared, the final concentration was significantly lower than the initial (P = 0.018) with a 2-fold reduction reaching the small intestine. The digestive assay was repeated with the addition of CP in the small intestines to determine if anti-clostridial properties were maintained. Using a 10⁶ dose of spores, the reduction in CP was significant at P= 0.103. The results of the experiments indicate the probiotic is a candidate for treatment and control of necrotic enteritis due to its broad anti-clostridial properties and resilience in harsh environments.

Key Words: probiotic, poultry, necrotic enteritis, antibiotic alternatives, direct-fed microbial.

Authors: M. Trombetta *1, K. McGovern 1, A. Duff 1, H. Xue 2, D. Wang 2, L. Johnston 2, and L. Bielke 1 / 1 The Ohio State University, Wooster, OH, USA, 2 Amlan, Chicago, IL, USA.