Enteric permeability and inflammation associated with day of hatch Enterobacteriaceae inoculation.

Early exposure to Enterobacteriaceae may result in inappropriate microbial colonization of the gastrointestinal (GI) tract, induce mild GI inflammation, alter immune system development, and predispose poultry to opportunistic infection. Four experiments were conducted to test Enterobacteriaceae isolates Escherichia coli LG strain (LG), E. coli Huff strain (Huff), Salmonella Enteritidis LB (SE) and Salmonella Typhimurium (ST) on ability to induce GI inflammation. All 4 experiments included a noninoculated control, and day of hatch (DOH) oral inoculation of LG, Huff, SE and ST in experiment 1, LG and SE in experiment 2, and LG, Huff, SE, and ST in experiment 3. Experiment 4 included LG, Huff, a noninoculated control (NIC), and Clostridium perfringens only (NCP) wherein birds received oral C. perfringens challenge on d15-16 to induce necrotic enteritis. Body weight was measured, yolk sacs and spleens were collected, and blood was obtained for serum fluorescein isothiocyanate dextran (FITC-d) recovery and alpha-1-acid glycoprotein (A1GP) concentrations. Samples were taken weekly through 2 wk of age in experiments 1 and 2, or 4 wk of age in experiments 3 and 4. Increased FITC-d recovery was observed for LG and SE on d13 in experiment 2 (P < 0.05), and C. perfringens only birds on d27 in experiment 4 (P < 0.05) as compared to noninoculated controls. Each experiment resulted in notable differences in A1GP serum concentrations over time, with fluctuations in A1GP patterns through d14 based on DOH inoculation (P < 0.05). Over time, A1GP was increased for DOH inoculated birds from d 22 to 29, the fourth wk of life, and d 2-29, the entire experiment, vs. noninoculated controls in experiment 3 (P < 0.05). Similarly, NCP and LGCP showed increased A1GP from d 20 to 27 and d 6 to 27, vs. NIC in experiment 4 (P < 0.05). In experiment 4, C. perfringens challenge resulted in earlier A1GP response in DOH inoculated birds, d 17-20, as compared to NCP birds, d 20-27 (P < 0.05). These results suggest early Enterobacteriaceae exposure may influence early inflammatory state in the GI tract and may also alter patterns of inflammation and responsiveness to pathogens.

Evaluation of day of hatch exposure to various Enterobacteriaceae on inducing gastrointestinal inflammation in chicks through two weeks of age.

Inappropriate microbial colonization can induce gastrointestinal (GI) inflammation may predispose poultry to opportunistic infections and reduce growth performance. Four independent experiments were completed to test ability of select Enterobacteriaceae isolates to induce GI inflammation. Experiments 1 and 2 included a non-inoculated control (NC), and a low (L), medium (M), or high (H) day of hatch (DOH) oral inoculation level. In experiment 1, birds in L1, M1, and H1 received 102 to 104 CFU of a mixed dose of 2 species of Citrobacter and Salmonella Enteritidis LB (SE). In experiment 2, birds in L2, M2, and H2 received 103 to 105 CFU of E. coli LG (LG) and included NC. Body weight was recorded on d 0, 7, and 14, with blood collected for chicken serum alpha-1-acid glycoprotein (A1GP) measurements on d14. Neither experiment resulted in differences in BWG, however, A1GP was increased (P < 0.05) on d 14 when DOH inoculation dose 103 CFU/chick was used compared to NC. This observed increase in A1GP resulted in selection of 103 CFU/chick for DOH inoculation in experiments 3 and 4. Experiment 3 consisted of NC, E. coli Huff (Huff), and SE. On d 0, 7 and 15, BW was measured, with blood collected on d 15 for A1GP. Both d 15 A1GP and BWG from d 7 to 15 were reduced in inoculated chicks, Huff and SE, in experiment 3 (P < 0.05). Experiment 4 evaluated NC and LG with BW measured on d 0, 2, 7 and 14. Yolk sacs were evaluated for retention and bacterial enumeration, and blood for serum A1GP were collected on d 2 and 14. Experiment 4 resulted in no differences in yolk sac parameters or A1GP, whereas there was an increase in BWG for LG from d 0 to 14 (P < 0.05). When evaluated over time, serum A1GP increased between d 2 and d 14 by nearly 46% in LG, compared to negligible changes in NC (P = 0.111). Mild GI inflammation induced by early Enterobacteriaceae exposure may not drastically impact growth or inflammation parameters but may increase susceptibility to opportunistic infection necessitating further study of this model.

Impact of in ovo administered pioneer colonizers on intestinal proteome on day of hatch.

Pioneer colonization of the gastrointestinal tract (GIT) by bacteria is thought to have major influence on neonatal tissue development. Previous studies have shown in ovo inoculation of embryos with saline (S), species of Citrobacter (C, C2), or lactic acid bacteria (L) resulted in an altered microbiome on day of the hatch (DOH). The present study investigated GIT proteomic changes at DOH in relation to different inoculations. Embryos were inoculated in ovo with S or ∼102 cfu of C, C2, or L at 18 embryonic days. On DOH, the GIT was collected, and tissue proteins were extracted for analysis via tandem mass spectrometry. A total of 493 proteins were identified for differential comparison with S at P ≤ 0.10. Different levels were noted in 107, 39, and 78 proteins in C, C2, and L groups, respectively, which were uploaded to Ingenuity Pathway Analysis to determine canonical pathways and biological functions related to these changes. Three members of the cytokine family (interleukin [IL]-1ß, IL6, and Oncostatin M) were predicted to be activated in C2, indicated with Z-score ≥ 1.50, which suggested an overall proinflammatory GIT condition. This was consistent with the activation of the acute-phase response signaling pathway seen exclusively in C2 (Z-score = 2.00, P < 0.01). However, activation (Z-score = 2.00) of IL-13, upregulation of peroxiredoxin-1 and superoxide dismutase 1, in addition to activation of nitric oxide signaling in the cardiovascular system of the L treatment may predict a state of increased antioxidant capacity and decreased inflammatory status. The nuclear factor erythroid 2-related factor 2 (NRF2)-mediated oxidative stress response (Z-score = 2.00, P < 0.01) was predicted to be upregulated in C which suggested that chicks were in an inflammatory state and associated oxidative stress, but the impact of these pathways differed from that of C2. These changes in the proteome suggest that pioneer colonizing microbiota may have a strong impact on pathways associated with GIT immune and cellular development.

A Proteomic View of the Cross-Talk Between Early Intestinal Microbiota and Poultry Immune System.

Proteomics has been used to investigate cross-talk between the intestinal microbiome and host biological processes. In this study, an in ovo technique and a proteomics approach was used to address how early bacterial colonization in the gastrointestinal tract (GIT) could modulate inflammatory and immune responses in young broilers. Embryos at 18 embryogenic days were inoculated with saline (S), 102 CFU of Citrobacter freundii (CF), Citrobacter species (C2), or lactic acid bacteria mixture (L) into the amnion. At 10 days posthatch, ileum samples from 12 birds per treatment were selected for tandem mass spectrometry analysis. Our further findings indicated that treatment-specific influences on early GIT microbiota resulted in different immune responses in mature broilers. Predicted functional analyses revealed activation of inflammation pathways in broilers treated in ovo with L and CF. Exposure to L enhanced functional annotation related to activation, trafficking of immune cells, and skeletal growth based-network, while CF inhibited biological functions associated with immune cell migration and inflammatory response. These results highlighted that proper immune function was dependent on specific GIT microbiota profiles, in which early-life exposure to L-based probiotic may have modulated the immune functions, whereas neonatal colonization of Enterobacteriaceae strains may have led to immune dysregulation associated with chronic inflammation.

Research Note: Evaluating fecal shedding of oocysts in relation to body weight gain and lesion scores during Eimeria infection.

Coccidiosis has been a pervasive disease within the poultry industry, with test parameters used to measure effectiveness of treatment strategies often being subjective or influenced by non-disease-related activity. Four experiments were completed, which examined several test parameters of coccidiosis, including body weight gain (BWG), lesion scores, and oocysts per gram of feces (OPG). Each experiment included at least 2 parameters for measuring coccidial infection in chickens and turkeys. In experiment 1, an inoculated control was measured against 3 anticoccidial groups, whereas in experiments 2 to 4, noninoculated and inoculated controls were compared via BWG and OPG. Lesion scores were also included in experiments 1, 3, and 4. Experiment 4 resulted in high correlation, via Pearson correlation coefficient, between BWG and OPG (r = -0.69), very high correlation between OPG and lesion score (r = 0.86), and moderate correlation between BWG and lesion score (r = -0.49). Lesion scores proved to be effective in confirming Eimeria infection, although they did not correlate well with BWG or OPG. Each parameter tended to provide more useful information when lined up with the Eimeria life cycle. Incorporation of OPG, with BWG and lesion scores, as test parameters to measure coccidiosis intervention strategies, provides a global description of disease that may not otherwise be observed with the 2 latter measurements alone.

Preliminary studies on development of a novel subunit vaccine targeting Clostridium perfringens mucolytic enzymes for the control of necrotic enteritis in broilers.

Necrotic enteritis (NE) is a pervasive enteric disease responsible for large scale economic losses within the global poultry industry. The etiologic agent of NE is Clostridium perfringens (CP), an opportunistic pathogen that utilizes numerous extracellular toxins and glycoside hydrolases (GH) as key virulence and nutrient acquisition factors. Notably, some GH, mucinases, degrade components of mucin in the gastrointestinal tract as an energy source. Targeting this mechanism may serve to reduce the incidence of disease associated with CP. Two experiments were completed that evaluated mucinase vaccine targets sourced from conserved peptide sequences of carbohydrate binding module 32 of CP mucinases. In experiment 1, 37 antigen peptides were synthetically generated and used to produce hyper-immune sera, which was then evaluated for ability to obstruct CP growth in vitro. Total CFU of CP were measured at 4, 6, and 8 h incubation to determine growth rate. Peptides 4, 5, 22, 24, and 30 were selected for further in vivo testing based on conservation or the ability to inhibit CP growth by over 50% at 6 and 8 h. In experiment 2, the aforementioned peptides were conjugated to an agonistic, CD40-targetting antibody and evaluated in vivo. Broilers were given an Eimeria maxima and CP in order to induce NE and assess vaccine efficacy. Treatments included a non-vaccinated non-inoculated control, non-vaccinated inoculated control (NVIC), vaccination with peptide 4, 5, 22, 24, or 30 (VP4-VP30), or a combination of all 5 peptides (MC). There was a significant increase (P < 0.05) in the percent change in BWG relative to NVIC for vaccination with peptide 22 and MC of 18.54 and 17.43%, respectively. MC vaccinated group had the lowest lesions with a mean score of 0.63 ± 0.18. These results suggest the MC combination was the most successful in alleviating overall performance losses associated with NE-infected broilers and encourage future testing of MC in the development of an NE vaccine.

Evaluation of the impact of in ovo administered bacteria on microbiome of chicks through 10 days of age.

Initial inoculation and colonization of the chicken gastrointestinal tract (GIT) by microbiota have been suggested to have a major influence on the growth performance and health of birds. Commercial practices in chicken production may alter or delay microbial colonization by pioneer colonizing bacteria that can have an impact on the development and maturation of the GIT and intestinal microflora. The objective of this study was to compare the impact of apathogenic Gram-negative isolates or lactic acid bacteria (LAB) as pioneer colonizers on the microbiome at the day of hatch (DOH) and evaluate the influence through 10 D of age on ceca. At 18 embryonic days (E), the amnion of embryos was inoculated with either saline (S), approximately 102 CFU of LAB (L), Citrobacter freundii (C), or Citrobacter species (C2). Once DNA was isolated from mucosal and digesta contents, samples underwent 2 × 300 paired-end Illumina MiSeq library preparation for microbiome analysis. An increased abundance of Lactobacillaceae family and Lactobacillus genus was observed in the L group at DOH (P < 0.05), whereas the abundance of Enterococcaceae and Enterococcus was numerically decreased. While Lactobacillus salivarius was one of the pioneer colonizers in the L group at 18E, the population decreased by 10 D (39.59 to 0.09%) and replaced with a population of undefined Lactobacillus (10.36%) and Lactobacillus reuteri (3.63%). Results suggest that L treatment may have accelerated a mature microbiota. Enterobacteriaceae was the dominant family (57.44%) in C group at DOH (P < 0.05). The C2 group only showed some abundance of the C2 species (7.92%) at DOH but had the highest overall abundance of undefined Lactobacillus in the ceca by 10 D (25.28%). Taken together, different isolates provided in ovo can have an impact on the initial microbiome of the GIT, and some of these differences in ceca remain notable at 10 D.

Comparison of multiple methods for induction of necrotic enteritis in broilers: II. Impact on the growth curve.

Necrotic enteritis (NE) is a disease that has gained relevance in the poultry industry with both immediate and sustained effects on BW of broilers. The objective of the 3 experiments was to evaluate the impact of NE, induced by methods that reflect common broiler production systems, on the growth curve throughout the growth period. In addition, the impact of Eimeria maxima (EM) on NE, as well as the long-term impact of Clostridium perfringens (CP) on BW, were analyzed. In experiment 1, a dual infection model of EM and CP was compared to a non-challenged control, while experiment 2 evaluated 2 different strains of EM dual infection, as well as 6 CP-only groups. Similarly, experiment 3 tested dual infection and both high and low dose CP-only groups. Both NetB and non-NetB strains of CP were used to evaluate whether NetB toxin may potentially play a role in NE induction. In all 3 experiments, BW was measured immediately before infection on day 16, then weekly through the end of the test period. In all 3 experiments, a decrease (p < 0.05) in BW was observed immediately following the acute NE disease period of day 21 to day 23, with a negative impact also observed of BW gain during NE disease period (p < 0.05). A long-term effect on BW was most clearly detected in the EM + CP dual infection models, as well as when high levels of CP-only were administered. In these cases, BW was impacted long-term, with a requisite week or more to return to a BW similar to the non-challenged control. The separation in BW, though not significant, was nearly parallel with the non-challenged control throughout the growth period, indicating a shift in the growth curve. In addition to showing the long-term impact of various forms of NE on broiler growth, these shifts in the growth curve can be used to measure the effects of treatments on prevention and recovery of broilers impacted by NE.