Exploring the underlying mechanisms of enteritis impact on golden pompano (Trachinotus ovatus) through multi-omics analysis.

Enteritis posed a significant health challenge to golden pompano (Trachinotus ovatus) populations. In this research, a comprehensive multi-omics strategy was implemented to elucidate the pathogenesis of enteritis by comparing both healthy and affected golden pompano. Histologically, enteritis was characterized by villi adhesion and increased clustering after inflammation. Analysis of the intestinal microbiota revealed a significant increase (P < 0.05) in the abundance of specific bacterial strains, including Photobacterium and Salinivibrio, in diseased fish compared to the healthy group. Metabolomic analysis identified 5479 altered metabolites, with significant impacts on terpenoid and polyketide metabolism, as well as lipid metabolism (P < 0.05). Additionally, the concentrations of several compounds such as calcitetrol, vitamin D2, arachidonic acid, and linoleic acid were significantly reduced in the intestines of diseased fish post-enteritis (P < 0.05), with the detection of harmful substances such as Efonidipine. In transcriptomic profiling, enteritis induced 68 upregulated and 73 downregulated genes, predominantly affecting steroid hormone receptor activity (P < 0.05). KEGG pathway enrichment analysis highlighted upregulation of SQLE and CYP51 in steroidogenesis, while the HSV-1 associated MHC1 gene exhibited significant downregulation. Integration of multi-omics results suggested a potential pathogenic mechanism: enteritis may have resulted from concurrent infection of harmful bacteria, specifically Photobacterium and Salinivibrio, along with HSV-1. Efonidipine production within the intestinal tract may have blocked certain calcium ion channels, leading to downregulation of MHC1 gene expression and reduced extracellular immune recognition. Upregulation of SQLE and CYP51 genes stimulated steroid hormone synthesis within cells, which, upon binding to G protein-coupled receptors, influenced calcium ion transport, inhibited immune activation reactions, and further reduced intracellular synthesis of anti-inflammatory substances like arachidonic acid. Ultimately, this cascade led to inflammation progression, weakened intestinal peristalsis, and villi adhesion. This study utilized multi-level omics detection to investigate the pathological symptoms of enteritis and proposed a plausible pathogenic mechanism, providing innovative insights into enteritis verification and treatment in offshore cage culture of golden pompano.

Intestinal microbiota and gene expression alterations in leopard coral grouper (Plectropomus leopardus) under enteritis.

Enteritis poses a significant threat to fish farming, characterized by symptoms of intestinal and hepatic inflammation, physiological dysfunction, and dysbiosis. Focused on the leopard coral grouper (Plectropomus leopardus) with an enteritis outbreak on a South China Sea farm, our prior scrutiny did not find any abnormalities in feeding or conventional water quality factors, nor were any specific pathogen infections related to enteritis identified. This study further elucidates their intestinal flora alterations, host responses, and their interactions to uncover the underlying pathogenetic mechanisms and facilitate effective prevention and management strategies. Enteritis-affected fish exhibited substantial differences in intestinal flora compared to control fish (P = 0.001). Notably, norank_f_Alcaligenaceae, which has a negative impact on fish health, predominated in enteritis-affected fish (91.76 %), while the probiotic genus Lactococcus dominated in controls (93.90 %). Additionally, certain genera with pathogenesis potentials like Achromobacter, Sphingomonas, and Streptococcus were more abundant in diseased fish, whereas Enterococcus and Clostridium_sensu_stricto with probiotic potentials were enriched in control fish. At the transcriptomic level, strong inflammatory responses, accompanied by impaired metabolic functions, tissue damage, and iron death signaling activation were observed in the intestines and liver during enteritis. Furthermore, correlation analysis highlighted that potential pathogen groups were positively associated with inflammation and tissue damage genes while presenting negatively correlated with metabolic function-related genes. In conclusion, dysbiosis in the intestinal microbiome, particularly an aberrantly high abundance of Alcaligenaceae with pathogenic potential may be the main trigger for this enteritis outbreak. Alcaligenaceae alongside Achromobacter, Sphingomonas, and Streptococcus emerged as biomarkers for enteritis, whereas some species of Lactococcus, Clostridium_sensu_stricto, and Enterococcus showed promise as probiotics to alleviate enteritis symptoms. These findings enhance our understanding of enteritis pathogenesis, highlight intestinal microbiota shifts in leopard coral grouper, and propose biomarkers for monitoring, probiotic selection, and enteritis management.

Supplementation with organic yeast-derived selenium provides immune protection against experimental necrotic enteritis in broiler chickens.

Necrotic enteritis (NE) is a potentially fatal poultry disease that causes enormous economic losses in the poultry industry worldwide. The study aimed to evaluate the effects of dietary organic yeast-derived selenium (Se) on immune protection against experimental necrotic enteritis (NE) in commercial broilers. Chickens were fed basal diets supplemented with different Se levels (0.25, 0.50, and 1.00 Se mg/kg). To induce NE, Clostridium perfringens (C. perfringens) was orally administered at 14 days of age post hatch. The results showed that birds fed 0.25 Se mg/kg exhibited significantly increased body weight gain compared with the non-supplemented/infected birds. There were no significant differences in gut lesions between the Se-supplemented groups and the non-supplemented group. The antibody levels against α-toxin and NetB toxin increased with the increase between 0.25 Se mg/kg and 0.50 Se mg/kg. In the jejunal scrapings and spleen, the Se-supplementation groups up-regulated the transcripts for pro-inflammatory cytokines IL-1ß, IL-6, IL-8, iNOS, and LITAF and avian ß-defensin 6, 8, and 13 (AvBD6, 8 and 13). In conclusion, supplementation with organic yeast-derived Se alleviates the negative consequences and provides beneficial protection against experimental NE.

Effect of 125% and 135% arginine on the growth performance, intestinal health, and immune responses of broilers during necrotic enteritis challenge.

The objective of this study was to evaluate the effects of 25% and 35% arginine supplementation in partially alleviating the effects of necrotic enteritis (NE) challenge on the production performance, intestinal integrity, and relative gene expression of tight junction proteins and inflammatory cytokines in broilers. Four hundred and eighty 1-day-old chicks were randomly allocated to the 4 treatments- Uninfected + Basal, NE + Basal, NE + Arg 125%, and NE + Arg 135%. NE was induced by inoculating 1 × 104Eimeria maxima sporulated oocysts on d 14 and 1 × 108 CFU/bird C. perfringens on d 19, 20, and 21 of age by oral gavage. The NE challenge significantly decreased body weight gain (BWG) (p < 0.05) and increased the feed conversion ratio (FCR) (p < 0.05). On d 21, the NE challenge also increased the jejunal lesion score (p < 0.05) and relative gene expression of IL-10 and decreased the expression of the tight junction proteins occludin (p < 0.05) and claudin-4 (p < 0.05). The 125% arginine diet significantly increased intestinal permeability (p < 0.05) and the relative gene expression of iNOS (p < 0.05) and IFN-γ (p < 0.05) on d 21 and the bile anti-C. perfringens IgA concentration by 39.74% (p < 0.05) on d 28. The 135% arginine diet significantly increased the feed intake during d 0 - 28 (p < 0.05) and 0 to 35 (p < 0.05) and increased the FCR on d 0 to 35 (p < 0.05). The 135% and 125% arginine diet increased the spleen CD8+: CD4+ T-cell ratio on d 28 (p < 0.05) and 35 (p < 0.05), respectively. The 135% arginine diet increased the CT CD8+:CD4+ T-cell ratio on d 35 (p < 0.05). In conclusion, the 125% and 135% arginine diets did not reverse the effect of the NE challenge on the growth performance. However, the 125% arginine diet significantly increased the cellular and humoral immune response to the challenge. Hence, the 125% arginine diet could be used with other feed additives to improve the immune response of the broilers during the NE challenge.

LTA and PGN from Bacillus siamensis can alleviate soybean meal-induced enteritis and microbiota dysbiosis in Lateolabrax maculatus.

An eight-week feeding trial was designed to assess which component of commensal Bacillus siamensis LF4 can mitigate SBM-induced enteritis and microbiota dysbiosis in spotted seabass (Lateolabrax maculatus) based on TLRs-MAPKs/NF-кB signaling pathways. Fish continuously fed low SBM (containing 16 % SBM) and high SBM (containing 40 % SBM) diets were used as positive (FM group) and negative (SBM group) control, respectively. After feeding high SBM diet for 28 days, fish were supplemented with B. siamensis LF4-derived whole cell wall (CW), cell wall protein (CWP), lipoteichoic acid (LTA) or peptidoglycan (PGN) until 56 days. The results showed that a high inclusion of SBM in the diet caused enteritis, characterized with significantly (P < 0.05) decreased muscular thickness, villus height, villus width, atrophied and loosely arranged microvillus. Moreover, high SBM inclusion induced an up-regulation of pro-inflammatory cytokines and a down-regulation of occludin, E-cadherin, anti-inflammatory cytokines, apoptosis related genes and antimicrobial peptides. However, dietary supplementation with CW, LTA, and PGN of B. siamensis LF4 could effectively alleviate enteritis caused by a high level of dietary SBM. Additionally, CWP and PGN administration increased beneficial Cetobacterium and decreased pathogenic Plesiomonas and Brevinema, while dietary LTA decreased Plesiomonas and Brevinema, suggesting that CWP, LTA and PGN positively modulated intestinal microbiota in spotted seabass. Furthermore, CW, LTA, and PGN application significantly stimulated TLR2, TLR5 and MyD88 expressions, and inhibited the downstream p38 and NF-κB signaling. Taken together, these results suggest that LTA and PGN from B. siamensis LF4 could alleviate soybean meal-induced enteritis and microbiota dysbiosis in L. maculatus, and p38 MAPK/NF-κB pathways might be involved in those processes.

Effect of arginine supplementation on the growth performance, intestinal health, and immune responses of broilers during necrotic enteritis challenge.

The objective of this study was to evaluate the effect of 25% arginine supplementation as a functional amino acid in partially alleviating the detrimental effects of necrotic enteritis (NE) on the growth performance, serum biochemistry, gut integrity, and the relative gene expression of tight junction proteins and inflammatory cytokines in broilers during NE. Three hundred and sixty 1-day-old chicks were randomly allocated to 4 treatments in a 2 × 2 factorial arrangement -basal diet and 125% arginine diet, with or without NE challenge. NE was induced by inoculating 1 × 104Eimeria maxima sporulated oocysts on d 14 and 1 × 108 CFU/bird C. perfringens on d 19, 20, and 21. The NE challenge had a significant effect on the BWG (p < 0.05), FCR (p < 0.05), serum AST (p < 0.05), GLU (p < 0.05), and K+ (p < 0.05) levels, and intestinal permeability (p < 0.05) and jejunal lesion score (p < 0.05). A significant challenge × diet interaction effect was observed in the cecal tonsil CD8+: CD4+ T-cell ratio on d 21 (p < 0.05) and 28 (p < 0.05) and spleen CD8+: CD4+ T-cell ratio on d 21 (p < 0.05) and 35 (p < 0.05). Arginine supplementation significantly increased the CD8+: CD4+ T-cell ratio in uninfected birds but decreased the CD8+: CD4+ T-cell ratio in infected birds. On d 21, a significant interaction effect was observed on the relative expression of the iNOS gene (p < 0.05). Arginine supplementation significantly downregulated the expression of the iNOS gene in infected birds. A significant effect of the challenge (p < 0.05) was observed on the relative gene expression of the ZO-1 gene in the jejunum. NE challenge significantly downregulated the expression of the ZO-1 gene on d 21. In conclusion, arginine supplementation did not alleviate the depression in growth performance and disease severity during the NE challenge. However, arginine downregulated the expression of inflammatory cytokines and enzymes, preventing inflammatory injury to the tissues during NE. Hence, arginine might be supplemented with other alternatives to downregulate inflammatory response during NE in poultry.

Genetic diversity and phylogenetic relationships of Clostridium perfringens strains isolated from mastitis and enteritis in Egyptian dairy farms.

BACKGROUND: Clostridium perfringens, a common environmental bacterium, is responsible for a variety of serious illnesses including food poisoning, digestive disorders, and soft tissue infections. Mastitis in lactating cattle and sudden death losses in baby calves are major problems for producers raising calves on dairy farms. The pathogenicity of this bacterium is largely mediated by its production of various toxins. RESULTS: The study revealed that Among the examined lactating animals with a history of mastitis, diarrheal baby calves, and acute sudden death cases in calves, C. perfringens was isolated in 23.5% (93/395) of the total tested samples. Eighteen isolates were obtained from mastitic milk, 59 from rectal swabs, and 16 from the intestinal contents of dead calves. Most of the recovered C. perfringens isolates (95.6%) were identified as type A by molecular toxinotyping, except for four isolates from sudden death cases (type C). Notably, C. perfringens was recovered in 100% of sudden death cases compared with 32.9% of rectal swabs and 9% of milk samples. This study analyzed the phylogeny of C. perfringens using the plc region and identified the plc region in five Egyptian bovine isolates (milk and fecal origins). Importantly, this finding expands the known data on C. perfringens phospholipase C beyond reference strains in GenBank from various animal and environmental sources. CONCLUSION: Phylogenetic analyses of nucleotide sequence data differentiated between strains of different origins. The plc sequences of Egyptian C. perfringens strains acquired in the present study differed from those reported globally and constituted a distinct genetic ancestor.

Research Note: Bovine lactoferrin in chickens: an investigation into its viability as an antibiotic alternative.

Finding effective antibiotic alternatives is crucial to managing the re-emerging health risk of Clostridium perfringens (CP) type A/G-induced avian necrotic enteritis (NE), a disease that has regained prominence in the wake of governmental restrictions on antibiotic use in poultry. Known for its antimicrobial and immunomodulatory effects, the use of bovine lactoferrin (bLF) in chickens is yet to be fully explored. In this study, we hypothesized that bLF can accumulate in the small intestines of healthy chickens through gavage and intramuscular supplementation and serves as a potential antibiotic alternative. Immunohistochemistry located bLF in various layers of the small intestines and ELISA testing confirmed its accumulation. Surprisingly, sham-treated chickens also showed the presence of bLF, prompting a western blotting analysis that dismissed the notion of cross-reactivity between bLF and the avian protein ovotransferrin. Although the significance of the route of administration remains inconclusive, this study supports the hypothesis that bLF is a promising and safe antibiotic alternative with demonstrated resistance to the degradative environment of the chicken intestines. Further studies are needed to determine its beneficial pharmacological effects in CP-infected chickens.

The effect of butyric acid and nucleotides supplementation on broiler (Gallus gallus domesticus) growth performance, immune status, intestinal histology, and serum parameters.

Background: Butyric acid and its derivatives support the immune system, lessen inflammation, and lessen oxidative stress in broilers in addition to preserving gut homeostasis and epithelial integrity. Broiler performance has also been demonstrated to rise with the addition of nucleotides to the diet. Aim: The purpose of the study was to ascertain the effects of butyric acid and nucleotides added to feed on the overall performance, immunity, oxidant/antioxidant enzyme levels, intestinal histology, and hepatic functions of broilers. Methods: Four experimental groups of thirty chickens, each were used in the present study. The groups were assigned as a control group that received normal diet without additives, butyrate (B) group received the diet supplemented with butyric acid (250 g/ton feed), nucleotides (N) group received the diet supplemented with nucleotides (200 g/ton feed), and the fourth group received the diet supplemented with a combination of butyrate and nucleotide (BN) (250 g/ton B feed, and 200 g/ton N feed, respectively). Necrotic enteritis was produced in ten birds from each group to assess the immune-modulatory effect of these supplements, antioxidant status, intestinal histology, and liver functions were measured in all experimental groups. Results: The addition of butyric acid and nucleotides to feed enhanced body weight, growth performance, hepatic functions, and antioxidant capabilities. Histological sections of the gut from challenged or unchallenged (with necrotic enteritis) groups in the BN group showed considerable improvement, as shown by strong proliferation in intestinal crypts and villus enterocytes. Conclusion: Nucleotides and butyric acid can be added to broiler feeding regimens to enhance growth and health.

Antibiotic resistance and virulence genes profile of Non typhodial Salmonella species isolated from poultry enteritis in India.

Salmonella species (spp) is the most important gastrointestinal pathogen present ubiquitously. Non typhoidal Salmonella (NTS) is commonly associated with gastroenteritis in humans. Layer birds once get infection with NTS, can become persistently infected with Salmonella Typhimurium and intermittently shed the bacteria. It results in a high risk of potential exposure of eggs to the bacteria. The current study was conducted to determine the serotype diversity, presence of virulence genes, antibiotic resistance pattern, and genes of NTS from poultry enteritis. Out of 151 intestinal swabs from poultry total 118 NTS were isolated, which were characterized serologically as S. Typhimurium (51 strains), S. Weltevreden (57 strains) and untypable (10 strains). Most effective antibiotics were amikacin, gentamycin and ceftriaxone (33.05%) followed by ampicillin, azithromycin and ciprofloxacin (16.69%), co-trimoxazole (13.55%), and tetracycline (6.78%). Multidrug resistance recorded in 17.70% (N = 21/118) strains. Antimicrobial-resistant genes i.e. blaTEM, blaSHV, blaCTX-M, tet(A), tet(B), tet(C), sul1, sul2, sul3. blaTEM and tet(A) were present in 95% (20/21). Eleven virulence genes i.e. invA, hilA, sivH, tolC, agfA, lpfA, spaN, pagC, spiA, iroN and fliC 2 were present in all the 30 isolates. While, sopE was present in only 2 isolates, NTS strains with characteristics of pathogenicity and multidrug resistance from poultry enteritis were detected. Multidrug resistance showed the necessity of prudent use of antibiotics in the poultry industry.

Comparison of necrotic enteritis effects on growth performance and intestinal health in two different meat-type chicken strains Athens Canadian Random Bred and Cobb 500.

Chickens have undergone genetic improvements in the past few decades to maximize growth efficiency. However, necrotic enteritis (NE), an enteric disease primarily caused by C. perfringens, remains a significant problem in poultry production. A study investigated the differences in intestinal health between the nonselected meat-type chicken Athens Canadian Random Bred (ACRB) and the modern meat-type Cobb 500 broilers (Cobb) when challenged with experimental NE. The study utilized a 2 × 3 factorial arrangement, consisting of two main effects of chicken strain and NE challenge model (nonchallenged control, NC; NE challenge with 2,500/12,500 Eimeria maxima oocysts + 1 × 109C. perfringens, NE2.5/NE12.5). A total of 432 fourteen-day-old male ACRB and Cobb were used until 22 d (8 d postinoculation with E. maxima on d 14, dpi), and the chickens were euthanized on 6 and 8 dpi for the analysis. All data were statistically analyzed using a two-way ANOVA, and Student's t-test or Tukey's HSD test was applied when P < 0.05. The NE12.5 group showed significant decreases in growth performance and relative growth performance from d 14 to 20, regardless of chicken strain (P < 0.01). The ACRB group exhibited significant decreases in relative body weight and relative body weight gain compared to the Cobb group from d 14 to 22 (P < 0.01). On 6 and 8 dpi, both NE challenge groups showed significant decreases in intestinal villus height to crypt depth ratio, jejunal goblet cell count, and jejunal MUC2 and LEAP2 expression (P < 0.01). Additionally, the NE12.5 group had significantly higher intestinal NE lesion score, intestinal permeability, fecal E. maxima oocyst count, intestinal C. perfringens count, and jejunal IFNγ and CCL4 expression compared to the NC group (P < 0.05). In conclusion, NE negatively impacts growth performance and intestinal health in broilers, parameters regardless of the strain.

Treatment of chickens with lactobacilli prior to challenge with Clostridium perfringens modifies innate responses and gut morphology.

Necrotic enteritis caused by Clostridium perfringens (CP), is a common enteric disease of poultry that has been previously controlled by in-feed antibiotics. However, due to the rapid emergence of antimicrobial resistance, alternatives to antibiotics such as probiotics have received considerable attention because of their immunomodulatory and intestinal health benefits. The present study investigated the effects of probiotic lactobacilli on gut histomorphology and intestinal innate responses in chickens. Day-old male broiler chickens were treated with 1 × 107 or 1 × 108 colony-forming units (CFU) of a lactobacilli cocktail on days 1, 7, 14, and 20 post-hatch, while control groups were not treated with lactobacilli. On day 21, birds in all groups (except the negative control) were challenged with 3 × 108 CFU of CP for 3 days. Intestinal tissue samples were collected before and after the CP challenge to assess gene expression and for histomorphological analysis. Lactobacilli treatment at a dose of 1 × 108 CFU conferred partial protection against NE by lowering lesion scores, increasing villus height in the ileum and reducing crypt depth in the jejunum. In addition, 1 × 108 CFU of lactobacilli enhanced the expression of Toll-like receptor (TLR) 2, interferon-gamma (IFN-γ), interleukin (IL)-10, IL-12, and IL-13 in both the jejunum and ileum at different timepoints and subsequently decreased the expression of transforming growth factor beta (TGF-ß) and IL-1ß post-CP challenge. In conclusion, the results indicate that treatment with lactobacilli mitigated NE in a dose-dependent manner via improvement of intestinal morphology and modulation of innate immune response in chickens.

Bacillus subtilis HW2 enhances growth performance and alleviates gut injury via attenuation of endoplasmic reticulum stress and regulation of gut microbiota in broilers under necrotic enteritis challenge.

This study investigated the effects of Bacillus subtilis HW2 on the growth performance, immune response, endoplasmic reticulum (ER) stress, and intestinal health in broilers with necrotic enteritis. Three hundred 1-day-old male Cobb 500 broilers (33.88 ± 2.34 g) were randomly allocated to 5 groups including non-infected control (NC group), basal diet + necrotic enteritis challenge (NE group), basal diet + 1 × 106 CFU/g B. subtilis HW2 + necrotic enteritis challenge (L-Pro group), basal diet + 5 × 106 CFU/g B. subtilis HW2 + necrotic enteritis challenge (M-Pro group), and basal diet + 1 × 107 CFU/g B. subtilis HW2 + necrotic enteritis challenge (H-Pro group), with 6 replicates per group. All broilers except NC group were orally given with sporulated coccidian oocysts at day 14 and Clostridium perfringens from days 19 to 21. Results showed that L-Pro and M-Pro groups improved growth performance and intestinal morphology in necrotic enteritis-challenged broilers, and L-Pro, M-Pro, and H-Pro groups improved intestinal barrier function and immune response and decreased ER stress in necrotic enteritis-challenged broilers. Analysis of the gut microbiota revealed that L-Pro group increased the abundances of Alistipes, Coprobacter, Barnesiella, and Limosilactobacillus, decreased Erysipelatoclostridium abundance on day 42 in necrotic enteritis-challenged broilers. M-Pro group increased Turicibacter abundance on day 28 and the abundances of Alistipes, Barnesiella, and Limosilactobacillus on day 42 in necrotic enteritis-challenged broilers. H-Pro group decreased Romboutsia abundance on day 28 and unidentified_Clostridia abundance on day 42 in necrotic enteritis-challenged broilers. Analysis of short-chain fatty acids (SCFAs) revealed higher isobutyric acid and isovaleric acid levels in L-Pro and M-Pro groups than NE group. Correlation analysis revealed the correlations between the biochemical parameters and gut microbiota as well as SCFAs, especially Romboutsia, Barnesiella, Coprobacter, isobutyric acid, and isovaleric acid. Overall, our results indicated that B. subtilis HW2 supplementation could ameliorate necrotic enteritis infection-induced gut injury. The optimal dietary supplementation dosage of Bacillus subtilis HW2 was 5 × 106 CFU/g.

Natural intraepithelial lymphocyte populations rise during necrotic enteritis in chickens.

Intraepithelial lymphocytes (IEL) reside in the epithelium at the interface between the contents of the intestinal lumen and the sterile environment of the lamina propria. Because of this strategic location, IEL play a crucial role in various immunological processes, ranging from pathogen control to tissue stability. In mice and humans, IEL exhibit high diversity, categorized into induced IEL (conventional CD4 and CD8αß T cells) and natural IEL (TCRαßCD8αα, TCRγδ, and TCRneg IEL). In chickens, however, the subpopulations of IEL and their functions in enteric diseases remain unclear. Thus, we conducted this study to investigate the role of IEL populations during necrotic enteritis (NE) in chickens. At 14 days of age, sixty-three Specific-pathogen-free (SPF) birds were randomly assigned to three treatments: Control (sham challenge), Eimeria maxima challenge (EM), and Eimeria maxima + Clostridium Perfringens (C. Perfringens) co-challenge (EM/CP). The EM and EM/CP birds were infected with Eimeria maxima at day 14 of age, and EM/CP birds were additionally orally inoculated with C. perfringens at days 18 and 19 of age. Birds were weighed at days 18, 20, and 26 of age to assess body weight gain (BWG). At 20 days of age (1 day-post C. perfringens infection; dpi), and 26 days of age (7 dpi), 7 birds per treatment were euthanized, and jejunum was harvested for gross lesion scores, IEL isolation, and gene expression. The EM/CP birds exhibited subclinical NE disease, lower BWG and shorter colon length. The Most changes in the IEL populations were observed at 1 dpi. The EM/CP group showed substantial increases in the total number of natural IEL subsets, including TCRαß+CD4-CD8-, TCRαß+CD8αα+, TCRγδ+, TCRneg and innate CD8α (iCD8α) cells by at least two-fold. However, by 7 dpi, only the number of TCRαß+CD4-CD8- and TCRαß+CD8αα+ IEL maintained their increase in the EM/CP group. The EM/CP group had significantly higher expression of proinflammatory cytokines (IL-1ß and IFN-γ) and Osteopontin (OPN) in the jejunum at 1 dpi. These findings suggest that natural IEL with innate and innate-like functions might play a critical role in the host response during subclinical NE, potentially conferring protection against C. perfringens infection.

Research Note: Clostridium perfringens NetB and CnaA neutralizing nanobodies in feed reduce the incidence of poultry necrotic enteritis.

Necrotic enteritis is a devastating disease to poultry caused by the bacterium Clostridium perfringens. As a novel approach to combating poultry necrotic enteritis, we identified and characterized several hundred single domain antibody fragments (or nanobodies) capable of binding either the NetB toxin or the collagen-binding adhesin (CnaA) of C. perfringens. Many of the nanobodies could neutralize the in vitro functions of NetB or CnaA with inhibitory concentrations in the nanomolar range. The nanobodies were also screened for proteolytic stability in an extract derived from gastrointestinal tract fluids of chickens. A collection of 6 nanobodies (4 targeting NetB and 2 targeting CnaA) with high neutralizing activity and high gastrointestinal tract extract stability were expressed and secreted by Pichia pastoris or Bacillus subtilis. Chickens were given a feed with 1 of the 2 nanobody-containing groups: 1) nanobody-containing P. pastoris supernatants that were semi-purified, lyophilized, and enterically coated, or 2) B. subtilis spores from strains containing the nanobody genes. Compared to untreated chickens (23.75% mortality), mortality of chickens receiving feed modified with the P. pastoris and B. subtilis products decreased to 11.25 and 7.5%, respectively. These results offer a new opportunity to improve the control of poultry necrotic enteritis by incorporating highly specific nanobodies or bacteria expressing these nanobodies directly into chicken feed.

Bromelain can reduce the negative effects of a subclinical necrotic enteritis in broiler chickens.

This study was conducted to examine the efficacy of a bromelain-based supplementation coded ANR-pf on growth performance and intestinal lesion of broiler chickens under necrotic enteritis (NE) challenge. A total of 540 Ross 308 day-old male chicks were randomly allocated into 6 treatments of 6 replicates. The bromelain formulation was delivered to chickens through gavaging or in drinking water method twice, on d 8 and 13. Nonchallenged groups included 1) without or 2) with the specific bromelain formulation gavaged at 0.8 mL/kg. NE-challenged groups included 3) without the specific bromelain formulation; 4) gavaged with 0.4 mL/kg; 5) gavaged with 0.8 mL/kg and 6) supplemented with 0.8 mL/kg via drinking water. Birds were challenged with Eimeria spp. on d 9 and Clostridium perfringens (NE-18 strain) on d 14 and 15. On d 14 and 19, fresh faecal contents were collected for the determination of oocyst counts. Intestinal lesion scores were determined on d16. Performance and mortality were recorded throughout the entire experiment. Among challenged groups, birds received additive via drinking water had higher weight gain (WG) compared to the remaining groups (P < 0.001) in the grower phase and had lower FCR compared to 0.4 mL/kg inoculated group in the grower and finisher phases (P < 0.001). Bromelain supplementation via drinking water improved the WG of challenged birds, similar to that of the nonchallenged birds (P < 0.001), and lowered FCR compared to other challenged groups (P < 0.001). Nonchallenged birds and birds that received bromelain formulation in drinking water did not have lesions throughout the small intestine whereas challenged birds, either un-supplemented or supplemented with bromelain via inoculation route recorded similar lesion score levels in the jejunum. At d 19, birds received bromelain in drinking water had lower fecal oocyst numbers compared to challenged birds without additive (P < 0.001). In conclusion, bromelain administration via drinking water could ameliorate the negative impacts of NE-infection in broilers by improving performance, lowering the oocyst numbers and lesion scores.

Coccidiosis and necrotic enteritis model may have a greater impact than dietary anti-interleukin-10 on broiler chicken systemic immunometabolic responses.

Dietary egg yolk-derived anti-interleukin (IL)-10 may preserve broiler chicken performance during coccidiosis due to Eimeria spp. infection while effects on secondary Clostridium perfringens (necrotic enteritis) are unknown. Some necrotic enteritis models implement Salmonella Typhimurium to improve repeatability; however, Salmonella upregulation of IL-10 may be a confounder when evaluating anti-IL-10. The study objective was to investigate anti-IL-10 effects on systemic cytokine concentrations and immunometabolism during E. maxima ± C. perfringens challenge in models ± S. Typhimurium. Three 25 d replicate studies using Ross 308 chicks were conducted in wire-floor cages (32 cages/ replicate) with chicks assigned to diets ± 0.03% anti-IL-10. 640 chicks (20/ cage; replicates 1 and 2) were inoculated with sterile saline ± 1×108 colony forming units (CFU) S. Typhimurium while 480 chicks (15/ cage) were placed in replicate 3. In all replicates, blood samples were collected on d 14 (6 chicks/treatment) before administering 15,000 sporulated E. maxima M6 oocysts to S. Typhimurium-inoculated (replicates 1 and 2) or challenge-designated chicks (replicate 3). Half the E. maxima-challenged chicks received 1×108 CFU C. perfringens on d 18 and 19. Blood samples were collected at 1, 3, 7, and 11 d post-inoculation (dpi) with E. maxima and 1, 3, and 7 dpi with secondary C. perfringens. Plasma cytokines were determined by ELISA while immunometabolic assays evaluated peripheral blood mononuclear cell ATP production and glycolytic rate responses. Data were analyzed with diet and challenge fixed effects plus associated interactions (SAS 9.4; P ≤ 0.05). Replicates 1 and 2 showed few immunometabolic responses within 3 dpi with E. maxima, but 25 to 31% increased ATP production and 32% increased compensatory glycolysis at 1 dpi with C. perfringens in challenged vs. unchallenged chicks (P ≤ 0.04). In replicate 3, total ATP production and compensatory glycolysis were increased 25 and 40%, respectively, by the E. maxima main effect at 1dpi (P ≤ 0.05) with unobserved responsiveness to C. perfringens. These outcomes indicate that model type had greater impacts on systemic immunity than anti-IL-10.

Clinical and inflammatory response to antiviral treatments in dogs with parvoviral enteritis.

BACKGROUND: Canine parvoviral enteritis (CPE) is a fatal disease worldwide. The treatment of CPE is based mainly on supportive and symptomatic treatment. Antiviral addition to the treatment may result in a higher survival. OBJECTIVES: This study evaluated the effects of antiviral treatments with a standardized treatment (ST) on the clinical and inflammatory response of dogs with naturally occurring CPE. METHODS: Twenty-eight dogs with CPE caused by canine parvovirus type 2 were divided randomly into treatment groups. The ST group received fluid, antibiotic, antiemetic, and deworming treatments. The antiviral treatment groups received the same ST with an additional antiviral drug, recombinant feline interferon omega (rFeIFN-ω), oseltamivir (OSEL) or famciclovir (FAM). RESULTS: Compared to the healthy control, the tumor necrosis factor-α, interleukin-1ß, interferon (IFN)-α, IFN-γ, haptoglobin, and C-reactive protein values were high (p < 0.05) on day zero. At presentation, mild lymphopenia, neutropenia, and a high neutrophil to lymphocyte (LYM) ratio (NLR) were also observed. Adding rFeIFN-ω to the ST produced the best improvement in the clinical score with a decreased NLR, while leucocytes remained low and inflammatory markers stayed high on day three. The survival rates of the groups were 85.7% in ST+IFN, 71.4% in ST+OSEL, 71.4% in ST+FAM, and 57.1% in ST groups on day seven. CONCLUSIONS: Antiviral drugs may be valuable in treating CPE to improve the clinical signs and survival. In addition, the decrease in NLR in favor of LYM may be an indicator of the early prognosis before the improvement of leukocytes, cytokines, and acute phase proteins in CPE.

Elucidating the Varying Impact of Necrotic Enteritis Using Performance and Health Indicators in Broiler Infection Models.

Necrotic enteritis (NE) continues to be a significant burden to the poultry industry, compounded by pressure to reduce antibiotic use. Researchers use NE-challenge models to study the infection biology of NE and as screening tools to develop potential novel interventions. Currently, data are limited comparing such models between research establishments, and few indicate which quantitative metrics provide the most accurate measure for determining the efficacy of interventions. We compared data from 10 independent NE-challenge trials incorporating six challenge models employed in four geographical regions to determine the extent of variability in bird responses and to determine, using principal component analysis (PCA), which variables discriminated most effectively between nonchallenged control (NC) and challenged control (CC) groups. Response variables related to growth performance (weight gain, feed intake, feed conversion), health (mortality, lesion scores, NE induction rate), and, in three trials only, gut integrity (tight junction protein claudin-1, claudin-2, and zonula occludens-1 expression, coccidia counts, and intestinal permeability [assessed by FITC-dextran assay]). Treatments included a CC, which varied between trials (for example, in Eimeria predisposition, Clostridium perfringens strain, and days of inoculation), and a NC. The degree of response to challenge in CC birds varied significantly among models and trials. In all trials, lesion scores 1 to 4 days postchallenge were increased in CC vs. NC birds and varied both within and among models (by 0.29-1.17 points and 0.05-2.50 points, respectively). In addition, NE-related mortality at day 28 was increased in CC vs. NC, both within and among models (by 1.79%-4.72% and 0.02%-16.70%, respectively), and final (day 35 or 42) body weight was reduced by 3.9%-14.4% and overall FCR increased by up to 27% across trials (P , 0.05). A PCA on the combined dataset including only performance indicators failed to adequately differentiate NC and CC groups. However, the combination of performance and gut integrity variables and standardization of data by trial and phase achieved greater resolution between groups. This indicated that the inclusion of both types of variables in future NE-challenge studies would enable the generation of more robust predictions about intervention efficacy from different types of infection models. A final PCA based on a subset of key indicator variables, including body weight, feed intake, feed conversion ratio, mortality, and lesion score, achieved a good level of separation between NC and CC status of birds and could, with further research, be a useful supplement to existing approaches for assessing and predicting the NE status of birds in the field.

Determinación del impacto variable de la enteritis necrótica mediante indicadores de rendimiento y salud en modelos de infección de pollos de engorde. La enteritis necrótica (EN) sigue siendo un problema importante para la industria avícola, que se ha agudizado por la presión para reducir el uso de antibióticos. Los investigadores utilizan modelos de desafío de enteritis necrótica para estudiar la biología de la infección de este problema y como herramientas de detección para desarrollar posibles intervenciones novedosas. Actualmente, los datos que comparan dichos modelos entre grupos de investigación son limitados y pocos indican qué métricas cuantitativas proporcionan la medida más precisa para determinar la eficacia de las intervenciones. Comparamos datos de 10 ensayos independientes de desafío para enteritis necrótica que incorporan seis modelos de desafío empleados en cuatro regiones geográficas para determinar el grado de variabilidad en las respuestas de las aves y determinar, utilizando el análisis de componentes principales (PCA), qué variables discriminaron más efectivamente entre el control no desafiado (NC) y grupos de control desafiados (CC). Variables de respuesta relacionadas con el rendimiento del crecimiento (aumento de peso, consumo de alimento, conversión alimenticia), salud (mortalidad, puntuaciones de lesiones, tasa de inducción de enteritis necrótica) y, en sólo tres ensayos, la integridad intestinal (proteína de unión estrecha claudina-1, claudina-2, y expresión de zonula occludens-1, recuentos de coccidias y permeabilidad intestinal [evaluada mediante ensayo FITC-dextrano]). Los tratamientos incluyeron un control desafiado, que fue variable entre los ensayos (por ejemplo, en la predisposición a Eimeria, la cepa de Clostridium perfringens y los días de inoculación) y un control no desafiado. El grado de respuesta al desafío en aves del grupo control desafiado varió significativamente entre modelos y ensayos. En todos los ensayos, las puntuaciones de lesiones de 1 a 4 días después del desafío aumentaron en las aves del grupo control desafiado en comparación con el control no desafiado y variaron tanto dentro como entre los modelos (entre 0.29 y 1.17 puntos y entre 0.05 y 2.50 puntos, respectivamente). Además, la mortalidad relacionada con enteritis necrótica en el día 28 aumentó en el control desafiado en comparación con el control no desafiado, tanto dentro como entre modelos (entre un 1.79% y un 4.72% y entre un 0.02% y un 16.70%, respectivamente), y el peso corporal final (día 35 o 42). se redujo entre un 3.9% y un 14.4% y la conversión alimenticia en general aumentó hasta un 27% en todos los ensayos (P ,0.05). El análisis de componentes principales sobre el conjunto de datos combinado que incluye solo indicadores de desempeño no logró diferenciar adecuadamente los grupos control no desafiado y desafiado. Sin embargo, la combinación de variables de rendimiento e integridad intestinal y la estandarización de los datos por ensayo y fase lograron una mayor resolución entre los grupos. Esto indicó que la inclusión de ambos tipos de variables en futuros estudios de desafío para enteritis necrótica permitiría la generación de predicciones más sólidas sobre la eficacia de la intervención a partir de diferentes tipos de modelos de infección. Un análisis de componentes principales final basado en un subconjunto de variables indicadoras clave, incluido el peso corporal, el consumo de alimento, el índice de conversión alimenticia, la mortalidad y la puntuación de las lesiones, logró un buen nivel de separación entre el estado de las aves no desafiadas y desafiadas y podría, con más investigación, ser un complemento útil a los enfoques existentes para evaluar y predecir el estado de enteritis necrótica de las aves en el campo.

Cryptosporidium-associated enteritis in captive koalas (Phascolarctos cinereus).

Cryptosporidium spp. sporadically infect a range of Australian native mammals including koalas, red kangaroos, eastern grey kangaroos, bilbies and brush tailed possums and can range from asymptomatic to fatal infections. Traditionally considered a disease of the young or immuno-compromised, and resulting in profuse diarrhoea in other species, here we report an atypical clinical syndrome associated with Cryptosporidium in a captive population of koalas. All affected animals were in-contact adults, and demonstrated anorexia, dehydration and abdominal pain in the absence of diarrhoea. Following euthanasia on welfare grounds, Cryptosporidium infection was confirmed postmortem in three of four symptomatic animals via faecal floatation and/or intestinal histopathology, with enteritis also diagnosed in the fourth koala. Further screening of the captive colony found the outbreak had been contained. Based on sequencing the cause of the infection was C. fayeri, but the source was undetermined. In conclusion, Cryptosporidium should be considered as a possible cause of generalised illness in koalas.