Effect of in-water administration of quorum system inhibitors in broilers’ productive performance and intestinal microbiome in a mild necrotic enteritis challenge

Nigella sativa as an antibiotic alternative to promote growth and enhance health of broilers challenged with Eimeria maxima and Clostridium perfringens

Subverting the host immune system is a major task for any given pathogen to assure its survival and proliferation. For the opportunistic human pathogen Bacillus cereus (Bc), immune evasion enables the establishment of potent infections. In various species of the Bc group, the pleiotropic regulator PlcR and its cognate cell-cell signaling peptide PapR7 regulate virulence gene expression in response to fluctuations in population density, i.e., a quorum-sensing (QS) system. However, how QS exerts its effects during infections and whether PlcR confers the immune evading ability remain unclear. Herein, we report how interception of the QS communication in Bc obliterates the ability to affect the host immune system. Here, we designed a peptide-based QS inhibitor that suppresses PlcR-dependent virulence factor expression and attenuates Bc infectivity in mouse models. We demonstrate that the QS peptidic inhibitor blocks host immune system-mediated eradication by reducing the expression of PlcR-regulated major toxins similarly to the profile that was observed for isogenic strains. Our findings provide evidence that Bc infectivity is regulated by QS circuit-mediated destruction of host immunity, thus reveal a interesting strategy to limit Bc virulence and enhance host defense. This peptidic quorum-quenching agent constitutes a readily accessible chemical tool for studying how other pathogen QS systems modulate host immunity and forms a basis for development of anti-infective therapeutics.

Can dietary zinc diminish the impact of necrotic enteritis on growth performance of broiler chickens by modulating the intestinal immune-system and microbiota?

Clostridium perfringens is the main pathogen of chicken necrotic enteritis (NE) causing huge economic losses in the poultry industry. Although dietary secondary bile acid deoxycholic acid (DCA) reduced chicken NE, the accumulation of conjugated tauro-DCA (TDCA) raised concerns regarding DCA efficacy. In this study, we aimed to deconjugate TDCA by bile salt hydrolase (BSH) to increase DCA efficacy against the NE pathogen C. perfringens. Assays were conducted to evaluate the inhibition of C. perfringens growth, hydrogen sulfide (H2S) production, and virulence gene expression by TDCA and DCA. BSH activity and sequence alignment were conducted to select the bsh gene for cloning. The bsh gene from Bifidobacterium longum was PCR-amplified and cloned into plasmids pET-28a (pET-BSH) and pDR111 (pDR-BSH) for expressing the BSH protein in E. coli BL21 and Bacillus subtilis 168 (B-sub-BSH), respectively. His-tag-purified BSH from BL21 cells was evaluated by SDS-PAGE, Coomassie blue staining, and a Western blot (WB) assays. Secretory BSH from B. subtilis was analyzed by a Dot-Blot. B-sub-BSH was evaluated for the inhibition of C. perfringens growth. C. perfringens growth reached 7.8 log10 CFU/mL after 24 h culture. C. perfringens growth was at 8 vs. 7.4, 7.8 vs. 2.6 and 6 vs. 0 log10 CFU/mL in 0.2, 0.5, and 1 mM TDCA vs. DCA, respectively. Compared to TDCA, DCA reduced C. perfringens H2S production and the virulence gene expression of asrA1, netB, colA, and virT. BSH activity was observed in Lactobacillus johnsonii and B. longum under anaerobe but not L. johnsonii under 10% CO2 air. After the sequence alignment of bsh from ten bacteria, bsh from B. longum was selected, cloned into pET-BSH, and sequenced at 951 bp. After pET-BSH was transformed in BL21, BSH expression was assessed around 35 kDa using Coomassie staining and verified for His-tag using WB. After the subcloned bsh and amylase signal peptide sequence was inserted into pDR-BSH, B. subtilis was transformed and named B-sub-BSH. The transformation was evaluated using PCR with B. subtilis around 3 kb and B-sub-BSH around 5 kb. Secretory BSH expressed from B-sub-BSH was determined for His-tag using Dot-Blot. Importantly, C. perfringens growth was reduced greater than 59% log10 CFU/mL in the B-sub-BSH media precultured with 1 vs. 0 mM TDCA. In conclusion, TDCA was less potent than DCA against C. perfringens virulence, and recombinant secretory BSH from B-sub-BSH reduced C. perfringens growth, suggesting a new potential intervention against the pathogen-induced chicken NE.

Several factors predisposing to necrotic enteritis (NE) have been identified, including diet and Eimeria spp. infestations. Coccidiosis vaccines are indicated to decrease the intestinal lesions caused by specific Eimeria species that are a known predisposing factor to NE and, consequently, these vaccines could be a holistic approach to the control of NE disease and an alternative solution to coccidiostats. Besides, feed additives have also gained special attention from the poultry industry as an alternative solution to antibiotics to prevent NE as well as other bacterial enteritis.Then, the combination of vaccination against coccidiosis and the supplementation of the diet with feed additives could be a composite approach to the control of NE problems triggered by Eimeria spp. infestation. The objective of this study was to test the efficacy of an attenuated coccidiosis vaccine (EVANT) in combination with different feed additives to prevent the loss of production performance and intestinal lesions in broilers challenged with NE.Healthy day-old broilers (n = 960) were randomly allocated to 6 groups (8 cages/group). Groups 1–2 were left unvaccinated. Groups 3–6 were vaccinated following the manufacturer's instructions. Chickens were grown using a diet favoring the intestinal proliferation of Clostridium perfringens. Moreover, the diets of groups 4–6 were supplemented with medium chain fatty acids (MCFA), butyric acid or phytogenic feed additives (PFA), respectively. A NE infection model was used to challenge groups 2–6; chickens were orally infected with Eimeria maxima (4,500 oocysts) and then C. perfringens (108 CFU) at 15 and 20 d, respectively. Birds were monitored and productive parameters recorded until 42 d; intestinal lesions were scored.Results showed that coccidiosis vaccination, with or without the addition of feed additives, decreased intestinal lesions associated with NE and improved the performance of the birds. Besides, the addition of MCFA to the diet decreased intestinal lesions associated to NE in vaccinated animals compared to all treatment groups. Moreover, the same additive improved the feed conversion rate. Therefore, vaccination with a live attenuated coccidiosis vaccine together with in-feed inclusion of MCFA might be a solution to reduce NE in broilers raised antimicrobial- and coccidiostat-free.

Challenge models are needed to understand the pathogenesis of necrotic enteritis (NE) and provide the basis of evaluating nonantibiotic feed-additive interventions. In the category of nonantibiotic feed additives, the application of probiotics to improve intestinal health and growth performance of broiler chickens in the face of an NE challenge has been well described. However, it is crucial to evaluate the consistency of specific probiotics for mitigating the disease challenge and improving performance. Therefore, a meta-analysis of five independent research trials was conducted with the objective of evaluating the effect of Bacillus subtilis DSM 32315 (probiotic) on body weight gain (BWG), feed conversion ratio (FCR), NE mortality, and lesion score (LS) of broiler chickens challenged with NE. These independent studies were conducted in three countries (the United States, Thailand, and Finland). The statistical analysis used fixed and random effects to estimate the mean effect size (MES) of the difference between NE-challenged birds (control) and NE-challenged probiotic-fed birds and the 95% confidence interval of MES. A meta-regression was performed to evaluate heterogeneity (MES variance) among studies. The statistical analysis was performed using a robust variance estimation strategy with a SAS macro. Probiotic-supplemented birds had a significantly higher BWG (MES = 1.04, P = 0.009) and a significantly lower FCR (MES = -1.39, P = 0.020), NE mortality (MES = -1.15, P = 0.012), and LS (MES = -1.29, P = 0.045). Response variables of BWG (Q = 2.81, P = 0.560) and NE mortality (Q = 5.60, P = 0.354) did not present heterogeneity. Heterogeneity was found for FCR (Q = 10.34, P = 0.035) and LS (Q = 16.13, P = 0.001). Overall, dietary supplementation of B. subtilis DSM 32315 significantly improved BWG and reduced FCR, mortality, and LS in a repeatable large-scale manner.

Coccidia-induced mucogenesis promotes the onset of necrotic enteritis by supporting Clostridium perfringens growth

Over the last decades, massive misuse of antibiotics prompted the apparition of resistances in many microorganisms such as ESKAPEE pathogens responsible for various nosocomial infections. Indeed, the selective pressure put on sensitive bacteria by conventional antimicrobial molecules that cause their death promotes resistant strain survival. The development of antivirulence agents that could attenuate bacteria pathogenicity without affecting their growth, seems to be a new promising therapeutic strategy. This could facilitate the host’s defence by immune system and restore the associated efficiency of conventional treatments. The inhibition of quorum sensing (QS) that refers to bacterial communication systems, could disrupt, especially in P. aeruginosa, virulence pathways (synthesis of pyocyanin or rhamnolipids) and intra/inter-species protective interactions (biofilm formation). Among a pool of promising pharmacological targets provided by QS, the interest of Pseudomonas Quinolone Signal receptor (PqsR) that regulates virulence gene expression in response to environmental factors and population density once activated by its natural ligand (PQS), has emerged for the development of inhibitors. Several 2-heptyl-4-quinolone analogues of PQS, recently described in the literature, revealed efficient as PqsR antagonists. Taking these studies into account, we aim to develop a new 2‑heteroaryl‑4‑quinolone family potentially active against ESKAPEE pathogens as QS inhibitors. The synthesis pathways based on pallado-catalyzed coupling reactions, but also the physicochemical and biological evaluations of these compounds (MIC in various ESKAPEE strains and biofilm formation inhibitory properties) will be described in the presentation.

Clostridium perfringens possesses at least two functional quorum sensing (QS) systems, i.e. an Agr-like system and a LuxS-dependent AI-2 system. Both of those QS systems can reportedly control in vitro toxin production by C. perfringens but their importance for virulence has not been evaluated. Therefore, the current study assessed whether these QS systems might regulate the pathogenicity of CN3685, a C. perfringens type C strain. Since type C isolates cause both haemorrhagic necrotic enteritis and fatal enterotoxemias (where toxins produced in the intestines are absorbed into the circulation to target other internal organs), the ability of isogenic agrB or luxS mutants to cause necrotizing enteritis in rabbit small intestinal loops or enterotoxemic lethality in mice was evaluated. Results obtained strongly suggest that the Agr-like QS system, but not the LuxS-dependent AI-2 QS system, is required for CN3685 to cause haemorrhagic necrotizing enteritis, apparently because the Agr-like system regulates the production of beta toxin, which is essential for causing this pathology. The Agr-like system, but not the LuxS-mediated AI-2 system, was also important for CN3685 to cause fatal enterotoxemia. These results provide the first direct evidence supporting a role for any QS system in clostridial infections.

The efficacy of bacitracin methylene disalicylate when fed in combination with narasin in the management of necrotic enteritis in broiler chickens

Effect of a Competitive Exclusion Culture in a Necrotic Enteritis Challenge Model in Broilers

The ban on antibiotics in the poultry diet resulted in re-emergence of several infectious diseases including necrotic enteritis (NE). These infectious diseases are leading to poor health and welfare as well as production and economic loss. Synbiotic could be a potential candidate to replace the antibiotics in poultry diet. Therefore, a 35-day study was conducted to compare the efficacy of synbiotic (PoultryStar®ME) and antibiotic (Stafac®50, Virginiamycin) supplementation during an experimentally induced necrotic enteritis infection. A total of 360 day-old chicks were randomly assigned to four treatment groups: Antibiotic, Challenge + Antibiotic, Synbiotic, and Challenge + Synbiotic, each with 6 replicates. The treatment groups referred as “Challenge + Antibiotic” and “Challenge + Synbiotic” were challenged, while their respective non-challenged treatment groups were “antibiotic” and “synbiotic”. NE in birds was induced by gavaging 1 × 104 oocysts of Eimeria maxima on day 14 (D14) and 1 × 108 CFU/mL of Clostridium perfringens on D19, 20, and 21. Both synbiotic and antibiotic supplementation during the NE challenge did not improve BW gain, feed intake, and feed conversion ratio at the end of the experiment (D0-35). However, antibiotic supplementation reduced mortality during the week of the challenge (D14-21) (P < 0.001). At D21, both synbiotic and antibiotic supplementation during the NE challenge did not decrease the intestinal lesion score (P < 0.001) compared to their respective non-challenged treatment groups. At D21, synbiotic supplementation during the NE challenge did not decrease intestinal permeability (P = 0.04) compared to the synbiotic group. At D21, antibiotic supplementation during the NE challenge increased the CD4+:CD8+ T cells (P < 0.001) in the cecal tonsil. It can be concluded that synbiotic supplementation elicited an immune response, decreasing the inflammatory response in the intestine and ameliorating the NE infection. Therefore, synbiotic could be a potential alternative to replace antibiotics in the poultry industry, but their efficacy needs to be improved through blending additional probiotics and prebiotics, and further exploration is required.

BackgroundThe poultry industry is in need of effective antibiotic alternatives to control outbreaks of necrotic enteritis (NE) due to Clostridium perfringens.MethodsThis study was conducted to investigate the effects of feeding Bacillus coagulans on the growth performance and gut health of broiler chickens with C. perfringens-induced NE. Two hundred and forty 1-day-old broiler chicks were randomly assigned to a 2 × 2 factorial arrangement with two dietary B. coagulans levels (0 or 4 × 109 CFU/kg of diet) and two disease challenge statuses (control or NE challenged).ResultsNE-induced reduction in body weight gain was relieved by the addition of B. coagulans into broiler diets compared with the NE-infected birds. NE infection damaged intestinal morphological structure, promoted intestinal C. perfringens growth and liver invasion, and enhanced anti-C. perfringens specific sIgA concentrations in the gut and specific IgG levels in serum compared with the uninfected birds. NE infection significantly (P < 0.05) decreased mucin-2 (at 14 d post-infection (DPI), toll -like receptor 2 (TLR2, at 7 and 14 DPI), TLR4 (at 7 and 14 DPI), tumor necrosis factor super family 15 (TNFSF15, at 7 and 14 DPI), lysozyme (LYZ, at 14 DPI) and fowlicidin-2 (at 7 and 14 DPI) mRNA levels, whereas it dramatically (P = 0.001) increased IFN-γ mRNA levels at 7 DPI. However, challenged birds fed diets supplemented with B. coagulans showed a significant (P < 0.01) decrease in gut lesion scores, decreased C. perfringens numbers in the cecum and liver, and an increase in fowlicidin-2 mRNA levels in compared with the uninfected birds. In addition, compared with the non-supplemented group, dietary inclusion of B. coagulans improved intestinal barrier structure, further increased specific sIgA levels and alkaline phosphatase (IAP) activity in the jejunum, enhanced the expression of jejunum lysozyme mRNA, and inhibited the growth, colonization, and invasion of C. perfringens; in contrast, it reduced serum-specific IgG concentrations and jejunum IFN-γ mRNA levels.ConclusionThese results indicated that dietary B. coagulans supplementation appeared to be effective in preventing the occurrence and reducing the severity of C. perfringens-induced NE in broiler chickens.

Necrotic enteritis (NE) in poultry is an economically important disease caused by Clostridium perfringens type A bacteria. A global trend on restricting the use of antibiotics as feed supplements in food animal production has caused a spike in the NE incidences in chickens, particularly in broiler populations. Amongst several non-antibiotic strategies for NE control tried so far, probiotics seem to offer promising avenues. The current review focuses on studies that have evaluated probiotic effects on C. perfringens growth and NE development. Several probiotic species, including Lactobacillus, Enterococcus, Bacillus, and Bacteroides bacteria as well as some yeast species have been tested in chickens against C. perfringens and NE development. These findings have shown to improve bird performance, reduce C. perfringens colonization and NE-associated pathology. The underlying probiotic mechanisms of NE control suggest that probiotics can help maintain a healthy gut microbial balance by modifying its composition, improve mucosal integrity by upregulating expression of tight-junction proteins, and modulate immune responses by downregulating expression of inflammatory cytokines. Collectively, these studies indicate that probiotics can offer a promising platform for NE control and that more investigations are needed to study whether these experimental probiotics can effectively prevent NE in commercial poultry operational settings.

Pseudomonas aeruginosa is a major opportunistic pathogen that causes severe acute lung infection. The ability of P. aeruginosa to precisely self-regulate its virulence factors is crucial for enhancing pathogenicity and establishing infection. However, how the bacterium dynamically adjusts virulence gene expression in response to host interaction remains to be elucidated. Here, transcriptome profiling revealed that infection of A549 alveolar epithelial cells significantly upregulated virulence genes associated with the type III secretion system (T3SS) and quorum sensing (QS) in P. aeruginosa PAO1, compared to a standard laboratory growth condition. In contrast, genes of the amiEBCRS operon were markedly downregulated during infection. Notably, AmiL, the leader small RNA (sRNA) of the operon, exhibited consistently reduced expression across multiple P. aeruginosa infection models, including A549 cells, mouse lungs, and clinical isolates. AmiL expression was primarily repressed by succinate-mediated carbon catabolite repression (CCR) during the early stage of infection, and later by the transcription factor ArgR as succinate levels declined. Functional analyses showed AmiL bound to the mRNAs of the T3SS effector gene exoT and the hydrogen cyanide synthesis gene hcnC, repressing their expression and thereby attenuating PAO1 virulence. Furthermore, deletion of amiL gene enhanced the cytotoxicity of PAO1 in A549 cells, Galleria mellonella larvae, and a mouse lung infection model, resulting in more severe tissue damage and increased mortality. Therefore, our findings demonstrate that P. aeruginosa strategically downregulates AmiL in response to carbon source changes to enhance its virulence and pathogenicity during acute lung infection, and highlight a potential target for antibacterial therapy.