Explore the vast range of titles available.

Clostridium perfringens, a prevalent Gram-positive bacterium, causes necrotic diseases associated with abundant life loss and economic burdens of billions of USD. The mechanism of C. perfringens-induced necrotic diseases remains largely unknown, in part, because of the lack of effective animal models and the presence of a large array of exotoxins and diverse disease manifestations from the skin and deep tissues to the gastrointestinal tract. In the light of the advancement of medical and veterinary research, a large body of knowledge is accumulating on the factors influencing C. perfringens-induced necrotic disease onset, development, and outcomes. Here, we present an overview of the key virulence factors of C. perfringens exotoxins. Subsequently, we focus on comprehensively reviewing C. perfringens-induced necrotic diseases such as myonecrosis, acute watery diarrhea, enteritis necroticans, preterm infant necrotizing enterocolitis, and chicken necrotic enteritis. We then review the current understanding on the mechanisms of myonecrosis and enteritis in relation to the immune system and intestinal microbiome. Based on these discussions, we then review current preventions and treatments of the necrotic diseases and propose potential new intervention options. The purpose of this review is to provide an updated and comprehensive knowledge on the role of the host–microbe interaction to develop new interventions against C. perfringens-induced necrotic diseases.

Campylobacter jejuni is a prevalent foodborne pathogen mainly transmitting through poultry. It remains unknown how chicken-transmitted C. jejuni and microbiota impact on human campylobacteriosis. Campylobacter jejuni AR101 (Cj-P0) was introduced to chickens and isolated as passage 1 (Cj-P1). Campylobacter jejuni Cj-P1-DCA-Anaero was isolated from Cj-P0-infected birds transplanted with DCA-modulated anaerobic microbiota. Specific pathogen free Il10 −/− mice were gavaged with antibiotic clindamycin and then infected with Cj-P0, Cj-P1, or Cj-P1-DCA-Anaero, respectively. After 8 days post infection, Il10 −/− mice infected with Cj-P1 demonstrated severe morbidity and bloody diarrhea and the experiment had to be terminated. Cj-P1 induced more severe histopathology compared to Cj-P0, suggesting that chicken transmission increased C. jejuni virulence. Importantly, mice infected with Cj-P1-DCA-Anaero showed attenuation of intestinal inflammation compared to Cj-P1. At the cellular level, Cj-P1 induced more C. jejuni invasion and neutrophil infiltration into the Il10 −/− mouse colon tissue compared to Cj-P0, which was attenuated with Cj-P1-DCA-Anaero. At the molecular level, Cj-P1 induced elevated inflammatory mediator mRNA accumulation of Il17a , Il1β , and Cxcl1 in the colon compared to Cj-P0, while Cj-P1-DCA-Anaero showed reduction of the inflammatory gene expression. In conclusion, our data suggest that DCA-modulated anaerobes attenuate chicken-transmitted campylobacteriosis in mice and it is important to control the elevation of C. jejuni virulence during chicken transmission process.

Clostridium perfringens is a versatile pathogen, inducing diseases in the skin, intestine (such as chicken necrotic enteritis (NE)), and other organs. The classical sign of NE is the foul smell gas in the ballooned small intestine. We hypothesized that deoxycholic acid (DCA) reduced NE by inhibiting C. perfringens virulence signaling pathways. To evaluate the hypothesis, C. perfringens strains CP1 and wild-type (WT) HN13 and its mutants were cultured with different bile acids, including DCA and isoallolithocholic acid (isoalloLCA). Growth, hydrogen sulfide (H2S) production, and virulence gene expression were measured. Notably, isoalloLCA was more potent in reducing growth, H2S production, and virulence gene expression in CP1 and WT HN13 compared to DCA, while other bile acids were less potent compared to DCA. Interestingly, there was a slightly different impact between DCA and isoalloLCA on the growth, H2S production, and virulence gene expression in the three HN13 mutants, suggesting possibly different signaling pathways modulated by the two bile acids. In conclusion, DCA and isoalloLCA reduced C. perfringens virulence by transcriptionally modulating the pathogen signaling pathways. The findings could be used to design new strategies to prevent and treat C. perfringens-induced diseases.

Clostridium perfringens is the prevalent enteric pathogen in humans and animals including chickens, and it remains largely elusive on the mechanism of C. perfringens-induced enteritis because of limited animal models available. In this study, we investigated the role of C. perfringens sporulation proteins as vaccine candidates in chickens to reduce necrotic enteritis (NE). C. perfringens soluble proteins of vegetative cells (CP-super1 and CP-super2) and spores (CP-spor-super1 and CP-spor-super2) were prepared, and cell and chicken experiments were conducted. We found that deoxycholic acid reduced C. perfringens invasion and sporulation using the Eimeria maxima and C. perfringens co-infection necrotic enteritis (NE) model. C. perfringens enterotoxin (CPE) was detected in the CP-spor-super1&2. CP-spor-super1 or 2 induced cell death in mouse epithelial CMT-93 and macrophage Raw 264.7 cells. CP-spor-super1 or 2 also induced inflammatory gene expression and necrosis in the Raw cells. Birds immunized with CP-spor-super1 or 2 were resistant to C. perfringens-induced severe clinical NE on histopathology and body weight gain loss. CP-spor-super1 vaccine reduced NE-induced proinflammatory Ifnγ gene expression as well as C. perfringens luminal colonization and tissue invasion in the small intestine. Together, this study showed that CP-spor-super vaccines reduced NE histopathology and productivity loss.

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.

Necrotic enteritis (NE), mainly induced by the pathogens of Clostridium perfringens and coccidia, causes huge economic losses with limited intervention options in the poultry industry. This study investigated the role of specific bile acids on NE development. Day-old broiler chicks were assigned to six groups: noninfected, NE, and NE with four bile diets of 0.32% chicken bile, 0.15% commercial ox bile, 0.15% lithocholic acid (LCA), or 0.15% deoxycholic acid (DCA). The birds were infected with Eimeria maxima at day 18 and C. perfringens at day 23 and 24. The infected birds developed clinical NE signs. The NE birds suffered severe ileitis with villus blunting, crypt hyperplasia, epithelial line disintegration, and massive immune cell infiltration, while DCA and LCA prevented the ileitis histopathology. NE induced severe body weight gain (BWG) loss, while only DCA prevented NE-induced BWG loss. Notably, DCA reduced the NE-induced inflammatory response and the colonization and invasion of C. perfringens compared to NE birds. Consistently, NE reduced the total bile acids in the ileal digesta, while dietary DCA and commercial bile restored it. Together, this study showed that DCA and LCA reduced NE histopathology, suggesting that secondary bile acids, but not total bile acid levels, play an essential role in controlling the enteritis.

Campylobacter jejuni, a prevalent foodborne bacterial pathogen, is mainly transmitted from poultry with few effective prevention approaches. In this study, we aimed to investigate the role of microbiota on C. jejuni chicken colonization. Microbiota from specific pathogen-free (SPF) mouse stools were collected as SPF-Aerobe and SPF-Anaerobe. Birds were colonized with SPF-Aerobe or SPF-Anaerobe at day 0 and infected with C. jejuni AR101 at day 12. Notably, C. jejuni AR101 colonized at 5.3 and 5.6 log10 C. jejuni CFU/g chicken cecal digesta at days 21 and 28, respectively, while both SPF-Aerobe and SPF-Anaerobe microbiota reduced pathogen colonization. Notably, SPF-Aerobe and SPF-Anaerobe increased cecal phylum Bacteroidetes and reduced phylum Firmicutes compared to those in the nontransplanted birds. Interestingly, microbiota from noninfected chickens, SPF-Aerobe, or SPF-Anaerobe inhibited AR101 in vitro growth, whereas microbiota from infected birds alone failed to reduce pathogen growth. The bacterium Enterobacter102 isolated from infected birds transplanted with SPF-Aerobe inhibited AR101 in vitro growth and reduced pathogen gut colonization in chickens. Together, SPF mouse microbiota was able to colonize chicken gut and reduce C. jejuni chicken colonization. The findings may help the development of effective strategies to reduce C. jejuni chicken contamination and campylobacteriosis.

Clostridium perfringens is a predominant intestinal pathogen affecting both humans and animals, including chickens resulting in significant economic losses. In chapter I, I have overviewed the dynamics of the intestinal immune system, the composition of the intestinal microbiome, and how the microbiome influences inflammation in the development of Clostridium infection. The intricate interplay between immunity, microbiota, and their metabolites is likely critical in the pathogenesis of C. perfringens and uncovering these interactions may unveil new avenues for prevention and treatment. The objective of this chapter is to provide updated insights into the role of host-microbe interactions and their potential therapeutic applications in addressing C. perfringens infection.In chapter II, I focused on investigating one of the prominent Clostridium infections of chicken necrotic enteritis (NE), mainly caused by C. perfringens. The classical sign of NE is the foul smell gas in the ballooned small intestine. We hypothesized that deoxycholic acid (DCA) reduces NE by inhibiting C. perfringens virulence signaling pathways. To evaluate the hypothesis, C. perfringens strains CP1 and wild-type (WT) HN13 and its mutants were cultured with different bile acids, including DCA and isoallolithocholic acid (isoalloLCA). Growth, hydrogen sulfide (H2S) production, and virulence gene expression were measured. Notably, isoalloLCA was more potent in reducing growth, H2S production, and virulence gene expression in CP1 and WT HN13 compared to DCA, while other bile acids were less potent compared to DCA. Interestingly, there was a slightly different impact between DCA and isoalloLCA on the growth, H2S production, and virulence gene expression in the three HN13 mutants, suggesting possibly different signaling pathways modulated by the two bile acids.In chapter III, I investigated the effect of deconjugating taurodeoxycholic acids (TDCA) on reducing C. perfringens virulence. Although dietary secondary bile acid DCA reduces chicken NE, the accumulation of conjugated TDCA raised the concerns of its dietary efficacy. In this study, we aimed to deconjugate TDCA by recombinant bile salt hydrolase (BSH) to increase DCA efficacy against 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 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 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 Western Blot assays. Secretory BSH from B. subtilis was analyzed by 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 virulence gene expression of asrA1, netB, colA, and virT. BSH activity was observed in L. jonsonii and B. longum under anaerobic conditions but not L. jonsonii under 10% CO2. After 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 subcloned bsh and amylase signal peptide sequence 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, despite the urgent need for antimicrobial free alternatives in agricultural and healthcare industries, few interventions are available. The findings from my research could be used to design new strategies to prevent and treat C. perfringens-induced enteritis or other diseases.

Clostridium perfringens is a versatile pathogen, inducing diseases in the skin, intestine (such as chicken necrotic enteritis (NE)), and other organs. The classical sign of NE is the foul smell gas in the ballooned small intestine. We hypothesized that deoxycholic acid (DCA) reduced NE by inhibiting C. perfringens virulence signaling pathways. To evaluate the hypothesis, C. perfringens strains CP1 and wild-type (WT) HN13 and its mutants were cultured with different bile acids, including DCA and isoallolithocholic acid (isoalloLCA). Growth, hydrogen sulfide (H[sub.2] S) production, and virulence gene expression were measured. Notably, isoalloLCA was more potent in reducing growth, H[sub.2] S production, and virulence gene expression in CP1 and WT HN13 compared to DCA, while other bile acids were less potent compared to DCA. Interestingly, there was a slightly different impact between DCA and isoalloLCA on the growth, H[sub.2] S production, and virulence gene expression in the three HN13 mutants, suggesting possibly different signaling pathways modulated by the two bile acids. In conclusion, DCA and isoalloLCA reduced C. perfringens virulence by transcriptionally modulating the pathogen signaling pathways. The findings could be used to design new strategies to prevent and treat C. perfringens-induced diseases.

The Comniphoramohnol (myrrha) has been used as a traditional medicine for centuries in different cultures. An ethanol extract of myrrha was evaporated under vacuum to obtain an oil. A 20% solution of this oil in ethanol was used to determine antimicrobial activity against the Gram-positive bacteria S. aureus, the Gram-negative bacteria, E. coil, MV 10Na1, and fungi, (yeast), Saccharomyces cerevisiae. The MIC for E. coil and S. aureus were determined on phosphate buffer since the oil did not show antibiotic activity on growing cells. The MIC of myrrha oil in phosphate buffer for E. coil was 0.56% (5.6 mg/ml) and for S. aureus was 0.1% (1 Ong/ml). However, the oil could be used to kill cells in a nutrient-rich medium provided growth of the bacteria is first stopped using a bacteriostatic antibiotic such as Chloramphenicol. The results show that chloramphenicol enhanced the antimicrobial activity of myrrha oil. Zone of inhibition test shows myrrha extract has antibacterial property against S. aureus. Furthermore, the antifungal activity of myrrha extract was prodigious since most of the cells were killed in 10 minutes at a dose of 0.05% (0.5 mg/ml) of myrrha extract. Repeated attempts to obtain an E. coil or S. aureus strain that is resistant to myrrha oil were unsuccessful. A possible explanation of this can be that myrrha oil is a membrane acting antibiotic. In conclusion, the results of this study suggest that myrrha extract could be a promising antibacterial and antifungal drug.