An atypical lipoteichoic acid from Clostridium perfringens elicits a broadly cross-reactive and protective immune response

Clostridium perfringens is a leading cause of food-poisoning and causes avian necrotic enteritis, posing a significant problem to both the poultry industry and human health. No effective vaccine against C. perfringens is currently available. Using an antiserum screen of mutants generated from a C. perfringens transposon-mutant library, here we identified an immunoreactive antigen that was lost in a putative glycosyltransferase mutant, suggesting that this antigen is likely a glycoconjugate. Following injection of formalin-fixed whole cells of C. perfringens HN13 (a laboratory strain) and JGS4143 (chicken isolate) intramuscularly into chickens, the HN13-derived antiserum was cross-reactive in immunoblots with all tested 32 field isolates, whereas only 5 of 32 isolates were recognized by JGS4143-derived antiserum. The immunoreactive antigens from both HN13 and JGS4143 were isolated, and structural analysis by MALDI-TOF-MS, GC-MS, and 2D NMR revealed that both were atypical lipoteichoic acids (LTAs) with poly-(β1→4)-ManNAc backbones substituted with phosphoethanolamine. However, although the ManNAc residues in JGS4143 LTA were phosphoethanolamine-modified, a few of these residues were instead modified with phosphoglycerol in the HN13 LTA. The JGS4143 LTA also had a terminal ribose and ManNAc instead of ManN in the core region, suggesting that these differences may contribute to the broadly cross-reactive response elicited by HN13. In a passive-protection chicken experiment, oral challenge with C. perfringens JGS4143 lead to 22% survival, whereas co-gavage with JGS4143 and α-HN13 antiserum resulted in 89% survival. This serum also induced bacterial killing in opsonophagocytosis assays, suggesting that HN13 LTA is an attractive target for future vaccine-development studies.

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Clostridium perfringens – epidemiological importance and diagnostics

Medycyna Weterynaryjna ◽

10.21521/mw.6161 ◽

2019 ◽

Vol 75 (01) ◽

pp. 6161-2019

Author(s):

NINA KOZIEŁ ◽

ELŻBIETA KUKIER ◽

KRZYSZTOF KWIATEK

Keyword(s):

Clostridium Perfringens ◽

Extracellular Enzymes ◽

Food Poisoning ◽

Immunocompromised Host ◽

Necrotic Enteritis ◽

Virulence Determinants ◽

Gastrointestinal Infections ◽

Food Borne ◽

Disease Diagnostics ◽

Enteritis Necroticans

Clostridium perfringens is one of the most widespread anaerobic spore forming bacteria found in the environment. The toxotype A of the species inhabits the gastrointestinal tract of birds and mammals exhibiting pathogenic properties in the immunocompromised host. The virulence determinants of C. perfringens are toxins and extracellular enzymes which cause gas gangrene, enteritis necroticans, food poisoning, and non-food borne gastrointestinal infections in humans. The young animals suffer from enterotoxaemia, dysentery and necrotic enteritis due to the anaerobic spore forming bacilli. This article reviews the epidemiological significance of C. perfringens and its disease diagnostics, taking into account all known to date virulence determinants of the microorganism.

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ROLE OF CLOSTRIDIUM PERFRINGENS IN PATHOGENICITY OF SOME DOMESTIC ANIMALS

JOURNAL OF ADVANCES IN AGRICULTURE ◽

10.24297/jaa.v7i3.6325 ◽

2017 ◽

Vol 7 (3) ◽

pp. 1117-1121 ◽

Cited By ~ 1

Author(s):

Mohammad Reza Mohammadabadi

Keyword(s):

Clostridium Perfringens ◽

Gastrointestinal Disease ◽

Food Poisoning ◽

Small Ruminants ◽

Necrotic Enteritis ◽

Economic Stability ◽

Native Breed ◽

The World ◽

The Tropics

Clostridium perfringens, is an anaerobic, gram-positive, pathogenic and spore-forming bacillus and broadly gave out in our territory. This bacterium has spore formation capability and creating gangrene and gastrointestinal disease, for example food poisoning and necrotic enteritis in human, whilst in other animals, gastrointestinal and enterotoxemic diseases more happening. Prevalence of necrotic enteritis, created by C. perfringens, has been often stated in sheep, chickens and ostrich throughout the world. The most critical problem for epidemiological investigations and vaccines improvement is accurate recognition of C. perfringens variants. Moreover, Small ruminants, especially native breed types, play an important role to the livelihoods of a considerable part of human population in the tropics from socio-economic aspects. Therefore, integrated attempt in terms of management and genetic improvement to enhance production is of crucial importance. Poultry provide humans with companionship, food and fiber in the form of eggs, meat and feathers. Many people love to raise and show chickens and other poultry species at fairs and other poultry shows. Others just love to raise them for backyard pets and for fresh eggs every day. In the last few years, ostrich farming has progressed dramatically and the world ostrich industry has achieved some economic stability. There is considerable scope for improvement in the areas of artificial incubation, chick nutrition, environmental requirements and selective breeding. Hence, the aim of this paper was to study role of Clostridium perfringens in pathogenicity of sheep, broilers and Ostrich. In conclusion, recognition of toxins producing by C. perfringens is very momentous because their toxin types are related to particular gastric and intestinal animal sickness and PCR has become an essential research and diagnostic tool, being a powerful technique with a vast and increasing range of applications. Hence, it is better that animal breeders identify different types of C. perfringens using PCR technique to prevent the damage caused by this bacterium.

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A Live Oral Recombinant Salmonella enterica Serovar Typhimurium Vaccine Expressing Clostridium perfringens Antigens Confers Protection against Necrotic Enteritis in Broiler Chickens

Clinical and Vaccine Immunology ◽

10.1128/cvi.00406-09 ◽

2009 ◽

Vol 17 (2) ◽

pp. 205-214 ◽

Cited By ~ 39

Author(s):

R. R. Kulkarni ◽

V. R. Parreira ◽

Y.-F. Jiang ◽

J. F. Prescott

Keyword(s):

B Cell ◽

Clostridium Perfringens ◽

Salmonella Enterica ◽

Broiler Chickens ◽

Hypothetical Protein ◽

Effective Vaccine ◽

Necrotic Enteritis ◽

B Cell Epitopes ◽

Serovar Typhimurium ◽

Resultant Plasmid

ABSTRACT Necrotic enteritis (NE) in broiler chickens is caused by Clostridium perfringens, and there is currently no effective vaccine for NE. We previously showed that in broiler chickens protection against NE can be achieved through intramuscular immunization with alpha toxin (AT) and hypothetical protein (HP), and we subsequently identified B-cell epitopes in HP. In the present study, we identified B-cell epitopes in AT recognized by chickens immune to NE. The gene fragments encoding immunodominant epitopes of AT as well as those of HP were codon optimized for Salmonella and cloned into pYA3493, and the resultant plasmid constructs were introduced into an attenuated Salmonella enterica serovar Typhimurium χ9352 vaccine vehicle. The expression of these C lostridium perfringens proteins, alpha toxoid (ATd) and truncated HP (HPt), was confirmed by immunoblotting. The protection of broiler chickens against experimentally induced NE was assessed at both the moderate and the severe levels of challenge. Birds immunized orally with Salmonella expressing ATd were significantly protected against moderate NE, and there was a nonsignificant trend for protection against severe challenge, whereas HPt-immunized birds were significantly protected against both severities of challenge. Immunized birds developed serum IgY and mucosal IgA and IgY antibody responses against Clostridium and Salmonella antigens. In conclusion, this study identified, for the first time, the B-cell epitopes in AT from an NE isolate recognized by chickens and showed the partial protective ability of codon-optimized ATd and HPt against NE in broiler chickens when they were delivered orally by using a Salmonella vaccine vehicle.

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Antibacterial activity of Bacillus species-derived surfactin on Brachyspira hyodysenteriae and Clostridium perfringens

AMB Express ◽

10.1186/s13568-019-0914-2 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 4

Author(s):

Yi-Bing Horng ◽

Yu-Hsiang Yu ◽

Andrzej Dybus ◽

Felix Shih-Hsiang Hsiao ◽

Yeong-Hsiang Cheng

Keyword(s):

Antibacterial Activity ◽

Clostridium Perfringens ◽

Animal Husbandry ◽

Feed Additives ◽

Bacillus Species ◽

Necrotic Enteritis ◽

Growth Promoters ◽

Bacterial Killing ◽

Brachyspira Hyodysenteriae ◽

Potential Alternative

AbstractSwine dysentery and necrotic enteritis are a bane to animal husbandry worldwide. Some countries have already banned the use of antibiotics as growth promoters in animal production. Surfactin is a potential alternative to antibiotics and antibacterial agents. However, the antibacterial activity of Bacillus species-derived surfactin on Brachyspira hyodysenteriae and Clostridium perfringens are still poorly understood. In the current study, the antibacterial effects of surfactin produced from Bacillus subtilis and Bacillus licheniformis on B. hyodysenteriae and C. perfringens were evaluated. Results showed that multiple surfactin isoforms were detected in B. subtilis, while only one surfactin isoform was detected in B. licheniformis fermented products. The surfactin produced from B. subtilis exhibited significant antibacterial activity against B. hyodysenteriae compared with surfactin produced from B. licheniformis. B. subtilis-derived surfactin could inhibit bacterial growth and disrupt the morphology of B. hyodysenteriae. Furthermore, the surfactin produced from B. subtilis have the highest activity against C. perfringens growth. In contrast, B. licheniformis fermented product-derived surfactin had a strong bacterial killing activity against C. perfringens compared with surfactin produced from B. subtilis. These results together suggest that Bacillus species-derived surfactin have potential for development as feed additives and use as a possible substitute for antibiotics to prevent B. hyodysenteriae and C. perfringens-associated disease in the animal industry.

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Avian Macrophage Responses to Virulent and Avirulent Clostridium perfringens

Pathogens ◽

10.3390/pathogens11010100 ◽

2022 ◽

Vol 11 (1) ◽

pp. 100

Author(s):

Raveendra R. Kulkarni ◽

Carissa Gaghan ◽

Javid Mohammed

Keyword(s):

Nitric Oxide ◽

Clostridium Perfringens ◽

Mononuclear Cells ◽

Surface Expression ◽

No Production ◽

Necrotic Enteritis ◽

Whole Cells ◽

Mhc Ii ◽

Cecal Tonsil ◽

Secretory Products

The present study evaluated the avian macrophage responses against Clostridium perfringens that varied in their ability to cause necrotic enteritis in chickens. Strains CP5 (avirulent-netB+), CP1 (virulent-netB+), and CP26 (highly virulent-netB+tpeL+) were used to evaluate their effect on macrophages (MQ-NCSU cells) and primary splenic and cecal tonsil mononuclear cells. The bacilli (whole cells) or their secretory products from all three strains induced a significant increase in the macrophage transcription of Toll-like receptor (TLR)21, TLR2, interleukin (IL)-1β, inducible nitric oxide synthase (iNOS), and CD80 genes as well as their nitric oxide (NO) production and major histocompatibility complex (MHC)-II surface expression compared to an unstimulated control. The CP1 and CP26-induced expression of interferon (IFN)γ, IL-6, CD40 genes, MHC-II upregulation, and NO production was significantly higher than that of CP5 and control groups. Furthermore, splenocytes and cecal tonsillocytes stimulated with bacilli or secretory products from all the strains showed a significant increase in the frequency of macrophages, their surface expression of MHC-II and NO production, while CP26-induced responses were significantly higher for the rest of the groups. In summary, macrophage interaction with C. perfringens can lead to cellular activation and, the ability of this pathogen to induce macrophage responses may depend on its level of virulence.

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A rapid qualitative assay for detection of Clostridium perfringens in canned food products

Acta Biochimica Polonica ◽

10.18388/abp.2015_1169 ◽

2017 ◽

Vol 64 (2) ◽

Cited By ~ 1

Author(s):

Gayatri Ashwinkumar Dave

Keyword(s):

Phospholipase C ◽

Clostridium Perfringens ◽

Colorimetric Assay ◽

Food Poisoning ◽

The United States ◽

Necrotic Enteritis ◽

Yellow Colour ◽

Negative Results ◽

Reporter Assays ◽

Food Borne Infections

Clostridium perfringens (MTCC 1349) is a Gram-positive, anaerobic, endospore forming, and rod-shaped bacterium. This bacterium produces a variety of toxins under strict anaerobic environment. C. perfringens can grow at temperatures ranging between 20°C and 50°C. It is the major causetive agent for gas gangrene, cellulitis, septicemia, necrotic enteritis and food poisoning, which are common toxin induced conditions noted in human and animals. C. perfringens can produce produce four major types of toxins that are used for the classification of strains, classified under type A–E. Across the globe many countries, including the United States, are affected by C. perfringens food poisonings where it is ranked as one of the most common causes of food borne infections. To date, no direct one step assay for the detection of C. perfringens has been developed and only few methods are known for accurate detection of C. perfringens. Long detection and incubation time is the major consideration of these reporter assays. The prensent study proposes a rapid and reliable colorimetric assay for the detection of C. perfringens. In principale, this assay detects the para nitrophenyl (yellow colour end product) liberated due to the hydrolysis of paranitrophenyl phosphetidyl choline (PNPC) through phospholipase C (lecithinase). Constitutive secretion of phospholipase C is a charactristic feature of C. perfringens. This assay detects the presence of the extracellular lecithinse through the PNPC impragnated impregnated probe. The probe is impregnated with peranitrophenyl phosphotidyl choline ester, which is colourless substrate used by lecithinase. The designed assay is specific towards PNPC and detectes very small quantites of lecithinase under conditions used. The reaction is substrate specific, no cross reaction was observed upon incubation with other substrates. In addition, this assay gave negative results with other clostridium strains, no cross reactions were observed with other experimental strains like C. tetani, C. botulinum, C. acetobutyricum, Bacillus subtilis, and Escherichia coli. This assay is extramly rapid and provides reliable and reproducible results within one hour of incubation at 37°C.

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A Burkholderia pseudomallei Outer Membrane Vesicle Vaccine Provides Protection against Lethal Sepsis

Clinical and Vaccine Immunology ◽

10.1128/cvi.00119-14 ◽

2014 ◽

Vol 21 (5) ◽

pp. 747-754 ◽

Cited By ~ 50

Author(s):

Wildaliz Nieves ◽

Hailey Petersen ◽

Barbara M. Judy ◽

Carla A. Blumentritt ◽

Kasi Russell-Lodrigue ◽

...

Keyword(s):

Outer Membrane ◽

Burkholderia Pseudomallei ◽

Vaccine Development ◽

Outer Membrane Vesicles ◽

Effective Vaccine ◽

High Morbidity ◽

Bacterial Killing ◽

Gram Negative ◽

Content Type ◽

Lethal Sepsis

ABSTRACTThe environmental Gram-negative encapsulated bacillusBurkholderia pseudomalleiis the causative agent of melioidosis, a disease associated with high morbidity and mortality rates in areas of Southeast Asia and northern Australia in which the disease is endemic.B. pseudomalleiis also classified as a tier I select agent due to the high level of lethality of the bacterium and its innate resistance to antibiotics, as well as the lack of an effective vaccine. Gram-negative bacteria, includingB. pseudomallei, secrete outer membrane vesicles (OMVs) which are enriched with multiple protein, lipid, and polysaccharide antigens. Previously, we demonstrated that immunization with multivalentB. pseudomallei-derived OMVs protects highly susceptible BALB/c mice against an otherwise lethal aerosol challenge. In this work, we evaluated the protective efficacy of OMV immunization against intraperitoneal challenge with a heterologous strain because systemic infection with phenotypically diverse environmentalB. pseudomalleistrains poses another hazard and a challenge to vaccine development. We demonstrated thatB. pseudomalleiOMVs derived from strain 1026b afforded significant protection against septicemic infection withB. pseudomalleistrain K96243. OMV immunization induced robust OMV-, lipopolysaccharide-, and capsular polysaccharide-specific serum IgG (IgG1, IgG2a, and IgG3) and IgM antibody responses. OMV-immune serum promoted bacterial killingin vitro, and passive transfer ofB. pseudomalleiOMV immune sera protected naive mice against a subsequent challenge. These results indicate that OMV immunization provides antibody-mediated protection against acute, rapidly lethal sepsis in mice.B. pseudomallei-derived OMVs may represent an efficacious multivalent vaccine strategy against melioidosis.

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Comparative Genome Analysis and Spore Heat Resistance Assay Reveal a New Component to Population Structure and Genome Epidemiology Within Clostridium perfringens Enterotoxin-Carrying Isolates

Frontiers in Microbiology ◽

10.3389/fmicb.2021.717176 ◽

2021 ◽

Vol 12 ◽

Author(s):

Kaisa Jaakkola ◽

Kira Virtanen ◽

Päivi Lahti ◽

Riikka Keto-Timonen ◽

Miia Lindström ◽

...

Keyword(s):

Population Structure ◽

Heat Resistance ◽

Clostridium Perfringens ◽

Genome Analysis ◽

Food Poisoning ◽

Genetic Lineage ◽

Necrotic Enteritis ◽

Genetic Lineages ◽

Pan Genome ◽

Antibiotic Associated Diarrhea

Clostridium perfringens causes a variety of human and animal enteric diseases including food poisoning, antibiotic-associated diarrhea, and necrotic enteritis. Yet, the reservoirs of enteropathogenic enterotoxin-producing strains remain unknown. We conducted a genomic comparison of 290 strains and a heat resistance phenotyping of 30 C. perfringens strains to elucidate the population structure and ecology of this pathogen. C. perfringens genomes shared a conserved genetic backbone with more than half of the genes of an average genome conserved in >95% of strains. The cpe-carrying isolates were found to share genetic context: the cpe-carrying plasmids had different distribution patterns within the genetic lineages and the estimated pan genome of cpe-carrying isolates had a larger core genome and a smaller accessory genome compared to that of 290 strains. We characterize cpe-negative strains related to chromosomal cpe-carrying strains elucidating the origin of these strains and disclose two distinct groups of chromosomal cpe-carrying strains with different virulence characteristics, spore heat resistance properties, and, presumably, ecological niche. Finally, an antibiotic-associated diarrhea isolate carrying two copies of the enterotoxin cpe gene and the associated genetic lineage with the potential for the emergence of similar strains are outlined. With C. perfringens as an example, implications of input genome quality for pan genome analysis are discussed. Our study furthers the understanding of genome epidemiology and population structure of enteropathogenic C. perfringens and brings new insight into this important pathogen and its reservoirs.

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Multivalent poultry vaccine development using Protein Glycan Coupling Technology

Microbial Cell Factories ◽

10.1186/s12934-021-01682-4 ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Marta Mauri ◽

Thippeswamy H. Sannasiddappa ◽

Prerna Vohra ◽

Ricardo Corona-Torres ◽

Alexander A. Smith ◽

...

Keyword(s):

Vaccine Development ◽

Low Cost ◽

Food Poisoning ◽

Live Vaccine ◽

Protein Glycosylation ◽

Multiple Infections ◽

Necrotic Enteritis ◽

Bacterial Cells ◽

Track Record ◽

Poultry Vaccine

Abstract Background Poultry is the world's most popular animal-based food and global production has tripled in the past 20 years alone. Low-cost vaccines that can be combined to protect poultry against multiple infections are a current global imperative. Glycoconjugate vaccines, which consist of an immunogenic protein covalently coupled to glycan antigens of the targeted pathogen, have a proven track record in human vaccinology, but have yet to be used for livestock due to prohibitively high manufacturing costs. To overcome this, we use Protein Glycan Coupling Technology (PGCT), which enables the production of glycoconjugates in bacterial cells at considerably reduced costs, to generate a candidate glycan-based live vaccine intended to simultaneously protect against Campylobacter jejuni, avian pathogenic Escherichia coli (APEC) and Clostridium perfringens. Campylobacter is the most common cause of food poisoning, whereas colibacillosis and necrotic enteritis are widespread and devastating infectious diseases in poultry. Results We demonstrate the functional transfer of C. jejuni protein glycosylation (pgl) locus into the genome of APEC χ7122 serotype O78:H9. The integration caused mild attenuation of the χ7122 strain following oral inoculation of chickens without impairing its ability to colonise the respiratory tract. We exploit the χ7122 pgl integrant as bacterial vectors delivering a glycoprotein decorated with the C. jejuni heptasaccharide glycan antigen. To this end we engineered χ7122 pgl to express glycosylated NetB toxoid from C. perfringens and tested its ability to reduce caecal colonisation of chickens by C. jejuni and protect against intra-air sac challenge with the homologous APEC strain. Conclusions We generated a candidate glycan-based multivalent live vaccine with the potential to induce protection against key avian and zoonotic pathogens (C. jejuni, APEC, C. perfringens). The live vaccine failed to significantly reduce Campylobacter colonisation under the conditions tested but was protective against homologous APEC challenge. Nevertheless, we present a strategy towards the production of low-cost “live-attenuated multivalent vaccine factories” with the ability to express glycoconjugates in poultry.

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Supplementing Garlic Nanohydrogel Optimized Growth, Gastrointestinal Integrity and Economics and Ameliorated Necrotic Enteritis in Broiler Chickens Using a Clostridium perfringens Challenge Model

Animals ◽

10.3390/ani11072027 ◽

2021 ◽

Vol 11 (7) ◽

pp. 2027

Author(s):

Doaa Ibrahim ◽

Tamer Ahmed Ismail ◽

Eman Khalifa ◽

Shaimaa A. Abd El-Kader ◽

Dalia Ibrahim Mohamed ◽

...

Keyword(s):

Growth Performance ◽

Clostridium Perfringens ◽

Broiler Chickens ◽

Body Weight Gain ◽

Economic Benefits ◽

Production Costs ◽

Broiler Chicks ◽

Necrotic Enteritis ◽

Feed Conversion ◽

Intestinal Lesion

Necrotic enteritis (NE) caused by Clostridium perfringens (C. perfringens) results in impaired bird growth performance and increased production costs. Nanotechnology application in the poultry industry to control NE outbreaks is still not completely clarified. Therefore, the efficacy of dietary garlic nano-hydrogel (G-NHG) on broilers growth performance, intestinal integrity, economic returns and its potency to alleviate C. perfringens levels using NE challenge model were addressed. A total of 1200 male broiler chicks (Ross 308) were assigned into six groups; four supplemented with 100, 200, 300 or 400 mg of G-NHG/kg diet and co-challenged with C. perfringens at 21, 22 and 23 d of age and two control groups fed basal diet with or without C. perfringens challenge. Over the total growing period, the 400 mg/kg G-NHG group had the most improved body weight gain and feed conversion efficiency regardless of challenge. Parallel with these results, the mRNA expression of genes encoding digestive enzymes (alpha 2A amylase (AMY2A), pancreatic lipase (PNLIP) and cholecystokinin (CCK)) and intestinal barriers (junctional adhesion molecule-2 (JAM-2), occludin and mucin-2 (Muc-2)) were increased in groups fed G-NHG at higher levels to be nearly similar to those in the unchallenged group. At 14 d post challenge, real-time PCR results revealed that inclusion of G-NHG led to a dose-dependently decrease in the C. perfringens population, thereby decreasing the birds’ intestinal lesion score and mortality rates. Using 400 mg/kg of G-NHG remarkably ameliorated the adverse effects of NE caused by C. perfringens challenge, which contributed to better growth performance of challenged birds with rational economic benefits.

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