In vivo mRNA expression of a multi-mechanistic mAb combination protects against Staphylococcus aureus infection.

Single monoclonal antibodies (mAbs) can be expressed in vivo through gene delivery of their mRNA formulated with lipid nanoparticles (LNPs). However, delivery of a mAb combination could be challenging due to the risk of heavy and light variable chain mispairing. We evaluated the pharmacokinetics of a three mAb combination against Staphylococcus aureus first in single chain variable fragment scFv-Fc and then in immunoglobulin G 1 (IgG1) format in mice. Intravenous delivery of each mRNA/LNP or the trio (1 mg/kg each) induced functional antibody expression after 24 h (10-100 µg/mL) with 64%-78% cognate-chain paired IgG expression after 3 days, and an absence of non-cognate chain pairing for scFv-Fc. We did not observe reduced neutralizing activity for each mAb compared with the level of expression of chain-paired mAbs. Delivery of the trio mRNA protected mice in an S. aureus-induced dermonecrosis model. Intravenous administration of the three mRNA in non-human primates achieved peak serum IgG levels ranging between 2.9 and 13.7 µg/mL with a half-life of 11.8-15.4 days. These results suggest nucleic acid delivery of mAb combinations holds promise and may be a viable option to streamline the development of therapeutic antibodies.

Ampelopsin attenuates Staphylococcus aureus Alpha-Toxin-Induced Lung Injury.

Staphylococcus aureus is a prevalent cause of lung infections in hospitals and communities, and can cause a wide spectrum of human infections. Due to the bottleneck caused by antibiotic resistance and substantial increases in morbidity and mortality, targeting the virulence factors released by S. aureus as an alternative prevention and treatment method has become a promising approach. Ampelopsin, a component of vine tea, has promising potential for treating S. aureus-induced acute lung injury. In this study, the effects of ampelopsin were investigated on a mouse model of acute lung injury established using S. aureus 8325-4 and the α-hemolysin (hla) silent strain DU1090. The hla silent strain did not cause mortality in mice, whereas lethal and sublethal concentrations of S. aureus 8325-4 caused high mortality. Notably, ampelopsin treatment protected against mortality stemming from S. aureus infection. Ampelopsin yielded enhancements in lung barrier function, decreased total protein leakage in the alveolar lavage fluid, and modulated inflammatory signaling pathway-related proteins, thereby reducing the release of pro-inflammatory factors and improving respiratory dysfunction. Moreover, ampelopsin prevented the upregulation of ADAM10 activity, leading to E-cadherin mucin cleavage. In conclusion, our findings establish the key role of alpha -toxin in infectious lung injury in S. aureus and provide support for ampelopsin as an effective therapeutic approach to improve lung injury.

The in vitro effect of lactose on Clostridium perfringens alpha toxin production and the implications of lactose consumption for in vivo anti-alpha toxin antibody production.

Necro-hemorrhagic enteritis in calves, caused by Clostridium perfringens type A, is a fatal disease, mostly affecting calves in intensive rearing systems. The lack of development of active immunity against α toxin, an essential virulence factor in the pathogenesis, has been proposed as a main trigger. In this experimental study, the effect of a set of milk replacer components on α toxin production, and the effect of lactose on in vivo antibody production, were investigated. For the latter, Holstein-Friesian bull calves (n = 18) were fed an all liquid diet that contained either a milk replacer with high-lactose content (45% DM) or the same milk replacer that was lactase treated, resulting in a lactose-free equivalent. Antibody levels against α toxin were monitored from 2 to 12 wk of age. In the in vitro part of the study, a concentration-dependent inhibitory effect of lactose on in vitro C. perfringens α toxin activity was observed, whereas protein did not influence α toxin activity. The in vivo experiment then showed from the age of 10 wk onwards, that anti-α toxin antibody levels of high-lactose animals declined, whereas antibody levels of the animals consuming lactose-free milk replacer remained the same throughout the trial. This points to a natural decline in maternal immunity of lactose-consuming animals, that is not compensated by the development of an active immunity, resulting in inferior protection. This study suggests that dietary lactose reduces C. perfringens α toxin production in vivo, which may lead to a decreased antigen presentation and thus lower serum antibody levels against the toxin. Consequently, any event causing massive α toxin production puts lactose-consuming calves at higher risk of developing necro-hemorrhagic enteritis.

Anti-Alpha-Toxin Antibody Responses and Clinical Outcomes of Staphylococcus aureus Bacteremia.

BACKGROUND: Alpha-toxin (AT), a major virulence factor of Staphylococcus aureus, is an important immunotherapeutic target to prevent or treat invasive S. aureus infections. Previous studies have suggested that anti-AT antibodies (Abs) may have a protective role against S. aureus bacteremia (SAB), but their function remains unclear. Therefore, we aimed to investigate the association between serum anti-AT Ab levels and clinical outcomes of SAB. METHODS: Patients from a prospective SAB cohort at a tertiary-care medical center (n = 51) were enrolled in the study from July 2016 to January 2019. Patients without symptoms or signs of infection were enrolled as controls (n = 100). Blood samples were collected before the onset of SAB and at 2- and 4-weeks post-bacteremia. Anti-AT immunoglobin G (IgG) levels were measured using an enzyme-linked immunosorbent assay. All clinical S. aureus isolates were tested for the presence of hla using polymerase chain reaction. RESULTS: Anti-AT IgG levels in patients with SAB before the onset of bacteremia did not differ significantly from those in non-infectious controls. Pre-bacteremic anti-AT IgG levels tended to be lower in patients with worse clinical outcomes (7-day mortality, persistent bacteremia, metastatic infection, septic shock), although the differences were not statistically significant. Patients who needed intensive care unit care had significantly lower anti-AT IgG levels at 2 weeks post-bacteremia (P = 0.020). CONCLUSION: The study findings suggest that lower anti-AT Ab responses before and during SAB, reflective of immune dysfunction, are associated with more severe clinical presentations of infection.

A bivalent vaccine against avian necrotic enteritis and coccidiosis.

AIMS: In this study, we attempted to design a recombinant vaccine harbouring domain with a key role in enterocyte attachment and cell invasion in necrotic enteritis (NE) and coccidiosis. METHODS AND RESULTS: In this study, we investigated whether a recombinant protein consisting of necrotic enteritis B-like toxin, C-terminal domain of alpha-toxin, apical membrane antigen 1 (AMA1), and Rhoptry neck protein 2 (RON2) which we call "NeCoVac" hereafter, can improve protection against both diseases compared to vaccination with each antigen in previous studies. Birds intestinal lesion scores and specific antibody levels were measured to determine protection after oral gavage challenges with virulent Clostridium perfringens and LIVACOX® T. Birds immunized with NeCoVac were protected up to 84% against NE and coccidiosis compared to unimmunized and even positive groups (groups treated with LIVACOX® T [coccidiosis live vaccine] and tylosin as routine veterinary interventions) (p < 0.05). CONCLUSIONS: Our findings suggest that vaccination with NeCoVac is highly efficient in protecting birds from NE, coccidiosis and a combination of both diseases. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study is the first one to describe the combinatorial use of AMA1 and RON2 against coccidiosis, and the first report using NeCoVac against NE and coccidiosis together.

Isolation, toxinotyping and antimicrobial susceptibility testing of Clostridium perfringens isolated from Pakistan poultry.

BACKGROUND AND OBJECTIVES: Clostridium perfringens is a causative agent of enteric infections in animals including poultry by producing twenty different types of toxins. A single strain produces only a subset of these toxins, which form the basis of its classification into seven toxinotypes (A-G). C. perfringens toxinotype A is a widespread cause of necrotic enteritis (NE) in poultry. The current study was conducted to determine the prevalence of different toxins and antimicrobial susceptibility of C. perfringens isolated from Pakistan NE affected poultry. METHODS: A total of 134 intestinal samples of the diseased birds were collected postmortem and processed for isolation of C. perfringens using tryptose sulphite cycloserine (TSC) agar supplemented with d-cycloserine. Isolates were confirmed by Gram's staining, biochemical and molecular analyses. Toxinotyping was performed by multiplex PCR. Antimicrobial susceptibility profile of isolates was performed by Kirby Bauer disc diffusion method. RESULTS: A total of 34 strains of C. perfringens were isolated from 134 samples with prevalence rate of 25.37%. All the isolated strains were toxinotype A, as they were positive for alpha toxin (CPA) and negative for other tested toxins such as beta (CPB), epsilon (ETX), iota (ITX), enterotoxin (CPE), toxin perfringens large (TpeL) and necrotic B-like toxin (NetB). Interestingly, all the isolated strains of C. perfringens were multidrug resistant. The highest resistance was observed against Neomycin, Trimethoprim, Tetracycline and Lincomycin which are routinely used at Pakistan poultry production. CONCLUSION: C. perfringens toxinotype A is prevalent in Pakistan poultry. Incidence of C. perfringens with prevalence rate of 25.37% can pose serious threat to Pakistan's poultry industry given that all the isolated strains were multidrug resistant. Our findings highlight the need for new antibiotics and antibiotic alternatives to overcome multidrug resistance.

Structure-based discovery of a small-molecule inhibitor of methicillin-resistant Staphylococcus aureus virulence.

The rapid emergence and dissemination of methicillin-resistant Staphylococcus aureus (MRSA) strains poses a major threat to public health. MRSA possesses an arsenal of secreted host-damaging virulence factors that mediate pathogenicity and blunt immune defenses. Panton-Valentine leukocidin (PVL) and α-toxin are exotoxins that create lytic pores in the host cell membrane. They are recognized as being important for the development of invasive MRSA infections and are thus potential targets for antivirulence therapies. Here, we report the high-resolution X-ray crystal structures of both PVL and α-toxin in their soluble, monomeric, and oligomeric membrane-inserted pore states in complex with n-tetradecylphosphocholine (C14PC). The structures revealed two evolutionarily conserved phosphatidylcholine-binding mechanisms and their roles in modulating host cell attachment, oligomer assembly, and membrane perforation. Moreover, we demonstrate that the soluble C14PC compound protects primary human immune cells in vitro against cytolysis by PVL and α-toxin and hence may serve as the basis for the development of an antivirulence agent for managing MRSA infections.

Multimechanistic Monoclonal Antibody Combination Targeting Key Staphylococcus aureus Virulence Determinants in a Rabbit Model of Prosthetic Joint Infection.

In a rabbit model of methicillin-resistant Staphylococcus aureus prosthetic joint infection (PJI), prophylaxis with AZD6389*-a combination of three monoclonal antibodies targeting alpha-hemolysin, bicomponent cytotoxins (LukSF/LukED/HlgAB/HlgCB), and clumping factor A-resulted in significant reductions in joint swelling, erythema, intra-articular pus, and bacterial burden in synovial tissues and biofilm-associated prosthetic implants compared with isotype-matched control IgG. Targeting specific staphylococcal virulence factors may thus have potential clinical utility for prevention of PJI.

Clostridium perfringens-Associated Necrotic Enteritis-Like Disease in Coconut Lorikeets (Trichoglossus haematodus).

Several outbreaks of necrotic enteritis-like disease in lorikeets, from which Clostridium perfringens was consistently isolated, are described. All lorikeets had acute, segmental, or multifocal fibrinonecrotizing inflammatory lesions in the small and/or the large intestine, with intralesional gram-positive rods. The gene encoding C. perfringens alpha toxin was detected by PCR (polymerase chain reaction) on formalin-fixed, paraffin-embedded (FFPE) tissues in 20 out of 24 affected lorikeets (83%), but it was not amplified from samples of any of 10 control lorikeets (P < .0001). The second most prevalent C. perfringens toxin gene detected was the beta toxin gene, which was found in FFPE from 7 out of 24 affected lorikeets (29%). The other toxin genes were detected inconsistently and in a relatively low number of samples. These cases seem to be associated with C. perfringens, although the specific type involved could not be determined.

Clonal diversity of Clostridium perfringens human clinical isolates with various toxin gene profiles based on multilocus sequence typing and alpha-toxin (PLC) typing.

OBJECTIVES: Clostridium perfringens is a common anaerobic pathogen causing enteritis/enterocolitis and wound infections in humans. We analyzed clonal diversity and toxin gene prevalence in C. perfringens clinical isolates from humans in northern Japan. METHODS: Prevalence of nine toxin genes was analyzed for 585 C. perfringens isolates from patients collected for 20-month period between May 2019 and December 2020 by molecular methods. Sequence type (ST) based on multilocus sequence typing (Xiao's scheme) and alpha-toxin (PLC) sequence type were determined for a total of 124 isolates selected in the present study along with those in our previous study (2017-2018). RESULTS: Toxinotypes A (68.2%) was the most frequent, followed by F (31.6%), and G (0.2%), while additional toxin genes encoding binary enterotoxin (BEC/CPILE) and beta2 toxin were identified in one and six isolates, respectively. Among the 124 isolates with various toxin gene profiles, 62 STs including 53 novel types were identified, revealing the presence of six clonal complexes (CCs) consisting of 27 STs. Most of enterotoxin gene (cpe)-positive isolates belonged to CC36, CC41, and CC117. Based on 22 key amino acids in alpha toxin sequence, four PLC types (I-IV) including 21 subtypes were classified, and their relation to individual STs/CCs was clarified. Two isolates harboring bec/cpile belonged to different STs (ST95, ST131) and PLC types (If, IVb), indicating distribution of this toxin gene to distinct lineages. CONCLUSIONS: The present study revealed the diversity in C. perfringens clones of human origin with various toxin gene profiles represented by ST/CC and PLC type.

Immunization of rabbits with recombinant Clostridium perfringens alpha toxins CPA-C and CTB-CPA-C in a bicistronic design expression system confers strong protection against challenge.

The Clostridium perfringens alpha toxin (CPA), encoded by the plc gene, is the causative pathogen of gas gangrene, which is a lethal infection. In this study, we used an E. coli system for the efficient production of recombinant proteins and developed a bicistronic design (BCD) expression construct consisting of two copies of the C-terminal (247-370) domain of the alpha toxin (CPA-C) in the first cistron, followed by Cholera Toxin B (CTB) linked with another two copies of CPA-C in the second cistron that is controlled by a single promoter. Rabbits were immunized twice with purified proteins (rCPA-C rCTB-CPA-C) produced in the BCD expression system, with an inactivated recombinant E. coli vaccine (RE), C. perfringens formaldehyde-inactivated alpha toxoid (FA-CPA) and C. perfringensl-lysine/formaldehyde alpha toxoid (LF-CPA) vaccines. Following the second vaccination, 0.1 mL of pooled sera of the RE-vaccinated rabbits could neutralize 12× mouse LD100 (100% lethal dose) of CPA, while that of the rCPA-C rCTB-CPA-C-vaccinated rabbits could neutralize 6× mouse LD100 of CPA. Antibody titers against CPA were also assessed by ELISA, reaching titers as high as 1:2048000 in the RE group; this was significantly higher compared to the C. perfringens alpha toxoid vaccinated groups (FA-CPA and LF-CPA). Rabbits from all vaccinated groups were completely protected from a 2× rabbit LD100 of CPA challenge. These results demonstrate that the recombinant proteins are able to induce a strong immune responses, indicating that they may be potentially utilized as targets for novel vaccines specifically against the C. perfringens alpha toxin.

Molecular structure and function of the carboxy-terminus of the alpha-toxin from Clostridium perfringens type A.

In order to interpret the molecular structure and biological characteristics of Clostridium perfringens alpha-toxin (CPA), the CPA251-370 gene was cloned and the 120 amino acid carboxy terminal of CPA (CPA251-370) was obtained. The secondary and three-dimensional (3D) structures of CPA251-370 were predicted. The secondary structure of CPA251-370 consisted primarily of 35.48% ß-sheets and 44.35% random coils. Compared with the CPA toxin consisting of 10 α-helices and eight ß-sheets, the 3D structure of CPA251-370 only contained eight ß-sheets. The circular dichroism (CD) spectrum detection showed that the CD spectrum of CPA251-370 changed slightly compared with the CD spectrum of CPA. Biological activity assays showed that CPA251-370 had lost the phospholipase C (PLC) activity and haemolytic activity of CPA. More importantly, the mice immunized with CPA251-370 were protected against a challenge with 1 MLD C. perfringens type A strain C57-1. This study laid a solid foundation for explaining the relationship between molecular structure and biological characteristics of CPA in the future. Our research also provides CPA251-370 as a candidate strains for genetic engineering subunit vaccines of C. perfringens type A.

Staphylococcus aureus pore-forming toxins: The interface of pathogen and host complexity.

Staphylococcus aureus is a prominent human pathogen capable of infecting a variety of host species and tissue sites. This versatility stems from the pathogen's ability to secrete diverse host-damaging virulence factors. Among these factors, the S. aureus pore-forming toxins (PFTs) α-toxin and the bicomponent leukocidins, have garnered much attention for their ability to lyse cells at low concentrations and modulate disease severity. Although many of these toxins were discovered nearly a century ago, their host cell specificities have only been elucidated over the past five to six years, starting with the discovery of the eukaryotic receptor for α-toxin and rapidly followed by identification of the leukocidin receptors. The identification of these receptors has revealed the species- and cell type-specificity of toxin binding, and provided insight into non-lytic effects of PFT intoxication that contribute to disease pathogenesis.

Contribution of hla Regulation by SaeR to Staphylococcus aureus USA300 Pathogenesis.

The SaeRS two-component system in Staphylococcus aureus is critical for regulation of many virulence genes, including hla, which encodes alpha-toxin. However, the impact of regulation of alpha-toxin by Sae on S. aureus pathogenesis has not been directly addressed. Here, we mutated the SaeR-binding sequences in the hla regulatory region and determined the contribution of this mutation to hla expression and pathogenesis in strain USA300 JE2. Western blot analyses revealed drastic reduction of alpha-toxin levels in the culture supernatants of SaeR-binding mutant in contrast to the marked alpha-toxin production in the wild type. The SaeR-binding mutation had no significant effect on alpha-toxin regulation by Agr, MgrA, and CcpA. In animal studies, we found that the SaeR-binding mutation did not contribute to USA300 JE2 pathogenesis using a rat infective endocarditis model. However, in a rat skin and soft tissue infection model, the abscesses on rats infected with the mutant were significantly smaller than the abscesses on those infected with the wild type but similar to the abscesses on those infected with a saeR mutant. These studies indicated that there is a direct effect of hla regulation by SaeR on pathogenesis but that the effect depends on the animal model used.

Linezolid Attenuates Lethal Lung Damage during Postinfluenza Methicillin-Resistant Staphylococcus aureus Pneumonia.

Postinfluenza methicillin-resistant Staphylococcus aureus (MRSA) infection can quickly develop into severe, necrotizing pneumonia, causing over 50% mortality despite antibiotic treatments. In this study, we investigated the efficacy of antibiotic therapies and the impact of S. aureus alpha-toxin in a model of lethal influenza virus and MRSA coinfection. We demonstrate that antibiotics primarily attenuate alpha-toxin-induced acute lethality, even though both alpha-toxin-dependent and -independent mechanisms significantly contribute to animal mortality after coinfection. Furthermore, we found that the protein synthesis-suppressing antibiotic linezolid has an advantageous therapeutic effect on alpha-toxin-induced lung damage, as measured by protein leak and lactate dehydrogenase (LDH) activity. Importantly, using a Panton-Valentine leucocidin (PVL)-negative MRSA isolate from patient sputum, we show that linezolid therapy significantly improves animal survival from postinfluenza MRSA pneumonia compared with vancomycin treatment. Rather than improved viral or bacterial control, this advantageous therapeutic effect is associated with a significantly attenuated proinflammatory cytokine response and acute lung damage in linezolid-treated mice. Together, our findings not only establish a critical role of alpha-toxin in the extreme mortality of secondary MRSA pneumonia after influenza but also provide support for the possibility that linezolid could be a more effective treatment than vancomycin to improve disease outcomes.

Staphylococcus aureus alpha toxin activates Notch in vascular cells.

Staphylococcus aureus infection is one of the leading causes of morbidity in hospitalized patients in the United States, an effect compounded by increasing antibiotic resistance. The secreted agent hemolysin alpha toxin (Hla) requires the receptor A Disintegrin And Metalloproteinase domain-containing protein 10 (ADAM10) to mediate its toxic effects. We hypothesized that these effects are in part regulated by Notch signaling, for which ADAM10 activation is essential. Notch proteins function in developmental and pathological angiogenesis via the modulation of key pathways in endothelial and perivascular cells. Thus, we hypothesized that Hla would activate Notch in vascular cells. Human umbilical vein endothelial cells were treated with recombinant Hla (rHla), Hla-H35L (genetically inactivated Hla), or Hank's solution (HBSS), and probed by different methods. Luciferase assays showed that Hla (0.01 µg/mL) increased Notch activation by 1.75 ± 0.5-fold as compared to HBSS controls (p < 0.05), whereas Hla-H35L had no effect. Immunocytochemistry and Western blotting confirmed these findings and revealed that ADAM10 and γ-secretase are required for Notch activation after inhibitor and siRNA assays. Retinal EC in mice engineered to express yellow fluorescent protein (YFP) upon Notch activation demonstrated significantly greater YFP intensity after Hla injection than controls. Aortic rings from Notch reporter mice embedded in matrix and incubated with rHla or Hla-H35L demonstrate increased Notch activation occurs at tip cells during sprouting. These mice also had higher skin YFP intensity and area of expression after subcutaneous inoculation of S. aureus expressing Hla than a strain lacking Hla in both EC and pericytes assessed by microscopy. Human liver displayed strikingly higher Notch expression in EC and pericytes during S. aureus infection by immunohistochemistry than tissues from uninfected patients. In sum, our results demonstrate that the S. aureus toxin Hla can potently activate Notch in vascular cells, an effect which may contribute to the pathobiology of infection with this microorganism.

Efficacy of a Multimechanistic Monoclonal Antibody Combination against Staphylococcus aureus Surgical Site Infections in Mice.

Surgical site infections (SSIs) are commonly caused by Staphylococcus aureus We report that a combination of three monoclonal antibodies (MEDI6389) that neutralize S. aureus alpha-toxin, clumping factor A, and four leukocidins (LukSF, LukED, HlgAB, and HlgCB) plus vancomycin had enhanced efficacy compared with control antibody plus vancomycin in two mouse models of S. aureus SSI. Therefore, monoclonal antibody-based neutralization of multiple S. aureus virulence factors may provide an adjunctive perioperative approach to combat S. aureus SSIs.

Staphylococcus aureus alpha-hemolysin impairs corneal epithelial wound healing and promotes intracellular bacterial invasion.

Colonization by Staphylococcus aureus (S. aureus) has been implicated in many infectious and wound healing disorders. This study was performed to characterize the pathogenic role of S. aureus alpha-hemolysin (alpha-toxin) in corneal epithelial wound healing and infectious keratitis in the setting of a corneal wound. The effect of wild-type and isogenic Hla mutant (α-hemolysin gene deleted) S. aureus bacteria and conditioned media on corneal epithelial wound healing was tested in vitro using a scratch assay and in vivo using a murine epithelial debridement model. The invasiveness of wild-type and Hla mutant S. aureus was evaluated in vitro in human corneal epithelial cells and in vivo in a murine model of infectious keratitis following total epithelial debridement. S. aureus and its conditioned media significantly delayed epithelial wound closure both in vitro (P < 0.05) and in vivo (P < 0.05). The effect of S. aureus on wound healing was significantly diminished with the Hla mutant strain (P < 0.05). Likewise, compared to the wild-type strain, the Hla mutant strain demonstrated significantly reduced ability to invade corneal epithelial cells in vitro (P < 0.05) and infect murine corneas following total epithelial debridement in vivo (P < 0.05). In conclusion, S. aureus alpha-hemolysin plays a major role in the pathologic modulation of corneal epithelial wound healing and the intracellular invasion of the bacteria. Limiting colonization by S. aureus and/or blocking alpha-hemolysin may provide a therapeutic approach for corneal wound healing and infectious disorders.

Inactivated recombinant Escherichia coli as a candidate vaccine against Clostridium perfringens alpha toxin in sheep.

Clostridium perfringens type A is the causative agent of gas gangrene and gastroenteric ("yellow lamb disease") disease in ruminants, with C. perfringens alpha toxin (CPA) being the main virulence factor in the pathogenesis of these illnesses. In the present study, we have developed recombinant Escherichia coli bacteria expressing rCPA and used it to vaccinate rabbits and sheep. Doses of up to 200 µg of rCPA used for inoculation, induced 13.82 IU.mL-1 of neutralizing antitoxin in rabbits, which is three times higher than that recommended by the USDA (4 IU.mL-1). In sheep, recombinant bacteria induced antitoxin titers of 4 IU.mL-1, 56 days after the first dose. rCPA which was expressed, mainly, in inclusion bodies, was not found to influence the immunogenicity of the vaccine. The recombinant Escherichia coli bacterin, produced simply and safely, is capable of affording protection against diseases caused by C. perfringens CPA. The current findings represent a novel production method for CPA vaccines potentially applicable to veterinary medicine.

A sandwich duplex immuno PCR for rapid and sensitive identification of Clostridium perfringens alpha and enterotoxin.

The prevalence and lethality associated with C. perfringens alpha (CPA) and enterotoxin (CPE) toxaemia necessitate the need for rapid and definitive detection systems to initiate management measures. In the present study, a sandwich duplex immuno-capture PCR (SD-IPCR) was developed by employing IgY antibodies against a bivalent protein r-Cpae derived from CPA and CPE for antigen capture and reporter antibodies against truncated CPA or CPE conjugated to oligomers of distinguishable size for antigen revealing and signal amplification. The avian immunoglobulin's (IgY) were devoid of reactivity with S. aureus protein A (SpA), a commensal that often co-exists with C. perfringens. The assay was specific, had a detection limit (LOD) of 1 pg/ml for both CPA and CPE in PBS and improved the LOD by 104 folds compared to an analogous sandwich ELISA with same set of antibodies. In spiking studies, a ten-fold reduction in LOD was observed in case of intestinal tissue samples (10 pg/ml) however, no change in LOD was observed when SD-IPCR was applied on to faecal, serum or muscle tissue samples. Of the 136 natural samples examined, the SD-IPCR could detect CPA and CPE in 29.4% and 35.3% samples, while the sandwich ELISAs could detect the same in 25.7% and 25% samples respectively owing to the relatively lesser sensitivity. The LOD and specificity of the SD-IPCR demonstrates its applicability as an efficient and rapid platform for direct detection CPA and CPE from diverse samples matrices in clinical microbiological and meat testing laboratories.