scholarly journals Detecting toxin genes of Clostridium perfringens isolated from diarrhea piglets using multiplex PCR

2018 ◽  
Vol 17 (06) ◽  
pp. 24-30
Author(s):  
Phat X. Dinh
Keyword(s):  
Multiplex Pcr ◽  
Clostridium Perfringens ◽  
Gene Sequencing ◽  
Clinical Samples ◽  
Alpha Toxin ◽  
Toxin Genes ◽  
Intestinal Lesions ◽  
Field Samples ◽  
Beta Toxin ◽  
Mpcr Assay

Clostridium perfringens is currently classified into five types (A, B, C, D, E) based on the different toxins produced. Type A and C are known as the causative agent of enteritis and enterotoxaemia in newborn and young piglets with severe intestinal lesions including edema, hemorrhage and necrosis. A multiplex PCR (mPCR) was developed in order to quickly and early determine the presence of genotypes of C. perfringens based on their genes of cpa, cpb, cpb2 and cpe encoding alpha toxin, beta toxin, beta2 toxin and enterotoxin with predicted products of 324 bp, 196 bp, 107 bp and 257 bp respectively. The detection limit of the mPCR assay was 1 × 103 copies/reaction for each gene. Sequencing of mPCR products performed with clinical samples collected from C. perfringens suspected pigs showed that the mPCR test functioned specifically. In conclusion, the developed mPCR test successfully detected the presence of genes cpa, cpb, cpb2 and cpe in the examined samples. Analysis of the bacteria isolated from field samples of diarrheal piglets collected in this study indicated that C. perfringens carrying gene cpa counted for 96.66% and 3.33% was identified as C. perfringens carrying genes cpa and cpb concurrently. Gene cpe was not found in this study, while gene cpb2 was detected coincidently in 73.33% of the samples with cpa gene. The results indicate that the prevalence of these four toxin genes is cpa, cpb2, cpb and cpe in decending order.

scholarly journals Humoral Immune Response Evaluation in Horses Vaccinated with Recombinant Clostridium perfringens Toxoids Alpha and Beta for 12 Months

Toxins ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 566
Author(s):  
Nayra F. Q. R. Freitas ◽  
Denis Y. Otaka ◽  
Cleideanny C. Galvão ◽  
Dayane M. de Almeida ◽  
Marcos R. A. Ferreira ◽  
...  
Keyword(s):  
Immune Response ◽  
Clostridium Perfringens ◽  
Humoral Immune Response ◽  
Alpha Toxin ◽  
Response Evaluation ◽  
Humoral Immune ◽  
Beta Toxin ◽  
The Ideal

In horses, Clostridium perfringens is associated with acute and fatal enterocolitis, which is caused by a beta toxin (CPB), and myonecrosis, which is caused by an alpha toxin (CPA). Although the most effective way to prevent these diseases is through vaccination, specific clostridial vaccines for horses against C. perfringens are not widely available. The aim of this study was to pioneer the immunization of horses with three different concentrations (100, 200 and 400 µg) of C. perfringens recombinant alpha (rCPA) and beta (rCPB) proteins, as well as to evaluate the humoral immune response over 360 days. Recombinant toxoids were developed and applied to 50 horses on days 0 and 30. Those vaccines attempted to stimulate the production of alpha antitoxin (anti-CPA) and beta antitoxin (anti-CPB), in addition to becoming innocuous, stable and sterile. There was a reduction in the level of neutralizing anti-CPA and anti-CPB antibodies following the 60th day; therefore, the concentrations of 200 and 400 µg capable of inducing a detectable humoral immune response were not determined until day 180. In practical terms, 200 µg is possibly the ideal concentration for use in the veterinary industry’s production of vaccines against the action of C. perfringens in equine species.

scholarly journals Evaluation of two PCR-based procedures for typing Clostridium perfringens : research communication

2000 ◽  
Vol 71 (2) ◽  
Author(s):  
B. Dungu ◽  
M.M. Henton ◽  
A. Bosman ◽  
H. Fourie ◽  
G. Viljoen
Keyword(s):  
Multiplex Pcr ◽  
Clostridium Perfringens ◽  
Intradermal Test ◽  
Clinical Samples ◽  
Chain Reaction ◽  
Skin Reactions ◽  
Large Numbers ◽  
Polymerase Chain ◽  
The Individual

Two polymerase chain reaction (PCR)-based procedures for typing Clostridium perfringens, which affects most domestic animals, were compared and evaluated for efficiency as substitute to the guinea-pig intradermal test routinely used in our laboratory, namely a multiplex PCR and a protocol based on the individual amplification of gene sequences specific for each toxin. Reference isolates of C. perfringens types A, B, C and D as well as cultures from clinical specimens were tested. The sensitivity and specificity of the PCR was confirmed on reference isolates. There was similarity in results on 43 of the 46 samples typed by all 3 methods. Clear results were obtained by PCR on 5 clinical samples that showed either equivocal or weak skin reactions in guinea-pigs. The multiplex PCR protocol, in combination with the evaluation of bacterial growth, is a better alternative to in vivo toxin typing, since C. perfringens can only be incriminated as cause of a disease when it is present in large numbers in the intestine.

scholarly journals Characterization of Toxin Plasmids in Clostridium perfringens Type C Isolates

2010 ◽  
Vol 78 (11) ◽  
pp. 4860-4869 ◽  
Author(s):  
Abhijit Gurjar ◽  
Jihong Li ◽  
Bruce A. McClane
Keyword(s):  
Clostridium Perfringens ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Toxin Gene ◽  
Gene Encoding ◽  
Positive Type ◽  
Toxin Genes ◽  
Type C ◽  
Beta2 Toxin ◽  
Beta Toxin

ABSTRACT Clostridium perfringens type C isolates cause enteritis necroticans in humans or necrotizing enteritis and enterotoxemia in domestic animals. Type C isolates always produce alpha toxin and beta toxin but often produce additional toxins, e.g., beta2 toxin or enterotoxin. Since plasmid carriage of toxin-encoding genes has not been systematically investigated for type C isolates, the current study used Southern blot hybridization of pulsed-field gels to test whether several toxin genes are plasmid borne among a collection of type C isolates. Those analyses revealed that the surveyed type C isolates carry their beta toxin-encoding gene (cpb) on plasmids ranging in size from ∼65 to ∼110 kb. When present in these type C isolates, the beta2 toxin gene localized to plasmids distinct from the cpb plasmid. However, some enterotoxin-positive type C isolates appeared to carry their enterotoxin-encoding cpe gene on a cpb plasmid. The tpeL gene encoding the large clostridial cytotoxin was localized to the cpb plasmids of some cpe-negative type C isolates. The cpb plasmids in most surveyed isolates were found to carry both IS1151 sequences and the tcp genes, which can mediate conjugative C. perfringens plasmid transfer. A dcm gene, which is often present near C. perfringens plasmid-borne toxin genes, was identified upstream of the cpb gene in many type C isolates. Overlapping PCR analyses suggested that the toxin-encoding plasmids of the surveyed type C isolates differ from the cpe plasmids of type A isolates. These findings provide new insight into plasmids of proven or potential importance for type C virulence.

scholarly journals Multiplex PCR assay for identification of Corynebacterium pseudotuberculosis from pure cultures and for rapid detection of this pathogen in clinical samples

2007 ◽  
Vol 56 (4) ◽  
pp. 480-486 ◽  
Author(s):  
Luis G. C. Pacheco ◽  
Roberta R. Pena ◽  
Thiago L. P. Castro ◽  
Fernanda A. Dorella ◽  
Robson C. Bahia ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Corynebacterium Pseudotuberculosis ◽  
Clinical Samples ◽  
Rrna Gene ◽  
Sheep And Goats ◽  
Multiplex Pcr Assay ◽  
Pure Cultures ◽  
Biochemical Identification ◽  
Biochemical Testing ◽  
Mpcr Assay

Corynebacterium pseudotuberculosis is the aetiological agent of caseous lymphadenitis (CLA), a debilitating disease of sheep and goats. Accurate diagnosis of CLA primarily relies on microbiological examination, followed by biochemical identification of isolates. In an effort to facilitate C. pseudotuberculosis detection, a multiplex PCR (mPCR) assay was developed targeting three genes of this bacterium: the 16S rRNA gene, rpoB and pld. This method allowed efficient identification of 40 isolates of this bacterium that had been identified previously by biochemical testing. Analysis of taxonomically related species did not generate the C. pseudotuberculosis mPCR amplification profile, thereby demonstrating the assay's specificity. As little as 1 pg of C. pseudotuberculosis genomic DNA was detected by this mPCR assay, demonstrating the sensitivity of the method. The detection limit in clinical samples was estimated to be 103 c.f.u. C. pseudotuberculosis could be detected directly in pus samples from infected sheep and goats (n=56) with a high diagnostic sensitivity (94.6 %). The developed assay significantly improves rapid C. pseudotuberculosis detection and could supersede bacteriological culture for microbiological and epidemiological diagnosis of CLA.

scholarly journals Dissecting the Contributions of Clostridium perfringens Type C Toxins to Lethality in the Mouse Intravenous Injection Model

2006 ◽  
Vol 74 (9) ◽  
pp. 5200-5210 ◽  
Author(s):  
Derek J. Fisher ◽  
Mariano E. Fernandez-Miyakawa ◽  
Sameera Sayeed ◽  
Rachael Poon ◽  
Victoria Adams ◽  
...  
Keyword(s):  
Clostridium Perfringens ◽  
Intravenous Injection ◽  
Domestic Animals ◽  
Alpha Toxin ◽  
Perfringolysin O ◽  
Injection Model ◽  
Type C ◽  
Beta2 Toxin ◽  
Beta Toxin ◽  
Culture Supernatants

ABSTRACT The gram-positive anaerobe Clostridium perfringens produces a large arsenal of toxins that are responsible for histotoxic and enteric infections, including enterotoxemias, in humans and domestic animals. C. perfringens type C isolates, which cause rapidly fatal diseases in domestic animals and enteritis necroticans in humans, contain the genes for alpha toxin (plc), perfringolysin O (pfoA), beta toxin (cpb), and sometimes beta2 toxin (cpb2) and/or enterotoxin (cpe). Due to the economic impact of type C-induced diseases, domestic animals are commonly vaccinated with crude type C toxoid (prepared from inactivated culture supernatants) or bacterin/toxoid vaccines, and it is not clear which toxin(s) present in these vaccines actually elicits the protective immune response. To improve type C vaccines, it would be helpful to assess the contribution of each toxin present in type C supernatants to lethality. To address this issue, we surveyed a large collection of type C isolates to determine their toxin-producing abilities. When late-log-phase vegetative culture supernatants were analyzed by quantitative Western blotting or activity assays, most type C isolates produced at least three lethal toxins, alpha toxin, beta toxin, and perfringolysin O, and several isolates also produced beta2 toxin. In the mouse intravenous injection model, beta toxin was identified as the main lethal factor present in type C late-log-phase culture supernatants. This conclusion was based on monoclonal antibody neutralization studies and regression analyses in which the levels of alpha toxin, beta toxin, perfringolysin O, and beta2 toxin production were compared with lethality. Collectively, our results highlight the importance of beta toxin for type C-induced toxemia.

scholarly journals Prevalence and Genetic Diversity of Toxin Genes in Clinical Isolates of Clostridium perfringens: Coexistence of Alpha-Toxin Variant and Binary Enterotoxin Genes (bec/cpile)

Toxins ◽  
2019 ◽  
Vol 11 (6) ◽  
pp. 326 ◽  
Author(s):  
Asami Matsuda ◽  
Meiji Soe Aung ◽  
Noriko Urushibara ◽  
Mitsuyo Kawaguchiya ◽  
Ayako Sumi ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Clostridium Perfringens ◽  
Clinical Isolates ◽  
Nucleotide Sequencing ◽  
Toxin Gene ◽  
Alpha Toxin ◽  
Toxin Genes ◽  
Food Borne ◽  
Putative Origin ◽  
High Level

Clostridium perfringens (C. perfringens) is responsible for food-borne gastroenteritis and other infectious diseases, and toxins produced by this bacterium play a key role in pathogenesis. Although various toxins have been described for C. perfringens isolates from humans and animals, prevalence of individual toxins among clinical isolates has not yet been well explored. In the present study, a total of 798 C. perfringens clinical isolates were investigated for prevalence of eight toxin genes and their genetic diversity by PCR, nucleotide sequencing, and phylogenetic analysis. Besides the alpha-toxin gene (plc) present in all the isolates, the most common toxin gene was cpe (enterotoxin) (34.2%), followed by cpb2 (beta2 toxin) (1.4%), netB (NetB) (0.3%), and bec/cpile (binary enterotoxin BEC/CPILE) (0.1%), while beta-, epsilon-, and iota-toxin genes were not detected. Genetic analysis of toxin genes indicated a high level of conservation of plc, cpe, and netB. In contrast, cpb2 was revealed to be considerably divergent, containing at least two lineages. Alpha-toxin among 46 isolates was classified into ten sequence types, among which common types were distinct from those reported for avian isolates. A single isolate with bec/cpile harbored a plc variant containing an insertion of 834-bp sequence, suggesting its putative origin from chickens.

Real-time multiplex PCR assays for reliable detection of Clostridium perfringens toxin genes in animal isolates

2008 ◽  
Vol 127 (1-2) ◽  
pp. 179-185 ◽  
Author(s):  
S. Albini ◽  
I. Brodard ◽  
A. Jaussi ◽  
N. Wollschlaeger ◽  
J. Frey ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Real Time ◽  
Clostridium Perfringens ◽  
Toxin Genes ◽  
Reliable Detection ◽  
Pcr Assays ◽  
Animal Isolates
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