RESUMO
Diarrhea caused by zoonotic pathogens is one of the most common diseases in dairy calves, threatening the health of young animals. Humans are also at risk, in particular children. To explore the pathogens causing diarrhea in dairy calves, the present study applied PCR-based sequencing tools to investigate the occurrence and molecular characteristics of three parasites (Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi) and three bacterial pathogens (Escherichia coli, Clostridium perfringens, and Salmonella spp.) in 343 fecal samples of diarrheic dairy calves from five farms in Lingwu County, Ningxia Hui Autonomous Region, China. The total positive rate of these pathogens in diarrheic dairy calves was 91.0% (312/343; 95% CI, 87.9-94.0), with C. perfringens (61.5%, 211/343; 95% CI, 56.3-66.7) being the dominant one. Co-infection with two to five pathogens was found in 67.3% (231/343; 95% CI, 62.4-72.3) of investigated samples. There were significant differences (p < 0.05) in the positive rates of Cryptosporidium spp. and diarrheagenic E. coli among farms, age groups, and seasons. Two Cryptosporidium species (C. parvum and C. bovis) and five gp60 subtypes of C. parvum (IIdA15G1, IIdA20G1, IIdA19G1, IIdA14G1, and a novel IIdA13G1) were identified. Two assemblages (assemblage E and zoonotic assemblage A) of G. duodenalis and six ITS genotypes of E. bieneusi (J, Henan-IV, EbpC, I, EbpA, and ESH-01) were observed. Four virulence genes (eaeA, stx1, stx2, and st) of diarrheagenic E. coli and one toxin type (type A) of C. perfringens were detected. Our study enriches our knowledge on the characteristics and zoonotic potential of diarrhea-related pathogens in dairy calves.
Title: Caractérisation moléculaire des protozoaires parasites zoonotiques courants et des bactéries responsables de diarrhée chez les veaux laitiers dans la région autonome Hui du Ningxia, en Chine. Abstract: La diarrhée causée par des agents pathogènes zoonotiques est l'une des maladies les plus courantes chez les veaux laitiers, menaçant la santé des jeunes animaux. Ceci est également un risque pour la santé humaine, en particulier les enfants. Pour explorer les agents pathogènes responsables de la diarrhée chez les veaux laitiers, cette étude a utilisé des outils de séquençage basés sur la PCR pour étudier l'occurrence et les caractères moléculaires de trois parasites (Cryptosporidium spp., Giardia duodenalis et Enterocytozoon bieneusi) et de trois agents pathogènes bactériens (Escherichia coli, Clostridium perfringens et Salmonella spp.) dans 343 échantillons fécaux de veaux laitiers diarrhéiques provenant de cinq fermes du comté de Lingwu, région autonome Hui du Ningxia, en Chine. Le taux total positif de ces pathogènes chez les veaux laitiers diarrhéiques était de 91,0 % (312/343; IC à 95 %, 87,994,0), et C. perfringens (61,5 %, 211/343; IC à 95 %, 56,366,7) était le plus répandu. Une co-infection avec deux à cinq pathogènes a été trouvée dans 67,3 % (231/343; IC à 95 %, 62,472,3) des échantillons étudiés. Il y avait des différences significatives (p < 0,05) dans les taux positifs de Cryptosporidium spp. et d'E. coli diarrhéogènes entre les fermes, les groupes d'âge et les saisons. Deux espèces de Cryptosporidium (C. parvum et C. bovis) et cinq sous-types de gp60 de C. parvum (IIdA15G1, IIdA20G1, IIdA19G1, IIdA14G1 et un nouveau, IIdA13G1) ont été identifiés. Deux assemblages (assemblage E et assemblage zoonotique A) de G. duodenalis et six génotypes ITS d'E. bieneusi (J, Henan-IV, EbpC, I, EbpA et ESH-01) ont été observés. Quatre gènes de virulence (eaeA, stx1, stx2 et st) d'E. coli diarrhéogènes et un type de toxine (type A) de C. perfringens ont été détectés. Notre étude enrichit les connaissances sur les caractères et le potentiel zoonotique des agents pathogènes liés à la diarrhée chez les veaux laitiers.
Assuntos
Doenças dos Bovinos , Criptosporidiose , Cryptosporidium , Diarreia , Enterocytozoon , Fezes , Giardia lamblia , Zoonoses , Animais , Bovinos , Diarreia/veterinária , Diarreia/parasitologia , Diarreia/microbiologia , Diarreia/epidemiologia , China/epidemiologia , Doenças dos Bovinos/parasitologia , Doenças dos Bovinos/epidemiologia , Doenças dos Bovinos/microbiologia , Cryptosporidium/genética , Cryptosporidium/isolamento & purificação , Cryptosporidium/classificação , Enterocytozoon/genética , Enterocytozoon/isolamento & purificação , Enterocytozoon/classificação , Giardia lamblia/genética , Giardia lamblia/isolamento & purificação , Giardia lamblia/classificação , Fezes/parasitologia , Fezes/microbiologia , Criptosporidiose/epidemiologia , Criptosporidiose/parasitologia , Escherichia coli/isolamento & purificação , Escherichia coli/genética , Escherichia coli/classificação , Giardíase/veterinária , Giardíase/epidemiologia , Giardíase/parasitologia , Coinfecção/veterinária , Coinfecção/epidemiologia , Coinfecção/parasitologia , Coinfecção/microbiologia , Microsporidiose/veterinária , Microsporidiose/epidemiologia , Clostridium perfringens/isolamento & purificação , Clostridium perfringens/genética , Clostridium perfringens/classificação , Salmonella/isolamento & purificação , Salmonella/genética , Salmonella/classificação , Humanos , Reação em Cadeia da Polimerase/veterinária , Indústria de LaticíniosRESUMO
INTRODUCTION: Chickens with Necrotic Enteritis (NE), caused by Clostridium perfringens, exhibit acute and chronic symptoms that are difficult to diagnose, leading to significant economic losses. Vaccination is the best method for controlling and preventing NE. However, only two vaccines based on the CPA and NetB toxins have been commercialized, offering partial protection, highlighting the urgent need for more effective vaccines. OBJECTIVE: This review aimed to identify promising antigens for NE vaccine formulation and discuss factors affecting their effectiveness. METHODS: A systematic review using five scientific databases identified 30 eligible studies through the Rayyan tool, which were included for quality review. RESULTS: We identified 25 promising antigens, including CPA, NetB, FBA, ZMP, CnaA, FimA, and FimB, categorized by their role in disease pathogenesis. This review discusses the biochemical, physiological, and genetic traits of recombinant antigens used in vaccine prototypes, their expression systems, and immunization potential in chickens challenged with virulent C. perfringens strains. Market supply challenges, immunogenic potential, vaccine platforms, adjuvants, and factors related to vaccination schedules-such as administration routes, dosing intervals, and age at immunization-are also addressed. Additionally, the study notes that vaccine formulations tested under mild challenges may not offer adequate field-level protection due to issues replicating aggressive conditions, strain virulence loss, and varied methodologies. CONCLUSIONS: An ideal NE vaccine should incorporate multiple antigens, molecular adjuvants, and delivery systems via in ovo and oral routes. The review underscores the challenges in developing and validating NE vaccines and the urgent need for a standardized protocol to replicate aggressive challenges accurately.
Assuntos
Vacinas Bacterianas , Galinhas , Infecções por Clostridium , Clostridium perfringens , Enterite , Doenças das Aves Domésticas , Animais , Antígenos de Bactérias/imunologia , Vacinas Bacterianas/imunologia , Vacinas Bacterianas/administração & dosagem , Galinhas/imunologia , Galinhas/microbiologia , Infecções por Clostridium/prevenção & controle , Infecções por Clostridium/veterinária , Infecções por Clostridium/imunologia , Clostridium perfringens/imunologia , Clostridium perfringens/genética , Enterite/prevenção & controle , Enterite/veterinária , Enterite/microbiologia , Enterite/imunologia , Necrose/veterinária , Doenças das Aves Domésticas/prevenção & controle , Doenças das Aves Domésticas/microbiologia , Vacinação/veterinária , Vacinação/métodos , Desenvolvimento de Vacinas/métodosRESUMO
Clostridium perfringens type A causes gas gangrene, which involves muscle infection. Both alpha toxin (PLC), encoded by the plc gene, and perfringolysin O (PFO), encoded by the pfoA gene, are important when type A strains cause gas gangrene in a mouse model. This study used the differentiated C2C12 muscle cell line to test the hypothesis that one or both of those toxins contributes to gas gangrene pathogenesis by releasing growth nutrients from muscle cells. RT-qPCR analyses showed that the presence of differentiated C2C12 cells induces C. perfringens type A strain ATCC3624 to upregulate plc and pfoA expression, as well as increase expression of several regulatory genes, including virS/R, agrB/D, and eutV/W. The VirS/R two component regulatory system (TCRS) and its coupled Agr-like quorum sensing system, along with the EutV/W TCRS (which regulates expression of genes involved in ethanolamine [EA] utilization), were shown to mediate the C2C12 cell-induced increase in plc and pfoA expression. EA was demonstrated to increase toxin gene expression. ATCC3624 growth increased in the presence of differentiated C2C12 muscle cells and this effect was shown to involve both PFO and PLC. Those membrane-active toxins were each cytotoxic for differentiated C2C12 cells, suggesting they support ATCC3624 growth by releasing nutrients from differentiated C2C12 cells. These findings support a model where, during gas gangrene, increased production of PFO and PLC in the presence of muscle cells causes more damage to those host cells, which release nutrients like EA that are then used to support C. perfringens growth in muscle.
Assuntos
Toxinas Bacterianas , Clostridium perfringens , Gangrena Gasosa , Fosfolipases Tipo C , Clostridium perfringens/genética , Clostridium perfringens/crescimento & desenvolvimento , Clostridium perfringens/metabolismo , Clostridium perfringens/fisiologia , Camundongos , Animais , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Linhagem Celular , Gangrena Gasosa/microbiologia , Fosfolipases Tipo C/genética , Fosfolipases Tipo C/metabolismo , Diferenciação Celular , Células Musculares/microbiologia , Células Musculares/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Percepção de QuorumRESUMO
BACKGROUND: Clostridium perfringens (C. perfringens) is an important zoonotic microorganism that can cause animal and human infections, however information about the prevalence status in wild birds of this pathogenic bacterium is currently limited. RESULT: In this study, 57 strains of C. perfringens were isolated from 328 fecal samples of wild birds. All the isolates were identified as type A and 70.18% of the isolates carried the cpb2 gene. Antimicrobial susceptibility testing showed that and 22.80% of the isolates were classified as multidrug-resistant strains. The MLST analysis of the 57 isolates from wild birds was categorized into 55 different sequence types (STs) and clustered into eight clonal complexes (CCs) with an average of 20.1 alleles and the Simpson Diversity index (Ds) of 0.9812, and revealed a high level of genetic diversity within the C. perfringens populations. Interestingly, the isolates from swan goose were clustered in the same CC while isolates from other bird species were more scattered suggesting that a potential difference in genetic diversity among the C. perfringens populations associated with different bird species. CONCLUSION: C. perfringens exhibits a wide range of host adaptations, varying degrees of antimicrobial resistance, and a high degree of genetic diversity in wild birds. Understanding the prevalence, toxin type, antimicrobial resistance, and genetic diversity of C. perfringens in wildlife populations is essential for developing effective strategies for disease control and management.
Assuntos
Animais Selvagens , Aves , Infecções por Clostridium , Clostridium perfringens , Farmacorresistência Bacteriana Múltipla , Variação Genética , Clostridium perfringens/genética , Clostridium perfringens/isolamento & purificação , Clostridium perfringens/efeitos dos fármacos , Animais , Aves/microbiologia , Farmacorresistência Bacteriana Múltipla/genética , Infecções por Clostridium/veterinária , Infecções por Clostridium/microbiologia , Infecções por Clostridium/epidemiologia , Animais Selvagens/microbiologia , Fezes/microbiologia , Tipagem de Sequências Multilocus/veterinária , Antibacterianos/farmacologia , Doenças das Aves/microbiologia , Doenças das Aves/epidemiologia , Testes de Sensibilidade Microbiana/veterináriaRESUMO
SUMMARYIn the 2018-revised Clostridium perfringens typing classification system, isolates carrying the enterotoxin (cpe) and alpha toxin genes but no other typing toxin genes are now designated as type F. Type F isolates cause food poisoning and nonfoodborne human gastrointestinal (GI) diseases, which most commonly involve type F isolates carrying, respectivefooly, a chromosomal or plasmid-borne cpe gene. Compared to spores of other C. perfringens isolates, spores of type F chromosomal cpe isolates often exhibit greater resistance to food environment stresses, likely facilitating their survival in improperly prepared or stored foods. Multiple factors contribute to this spore resistance phenotype, including the production of a variant small acid-soluble protein-4. The pathogenicity of type F isolates involves sporulation-dependent C. perfringens enterotoxin (CPE) production. C. perfringens sporulation is initiated by orphan histidine kinases and sporulation-associated sigma factors that drive cpe transcription. CPE-induced cytotoxicity starts when CPE binds to claudin receptors to form a small complex (which also includes nonreceptor claudins). Approximately six small complexes oligomerize on the host cell plasma membrane surface to form a prepore. CPE molecules in that prepore apparently extend ß-hairpin loops to form a ß-barrel pore, allowing a Ca2+ influx that activates calpain. With low-dose CPE treatment, caspase-3-dependent apoptosis develops, while high-CPE dose treatment induces necroptosis. Those effects cause histologic damage along with fluid and electrolyte losses from the colon and small intestine. Sialidases likely contribute to type F disease by enhancing CPE action and, for NanI-producing nonfoodborne human GI disease isolates, increasing intestinal growth and colonization.
Assuntos
Infecções por Clostridium , Clostridium perfringens , Enterotoxinas , Esporos Bacterianos , Clostridium perfringens/patogenicidade , Clostridium perfringens/genética , Humanos , Enterotoxinas/genética , Enterotoxinas/metabolismo , Infecções por Clostridium/microbiologia , Virulência , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/genética , Doenças Transmitidas por Alimentos/microbiologia , Animais , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Gastroenteropatias/microbiologiaRESUMO
Clostridium perfringens inhabits the guts of humans and animal species. C. perfringens can proliferate and express an arsenal of toxins, promoting the development of multiple gut illnesses. Healthy animals carrying C. perfringens represents a risk of transmission to other animals or humans through close contact and an increased likelihood of acquisition of toxin plasmids. The aim of this study was to evaluate the frequency of C. perfringens carriage in domestic and farm animals in the central highlands of Colombia. C. perfringens was detected in six animal species using PCR targeting alpha toxin (cpa) and 16S ribosomal RNA (16S-rRNA) genes from 347 fecal samples collected in two Departments: 177 from farm animals of Boyacá and 170 from domestic animals of both Cundinamarca and Boyacá. The overall frequency of C. perfringens detection was 22.1% (n = 77/347), with the highest frequency observed in cats 34.2% (n = 41/120), followed by dogs 30.0% (n = 15/50). The lowest frequency was detected in ruminants: goats 11.1% (n = 3/27), sheep 8.0% (n = 4/50) and cattle 6.0% (n = 6/50). Domestic animals showed a higher frequency of C. perfringens carriage than farm animals. This difference could be associated with dietary patterns, as domestic animals have diets rich in proteins and carbohydrates, while ruminants have low-carbohydrate diets, resulting in high production of endopeptidase-type enzymes and differences in pH due to the anatomy of gastrointestinal tract, which can influence bacterial proliferation. These findings indicate a potential risk of transmission of C. perfringens among animals and from animals to humans through close contact.
Assuntos
Infecções por Clostridium , Clostridium perfringens , Animais , Clostridium perfringens/genética , Clostridium perfringens/isolamento & purificação , Infecções por Clostridium/veterinária , Infecções por Clostridium/transmissão , Infecções por Clostridium/microbiologia , Colômbia/epidemiologia , Animais Domésticos/microbiologia , Portador Sadio/veterinária , Portador Sadio/microbiologia , Fezes/microbiologia , RNA Ribossômico 16S/genética , Bovinos , Humanos , Cabras , Ovinos , Zoonoses/transmissão , Zoonoses/microbiologia , GatosRESUMO
BACKGROUND: Clostridium perfringens, a common environmental bacterium, is responsible for a variety of serious illnesses including food poisoning, digestive disorders, and soft tissue infections. Mastitis in lactating cattle and sudden death losses in baby calves are major problems for producers raising calves on dairy farms. The pathogenicity of this bacterium is largely mediated by its production of various toxins. RESULTS: The study revealed that Among the examined lactating animals with a history of mastitis, diarrheal baby calves, and acute sudden death cases in calves, C. perfringens was isolated in 23.5% (93/395) of the total tested samples. Eighteen isolates were obtained from mastitic milk, 59 from rectal swabs, and 16 from the intestinal contents of dead calves. Most of the recovered C. perfringens isolates (95.6%) were identified as type A by molecular toxinotyping, except for four isolates from sudden death cases (type C). Notably, C. perfringens was recovered in 100% of sudden death cases compared with 32.9% of rectal swabs and 9% of milk samples. This study analyzed the phylogeny of C. perfringens using the plc region and identified the plc region in five Egyptian bovine isolates (milk and fecal origins). Importantly, this finding expands the known data on C. perfringens phospholipase C beyond reference strains in GenBank from various animal and environmental sources. CONCLUSION: Phylogenetic analyses of nucleotide sequence data differentiated between strains of different origins. The plc sequences of Egyptian C. perfringens strains acquired in the present study differed from those reported globally and constituted a distinct genetic ancestor.
Assuntos
Infecções por Clostridium , Clostridium perfringens , Enterite , Variação Genética , Mastite Bovina , Leite , Filogenia , Animais , Clostridium perfringens/genética , Clostridium perfringens/isolamento & purificação , Clostridium perfringens/classificação , Clostridium perfringens/patogenicidade , Bovinos , Egito , Feminino , Infecções por Clostridium/microbiologia , Infecções por Clostridium/veterinária , Leite/microbiologia , Enterite/microbiologia , Enterite/veterinária , Mastite Bovina/microbiologia , Doenças dos Bovinos/microbiologia , Fezes/microbiologia , Fosfolipases Tipo C/genética , Indústria de Laticínios , Fazendas , Toxinas Bacterianas/genéticaRESUMO
l-Carnosine (l-Car), an endogenous dipeptide presents in muscle and brain tissues of various vertebrates, has a wide range of application values. The enzymatic preparation of l-Car is a promising synthetic method because it avoids the protection and deprotection steps. In the present study, a dipeptidase gene (CpPepD) from Clostridium perfringens with high l-Car synthetic activity was cloned and characterized. In an effort to improve the performance of this enzyme, we carried out site saturation mutagenesis using CpPepD as the template. By the o-phthalaldehyde (OPA)-derived high throughput screening method, mutant A171S was obtained with 2.2-fold enhanced synthetic activity. The enzymatic properties of CpPepD and mutant A171S were investigated. Under the optimized conditions, 63.94â¯mM (14.46â¯gâ¯L-1) or 67.02â¯mM (15.16â¯gâ¯L-1) l-Car was produced at the substrate concentrations of 6â¯M ß-Ala and 0.2â¯M l-His using wild-type or mutant A171S enzyme, respectively. Although the mutation enhanced the enzyme activity, the reaction equilibrium was barely affected.
Assuntos
Carnosina , Clostridium perfringens , Dipeptidases , Clostridium perfringens/enzimologia , Clostridium perfringens/genética , Carnosina/metabolismo , Carnosina/química , Carnosina/análogos & derivados , Dipeptidases/genética , Dipeptidases/metabolismo , Dipeptidases/química , Engenharia de Proteínas/métodos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Mutagênese Sítio-DirigidaRESUMO
C. perfringens type D strains are the leading cause of enterotoxaemia in ruminants such as goats, sheep, and cattle. However, there has been no prior research on the genomic characteristics of C. perfringens type D strains from various regions in China. Here, we investigated the antibiotic resistance, genomic characteristics, and phylogenetic relationship of C. perfringens type D isolates recovered from goat farms in Shaanxi, Gansu, and Ningxia provinces. The antibiotic resistance test indicated that the isolates displayed high minimum inhibitory concentration (MIC) values to sulfafurazole, whereas the other antibiotics tested, such as penicillin, enrofloxacin, and florfenicol, worked well on them. Additionally, only tetracycline resistance genes [tetA(P) and tetB(P)] were identified from the isolates. A collective of 13 toxin genes, including etx and cpe were detected among the isolates. Sequence comparison revealed that the etx and cpe genes shared high sequence identities, and they could coexist on a pCW3-like plasmid, representing a potential risk to both animal breeding and public health. Phylogenetic analysis using core genome multi-locus sequence typing (cgMLST) and core genome single nucleotide polymorphisms (SNPs) revealed the close genetic relationship and potential regional/transregional transmission of the C. perfringens type D isolates in Shaanxi and Gansu provinces. Furthermore, pan-genomic analysis suggested the functional differences at the protein-coding gene level, although isolates from the same source shared a close genetic relationship. In conclusion, this study indicated the antibiotic resistance, virulence markers, potential transregional transmission, and genomic diversity of C. perfringens type D strains from various regions in China, which could provide references for the prevention of C. perfringens foodborne diseases and further research.
Assuntos
Antibacterianos , Clostridium perfringens , Doenças das Cabras , Cabras , Filogenia , Animais , Clostridium perfringens/genética , Clostridium perfringens/efeitos dos fármacos , Clostridium perfringens/classificação , Clostridium perfringens/isolamento & purificação , Doenças das Cabras/microbiologia , Doenças das Cabras/epidemiologia , China/epidemiologia , Antibacterianos/farmacologia , Genoma Bacteriano , Testes de Sensibilidade Microbiana , Infecções por Clostridium/microbiologia , Infecções por Clostridium/veterinária , Infecções por Clostridium/epidemiologia , Tipagem de Sequências Multilocus , Fazendas , Genômica , Farmacorresistência Bacteriana/genética , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Clostridium perfringens has emerged as a growing public health concern due to its ability to cause various infections and its increasing resistance to antibiotics. To assess its current epidemiology in clinical settings, we conducted a survey involving 426 healthy individuals and 273 ICU inpatients at a provincial hospital in China. Our findings revealed a high prevalence of C. perfringens in healthy individuals (45.77%, 95% CI: 41.0%-50.6%) and ICU patients (12.82%, 95% CI: 9.1%-17.4%). The identified 220 C. perfringens isolates displayed substantial resistance to erythromycin (57.9%), clindamycin (50.7%), and tetracycline (32.0%), primarily attributed to the presence of erm(Q) (54.4%), lnu(P) (13.8%), tetB(P) (83.6%), and tetA(P) (66.7%). Notably, C. perfringens isolates from this particular hospital demonstrated a high degree of sequence type diversity and phylogenic variation, suggesting that the potential risk of infection primarily arises from the bacteria's gut colonization rather than clonal transmissions within the clinical environment. This study provides an updated analysis of the current epidemiology of C. perfringens in healthy individuals and ICU patients in China and emphasizes the need to optimize intervention strategies against its public health threat. IMPORTANCE: Clostridium perfringens is a bacterium of growing public health concern due to its ability to cause infections and its increasing resistance to antibiotics. Understanding its epidemiology in clinical settings is essential for intervention strategies. This study surveyed healthy individuals and ICU inpatients in a provincial hospital in China. It found a high prevalence of C. perfringens, indicating infection risk. The isolates also showed significant antibiotic resistance. Importantly, the study revealed diverse sequence types and phylogenetic variation, suggesting infection risk from intestinal colonization rather than clonal transmission in hospitals. This analysis emphasizes the need to optimize intervention strategies against this public health threat.
Assuntos
Antibacterianos , Portador Sadio , Infecções por Clostridium , Clostridium perfringens , Unidades de Terapia Intensiva , Humanos , Clostridium perfringens/genética , Clostridium perfringens/isolamento & purificação , Clostridium perfringens/efeitos dos fármacos , Clostridium perfringens/classificação , China/epidemiologia , Infecções por Clostridium/epidemiologia , Infecções por Clostridium/microbiologia , Infecções por Clostridium/transmissão , Masculino , Feminino , Pessoa de Meia-Idade , Adulto , Antibacterianos/farmacologia , Portador Sadio/microbiologia , Portador Sadio/epidemiologia , Idoso , Prevalência , Adulto Jovem , Filogenia , Intestinos/microbiologia , Testes de Sensibilidade Microbiana , Adolescente , Farmacorresistência BacterianaRESUMO
The CPR1953 and CPR1954 orphan histidine kinases profoundly affect sporulation initiation and Clostridium perfringens enterotoxin (CPE) production by C. perfringens type F strain SM101, whether cultured in vitro (modified Duncan-Strong sporulation medium (MDS)) or ex vivo (mouse small intestinal contents (MIC)). To help distinguish whether CPR1953 and CPR1954 act independently or in a stepwise manner to initiate sporulation and CPE production, cpr1953 and cpr1954 null mutants of SM101 were transformed with plasmids carrying the cpr1954 or cpr1953 genes, respectively, causing overexpression of cpr1954 in the absence of cpr1953 expression and vice versa. RT-PCR confirmed that, compared to SM101, the cpr1953 mutant transformed with a plasmid encoding cpr1954 expressed cpr1954 at higher levels while the cpr1954 mutant transformed with a plasmid encoding cpr1953 expressed higher levels of cpr1953. Both overexpressing strains showed near wild-type levels of sporulation, CPE toxin production, and Spo0A production in MDS or MIC. These findings suggest that CPR1953 and CPR1954 do not function together in a step-wise manner, e.g., as a novel phosphorelay. Instead, it appears that, at natural expression levels, the independent kinase activities of both CPR1953 and CPR1954 are necessary for obtaining sufficient Spo0A production and phosphorylation to initiate sporulation and CPE production.
Assuntos
Proteínas de Bactérias , Clostridium perfringens , Enterotoxinas , Histidina Quinase , Esporos Bacterianos , Clostridium perfringens/genética , Clostridium perfringens/enzimologia , Esporos Bacterianos/genética , Esporos Bacterianos/crescimento & desenvolvimento , Enterotoxinas/genética , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Histidina Quinase/genética , Histidina Quinase/metabolismo , Regulação Bacteriana da Expressão Gênica , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , CamundongosRESUMO
Clostridium perfringens causes multiple diseases in humans and animals. Its pathogenic effect is supported by a broad and heterogeneous arsenal of toxins and other virulence factors associated with a specific host tropism. Molecular approaches have indicated that most C. perfringens toxins produce membrane pores, leading to osmotic cell disruption and apoptosis. However, identifying mechanisms involved in cell tropism and selective toxicity effects should be studied more. The differential presence and polymorphisms of toxin-encoding genes and genes encoding other virulence factors suggest that molecular mechanisms might exist associated with host preference, receptor binding, and impact on the host; however, this information has not been reviewed in detail. Therefore, this review aims to clarify the current state of knowledge on the structural features and mechanisms of action of the major toxins and virulence factors of C. perfringens and discuss the impact of genetic diversity of toxinotypes in tropism for several hosts.
Assuntos
Toxinas Bacterianas , Infecções por Clostridium , Clostridium perfringens , Fatores de Virulência , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/toxicidade , Fatores de Virulência/genética , Fatores de Virulência/metabolismo , Humanos , Animais , Clostridium perfringens/genética , Clostridium perfringens/patogenicidade , Clostridium perfringens/metabolismo , Infecções por Clostridium/microbiologiaRESUMO
This study aimed to elucidate the distribution, transmission, and cross-contamination of Clostridium perfringens during the breeding and milking process from dairy farms. The prevalence of 22.3% (301/1351) yielded 494 C. perfringens isolates; all isolates were type A, except for one type D, and 69.8% (345/494) of the isolates carried atyp. cpb2 and only 0.6% (3/494) of the isolates carried cons. cpb2. C. perfringens detected throughout the whole process but without type F. 150 isolates were classified into 94 pulsed-field gel electrophoresis (PFGE) genotypes; among them, six clusters contained 34 PFGE genotypes with 58.0% isolates which revealed epidemic correlation and genetic diversity; four PFGE genotypes (PT57, PT9, PT61, and PT8) were the predominant genotypes. The isolates from different farms demonstrated high homology. Our study confirmed that C. perfringens demonstrated broad cross-contamination from nipples and hides of dairy cattle, followed by personnel and tools and air-introduced raw milk during the milking process. In conclusion, raw milk could serve as a medium for the transmission of C. perfringens, which could result in human food poisoning. Monitoring and controlling several points of cross-contamination during the milking process are essential as is implementing stringent hygiene measures to prevent further spread and reduce the risk of C. perfringens infection.
Assuntos
Infecções por Clostridium , Clostridium perfringens , Animais , Bovinos , Humanos , Clostridium perfringens/genética , Infecções por Clostridium/epidemiologia , Infecções por Clostridium/veterinária , Leite , Prevalência , Fazendas , Genótipo , CruzamentoRESUMO
AIMS: Indole and mucin are compounds found in the host environment as they are produced by the host or by the host-associated microbiota. This study investigated whether indole and mucin impact Clostridium perfringens growth and sporulation, as well as enterotoxin production and biofilm formation. METHODS AND RESULTS: There was no impact on growth of Cl. perfringens for up to 400 µM indole and 240 mg/l mucin, and neither indole nor mucin affected sporulation. Reverse-transcriptase qPCR showed that mucin strongly upregulated the expression of Cl. perfringens enterotoxin (up to 121-fold increase), whereas indole had a much more modest effect (2-fold). This was also reflected in increased Cl. perfringens enterotoxin levels in mucin-treated Cl. perfringens (as assessed by a reversed passive latex agglutination assay). Finally, mucin and indole significantly increased biofilm formation of Cl. perfringens, although the effect size was relatively small (less than 1.5 fold). CONCLUSION: These results indicate that Cl. perfringens can sense its presence in a host environment by responding to mucin, and thereby markedly increased enterotoxin production.
Assuntos
Clostridium perfringens , Enterotoxinas , Clostridium perfringens/genética , Enterotoxinas/genética , Mucinas/metabolismo , Esporos Bacterianos , BiofilmesRESUMO
Necrotic enteritis is a devastating disease to poultry caused by the bacterium Clostridium perfringens. As a novel approach to combating poultry necrotic enteritis, we identified and characterized several hundred single domain antibody fragments (or nanobodies) capable of binding either the NetB toxin or the collagen-binding adhesin (CnaA) of C. perfringens. Many of the nanobodies could neutralize the in vitro functions of NetB or CnaA with inhibitory concentrations in the nanomolar range. The nanobodies were also screened for proteolytic stability in an extract derived from gastrointestinal tract fluids of chickens. A collection of 6 nanobodies (4 targeting NetB and 2 targeting CnaA) with high neutralizing activity and high gastrointestinal tract extract stability were expressed and secreted by Pichia pastoris or Bacillus subtilis. Chickens were given a feed with 1 of the 2 nanobody-containing groups: 1) nanobody-containing P. pastoris supernatants that were semi-purified, lyophilized, and enterically coated, or 2) B. subtilis spores from strains containing the nanobody genes. Compared to untreated chickens (23.75% mortality), mortality of chickens receiving feed modified with the P. pastoris and B. subtilis products decreased to 11.25 and 7.5%, respectively. These results offer a new opportunity to improve the control of poultry necrotic enteritis by incorporating highly specific nanobodies or bacteria expressing these nanobodies directly into chicken feed.
Assuntos
Infecções por Clostridium , Enterite , Doenças das Aves Domésticas , Anticorpos de Domínio Único , Animais , Clostridium perfringens/genética , Infecções por Clostridium/prevenção & controle , Infecções por Clostridium/veterinária , Aves Domésticas , Incidência , Enterite/prevenção & controle , Enterite/veterinária , Galinhas , Doenças das Aves Domésticas/prevenção & controle , Doenças das Aves Domésticas/microbiologiaRESUMO
BACKGROUND: Clostridium perfringens type B and D strains produce epsilon toxin (ETX), which can lead to enterotoxemia, an extremely lethal disease that has significant consequences for the farming of domestic ruminants, specifically sheep and goats. The bacterin-toxoids/toxoids enterotoxemia vaccines need time-consuming detoxification steps. Genetically derived toxoids (GTs) can be the alternative vaccines against ETX-associated enterotoxemia. This study was aimed to design, synthesize, and evaluate of five epsilon toxin mutants of C. perfringens by site-directed mutagenesis (SDM). METHODS: In this study, five ETX mutants (H106P, I51C, V56C, A114C, and F118C), as ETX-GTs, were designed and synthesized by SDM, which were then cloned in pET-26b (+) and expressed in Escherichia coli /BL21 (DE3). The expression of recombinant ETX-GTs was evaluated by SDS-PAGE, blotting, and ELISA and their toxicity was evaluated by the residual toxicity test based on BP Pharmacopoeia, 2021. RESULTS: The findings showed that the ETX-GTs could be considered alternative vaccine candidates against ETX-associated enterotoxemia. CONCLUSIONS: These data suggest that I51C mutant could form the basis of an improved recombinant vaccine against enterotoxemia.
Assuntos
Clostridium perfringens , Enterotoxemia , Ovinos , Animais , Enterotoxemia/prevenção & controle , Clostridium perfringens/genética , Vacinas Sintéticas , ToxoidesRESUMO
Necrotic enteritis (NE) is a poultry intestinal disease caused by virulent strains of the bacterium Clostridium perfringens (C. perfringens). This anaerobic bacterium produces a wide range of enzymes and toxins in the gut which leads to NE development. It is generally accepted by the poultry veterinarians that netB-positive C. perfringens strains are virulent and netB-negative strains do not cause NE. However, NE pathogenesis remains unclear as contradictory results have been reported. The use of experimental in vivo models is a valuable tool to understand the pathogenesis of a disease. In this study, a chicken ligated loop model was used to determine the virulence status of 79 C. perfringens strains from various geographical locations, sources, and genotype profiles. According to our model and based on histologic lesion scoring, 9 C. perfringens strains were classified as commensal, 35 as virulent, and 34 as highly virulent. The virulence of only 1 C. perfringens strain could not be classified as its lesion score was variable (from <10 to >15). In general, NE lesions were more severe in intestinal loops inoculated with netB-positive C. perfringens strains than those inoculated with netB-negative strains. The prevalence of netB among strains classified as commensal, virulent, and highly virulent was 56% (5/9), 54%, (19/35), and 59% (20/34). These results suggest that NetB is not required to cause NE lesions and that other factors are also involved. The classification of the virulence status of C. perfringens strains should not be based solely on the presence or absence of this toxin. Therefore, the use of an in vivo model is essential to distinguish commensal from virulent strains of C. perfringens.
Assuntos
Galinhas , Doenças das Aves Domésticas , Animais , Clostridium perfringens/genética , Composição de Bases , Virulência , Filogenia , RNA Ribossômico 16S , Análise de Sequência de DNA/veterinária , Necrose/veterináriaRESUMO
BACKGROUND: Necrotic enteritis (NE) is a severe intestinal infection that affects both humans and poultry. It is caused by the bacterium Clostridium perfringens (CP), but the precise mechanisms underlying the disease pathogenesis remain elusive. This study aims to develop an NE broiler chicken model, explore the impact of the microbiome on NE pathogenesis, and study the virulence of CP isolates with different toxin gene combinations. METHODS: This study established an animal disease model for NE in broiler chickens. The methodology encompassed inducing abrupt protein changes and immunosuppression in the first experiment, and in the second, challenging chickens with CP isolates containing various toxin genes. NE was evaluated through gross and histopathological scoring of the jejunum. Subsequently, jejunal contents were collected from these birds for microbiome analysis via 16S rRNA amplicon sequencing, followed by sequence analysis to investigate microbial diversity and abundance, employing different bioinformatic approaches. RESULTS: Our findings reveal that CP infection, combined with an abrupt increase in dietary protein concentration and/or infection with the immunosuppressive variant infectious bursal disease virus (vIBDV), predisposed birds to NE development. We observed a significant decrease (p < 0.0001) in the abundance of Lactobacillus and Romboutsia genera in the jejunum, accompanied by a notable increase (p < 0.0001) in Clostridium and Escherichia. Jejunal microbial dysbiosis and severe NE lesions were particularly evident in birds infected with CP isolates containing cpa, netB, tpeL, and cpb2 toxin genes, compared to CP isolates with other toxin gene combinations. Notably, birds that did not develop clinical or subclinical NE following CP infection exhibited a significantly higher (p < 0.0001) level of Romboutsia. These findings shed light on the complex interplay between CP infection, the gut microbiome, and NE pathogenesis in broiler chickens. CONCLUSION: Our study establishes that dysbiosis within the jejunal microbiome serves as a reliable biomarker for detecting subclinical and clinical NE in broiler chicken models. Additionally, we identify the potential of the genera Romboutsia and Lactobacillus as promising candidates for probiotic development, offering effective alternatives to antibiotics in NE prevention and control.
Assuntos
Infecções por Clostridium , Enterite , Microbioma Gastrointestinal , Doenças das Aves Domésticas , Humanos , Animais , Clostridium perfringens/genética , Galinhas/genética , RNA Ribossômico 16S/genética , Disbiose , Jejuno/química , Jejuno/patologia , Enterite/microbiologia , Enterite/patologia , Enterite/veterinária , Infecções por Clostridium/veterinária , Infecções por Clostridium/microbiologia , Infecções por Clostridium/patologia , Doenças das Aves Domésticas/microbiologia , Doenças das Aves Domésticas/patologiaRESUMO
BACKGROUND AND AIMS: This study aimed to identify microbial drivers of inflammatory bowel disease [IBD], by investigating mucosal-associated bacteria and their detrimental products in IBD patients. METHODS: We directly cultured bacterial communities from mucosal biopsies from paediatric gastrointestinal patients and examined for pathogenicity-associated traits. Upon identifying Clostridium perfringens as toxigenic bacteria present in mucosal biopsies, we isolated strains and further characterized toxicity and prevalence. RESULTS: Mucosal biopsy microbial composition differed from corresponding stool samples. C. perfringens was present in eight of nine patients' mucosal biopsies, correlating with haemolytic activity, but was not present in all corresponding stool samples. Large IBD datasets showed higher C. perfringens prevalence in stool samples of IBD adults [18.7-27.1%] versus healthy controls [5.1%]. In vitro, C. perfringens supernatants were toxic to cell types beneath the intestinal epithelial barrier, including endothelial cells, neuroblasts, and neutrophils, while the impact on epithelial cells was less pronounced, suggesting C. perfringens may be particularly damaging when barrier integrity is compromised. Further characterization using purified toxins and genetic insertion mutants confirmed perfringolysin O [PFO] toxin was sufficient for toxicity. Toxin RNA signatures were found in the original patient biopsies by PCR, suggesting intestinal production. C. perfringens supernatants also induced activation of neuroblast and dorsal root ganglion neurons in vitro, suggesting C. perfringens in inflamed mucosal tissue may directly contribute to abdominal pain, a frequent IBD symptom. CONCLUSIONS: Gastrointestinal carriage of certain toxigenic C. perfringens may have an important pathogenic impact on IBD patients. These findings support routine monitoring of C. perfringens and PFO toxins and potential treatment in patients.
Assuntos
Toxinas Bacterianas , Clostridium perfringens , Fezes , Doenças Inflamatórias Intestinais , Mucosa Intestinal , Humanos , Clostridium perfringens/isolamento & purificação , Clostridium perfringens/genética , Clostridium perfringens/patogenicidade , Criança , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Doenças Inflamatórias Intestinais/microbiologia , Toxinas Bacterianas/genética , Fezes/microbiologia , Feminino , Masculino , Adolescente , Biópsia , Infecções por Clostridium/microbiologia , Proteínas HemolisinasRESUMO
Enterotoxaemia (ET) is a severe disease that affects domestic ruminants, including sheep and goats, and is caused by Clostridium perfringens type B and D strains. The disease is characterized by the production of Epsilon toxin (ETX), which has a significant impact on the farming industry due to its high lethality. The binding of ETX to the host cell receptor is crucial, but still poorly understood. Therefore, the structural features of goat Myelin and lymphocytic (MAL) protein were investigated and defined in this study. We induced the mutations in aromatic amino acid residues of ETX and substituted them with aliphatic residues at domains I and II. Subsequently, protein-protein interactions (PPI) were performed between ETX (wild)-MAL and ETX (mutated)-MAL protein predicting the domain sites of ETX structure. Further, molecular dynamics (MD) simulation studies were performed for both complexes to investigate the dynamic behavior of the proteins. The binding efficiency between 'ETX (wild)-MAL protein' and 'ETX (mutated)-MAL protein complex' interactions were compared and showed that the former had stronger interactions and binding efficiency due to the higher stability of the complex. The MD analysis showed destabilization and higher fluctuations in the PPI of the mutated heterodimeric ETX-MAL complex which is otherwise essential for its functional conformation. Such kind of interactions with mutated functional domains of ligands provided much-needed clarity in understanding the pre-pore complex formation of epsilon toxin with the MAL protein receptor of goats. The findings from this study would provide an impetus for designing a novel vaccine for Enterotoxaemia in goats.Communicated by Ramaswamy H. Sarma.