Cpa Gene Research Articles

Molecular Identification and Virulence Genes Detection of Cronobacter Spp Isolated from Clinical Cases in Infants Under Two Years in Mosul City, Iraq

Cronobacter spp. are facultative-anaerobic non-spore forming Gram-negative bacteria, that are part of the Enterobacteriaceae family. They are opportunistic organisms that occasionally cause severe infections such as meningitis, necrotizing enterocolitis, and bloodstream infections in newborns and young children. There is a limited amount of information available about the diversity, pathogenicity, and virulence of Cronobacter species isolated from different sources. This study was conducted to isolate and identify Cronobacter spp from different clinical cases in children under two years in Mosul city and determine some virulence genes which are indicators for their pathogenicity. One hundred and fifty clinical specimens (diarrhea, blood and cerebrospinal fluid) were collected and cultured on Enterobacter sakazakii agar (HiCrome) and trypton soy agar. Eight Cronobacter spp were isolated and presumptively identified according to their morphological characteristics on selective media and confirmed by 16S rRNA gene sequencing and direct sequencing of ropB gene. PCR was used to amplify five potential virulence genes (cpa, hly, sip, zpx, and ompA) in order to determine their presence in our strains. The results revealed that all strains exhibited ompA and zpx, however, only four C. sakazakii strains contained cpa and hly genes. The sip gene was found in only two C. malonaticus strains. Lastly, ropB gene were detected in all Cronobacter spp isolated. Identification of Cronobacter from clinical specimens helps in understanding the sources and risks associated with these pathogens. By determining virulence genes, the study provides insights into how these bacteria cause disease which is crucial for developing targeted interventions and treatments. understanding the presence and characteristics of Cronobacter spp. in clinical cases informs diagnostic and treatment strategies, potentially leading to better patient outcomes.

Disinfection of sulfate-reducing clostridia, including Clostridium perfringens, in sewage effluents using peracetic acid

ABSTRACT Peracetic acid (PAA) is being tested as an alternative to chlorine for sewage disinfection due to its advantages, such as the lack of disinfection byproducts produced and low impact on the discharge site ecosystem. In this study, the inactivating efficiency of PAA against Clostridium perfringens, a known chlorine-resistant microorganism that also causes numerous foodborne illnesses, was evaluated in sewage. PAA remained 80–90% after 120 min of contact with sewage, while chlorine was reduced to 3%. The inactivation curve for Escherichia coli showed a linear decrease with chlorine, while a stepwise reduction was observed with PAA. The inactivation efficacy of PAA on sulfate-reducing clostridia (SRC)-containing C. perfringens was −2.41 log at a CT value of 3,025 mg·min/L. Among the detected SRCs, the −log inactivation of C. perfringens was estimated from the percentage of cpa gene positivity, and its PAA inactivation efficacy was higher than that of SRC at −3.17 log. Although SRC contained PAA-resistant non-C. perfringens clostridia, the effectiveness of PAA for inactivating C. perfringens in the presence of organic matter indicates its effectiveness as a sewage disinfectant.

Clostridium perfringens in central Colombia: frequency, toxin genes, and risk factors

Clostridium perfringens is an opportunistic bacterium that causes intestinal diseases in both humans and animals. This study aimed to assess the frequency of C. perfringens and the presence of toxin-encoding genes in fecal samples from individuals with or without gastrointestinal symptoms in the Department of Boyacá, Colombia. Additionally, risk factors associated with carriage and disease development were analyzed. A total of 114 stool samples were analyzed using a molecular test based on specific polymerase chain reaction (PCR) targeting 16S-rRNA and alpha toxin (cpa) genes. For individuals with a positive result for the PCR test, stool samples were cultured on Tryptose Sulfite Cycloserine (TSC) agar. Two to five colonies forming units were selected based on phenotypic characteristics, resulting in 56 bacterial isolates. These isolates were then analyzed for toxin-coding genes associated with gastrointestinal diseases. In addition, sociodemographic and clinical data from 77 individuals were also analyzed. The overall frequency of C. perfringens was 19.3% (n = 22/114). The detection frequency in 77 individuals with clinical data was 16.6% (n = 5/30) among symptomatic individuals and 21.2% (n = 10/47) among asymptomatic individuals. All 56 isolates obtained carried the cpa gene, while cpb2 was present in 10.7% (n = 6/56); cpe and cpb genes were not detected. Notably, diabetes and autoimmune diseases are significantly associated with an increased risk of C. perfringens detection (adjusted OR 8.41: 95% CI 1.32–35.89). This study highlights an elevated frequency of C. perfringens and the presence of the cpb2 gene in asymptomatic individuals compared with their symptomatic counterparts. These findings offer insights into the distribution and virulence factors of C. perfringens at a micro-geographical level. This information supports the need for developing tailored prevention strategies based on local characteristics to promote active surveillance programs based on molecular epidemiology.

Prevalence and Antibiotic Resistance Profile of Clostridium perfringens Isolated from Pork and Chicken Meat in Vietnam.

Clostridium perfringens is one of the most important zoonotic pathogens as it can cause food poisoning in humans and necrotic enteritis in both animals and humans. Meat, especially pork and chicken meat, is considered the main vehicle for the transmission of C. perfringens from animals to humans. The purpose of this study was to determine the prevalence, toxinotype, and antimicrobial resistance profile of C. perfringens isolated from pork and chicken meat sold in Vietnam. The isolation results showed that 15/50 (30%) of pork samples and 8/50 (16%) of chicken meat samples were contaminated with C. perfringens. The isolates exhibited their highest resistance rate to tetracycline (21/23; 91.30%) and clindamycin (10/23; 43.48%). On the contrary, their lowest resistance rates were observed in response to imipenem (2/23; 8.70%) and cefoxitin (1/23; 4.35%). In particular, 34.78% (8/23) of C. perfringens isolates were identified to be multidrug-resistant strains. The results of toxin genotyping indicated that all isolates were positive for the cpa gene and belonged to type A.

Two gene clusters and their positive regulator SlMYB13 that have undergone domestication-associated negative selection control phenolamide accumulation and drought tolerance in tomato

Among plant metabolites, phenolamides, which are conjugates of hydroxycinnamic acid derivatives and polyamines, play important roles in plant adaptation to abiotic and biotic stresses. However, the molecular mechanisms underlying phenolamide metabolism and regulation as well as the effects of domestication and breeding on phenolamide diversity in tomato remain largely unclear. In this study, we performed a metabolite-based genome-wide association study and identified two biosynthetic gene clusters (BGC7 and BGC11) containing 12 genes involved in phenolamide metabolism, including four biosynthesis genes (two 4CL genes, one C3H gene, and one CPA gene), seven decoration genes (five AT genes and two UGT genes), and one transport protein gene (DTX29). Using gene co-expression network analysis we further discovered that SlMYB13 positively regulates the expression of two gene clusters, thereby promoting phenolamide accumulation. Genetic and physiological analyses showed that BGC7, BGC11 and SlMYB13 enhance drought tolerance by enhancing scavenging of reactive oxygen species and increasing abscisic acid content in tomato. Natural variation analysis suggested that BGC7, BGC11 and SlMYB13 were negatively selected during tomato domestication and improvement, leading to reduced phenolamide content and drought tolerance of cultivated tomato. Collectively, our study discovers a key mechanism of phenolamide biosynthesis and regulation in tomato and reveals that crop domestication and improvement shapes metabolic diversity to affect plant environmental adaptation.

Exploring the predictive power of jejunal microbiome composition in clinical and subclinical necrotic enteritis caused by Clostridium perfringens: insights from a broiler chicken model

BackgroundNecrotic 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.MethodsThis 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.ResultsOur 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.ConclusionOur 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.

Prevalence of Clostridium perfringens toxinotypes in antibiotic-associated diarrheal (AAD) patients in Iranian hospitals; can toxinotype D serve as a possible zoonotic agent for humans?

While Clostridium perfringens (C. perfringens) toxinotype F is known as the cause of 15% of antibiotic-associated diarrhea (AAD) and sporadic diarrhea (SD) cases, the association of the other C. perfringens toxinotypes with AAD/SD is not investigated. Therefore, the incidence of C. perfringens-associated diarrhea was investigated in hospitalized patients in six Iranian hospitals. A total of 151 stool specimens from AAD/SD patients were investigated for C. perfringens strains and the isolates were analyzed for the major (cpa, cpb, etx, and iap) and minor (cpe, cpb2, netb, PFO, and tpeL) toxin genes by PCR. C. perfringens isolation ratio was 28.5% (43 of 151 patients). C. perfringens isolation rates were not significant between different gender and age groups (p > 0.05), whereas it was significant between different wards and hospitals (p < 0.01). The cpa gene was detected in all C. perfringens isolates (n = 116). After that, the highest prevalence belonged to tpeL (87.1%), followed by pfo (84.5%), cpb2 (69.8%), cpe (55.2%), etx (12.9%), and netb (1.7%) genes. Based on these gene profiles, 35 (30.2%), 64 (55.2%), 15 (12.9%), and two (1.7%) isolates belonged to toxinotypes A, F, D, and G, respectively, and the other toxinotypes were not detected. This study persists in considering toxinotype F in Iranian AAD patients as it was the dominant C. perfringens toxinotype. Remarkably, the isolation of toxinotype D suggests it as a potential trigger in C. perfringens-associated AAD for the first time and highlights it as a possible zoonotic agent for humans.

Phenotypical Identification and Toxinotyping of Clostridium perfringens Isolates from Healthy and Enteric Disease-Affected Chickens.

Clostridium perfringens is a ubiquitous spore-forming anaerobic pathogen that is frequently associated with enteric disease in chickens. Moreover, enterotoxin-producing C. perfringens has high zoonotic potential as well as serious public health concerns due to the emanation of food-borne intoxication. The present study was designed to isolate, identify, and toxinotype C. perfringens from both healthy and cases of necrotic or ulcerative enteritis chickens. A total of 110 samples were collected from July 2019 to February 2021. Among the samples, 38 (34.5%, 95% CI: 26.39-43.83) were positive for C. perfringens and were obtained from broiler 21 (33.3%, 95% CI: 22.91-45.67), Sonali 9 (34.6%, 95% CI: 19.31-53.88), and layer 8 (38%, 95% CI: 20.68-59.20). C. perfringens was highly prevalent (35.7%, 95% CI: 25.48-47.44) in enteritis chickens compared with healthy ones. In multiplex PCR toxinotyping, 34 (89.4%) isolates were identified as C. perfringens type A by the presence of the alpha toxin gene (cpa). Moreover, in addition to the cpa gene, 3 (14.3%, 95% CI: 4.14-35.48) broiler and 1 (11.1%, 95% CI: 0.01-45.67) Sonali isolates harbored the enterotoxin gene (cpe) and were classified as type F. However, none of the isolates carried genes encoding beta (cpb), epsilon (etx), iota (iap), or beta-2 (cpb2) toxins. Multivariable logistic regression analysis identified the following variables such as; "previously used litter materials" (OR 21.77, 95% CI 2.22-212.66, p ≤ 0.008); intestinal lesions, "presence of ulceration" (OR 30.01, 95% CI 3.02-297.91, p ≤ 0.004); "ballooned with gas" (OR 24.74, 95% CI 4.34-140.86, p ≤ 0.001) and "use of probiotics" (OR 5.24, 95% CI 0.74-36.75, p ≤ 0.095) act as risk factors for C. perfringens colonization in chicken gut. This is the first study of molecular toxinotyping of C. perfringens from healthy and enteric-diseased chickens in Bangladesh, which might have a potential food-borne zoonotic impact on human health.

Toxin genotypes of Clostridium perfringens isolates from common quail (Coturnix coturnix) with or without acute necrotic enteritis

Clostridial diseases are one of the foremost causes of mortality in quails which occur by Clostridium colinum, Clostridium perfringens (C. perfringens), and Clostridium sordellii. C. perfringens genotypes responsible for quail enteritis are not well understood. In this study, the prevalence of C. perfringens genotypes was investigated in common quail (Coturnix coturnix) farms that suffered from acute necrotic enteritis (diarrhoeic) and compared with healthy (non-diarrhoeic) quails. Toward this end, C. perfringens isolates were collected and genotyped for 16s rRNA, cpa, cpb, cpb2, etx, iap, cpe, netB, and tpeL genes, using PCR. It was revealed that 42, 23, and 19 isolates belonged to toxinotypes A, F, and G, respectively, and the other toxinotypes were not obtained. The recovery ratio of C. perfringens from diarrhoeic farms roughly doubled in non-diarrhoeic farms (40.0% versus 21.5%, p = 0.03). Also, we observed a high isolation ratio of the cpb2 genotype (90.48%), which was significantly eminent in the diarrhoeic group (94.2%, p < 0.05). Although the prevalence of tpeL genes was low (15.48%), there was an interesting relationship between this gene and cpb2, so we did not obtain cpb2-tpeL-. This study showed the prevalence of types A, F, and G of C. perfringens in quail enteritis. Also, we reported tpeL+C. perfringens strains in quail for the first time and its frequent co-occurrence with the cpb2 gene. These results highlight the necessity of more accurate investigations of the C. perfringens genotype in different hosts to verify the exact role of these toxins in quail enteritis.

Prevalence and genotypic characterization of Clostridium perfringens associated with goat (Capra hircus) enterotoxemia in Southeast Iran

Goat enterotoxaemia, a worldwide disease with a high case-fatality rate (up to 100%), is mainly caused by Clostridium perfringens (C. perfringens) toxinotypes D, C, and B, respectively. Here, the prevalence and genotypic characteristics of C. perfringens in goats were investigated in southeast Iran during 2016–2020. In total, 5300 goats from 57 herds were examined, and 112 strains of C. perfringens were isolated from 142 goats (79 dysenteric and 63 healthy goats with a history of enterotoxemia). PCR tests were conducted on isolates using the 16SrRNA, cpa, cpb, etx, iap, cpe, and netb gene primers. Toxinotyping of C. perfringens isolates revealed that the existing toxinotypes were type D (26.8%), followed by types A and F (25.9% for each), then type B (15.2%), and type C (6.2%). The overall prevalence of C. perfringens at the animal and farm levels was 19.0% and 36.8%, respectively. The isolation rate of C. perfringens from dysenteric goats (30.4%) was significantly higher than that of the healthy goats (p = 0.000). Among the isolates, 44.6% (50 of 112) were PCR positive for the cpe gene. The majority of cpe-positive isolates were characterized as type F (58.0%), followed by types D (28.0%), B (8.0%), and C (6.0%). Dysenteric goats were most likely to yield cpe-positive isolates (94.1%). Our results show the high prevalence of Clostridium perfringens toxinotypes in goats and, for the first time, indicate the presence of type F and cpe-positive type B strains in goat enterotoxemia.

Detection of Clostridium perfringens and determination of enterotoxin genes (cpa and cpe) in traditional turkish chicken doner kebab

The demand for the fast-food industry in the world is increasing day by day. In this sense, chicken doner kebab becomes frequently preferred food source in daily life. At the same time, chicken doner kebab is both a good animal origin protein source and a cheaper option. Therefore, it is prepared and consumed in high amounts in Turkey. In this study, it was aimed to determine the presence of Clostridium perfringens and its toxin genes in traditional Turkish chicken doner kebabs purchased from restaurants and modified atmosphere packaged (MAP) samples collected from markets. For this purpose, 100 ready-to-cook and 100 ready-to-eat, totally 200 doner samples have been used as material. As a result, the prevalence of C. perfringens has been found 29%, 6% in ready-to-cook and ready-to-eat samples, respectively. The cpa gene was detected in all isolates. However, both cpa and cpe gene was found only in 4% of isolates.

Prevalence, Antibiotic Resistance, Toxin-Typing and Genotyping of Clostridium perfringens in Raw Beef Meats Obtained from Qazvin City, Iran.

Background: Clostridium perfringens is one of the highest prevailing spore-forming foodborne pathogens, which is widely distributed and causes severe disease and outbreaks in humans and animals. Raw meat and poultry are the main vehicles of this pathogen. In this study, we investigated the prevalence, antibiotic resistance pattern, toxin-encoding genes and genetic diversity of C. perfringens isolates from raw whole and minced meat samples purchased from local markets in Qazvin city, Iran (the source of beef cattle production was also located in Qazvin city, Iran). Methods: We used conventional culture-based and Kirby–Bauer disk diffusion and conventional and arbitrary primer PCR methods. Results: A total of 18 C. perfringens strains were isolated from 133 raw meat samples (13.53%). Up to 44.4 and 55.5% of these isolates were detected in raw minced and whole meat samples, respectively. We found that 72.2, 66.6, 61.1, 37.8 and 33.3% of the C. perfringens isolates were resistant to ampicillin, tetracycline, amoxicillin, ciprofloxacin and chloramphenicol antibiotics, respectively. Multidrug resistance was found in 38% of the isolates. Among the four main toxin genes evaluated, the Cpa gene was detected in all isolates, and 61.1% of the isolates were mostly recognized as type A C. perfringens. High levels of genetic diversity were observed among the isolates, and they were classified into five distinct groups. Conclusions: The isolates from whole meat samples were more resistant to antibiotics. However, toxin genes were more detected in the isolates from minced meat samples. Our findings suggest that contamination of raw meat products with multidrug resistant C. perfringens could be regarded as one of the concerning pathogens in these products. Comprehensive monitoring of C. perfringens isolates is strongly recommended.

Risk Factors, Antibiotic Profile, and Molecular Detection of Virulence and Antibiotic Resistance Genes of Enteric Bacteria in Diarrheic Calves in Egypt

Diarrhea caused by different bacterial infections in calves is a serious issue. In the present study, 215 diarrheic animals, including 175 buffalo and 40 cattle calves were examined. The incidence varied amongst farms, ranging from 0 to 27.9%. Among the affected calves, 37.2% were 1 to 7 days old, 55.8% were 7 days to 3 months of age, while 6.9% were more than 3 months old. Bacteriological, antibiogram, and PCR-based detection of specific virulence and antimicrobial resistance genes were performed. E. coli (85.5%), C. perfringens (8.8%), and Salmonella (3.7%) were bacterial infections recovered from affected calves. Various E. coli pathotypes, such as Shiga-toxin-producing E. coli (STEC), enteropathogenic E. coli (EPEC), extraintestinal E. coli (ExPEC), and enterotoxigenic E. coli (ETEC), caused diarrhea in these calves. The most prevalent virulence genes in E. coli were the sta and eaeA genes (60%). All Salmonella strains were found positive for the invA gene, while all C. perfringens strains were tested positive for the cpa gene. Most of the identified strains were resistant to clindamycin, erythromycin, and oxytetracycline. The isolated strains harbored blaTEM, qnrA and tetA. Approximately 96.2% of E. coli and 62.5% of Salmonella isolates were MDR to different antimicrobial classes. Moreover, 10.5% of C. perfringens isolates were extensive drug-resistant (XDR) to seven antimicrobial classes, while 84.2% of them were MDR to various antimicrobial classes. The findings of this study provide a better understanding regarding the epidemiological aspects of bacterial illnesses and for the development of prevention techniques for this problem. The antibiograms recorded in this study highlight the dangers of indiscriminate antibiotic usage in diarrheic calves.

Occurrence of potentially zoonotic and cephalosporin resistant enteric bacteria among shelter dogs in the Central and South-Central Appalachia

BackgroundAntimicrobial resistance and presence of zoonotic enteropathogens in shelter dogs pose a public health risk to shelter workers and potential adopters alike. In this study we investigated the prevalence of zoonotic bacterial pathogens and cephalosporin resistant (CefR) enteric bacteria in the feces of apparently healthy shelter dogs in the Cumberland Gap Region (CGR) in the US states of Kentucky, Tennessee and Virginia.ResultsFecal samples of 59 dogs from 10 shelters in the CGR of Central and South-Central Appalachia were screened for the presence of Campylobacter jejuni, Clostridium perfringens, Salmonella and CefR enteric bacteria. C. jejuni, C. perfringens were detected by PCR based assays. Culture and PCR were used for Salmonella detection. Of 59 dogs, fecal samples from 14 (23.7%) and 8 (13.6%) dogs tested positive for cpa and hipO genes of C. perfringens and C. jejuni, respectively. Salmonella was not detected in any of the tested samples by PCR or culture. CefR enteric bacteria were isolated on MacConkey agar supplemented with ceftiofur followed by identification using MALDI-TOF. Fecal samples from 16 dogs (27.1%) yielded a total of 18 CefR enteric bacteria. Majority of CefR isolates (14/18, 77.8%) were E. coli followed by, one isolate each of Enterococcus hirae, Acinetobacter baumannii, Acinetobacter pittii, and Pseudomonas aeruginosa. CefR enteric bacteria were tested for resistance against 19- or 24-antibiotic panels using broth microdilution method. Seventeen (94.4%) CefR bacteria were resistant to more than one antimicrobial agent, and 14 (77.8%) displayed multidrug resistance (MDR).ConclusionsThis study shows that shelter dogs within the CGR not only carry zoonotic bacterial pathogens, but also shed multidrug resistant enteric bacteria in their feces that may pose public health risks.

Characterisation of Clostridium perfringens isolated from chickens in Vietnam

The objective of this study was isolating and characterising Clostridium perfringens from chickens in Vietnam and identifying virulence factors involved with enteritis. Five hundred thirty-one faecal and sixty-eight intestinal samples were collected from healthy and diseased chickens for the C. perfringens isolation. The presence of virulence factors was determined by multiplex PCR. The netB gene of the selected isolates was sequenced and checked for its expression by SDS-PAGE. Two hundred seventy-two C. perfringens isolates were collected. All of them were shown to be positive for the cpa gene. The netB gene was detected in 26.56% of the C. perfringens isolates from the healthy chickens, while 43.45% of the isolates from the faeces and 45% of the isolates from the intestinal samples were positive for this gene in the diseased birds. All eight isolates positive to netB from the diseased chickens showed 100% identity in the netB sequence and produced the NetB toxin in vitro, whereas only two out of eight healthy chicken-derived isolates produced this toxin. Nine out of ten chickens experimentally infected with the C. perfringens netB-positive isolate showed typical signs of enteritis. The cpa gene was the most prevalent virulence factor identified in the bacteria C. perfringens, but the netB gene could be a major player responsible for necrotic enteritis progression in chickens.

A patho-microbiological study of tissue samples of the Greater Adjutant Leptoptilos dubius (Aves: Ciconiiformes: Ciconiidae) that died in Deeporbeel Wildlife Sanctuary, Assam, India

The Greater Adjutant is an IUCN Red List ‘Endangered’ scavenging stork. This study reports the findings of post-mortem, histopathology, and a series of microbiological tests conducted on the Greater Adjutant that died in Deeporbeel Wildlife Sanctuary, Assam. A post-mortem examination revealed extensive nodule forming parasitic (Balfouria monogama) infestations in the stomach and intestine. Generalised congestion and haemorrhages in multiple organs were also revealed by the histopathological findings. Bacteriological culture detected the presence of Escherichia coli, Enterococcus sp., and Clostridium perfringens (C. perfringens was confirmed by cpa gene PCR). Virus detection tests like HA and HI test for NDV and rapid antigen detection test for Avian Influenza virus were found to be negative; however, PCR of tissue samples from two Greater Adjutants for Flavivirus was found to be positive. Greater Adjutants may carry the above bacteria as commensals in their GI tract and may possibly act as a reservoir of Flavivirus. The actual cause of deaths, however, were confirmed by the forensic report to be due to organophosphorus toxicity.

Detection of Leishmania tropica Using Nested-PCR and Some of Their Virulence Factors in Thi-Qar Province, Iraq

Cutaneous leishmaniasis is one of endemic diseases in Iraq. It is considered as widely health problem and is an uncontrolled disease. The aim of the study is to identify of Leishmania species that cause skin lesions among patients in Thi-Qar Province, South of Iraq, also to detect some virulence factors of L. tropica. This study includes three local locations, Al-Hussein Teaching, Suq Al-Shyokh General and Al-Shatrah General Hospitals in Province for the period from the beginning of December 2018 to the end of September 2019. The samples were collected from 80 patients suffering from cutaneous leishmaniasis, both genders, different ages, various residence places and single and multiple lesions. Nested-PCR technique was used to amplify kinetoplast minicircle fragments DNA (kDNA). Conventional-PCR was performed for determination of some virulence factors (LPG1, GP63, CPA and PPG1 genes). The electrophoresis findings of kDNA gene showed two species of the parasite found in the study area, 65 samples were positive for cutaneous leishmaniasis, L. tropica at 750bp and L. major at 560bp. Generally, L. tropica (57.5%) was the most common specie and L. major (23.75%) appeared in a low level. There are no significant differences between the infections of males and females, while there are significant differences at the comparison between age groups. All virulence genes (LPG1, GP63, CPA and PPG1) appeared in all L. tropica isolates with high percentage (100%). L. tropica is the major specie which that caused CL in Thi-Qar province, while L. major appeared in low incidence. The virulence genes, which were reviewed, are necessary and important in pathogenesis of L. tropica.

Sequencing of Clostridium perfringens toxin genes (cpa, etx, iap) from Iraqi hospitals and detection by PCR of the genes encoding resistance to metronidazole, tetracycline, and clindamycin

PurposeThis cross sectional study was designed to investigate the molecular detection of C. perfringens toxins (alpha, epsilon, iota) and antibiotics resistance genes, as well as sequencing of their toxin genes. MethodsDifferent wound swabs were taken from 140 patients. PCR was applied for detection of clostridial toxins; alpha toxin (cpa) gene, epsilon toxin (etx) gene, and iota toxin (iap) gene. Metronidazole nim gene, tetracycline resistance genes; (tetQ, tetM, tetB, and tetW) and clindamycin resistance genes erm (A,B), were used for genotypic detection of antibiotic resistance. ResultsOut of 140 clinical samples collected, 7 isolates were detected using specific primer 16S–23S intergenic rRNA spacer gene of C. perfringens. Results showed presence of alpha toxin (cpa) genes in all clostridial isolates, Epsilon toxin (etx) genes in 2/7(28.4%), and iota toxin (iap) genes in 2/7 (28.4%). Results of antibiotic resistance genes showed that all isolates were not able to produce nim, tet W and Q, tet B, erm (A), erm (B) genes except for tetM. Gene sequence analysis for cpa gene showed that there were 3 mutations in the sample of this gene, the results of etx gene when had been sent 2 samples from this gene. The first sample showed that there was one mutation and the results of iap gene showed that there were no mutation in 2 samples of the iap gene. The samples had been showed identical 100%. ConclusionThe DNA sequence analysis of bacterial genome revealed several important feature including that may allow to confirm new the presence of toxin and to identify new or mutant toxins that may be missed by diagnostic PCR specifically target toxin encoding genes.

Phenotypic detection and genotyping of Clostridium perfringens associated with enterotoxemia in sheep in the Qassim Region of Saudi Arabia.

Background and Aim:Enterotoxemia caused by Clostridium perfringens toxinotypes is an often fatal disease of sheep of all ages, with a substantial economic loss to the sheep industry. This study was conducted to isolate C. perfringens from suspected cases of enterotoxemia in sheep in the central part of the Qassim Region, Saudi Arabia, and to determine the prevalent toxinotype by detecting alpha (cpA), beta (cpB), and epsilon (etX) toxin genes, which might help control this disease locally.Materials and Methods:A total of 93 rectal swabs and intestinal content samples were collected from diseased and animals suspected of having died of enterotoxemia in early 2020. Samples were subjected to bacteriological examination, biochemical analysis of isolates by VITEK 2, and molecular toxinotyping of isolates by LightCycler® real-time polymerase chain reaction (RT-PCR).Results:Our results revealed that only 14 isolates were confirmed by VITEK 2 as being C. perfringens, with excellent identification (probability of 95% and 97%). According to the toxinotyping of isolates by RT-PCR, all 14 isolates possessed both the cpA and etX toxin genes, while the cpB toxin gene was not detected in any of the isolates.Conclusion:Our findings demonstrated that C. perfringens type D was the only toxinotype found in the central part of the Qassim Region in 2020; moreover, according to the culture method, only 15% (14/93) of the suspected cases of enterotoxemia were confirmed to be caused by C. perfringens infection, which highlighted the importance of clinical and laboratory differential diagnosis of enterotoxemia in sheep.

Molecular Cloning of a New Bi-Functional Fusion Protein of Clostridium perfringens Type A Alpha and Clostridium septicum Alpha Toxin Genes in E. coli

Background: A synthetic construct bi-functional protein fusion includes two protein domains, or proteins bind by a fragment. The synthetic construct is designed to achieve better characterize and new functionality. Therefore, having proper cells is essential for cloning fusion genes. Clostridium perfringens type A produces the alpha-toxin and can cause gas gangrene and gastrointestinal diseases. C. septicum produces the alpha-toxin and can cause non-traumatic and traumatic gas gangrene. Objectives: The current study aimed to investigate molecular cloning of a new bi-functional fusion protein of C. perfringens alpha (cpa) and C. septicum alpha (csa) toxin genes in E. coli TOP10. In silico analysis was used for the chimeric fusion protein structural prediction. Methods: To produce chimeric fusion protein, the alpha-alpha (α-α) fusion gene was designed according to nucleotide sequences of cpa (KY584046.1) and csa (JN793989.2) genes. Tertiary structural prediction and validation of the fusion protein were determined by online software. In the new synthetic construction, α-α fusion protein genes are bind via the linker AEAAAKEAAAKA. The linker was introduced between the two domains by fusion PCR. The synthetic fusion gene was cloned into the pUC57cloning vector and then transferred into the host cell. Results: Analysis of the chimeric protein fusion is showed using the I-TASSER server as C-score equal to -2.68 as well as Rampage software in order to confirm the geometrical model as a natural like protein. Also, 1.0% agarose gel electrophoresis of fusion PCR product and sequencing analysis revealed a DNA fragment length of 2346 bp. Screening gel electrophoresis showed 996 bp length, which the designed linker was contained in it. Gel electrophoresis of extracted and purified recombinant plasmid (pUC57/αα) showed that a pUC57/αα of 5056 bp. The digested recombinant pUC57/αα showed one 2.3 kb (our fusion gene) band and one 2.7 kb (pUC57) band. Conclusions: This study presented a new approach for the fusion of cpa and csa genes based on the fusion PCR strategy. According to the latest information, this is the first time that α-α fusion gene is designed and cloned into a suitable cloning vector.