Abstract
Clostridium perfringens is one of the most common and important pathogens in livestock due to its ability to produce a diverse arsenal of toxins. Owing to C. perfringens economic importance, this study aimed to determine the types and toxins of C. perfringens in newly born lambs. A total of 200 lambs of less than one-month old were examined, including 100 lambs suffered from diarrhea, 60 freshly dead and 40 apparent healthy. C. perfringens was identified morphologically and biochemically using bacteriological culture in 103 of 200 samples (51.5%). Moreover, serological typing of C. perfringens isolates revealed three serotypes, C. perfringens type A (54.2%), C. perfringens type B (28.8%) and C. perfringens type D (16.9%). The highest prevalence rate for C. perfringens infection was observed in winter (58.25%) in comparison with other seasons. The findings of the present study confirm the presence of enterotoxmia among lambs in localities under study, causing economic losses. The proper vaccination schedule particularly against C. perfringens type A and B is highly recommended.
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Introduction
Clostridium perfringens is a Gram-positive, anaerobic bacteria that causes a variety of human and animal diseases1. It is frequently discovered in the gastrointestinal system of animals and is widely distributed in the environment (e.g., in soil and sewage)2.
Clostridium perfringens infections have been associated with severe diseases in young animals that may result in significant economic lossess. These diseases are distinguished by a rapid progression and high mortality rate, as well as symptoms of colic, hemorrhagic enteritis, convulsions, and neurological abnormalities3.
Clostridium perfringens is classified into five toxinotypes (A, B, C, D, and E) based on the synthesis of four main toxins: alpha (α), beta (β), epsilon (ε), and iota (ι). Each toxin type is contributed to particular intestinal diseases in different animal species4,5. All toxinotypes generate α-toxin, however type B and C strains produce β-toxin, type B and D strains yield ε-toxin, and type E strains produce the ι-toxin6,7,8. Two more significant toxins produced by C. perfringens are the enterotoxin and the β2-toxin, and both have been documented in the past ten years9.
All five species of C. perfringens can induce enterotoxemia. Different C. perfringens biotypes are responsible for various human and animal diseases. Type A strains causes gas gangrene and food poisoning in humans and are frequently detected as a normal component of the intestinal microflora of lambs10. In sheep, lambs, calves, piglets, and poultry, type C strains induce enterotoxemia and necrotic enteritis11,12. Dysentery and pulpy kidney disease are believed to be caused by type D strains in sheep and lambs13.
The usual procedures for isolating, cultivating, and typing C. perfringens, such as the mouse neutralization tests, are time-consuming, expensive, and necessitate the use of live animals and monovalent diagnostic sera. In addition to the ethical issues, the mouse neutralization test also has inconsistent and imprecise results14,15. ELISA is one of quick and simple methods that are utilized to detect the presence of C. perfringens toxins in the intestinal contents of sick animals.
It was thought prudent to verify the ELISA against the more often used method of detection, anaerobic culture using selective media. When applied to nonenriched samples, the ELISA had sensitivity and specificity of 86 and 98%, respectively, and was thus deemed suitable for use in future intergroup comparisons16.
It has been discovered that toxin genotyping is more trust worthy than traditional toxin typing. In order to determine the predominant C. perfringens types in a given geographic area, toxin genes rather than the poisons they generate must be identified. Classifying the isolates into toxigenic types using colony shape, biochemical characteristics, and measurement of fatty acids and organic acid end products of metabolism by gas–liquid chromatography is challenging17.
In Egypt, C. perfringens type A, B, and D have been reported in sheep with morbidity rate up to 25% and mortality rate 16.25%18. Moreover, Selim, et al.19 have been reported C. perfringens type A among diarrheic and recently dead calves.
To ensure a thorough understanding of the epidemiology of C. perfringens infections, detection of C. perfringens toxin types and subtypes is essential. It may also be helpful in the development of an efficient disease control strategy20.
Therefore, the present study aimed to determine the prevalence of C. perfringens in lambs and to investigate the C. perfringens toxino types using ELISA technique.
Materials and methods
Ethical approval
The study was performed according to guidelines and regulations of ethical committee of faculty of veterinary medicine, Benha University (BUFVTM29-9-2022). The study was conducting following ARRIVE guidelines.
Animals and sampling
The study was performed during June 2021–May 2022, to investigate the presence of C. perfringens among lambs raised in the Qalyubia and Menofia governorates. A total of 200 samples were randomly collected from lambs (100 fecal samples from lambs had profuse diarrhea, 40 fecal swabs from apparent healthy lambs and 60 intestinal content from recently dead lambs). Samples were placed in sterile, individual polyethylene bags, labelled, and shipped to the lab on ice for examination. The data of each examined lamb including locality, sex and season were reported.
Isolation and identification of C. perfringens
In accordance with previously described procedures of Smith and Holdeman21, each sample was inoculated into sterile cooked meat medium and anaerobically incubated at 37 °C for 24–48 h. A loopful from a tube that had been incubated was streaked into 200 g/ml of sheep blood agar for isolation and anaerobically incubated at 37 °C for 24–48 h. The colonies with doubled zone of hemolysis were further identified.
Based on Nagler’s reaction, C. perfringens type A alpha antitoxin was inoculated in one half of egg yolk agar plates, and allowed to dry in the incubator for 30 min. From the antitoxin-free half of the plate to the other, the suspected isolated organisms were streaked across it. The plates were evaluated using previously described procedure of Koneman, et al.22 after being incubated anaerobically at 37 °C for 24 h., for the appearance of opalescence and formation of pearly layers on the half of the plate without antitoxin.
The collected C. perfringens isolates were subjected to biochemical analysis using techniques that had already been reported in previous study of Koneman et al. 23. All the isolates were capable of producing H2S and reducing maltose, lactose, sucrose, nitrate, and negative for urease reduction.
Identification of C. perfringens toxins
The toxin antitoxin neutralization test was carried out according to Smith and Holdeman21. The test was conducted by mixing 0.3 ml of centrifuged supernatant from the cooked meat culture of each kind of C. perfringens with 0.1 ml of particular antisera (A, B, C, D, and E).
Additionally, according to the manufacturer’s instructions, the intestinal filtrate and broth culture supernatants of the isolated strains were tested using BIO K 270 - Multiscreen Ag ELISA Enterotoxaemia (Bio-X Diagnostics S.A, Belgium) to identify C. perfringens toxins (Alpha, Beta, and Epsilon).
The optical densities (ODs) were measured at 450 nm using a micro plate reader (clindiag MR-96, Belgium).
The measurement of each sample well was subtracted from the OD of the corresponding negative control to determine the net OD for each sample. Any sample that produced a difference in OD 0.150 was regarded as positive for the tested toxins. In accordance with the manufacturer’s QC data sheet, the limit of OD positivity for the alpha, beta, and epsilon toxins is 0.150. On the other hand, a sample was deemed negative if the OD difference was less than 0.150.
Statistical analysis
All data collected were entered into Microsoft excel spreadsheet. For analysis of the data SPSS version 16 software was used. Data were analyzed descriptively in the first step, using the frequency table and cross tabulation. Then the association of the different variables with the prevalence of C. perfringens at the animal level was analyzed using a Chi-square test.
Results
Isolation and identification of C. perfringens
A total of 103 out of 200 fecal samples (51.5%) were positive. The positive samples for C. perfringens showed characteristic features on blood agar like dewdrops, smooth greyish convex colonies with a double zone of hemolysis.
Prevalence of C. perfringens among different examined lambs
The prevalence rate for C. perfringens was significantly differed between examined Lambs (P < 0.01) and the highest rate was observed among diseased lambs 67% while the lowest rate was observed among apparent healthy lambs 17.5%, Table 1.
Concerning the seasonal effect on the prevalence of C. perfringens, the prevalence rate showed significant disparity (P < 0.01) between different seasons. The highest prevalence rate was observed in winter 33.33%, 82.97% and 55.17% in apparent healthy, diseased and dead lambs, respectively in comparison with other seasons, Table 1.
Typing of C. perfringens isolates
The toxigenic C. perfringens isolated were examined to determine the type of toxin based on lecithinase activity. The results were significantly differed (P < 0.01) between healthy, diseased and different organs of dead lambs, Table 2.
All identified isolates in healthy lambs were C. perfringens type A, while type A (50%), B (34.21) and D (15.78%) were investigated in diseased lambs, Table 2. In addition, the C. perfringnes type A (85.71%) and type D (14.21%) were identified for isolates from liver while type A and B (50%) were identified in isolates from kidney. However, C. perfringens type A (44.44%), B (22.22%) and D (33.33%) were identified among isolates from intestine, Table 2.
Identification of C. perfringens toxins by ELISA
By comparing the results from ELISA, there was a significant difference (P < 0.01) regarding type of C. perfringens among examined lambs. C. perfringens type A was the highest prevalent (43.69%) type, followed by type B (33.99%) and type D (22.33%), Table 3.
Discussion
C. perfringens is ranked among the most important pathogens in livestock and humans. It causes both histotoxic disease and intestinal infections. Toxins produced by C. Perfringens types are responsible for enteric diseases in sheep and goats and it has been suggested that economic losses related to C. perfringens infections may be resulted from all seven types of the bacterium5,24. It is well known that enterotoxaemia causes considerable economic losses to sheep industry due to a high fatality rate, decreased productivity, and increased treatment costs5,25.
Enterotoxemia is one of the most frequently occurring diseases in sheep and goats worldwide. Reports from countries around the world have reported prevalence rates of enterotoxemia ranging between 24.13% and 100% (El Idrissi and Ward, 1992, Greco et al., 2005).
The prevalence rate of C. perfringens in the present study was 17.5% among apparent healthy lambs which was lower than previous reported rate (39.71%)15, while it was 67% among diseased lambs, which come in line with previous rate (69.29%), reported by Kumar, et al.15. Moreover, the overall prevalence rate of C. perfringens in different examined lambs based on bacteriological examination was 51.5%. In comparison with the findings of previous studies, we observed the prevalence rate of C. perfringens was lower than those reported in Andra Pradesh, India (59.62%)15 and higher than those reported in Saudi Arabia (30.41%)26 and in Bangladesh (32.1%)27.
These prevalence rates may differ due to differences in experimental design, geographic or environmental characteristics, research length, the time of year the investigations were conducted, the use of sanitary precautions, and the diagnostic tests that were utilized28,29.
From the present findings, it is clear that the prevalence rate of C. perfringens was highest in winter season; it was 33.33%, 82.97% and 55.17% in apparent healthy, diseased and dead lambs, respectively. These findings are proportionally consistent with Selim, et al.19, they reported that the higher prevalence rate in winter 95.2% in clinically affected calves and 73.3% in dead calves. The higher incidence in winter may be attributed to poor hygienic conditions and lower temperature. These findings might be contributed to high humidity and changing in pasture during winter season in comparison with other seasons30.
Our results demonstrated that, typing of C. perfringens by intradermal inoculation test in Guinea pig revealed that the incidence of toxigenic and non-toxigenic strains were 57.3% and 42.7%, respectively. In contrast, Atwa, et al.31 found that typing of C. perfringens by intradermal inoculation test in albino Guinea pig revealed that the incidence of toxigenic and non-toxigenic strains were 81.9% and 18.1%, respectively in diarrheic calves.
In the current study, C. perfringens type A was more prevalent (43.69%), followed by type B (33.99%) and type D (22.33%). This result is closely identical to the finding of Nayel, et al.18 who stated that C. perfringens type A, B and D are the main types causing diseases in sheep in Egypt, but differed somewhat from a previous study that revealed the predominance of C. perfringens type A (67.2%), followed by type D (16.4%), then type B (13.4%) and type C (3%) recovered sheep, goats, cattle and camels in the Kingdom Saudi Arabia.
All C. perfringens isolates (n = 103) were examined by multiscreen Ag ELISA to identify type of toxin. The most predominant type was type A (43.68%), followed by type B (33.98%) and type D (22.33%). Regarding the toxin type detected in the tested isolates, types A and B were the dominant types detected by ELISA in lambs. However, previous studies; Fayez, et al.26,Moussa and Hessan32,Al-Humiany33 showed the high prevalence of C. perfringens type-A and D among enterotoxaemic sheep.
ELISA has the advantage of being simple and rapid to run, and it can provide additional information about the toxin profile of C. perfringens isolates if conditions are favorable for toxin synthesis at the time of sample collection and at a level detectable by the ELISA16.
Conclusion
C. perfringens is highly prevalent among lambs. The toxinotype A was more prevalent than toxinotype D in sheep. Absence of toxinotypes C, and E in the present study does not indicate the absence of these toxinotypes in the sheep.
Data availability
All data generated or analysed during this study are included in this published article.
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Conceptualization, methodology, formal analysis, investigation, resources, data curation, writing-original draft preparation, S.M., A.S., I.Z., A.M. and R.A.; writing-review and editing, S.M., A.S., I.Z., A.M. and R.A.; project administration, S.M., A.S., I.Z. and A.M.; funding acquisition, S.M., A.S., I.Z. and R.A. All authors have read and agreed to the published version of the manuscript.
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Moustafa, S., Zakaria, I., Moustafa, A. et al. Bacteriological and serological investigation of Clostridium perfringens in lambs. Sci Rep 12, 19715 (2022). https://doi.org/10.1038/s41598-022-21918-6
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DOI: https://doi.org/10.1038/s41598-022-21918-6
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