Egypt’s robust milk production contributes significantly to the livelihoods of rural communities and national well-being [26]. The data about C. perfringens based infection in dairy farms is limited. However, it is known that mastitis is a common problem in Egyptian dairy farms, leading to economic losses [12].

Mastitis is one of the most important problems in dairy cattle. C. perfringens type A infections pose a particular danger to postpartum dairy cows, potentially triggering aggressive mastitis with sudden symptoms and high fatality rates [27].

Most mastitis-infected dairy cows carried C. perfringens type A, but specific toxins linked to mastitis weren’t found. This suggests C. perfringens might contribute to uterine and mastitis issues, but more research is needed to confirm its role and identify the key factors involved [2, 28].

Since the beginning of Osman and his colleague’s work, there has been no reference data on the current situation of C. perfringens and mastitis cases in dairy farms in Egypt [12].

In the present work, the recovery results of C. perfringens have been demonstrated at a rate of 23.5% overall in examined samples from dairy farms. The occurrence of C. perfringens in milk samples was 9% suggesting the role of this pathogen in causing disease in dairy animals.

Despite the intensive vaccination programs on these dairy farms to control Clostridium infection, the recorded proportion of C. perfringens isolated in cases of mastitis is very high and alarming compared to what was identified in Belgian dairy cattle by [29].

On the other hand, clinical samples collected from diarrheal cases in baby calves, or intestinal contents in sudden death cases revealed an isolation rate of C. perfringens of 32.9% and 100%, respectively. The results maximize the role of circulating C. perfringens in dairy farm systems as a food-borne contaminant for lactating baby calves. Acute enteritis and fatal enterotoxemia in animals have been attributed to C. perfringens, and the pathogenicity of this organism is associated with enterotoxins [12, 27].

Toxin genotyping is considered a convenient and highly reliable tool for the molecular detection of all major toxin genes, such as (cpa), (cpb1), (etx), and (iap). The designed multiplex PCR was found to be a suitable tool for toxin genotyping of C. perfringens isolates and for detecting the presence of cpe in the tested isolates.

According to multiplex PCR results, the present study indicated that type A is the most frequently isolated genotype of C. perfringens. A total of 93 C. perfringens isolates were classified as type A toxin producers; all are enterotoxigenic strains (cpe+ve). However, those strains isolated from the sudden death cases were mainly of type A, except for only four strains that were of type C (4.3%) (Table 3).

Globally, about 5% of all C. perfringens isolates produce a toxin named C. perfringens enterotoxin (cpe) [30]. Most (cpe+) strains are classified as type A, although types C and D strains producing this enterotoxin are also common [31, 32].

These results were in agreement with 12, 13, and 27. Alpha toxin is produced at a high level in type A and is involved in the pathogenesis of various diseases in animals [16, 33]. It is the main lethal toxin of C. perfringens, a multifunctional phospholipase produced by almost all isolates. The toxin is hemolytic, necrotizing, and effectively lethal [34].

C. perfringens enterotoxin (CPE) is responsible for causing the gastrointestinal symptoms of several C. perfringens food-borne and non-food gastrointestinal diseases in humans. According to [35] the gene for CPE (cpe) is located either on the chromosomes of most C. perfringens type A food poisoning strains or on large conjugative plasmids of the remaining type A food poisoning and most, if not all, other CPE-producing strains.

Another theory stated that the enterotoxin CPE encoded by the cpe gene is the only toxin known to occur on both chromosomes and plasmids. Interestingly, cpe has been found as plasmid-borne in strains isolated from livestock or non-food-borne human gastrointestinal cases (i.e. antibiotic-associated diarrhea or sporadic diarrhea) [36, 37]. In addition, cpe is only released during the sporulation of C. perfringens [38].

However, previous research has found that dairy farms are not a significant source of cpe-positive isolates, which agrees with [39], who found a low recovery in ruminant-associated isolates (2.9%) and [40], who found a high incidence of cpe carriage among canine, equine, and food isolates with an incidence of 94.1%, 93.8%, and 86.7%, respectively.

There are limited studies on the analysis of the cpe sequence of strains of bovine origin, so, in this study, a primer set was designed for the amplification of the plc gene of five enterotoxogenic strains from two different sources of feces and milk.

Bovine necrohaemorrhagic enteritis caused by C. perfringens is an important cause of sudden death with necrohaemorrhagic lesions in the small intestine [28]. The disease frequently strikes calves without warning symptoms in good to excellent bodily health who are fed huge amounts of milk or milk substitute [41]. Although mortality is very close to 100%, the disease has a significant economic impact despite the relatively low morbidity. From this fact, we apply molecular sequencing for the plc region from C. perfringens strains of lactating animals, diarrheal calves, and sudden death cases to investigate the pathogenesis of enterotoxigenic strains in dairy farming chains.

Many studies have sequenced the cpe gene [42, 43]. In the present work, the plc gene was amplified by novel oligonucleotide primers and sequenced for five strains. The nucleotide sequences of the selected Phospholipase C amino acid sequences have main regions of identity with other queries of different C. perfringens isolates. The detected complete phospholipase C sequences (MN635790 & MN635792 ) are not completely identical for different isolates with maximum query coverage (Fig. 5).

That result of partial CDS sequence for plc gene confirms the complete homology of the milk and diarrheal strains, which subsequently related to a neco-hemorrhagic lesion of the intestine (Fig. 6). The results can be confirmed by the role of C. perfringens type A in clostridial abomasitis, which was confirmed when intraluminal administration of C. perfringens type A to neonatal calves induced clinical signs similar to naturally acquired disease [44]. C. perfringens type A strains were isolated almost exclusively from animals diagnosed with either necro-hemorrhagic enteritis [7, 45] or clostridial abomasitis [46, 47].

The evolutionary tree for plc gene partial CDS C. perfringens isolates and the obtained 27 referral nucleotide sequences from GenBank using the Maximum Parsimony method. The bootstrap consensus tree was inferred from 1000 replicates This analysis involved 22 nucleotide sequences. There were a total of 408 positions in the final dataset. Evolutionary analyses were conducted in MEGA11. Red labelled strains (The Egyptian strains in the present study)

The enterotoxin (cpe), which acts as a warning to food-posing strains in humans, plays a significant role in the development of intestinal sickness in many animal species, including human. In contrast to CPE-associated non-food-borne human gastrointestinal disorders, it has been shown that the majority, if not all, C. perfringens type A food poisoning isolates carry a plasmid-based copy of the cpe gene [36, 37].

House et al. 2014 [47] reported that C. perfringens type C can cause sudden death in neonatal calves less than 10 days old, which is completely confirmed with the current data as four isolates of sudden death cases harbored type C toxin genes. The recovery rate of type C was 4.3%, which is extremely high compared to the early work of Omer et al. 2020 [43] in Saudia Arabia with the percentage of type C records reaching 0.96%. Although several textbooks describe the occurrence of C. perfringens type C infection in calves worldwide [48, 49].

Neonates can pick up type C bacteria from a habitat that has been contaminated by sick animals or, less frequently, by asymptomatic carriers. CPB is incredibly sensitive to trypsin and other protease activity. Newborn animals suffer from a protease deficiency in the intestine layers and absorption into the system. Death may be caused directly by severe intestinal necrosis and diarrhea, indirectly by subsequent toxemia, or both directly and indirectly.