We also found numerous important genes involved in pathogenesis and virulence in the CP Del1 chromosome and plasmids (Additional file
1: Table S2). CP Del1 and CP4 strains share similar genes, with more genes absent in the strain ATCC13124 genome. Besides alpha-toxin phospholipase C (cpa plc), CP Del1 genome also had genes encoding necrotic enteritis B-like toxin (netB), Beta 2 toxin (cpb2), mu-toxin (nagH-K), sialidase (nanH, nanI and nanJ), collagenase (peptidase_U32), and perfringolysin O (thera toxin, pfo). Alpha toxin (cpa-encoded) is the most toxic extracellular enzyme produced by CP type A. It is present in all types and essential for virulence, and its core protein, phospholipase C, hydrolyzes both important constituents of eukaryotic cell membranes: phosphatidylcholine and sphingomyelin [
13,
14]. NetB is a pore-forming toxin critical for induction of NE in chickens with predisposing factors, such as preceding coccidial infection and dietary manipulation [
15,
16]. Sialidase cleaves terminal sialic acid residues in the alpha configuration linked to oligosaccharide chains present on proteins and lipids and functions as a nutritional factor to support bacterial growth and as a virulence factor during bacterial pathogenesis [
1].
Studies on the genetic diversity of
C. perfringens in mild NE-affected chickens indicated that more than 90% of all isolates from NE-specific organ lesions carried netB gene [
17]. However, immunization with vaccines containing recombinant NetB toxin only partially protected progeny chickens from necrotic enteritis challenges [
18]. Other virulence factors may additively contribute to the CP pathogenesis in NE induction. Global transcriptomic analysis performed on ligated intestinal loops in chickens following infection with a netB
+ CP strain revealed that numerous virulence factors were significantly expressed in vivo, such as cpa, netB and sialidase genes [
19]. Del1 strain is a virulent strain isolated from a field disease outbreak to be used as a challenge strain. Compared to CP4 strain, this Del1 strain contains some unique genes expressing adhensin, pilin of the toxin- coregulated-pilus (tcp cluster C–J), and programmed cell death toxin (PemK) which may be involved in multitude of pathogenesis functions, including adherence to eukaryotic cells, DNA uptake, and protein/toxin secretion, and therefore enhancing the pathogenesis and favoring the colonization and infection of
C. perfringens as a gastrointestinal pathogen in food animals. Sequencing analysis also indicated that Del1 strain did not contain genes expressing Tpel toxin and surface protein CPE1231 as the CP4 strain had. Previously, it was reported that Tpel protein enhanced bacterial virulence and severity of NE [
16]. Identifying bacterial important genes that encode toxin and virulence factors in genomics and transcriptomics approaches is critical for recombinant vaccine development, for example, cpa, netB, sialidase B, tpel, and PemK. In general, these genes contribute to toxin generation and bacterial colonization and growth. Strain Del1 is classified as Type A due to absence of beta, iota and epsilon toxins based on the definition of the presence of toxin genes in toxinotyping [
1].