Clostridium perfringens is an extremely versatile pathogen of humans and livestock, causing wound infections like gas gangrene , enteritis/enterocolitis (including one of the most common human food-borne illnesses), and enterotoxemia (where toxins produced in the intestine are absorbed and damage distant organs such as the brain). The virulence of this Gram-positive, spore-forming, anaerobe is largely attributable to its copious toxin production; the diverse actions and roles in infection of these toxins are now becoming established. Most C. perfringens toxin genes are encoded on conjugative plasmids, including the pCW3-like and the recently discovered pCP13-like plasmid families. Production of C. perfringens toxins is highly regulated via processes involving two-component regulatory systems, quorum sensing and/or sporulation-related alternative sigma factors. Non-toxin factors, such as degradative enzymes like sialidases, are also now being implicated in the pathogenicity of this bacterium. These factors can promote toxin action in vitro and, perhaps in vivo, and also enhance C. perfringens intestinal colonization, e.g. NanI sialidase increases C. perfringens adherence to intestinal tissue and generates nutrients for its growth, at least in vitro. The possible virulence contributions of many other factors, such as adhesins, the capsule and biofilms, largely await future study. Clostridium perfringens is considered to be anaerobic since it cannot grow in the presence of air [C. perfringens is also an unusual anaerobe by possessing an extremely rapid doubling time; its short generation time of <10 min [C. perfringens lacks flagella but exhibits gliding motility mediated by type IV pili, which also contribute to other potentially virulence-related functions like biofilm formation and adherence [The Gram-positive, spore-forming bacterium e of air . Howevere of air . C. perfdherence .C. perfringens is also a major pathogen of humans and livestock. The virulence of this bacterium can be ascribed, in large part, to its armory of ~20 potent toxins (C. perfringens isolates to one of the seven toxin types (A-G). The importance of toxins for C. perfringens pathogenicity is apparent from the association of specific toxin types with different disease niches (cpe), C. perfringens strains usually need to acquire a plasmid-borne toxin gene in order to cause GI disease, as discussed later.Although it has a ubiquitous environmental presence in decaying vegetation, soil, feces, and the normal gastrointestinal (GI) tract microbiota of humans and other animals, t toxins . Toxin pt toxins  that assC. perfringens pathogenicity, including the structure, action, and genetics of its toxins, which have been the traditional focus of C. perfringens research. Additionally, studies are now exploring the contributions of cell/tissue adherence, degradative enzymes, and sporulation to C. perfringens virulence, so those emerging topics will also be discussed.This review will briefly update current knowledge of C. perfringens. The role of proven, or strongly suggested, virulence factors in disease will then be discussed later in the molecular pathogenesis section.This section will briefly introduce the proven or potential virulence factors of Major Toxins Used for C. perfringens Isolate TypingAlpha toxin (CPA). Virtually all C. perfringens isolates produce CPA, a zinc-containing phospholipase C enzyme of 370 amino acids that consists of a membrane-binding C-domain composed of a β-sheet, a catalytic N-domain composed of α-helices, and a central loop domain containing a ganglioside GM1a binding site [Clostridium bifermentans, Clostridium novyi, Clostridium absonum and Clostridium barati [ing site . Based om barati .i type GTP-binding proteins, CPA can activate endogenous host enzymes with similar phospholipase and sphingomyelinase activities [CPA is thought to use ganglioside GM1 as a receptor . Throughtivities . In additivities . Differetivities .Beta toxin (CPB). C. perfringens type B and C isolates produce CPB as a pro-toxin of 336 amino acids, containing a 27-amino acid signal sequence that is cleaved during secretion. This results in a mature toxin of ~35 kDa [Staphylococcus aureus [s aureus .7 receptor is the CPB receptor for cultured THP-1 monocyte cells [7. In addition, siRNA knockdown of P2X7 in THP-1 cells was demonstrated to reduce CPB binding and CPB oligomer formation in those naturally CPB-sensitive cells. Moreover, it was shown that CPB specifically binds to immobilized P2X7 receptors in vitro and colocalizes with the P2X7 receptor on the THP-1 cell surface.There is some controversy regarding CPB receptors. An early study reported that the P2Xte cells . For exaMore recent studies reported that platelet endothelial cell adhesion molecule-1 (CD31 or PECAM-1) is a CPB receptor on endothelial cells . For exaOne possible resolution of those apparent discrepancies could be that CPB receptors vary among different cell types and/or species.2+, Na+ and Cl− into the cells, inducing cell swelling [CPB is an oligomerizing toxin that forms small functional pores in the plasma membrane of susceptible cells . These pswelling . In porcswelling . Efflux swelling . Cell deswelling . Neverthswelling .7 receptor triggers ATP release from cells through the ATP channel Pannexin 1 [The formation of CPB oligomers (but not CPB binding) in THP-1 cells was reduced by knockdown of Pannexin 1 production . On thisnnexin 1 ,17. It win vivo [in vitro [CPB is extremely sensitive to trypsin and other intestinal proteases . For thiin vivo . CPB varin vitro .C. perfringens enterotoxin (CPE). CPE, a 35 kDa single polypeptide, is produced by all type F strains and by some type C, D, and E strains. This toxin lacks primary amino acid sequence homology with other toxins but belongs structurally to the aerolysin β-PFT family [T family ,21. The T family .The cellular action of CPE starts with its binding to receptors, which include certain claudins . ClaudinOnce bound to a claudin receptor, CPE becomes localized in an ~90 kDa small complex that also contains a receptor claudin and the nonreceptor claudin-1 ,29. Appr2+ influx that induces a mild calpain activation to trigger a caspase 3-mediated apoptosis [2+ influx that induces a strong calpain activation and leads to MLKL-dependent necroptosis [The CPE pore is permeable to small molecules, particularly cations . Treatmepoptosis ,33, whilroptosis . RIP1 anroptosis . Dying Croptosis ,29. Formroptosis .Epsilon toxin (ETX). ETX, produced only by type B and D isolates, has structural similarity to the aerolysin toxin produced by Aeromonas sp., so it is classified as a β-PFT of the aerolysin family [n family ,37. Whiln family . After bn family ,40, folln family .C. perfringens strains can self-activate their ETX, an effect sometimes involving the production of sufficient amounts of λ-protease  to cleave between the 10th and the 11th amino acid residues from the N-terminus of the prototoxin [ETX is released as a weakly active prototoxin of ~33 kDa. Several intestinal proteases such as trypsin, α-chymotrypsin, and carboxypeptidases remove N-terminal and C-terminal residues from this protoxin, resulting in a mature, active protein that is 1000 times more toxic than the prototoxin ,43. Someototoxin ,44. UsinIota toxin (ITX). ITX, a binary toxin produced only by C. perfringens type E strains, is comprised of an enzyme component (Ia) and a binding component (Ib) [C. perfringens type E strains may also activate ITX [ent (Ib) . Removalent (Ib) . These pent (Ib) . In addivate ITX .The lipolysis-stimulated lipoprotein  is a cellular receptor for Ib . This toOnce bound to its receptor, the Ib binding component oligomerizes into heptamers that insert into the plasma membrane of target cells to form functional channels, facilitating the movement of ions and the translocation and endocytosis of the Ia enzymatic component ,50–52. Ain vitro [By ADP-ribosylating actin, iota toxin changes cell morphology and disorganizes intercellular tight and basolateral junctions, producing an increased paracellular permeability in cultured intestinal cells in vitro . Mechaniin vitro ,57.Necrotic enteritis B-like toxin (NetB). NetB, a 33 kDa single-chain protein [C. perfringens delta toxin and 38% and 31% similarity with CPB and S. aureus alpha hemolysin, respectively [ protein , is a meectively ,59. Likeectively .The NetB receptor has not yet been identified. NetB forms heptameric pores with an internal diameter of approximately 26 Å on susceptible cell membranes and the β-barrel channel of this pore has a strong preference for cations ,61.Other Toxins Not Used for C. perfringens Isolate TypingBEC toxin. BEC (binary enterotoxin of C. perfringens) is a novel binary clostridial toxin made by type A C. perfringens strains associated with some human foodborne gastrointestinal disease [ disease ,63. Like disease . The BECbecB null mutant lost fluid-accumulating activity in the suckling mouse model. However, complementation of this null mutant was not reported [C. perfringens in human foodborne gastroenteritis.It was shown that culture supernatants of BEC-positive strains cause fluid accumulation in rabbit intestinal loops . In addireported , so furtBeta2 toxin (CPB2). CPB2, a 28 kDa pore-forming toxin [C. perfringens isolate obtained from a piglet suffering from necrotic enteritis [cpb2) encoding this toxin has been detected in isolates from a wide variety of animals and humans with enteric disease, including humans, cattle, sheep and goats, horses, chicken, and swine [ng toxin , was firnteritis . Subseqund swine .C. perfringens types can produce CPB2, which has no significant amino acid homology with CPB [cpb2 variants, the “consensus” gene or the “atypical” gene [cpb2 genes were shown to have a frameshift that leads to lack of expression [Most or all with CPB . There aal” gene . The conpression . Howeverpression .The mode of action and receptor for CPB2 remain unclear at present.cpb2-positive C. perfringens strains are commonly isolated from healthy animals [The involvement of CPB2 in intestinal diseases of different animal species also remains unproven, since  animals . In the Delta toxin. Delta toxin is a 32 kDa single-chain protein produced by some type B and C strains. The delta toxin gene (cpd) appears to be located on plasmids that have not yet been characterized [S. aureus (32% identity) [cterized . This todentity) ,69.Ganglioside GM2 has been suggested as a potential cell-surface receptor for delta toxin ,70. DeltNetF toxin. NetF, a PFT made by some type A strains, is a 31.7 kDa single-chain protein that belongs to the α-hemolysin family of β-PFTs [S. aureus alpha-toxin [f β-PFTs . Like Neha-toxin .S. aureus alpha-toxin pore (2.8 nm) [Recently, it was shown that NetF binds to a sialoprotein(s) on the surface of biological membranes and form pores containing 6–8 NetF monomers . Osmotic(2.8 nm) ,73,74.Perfringolysin O (PFO). PFO  is produced by most C. perfringens strains, with the exception of type F strains that have a chromosomal cpe gene or type C Darmbrand strains [ strains . PFO is  strains ,76. The  strains . Four do strains . The hig strains .The model of PFO pore formation involves the binding of water-soluble PFO monomers to cholesterol in plasma membranes, which is mediated by the L1-L3 loops from domain 4 of PFO . The resTpeL toxin. Toxin perfringens large (TpeL), a ~ 205 kDa protein, belongs to the clostridial glucosylating toxin [Clostridioides difficile toxins A and B (TcdA/TcdB), Clostridium sordellii lethal and hemorrhagic toxins (TcsL and TcsH), and Clostridium novyi alpha toxin (Tcnα) [ng toxin ,82 familn (Tcnα) ,84.The cell-surface receptor for TpeL is the low-density lipoprotein receptor-related protein 1 (LRP1) . TpeL catpeL-positive type G strains from chickens with necrotic enteritis has led to speculation about its accessory role in necrotic enteritis [tpeL- and netB-positive type G strains are more virulent than netB-only type G strains [The timing of TpeL production is controversial, with some reports indicating TpeL is made during sporulation but others linking it to vegetative growth . Isolatinteritis . Subsequ strains .Whether TpeL contributes to pathogenicity, when produced, remains to be elucidated.C. perfringens produces a vast array of extracellular degradative enzymes, such as proteases (e.g. clostripain), hyaluronidase (mu toxin), collagenase, and endoglycosidases. Virulence contributions of these factors are now coming under intensive study. For example, it has been established that the endo-N-acetylgalactosamindase EngCP [se EngCP , but notse EngCP , is impose EngCP .C. perfringens degradative enzymes are the sialidases (neuraminidases), which generate free sialic acids  from various sialoglycoconjugates found on host cell surfaces or mucus [C. perfringens produces three sialidases named NanJ, NanI and NanH. NanI (77 kDa) and NanJ (129 kDa) are secreted exosialidases, while NanH (43 kDa) is a cytoplasmic sialidase, at least during early growth [The best-studied or mucus ,95. C. py growth .All three sialidases share the same family 33 carbohydrate-binding module, while the two exosialidases contain additional accessory carbohydrate-binding modules . Due to C. perfringens strains produce all three sialidases and, for those strains, NanI is their major sialidase [cpe gene and type C Darmbrand strains, which typically do not carry the nanI gene [nanI-negative strains do possess the nanH gene, sometimes along with the nanJ gene [Most ialidase ,97,98. TanI gene . HoweveranJ gene .C. perfringens in harsh environments. Of particular note are type C Darmbrand strains and type F strains with a chromosomal cpe gene, both of which cause human food-borne illnesses (discussion later in the C. perfringens disease/epidemiology section) and form especially resistant spores [Spores facilitate the survival of t spores ,101. Thet spores .cpe strains produce spores with a D100 (the time required at 100°C to reduce spore viability by one log) of ≥30-120 min [C. perfringens strains have a D100 value of <5 min [cpe disease strains also show exceptional resistance to other food environment stresses, e.g. when stored at the low temperatures found in refrigerators or freezers for 3–6 months, these spores exhibit an average log reduction in viability of only 0.3 or 0.6 at, respectively, 4°C or −20°C [To illustrate the exceptional heat resistance properties of their spores, type C Darmbrand strains or type F chromosomal -120 min . In contor −20°C . Similaror −20°C .C. perfringens spore resistance are the α/β-type small acid-soluble proteins (SASPs). These SASPs bind to spore DNA and provide protection from various environmental stresses. C. perfringens produces four major SASPs, each of which contributes to spore resistance against heat, chemicals, and UV radiation [Major contributors to adiation ,105.cpe type F food poisoning strains and type C Darmbrand isolates, both of which produce exceptionally resistant spores, represent a distinct genetic cluster within C. perfringens [C. perfringens isolates producing more sensitive spores [cpe type F strains or type C Darmbrand strains, i.e. in the same strain background, production of the Asp36 SASP4 variant significantly increases spore heat, nitrite, and cold resistance compared to the production of the Gly36 SASP4 variant [C. perfringens genome contains >70% AT, it is not surprising that SASP4, particularly the SASP4 Asp36 variant, plays such an important role in spore resistance.Multilocus Sequence Typing (MLST) analyses determined that the chromosomal fringens . Those te spores . Based u variant ,104,106.C. perfringens spore heat resistance [SASPs are not the only factor determining spore resistance properties. Factors such as spore coat thickness, spore core size, the concentration of DPA and metal ions, and the protoplast-to-sporoplast ratio have also been implicated in sistance ,108.C. perfringens proteins, including collagen adhesion protein (CNA) and fibrinogen-binding proteins FbpA and FbpB, have been suggested to function as adhesins during disease (see the molecular pathogenesis section).Several C. perfringens  became the first Gram-positive anaerobic bacterium within the phylum Firmicutes whose genome was fully sequenced [C. perfringens from a wide range of toxinotypes (A-G) revealed these strains contain a single circular chromosome of 2.9–4.1 Mb with relatively low G + C% content (averaging between 27.7% and 28.7%) and encoding between 2600 and 3800 predicted genes [C. perfringens genomes indicated the pangenome consists of 11,667 genes (12.6% core genes and 87.4% accessory genes), suggesting considerable genomic diversity among C. perfringens strains, putting it on a par with the genome of Escherichia coli [C. perfringens are associated with mobile genomic elements, such as insertion sequences (ISs), transposases, and prophages [C. perfringens virulence. In addition, the lack of hallmark differences in GC skew signs of horizontal gene transfer (HGT) and the fairly uniform nucleotide compositions of these variable regions suggests that, i) gene acquisition in these strains is not a recent event, ii) C. perfringens only maintains those acquired genes with a high degree of nucleotide composition similarity to the chromosome and/or iii) these genes were acquired only from very closely related organisms with low G+C % content [In 2002, equenced . A recened genes . The larhia coli . Most varophages ,112–115.C. perfringens genomes, along with unassembled short read sequences, revealed the presence of multiple putative novel toxin homologs with amino acid sequence identity to well-characterized C. perfringens toxins such as CPE, CPB, ETX, and ITX [Analyses of the 240 publicly available complete or draft  and ITX . Those oC. perfringens requires a number of essential nutrients and amino acids for proliferation [C. perfringens lacks genes required for the biosynthesis of many amino acids. It also lacks genes for the tricarboxylic acid cycle. However, the C. perfringens genome encodes degradative enzymes like sialidases and a complete set of enzymes for fermentation and glycolytic pathways, which facilities the utilization of complex host carbohydrates by their degradation into simple sugar components [C. perfringens carries more than 200 transport-related genes (e.g. ABC transporters) for sugars, amino acids, nucleotides, and anions/cations. These features enable this bacterium to overcome its inability to synthesize essential carbohydrates and amino acids by acquiring these molecules from host tissues [C. perfringens genome is the large number of encoded rRNA operons and tRNAs, which allows rapid production of secreted enzymes and toxins to support the exceptionally fast growth of the organism, an important aspect of virulence or outcompeting other bacteria involved in the decomposition of dead animals or tissue [feration . Genome mponents . In addi tissues ,110,118.r tissue ,119.C. perfringens toxin genes are carried on plasmids (see below), the chromosome does encode some proven (e.g. CPA and PFO) or potential  virulence factors [C. perfringens toxinotypes will provide a better understanding of the role of other chromosomal genes  in the pathogenesis of C. perfringens, as well as the genetic basis of host species adaptation by C. perfringens, where this occurs.Although many  factors ,112. TheC. perfringens pathogenicity, particularly when this bacterium causes diseases originating in the intestines [C. perfringens plasmids belong to three main families  and have been identified in all seven toxinotypes of C. perfringens [tcp locus shares a low degree of sequence homology with the conjugative transposon Tn916 that is found in a wide range of bacteria [tcp-conjugative plasmids, including TcpA (a coupling protein), TcpC and TcpH , TcpD and TcpE , TcpF (a putative ATPase), TcpG (a peptidoglycan hydrolase protein), and TcpK and TcpM (relaxosome proteins) [The pCW3-like plasmids share a common conjugation system designated as “fringens ,124–126.bacteria . This loroteins) ,127–133.tcp locus). Apart from this core region, each pCW3 family member also has a diverse accessory region [The pCW3-like plasmids contain an ~35 to 40 kb conserved core region that encodes 22 critical genetic elements required for efficient plasmid replication, maintenance, stability, partitioning, regulation, and transfer . The pCW3-like toxin plasmid family ranges in size from ~50 to ~140 kb in size. Several of these toxin plasmids have been completely sequenced [Almost all clinically important in genes ,135–143,plasmids . The cpbequenced ,144.Tablcpe) encoding CPE is unusual in that it can be located on either the chromosome or plasmids [cpe gene on the chromosome [1470 sequences that may represent the chromosomal integration of a cpe-carrying transposon [cpe-positive type C, D, and E strains carry their cpe gene on large pCW3-like conjugative plasmids. In type F strains, cpe plasmids mainly cluster into two sub-families [cpb2 gene) and the pCPF5603-like plasmids (which do carry the cpb2 gene).The gene  and complete or disrupted cpe genes are located near one another on pCW3-like plasmids [cpe and iap/ibp genes suggests that a possible hot-spot for IS insertion exists on pCW3-like plasmids.Interestingly, the variable and unique regions on pCW3-like toxin plasmids are often associated with mobile genomic elements, such as ISs and transposases ,125,126, element ,140. Intplasmids ,148; sinC. perfringens cell [C. perfringens tcp-conjugative plasmids may have up to 10 different types of partitioning system (ParMRCA-J) located in the core region of these plasmids [C. perfringens strain to carry multiple discrete plasmids. Another intriguing feature of pCW3-like plasmids is that their replication protein is highly similar and specific to C. perfringens plasmids [An interesting feature of pCW3-like plasmids is that multiple (up to five) related but independent plasmids of this family can stably co-exist within a single ens cell ,143,149.ens cell ,134. Recplasmids ,151 and plasmids ,121,126,cpe gene on their chromosome vs. those carrying cpe on pCW3-like plasmids [Since pCW3 has a copy number of ~5 , the toxplasmids .C. perfringens conjugative plasmid family are the pCP13-like plasmids [cpb2 and bec . The pCP13-like plasmids are conjugative plasmids that contain a newly described conjugative locus (~27 kb) designated the “pCP13 Clostridium perfringens” (pcp) transfer locus [pcp locus encodes almost all of the key components required for transformation homologues of a Gram-positive type four secretion system (T4SS). Unlike the tcp locus, which is only found in C. perfringens, the pcp-conjugative locus appears to be evolutionarily related to the conserved conjugation system existing in other pathogenic clostridial species, such as Clostridium botulinum, Clostridioides difficile, and Clostridium sordellii [A second distinct plasmids , which cer locus . The pcpordellii .C. perfringens plasmid family includes the pIP404-like plasmids. These plasmids are not conjugative. Compared to the pCW3-like and pCP13-like plasmids, the pIP404-like plasmids are relatively small and usually harbor a bacteriocin-encoding gene (bcn), but no toxin genes [The third in genes .C. perfringens genome encodes >20 TCRS [To control their virulence factor production, many pathogens use two-component regulatory systems (TCRS) that consist of a membrane sensor and a cytoplasmic transcriptional response regulator. Consistent with this theme, the >20 TCRS ,112 and C. perfringens TCRS. It is encoded by an operon and consists of a VirS membrane sensor histidine kinase and a VirR transcriptional regulator [The VirS/VirR TCRS is the best characterized egulator . By reguegulator . Furtheregulator ,158, or egulator , this TCegulator .nd extracellular loop of VirS in signal sensing (see quorum sensing section). When this signal is received, VirS autophosphorylates and then activates VirR by transferring a phosphate onto a conserved aspartate residue located in the N-terminal region of VirR [cpa gene encoding CPA, lack VirR boxes in their promoters but are indirectly controlled by small regulatory RNA molecules such as VR-RNA, whose promoter does contain VirR boxes [Computer modeling predicts that the VirS protein consists of seven predicted transmembrane domains, several exposed extracellular regions, and a C-terminal tail ,162. The of VirR ,162. The of VirR ,164. Thi of VirR ,159,164.rR boxes .C. perfringens virulence. This TCRS controls the expression of several virulence-associated genes, including several degradative enzymes such as clostripain and sialidases [C. perfringens virulence in the mouse myonecrosis model [A second TCRS named RevS/R has also recently been implicated in regulating alidases . Consistis model .C. perfringens also uses density-sensing QS systems to regulate virulence factor production. The Agr-like system is the most important QS system of this bacterium for virulence. This QS system was identified when it was shown to control CPA and PFO production [C. perfringens to cause gas gangrene [C. perfringens intestinal diseases [agrB null mutants [C. perfringens [oduction ,174. Congangrene . Subsequdiseases ,170,171. mutants ,171 showfringens , which cC. perfringens, the agr operon contains four genes [C. perfringens, the native AIP appears to be a 5-amino-acid peptide in a thiolactone ring [An Agr-like QS system is found in several Gram-positive pathogens and uses an autoinducing peptide (AIP) to signal a classical TCRS . In C. pur genes ,174, incone ring ,176.agr operon of the classical S. aureus Agr system also encodes the AgrA/AgrC TCRS, where AgrC is the membrane histidine kinase that binds the active AIP and AgrA is the response regulator phosphorylated by activated AgrC [C. perfringens agr operon does not encode an AgrA/AgrC homolog. Since the production of several C. perfringens toxins, including PFO, CPA, CPB, and NetB, is co-regulated by both the Agr-like QS and VirS/VirR TCRS, it was hypothesized that the VirS membrane sensor protein is an AIP receptor for the C. perfringens Agr-like QS system [nd extracellular loop of VirS was shown to be involved in AIP binding [C. perfringens toxin production [The ted AgrC . HoweverS system ; this pr binding . A modeloduction ,175 and C. perfringens also possesses a LuxS/AI-2 QS system that may contribute to regulating the production of CPA and PFO [ and PFO . However and PFO ,172.C. perfringens uses several other transcriptional regulators to control toxin production. CodY is a global transcriptional regulator that senses nutrient availability by binding GTP or branched-chain amino acids [C. perfringens type D strain CN3718, CodY binds to the promoter region of the etx gene and positively regulates ETX production [Besides TCRS and QS systems, no acids . CodY bioduction . In thatC. perfringens global regulatory protein, acting as a repressor of CcpA-regulated genes in the presence of increasing glucose levels [CcpA (catabolite control protein A), a member of the LacI/GalR family, functions as another e levels ,181. It e levels  and bothe levels .C. perfringens sialidases is controlled by a complex regulatory network that includes the VirR/VirS TCRS, Agr-like QS system, ReeS, and CodY [reeS null mutation does not significantly affect virulence in the mouse myonecrosis model [Production of and CodY ,179,182.and CodY . Productis model .nan operon that encodes the complete pathway for transporting and metabolizing sialic acid [nanI expression [However, the major regulator of sialidase production identified to date is NanR, which is present in the lic acid ,183. In C. perfringens pathogenicity by enabling survival in harsh conditions like cooking of foods. Spores can also be important for the transmission of diseases like gas gangrene and type F food poisoning [As mentioned earlier, sporulation plays an important role in oisoning . Sporulaoisoning .C. perfringens sporulation begins when one or more sporulation-specific orphan histidine kinase senses still unidentified signals and phosphorylates Spo0A, which is a transcriptional regulator essential for initiating C. perfringens sporulation, i.e. an spo0A knockout mutant is unable to form spores [cpe gene from upstream SigE- or SigK-dependent promoters [m spores . An orphm spores . That stm spores ,188. Spom spores ,188. Howromoters .C. perfringens sporulation is a complex process subject to both positive and negative regulations. Besides the direct sporulation pathway regulators described above, the transcriptional regulators CcpA and CodY impact C. perfringens sporulation and CPE production [abrB gene expression patterns between codY-null mutants of SM101 and CN3718 [C. perfringens sporulation through a mechanism requiring further study. The Agr-like quorum-sensing system also participates in controlling sporulation since inactivation of the agrB gene in non-foodborne human GI disease type F strain F5603 reduced sporulation ~15,000-fold due, at least in part, to significant decreases in sigF and sigG expression [virX gene. This regulatory RNA significantly inhibits sporulation and CPE production by type F food poisoning strain SM101 [virX expression is directly controlled by VirR/S TCRS [oduction ,181,189,oduction ,189. Thepression . Anotherin SM101 . While vR/S TCRS , there iR/S TCRS .C. perfringens pathogenesis [Germination of spores also plays an important role in ogenesis . For exaC. perfringens spore sensing small molecules named germinants that are strain-specific and include such factors as amino acids, KCl, and phosphate [Germination begins with the hosphate . Those ghosphate . This rehosphate . Germinahosphate .C. perfringenss type A [Clostridial myonecrosis or gas gangrene is a highly lethal, necrotizing infection of skeletal muscle and subcutaneous tissue that is most commonly caused by s type A . The curs type A . With prs type A . If untrs type A . Amputats type A . Clostris type A . In the s type A ,196.C. perfringens type A. The latter is the main cause of gas gangrene in horses [Gas gangrene involves invasion of traumatic wounds by type A vegetative cells or spores, followed by vegetative cell multiplication and toxin production (see next section), which then induce rapid, severe, and extensive necrosis in the affected tissue . The disn horses .Figure 4Infections by type A strains. Although several gastro-enteric syndromes of animals have reportedly been associated with type A strains [ strains , their r strains .Possible exceptions are the type A isolates encoding the recently discovered NetF toxin. Those strains are suggested to be associated with canine hemorrhagic gastroenteritis and necrotizing enterocolitis in foals . The linInfections by type B strains. Type B infection is classically a disease of sheep, although rare cases have been reported in cattle and horses [C. perfringens infection causing both intestinal lesions and enterotoxemia, where toxins produced in the intestine not only act on the intestines but are also absorbed into the circulation and then affect distant organs like the brain.d horses . In sheed horses . Lamb dyC. perfringens type B was isolated from the feces of a human patient with multiple sclerosis (MS) [Recently, sis (MS)  and ETX sis (MS) . These fsis (MS) ,207, altInfections by type C strains. Type C strains cause necrotizing enteritis  to initiate GI disease [C. perfringens type F food poisoning starts with the ingestion of food containing large numbers of type F vegetative cells [in vivo multiplication, those bacteria sporulate and produce CPE in the intestines. At the completion of this in vivo sporulation, the mother cell lyses, which releases CPE (as well as the mature spore) into the intestinal lumen. The released CPE then binds to the intestines and exerts its action (as described later).ve cells . After aC. perfringens type F food poisoning outbreaks in psychiatric facilities have led to fatalities in relatively young and physically healthy people [This food poisoning typically involves diarrhea and abdominal cramps that develop within 12–24 hours and then self-resolve within a day [y people . Those fC. perfringens type F isolates also cause ~5-15% of all non-foodborne human GI disease cases, which include antibiotic-associated diarrhea (AAD) and sporadic diarrhea [C. perfringens type F food poisoning. CPE-associated AAD often occurs in the nosocomial environment and develops after the intestines of patients (particularly the elderly) receiving antibiotics become colonized by type F strains in the nosocomial environment and those strains then produce CPE in vivo. In contrast to type F food poisoning isolates, which often carry a chromosomal cpe gene and produce highly resistant spores, nearly all type F strains causing non-foodborne GI illnesses carry a plasmid-borne cpe gene and make relatively sensitive spores [diarrhea . CPE-asse spores .Infections by type G strains. Type G strains are responsible for necrotic enteritis (NE), one of the most prevalent diseases of poultry worldwide. Worldwide economic losses due to NE are estimated to be ~ (US) 5 USD billion [ billion .Eimeria spp. is the most common predisposing factor for NE [Under natural conditions, infection by r for NE ,228. Typr for NE .NE usually occurs in the form of outbreaks in 2–6 week-old broiler chickens , a fact The disease may be sub-clinical, affecting mostly weight gain, or clinical. In the latter, clinical signs are varied and include one or more of the following: reluctance to move, diarrhea, decreased appetite or anorexia, huddling, and dehydration . OccasioIn cases of acute NE, gross lesions are mainly seen in the jejunum and ileum, although the duodenum and ceca may also be affected. These effects consist of gas distention of the intestine, which is full of dark brown, semi-liquid content with fibrin strands, with an ulcerated mucosa covered by a fibrino-necrotizing membrane . Rarely C. perfringens immunohistochemistry, are observed in both intestinal and hepatic lesions [Microscopically , birds w lesions ,233.FiguC. perfringens genes [C. perfringens molecular pathogenesis is now being elucidated, particularly regarding virulence contributions of host colonization and the local or distant effects of its various toxins during disease, as discussed below.Improved technology to introduce targeted null mutations in ns genes  has revoin vivo, is an important feature of some C. perfringens infections, e.g. type F strain nonfoodborne human GI diseases. C. perfringens colonization typically involves nutrient acquisition for growth/survival and adherence. For example, type F nonfoodborne GI disease can persist for up to several weeks even in the face of diarrhea, indicating that intestinal adherence and nutrient acquisition must be necessary for these type F strains to stably colonize the intestines in the presence of diarrhea. However, for acute diseases such as type F food poisoning, nonadherent C. perfringens cells present in the lumen likely mediate disease. For type C disease, the picture is more complicated. Walker et al. reported [The ability to colonize, i.e. persist and proliferate/survive reported  that adhC. perfringens uses several molecular strategies, including the production of sialidases. While C. perfringens produces three sialidases (see virulence section), NanI is produced by the type F nonfoodborne GI disease strains that persist in the intestinal tract but not by most of the type F food poisoning strains that are associated with acute infections. That correlation suggests an important contribution of NanI to C. perfringens chronic intestinal disease [To colonize the GI tract,  disease ,236. ThiC. perfringens strains cause diseases originating in the intestines, this sialidase is present in the intestinal lumen, where it encounters host proteases such as trypsin and chymotrypsin. Interestingly, NanI is proteolytically activated by trypsin, chymotrypsin, and mouse intestinal fluids [C. perfringens strains.When NanI-producing l fluids . Amino al fluids . Those oC. perfringens intestinal adherence, NanI contributions to intestinal colonization likely also involve increasing nutrient acquisition. Several in vitro studies have supported NanI contributions to the growth and survival of C. perfringens strains associated with intestinal infections. For example, NanI was shown to support the in vitro growth and survival of type F nonfoodborne disease strain F4969 in the presence of host mucin or cultured Caco-2 cells [C. perfringens [Besides their effects on increasing -2 cells , with thfringens ,189. Sevfringens ,236,238.In vitro studies suggest that NanI also contributes to C. perfringens intestinal adhesion. Compared to wild-type C. perfringens type D strain CN3718, an isogenic triple mutant that does not produce any sialidases exhibits significantly reduced ability to adhere to cultured human enterocyte-like Caco-2 cells [C. perfringens adherence by modifying the surface of intestinal cells, allowing, 1) the exposure of an unknown receptor used for binding and/or 2) the reduction of negative charges of sialic acids on this surface [-2 cells . Restori-2 cells . It is p surface ,239,240.Besides promoting colonization, NanI may also impact intestinal infections by affecting toxin activity and production. This sialidase increases the ETX sensitivity of MDCK cells, the CPB sensitivity of HUVEC cells, and the CPE sensitivity of Caco-2 cells, with those effects due to NanI causing an increase in toxin binding levels ,237. ThiC. perfringens intestinal colonization, the involvement of NanI  in the context of histotoxic infections by this bacterium is less clear. In a murine myonecrosis model, a nanJ and nanI double mutant of C. perfringens strain 13 remained virulent, suggesting that sialic acid metabolism is not necessary for bacterial growth or persistence in skeletal muscle [C. perfringens, contributions of NanI (such as generating nutrients) during early infection may have been masked [Despite its role in l muscle . Neverthn masked .C. perfringens colonization. For example, it has been proposed that CNA contributes to enteritis in pigs by promoting adhesion to damaged intestinal tissue, based upon the prevalence of the cna gene in strains isolated from porcine cases [C. perfringens strains associated with severe intestinal disease in chickens are more capable of binding to collagen types II, IV, and V [C. perfringens for binding, and two Fn-binding proteins have been identified on this bacterium, i.e. FbpA and FbpB [C. perfringens is able to firmly bind to collagen, especially types II and III [C. perfringens may take advantage of Fn to facilitate host cell contact and further colonization.Other factors such as adhesins are also emerging as potential contributors to ne cases . MoreoveV, and V ,243. Recand FbpB . In the  and III ,246. As  and III , C. perfType A strains. During gas gangrene, CPA plays an essential role [ial role ,249 due ial role ,251, myoial role , leukostial role ,253, plaial role , vasoconial role , and inhial role . CPA alsial role .C. perfringens from phagosomes [PFO is also involved synergistically with CPA in myonecrosis  by contragosomes ,260, andagosomes ,258.Type B infections. The role of toxins in the pathogenesis of type B infections is complex and not fully understood, the general dogma being that intestinal lesions are produced by CPB, while central nervous system lesions, when present, are mediated by ETX [d by ETX . Becaused by ETX .Type C infections. The enteropathogenicity of type C strains is mediated by CPB, a highly necrotizing toxin, as demonstrated by fulfilling molecular Koch’s postulates in rabbit small intestinal loop models [p models ,262. Synp models .It is currently unclear which cell type in the intestines is affected first by CPB. Some evidence suggests that, early during infection, CPB affects the enterocytes of the small and large intestine . For exaType D infections. Molecular Koch’s postulate analyses have shown that ETX is the main virulence factor for C. perfringens type D strains to produce clostridial enterotoxemia in sheep, goats, and sometimes in cattle [C. botulinum and C. tetani toxins, activated ETX is the third most lethal clostridial toxin [n cattle . After Cal toxin .The action of ETX at the site of its production, the intestine, has not been well characterized. However, ETX is involved in the development of fibrinonecrotic enterocolitis in cases of enterotoxemia in goats , but notType E infections. Molecular Koch’s postulates for C. perfringens type E and ITX have not been fulfilled in any animal species, and diagnostic criteria for the disease have not been defined.Type F infections. Studies with cpe null mutants confirm that CPE production is essential for the GI pathogenicity of type F strains associated with food poisoning or nonfoodborne GI diseases [diseases .in vivo [ex vivo [CPE affects the small intestine, particularly the ileum, of all tested mammalian species . As showin vivo  and huma[ex vivo .Figure 8Type G infections. Historically CPA was considered the main virulence factor when type G strains cause avian NE, but it is now clear that CPA is not essential for this disease [netB null mutant failed to produce lesions typical of NE in experimental chickens [ disease . Instead disease , as estachickens  and NetBC. perfringens strains, some uncertainty remains because not all NE isolates have been found to carry the netB gene and a few C. perfringens strains isolated from clinically normal chickens also have this gene [C. perfringens type A, but not G, strains were isolated. Furthermore, at least one study reported that a netB-negative C. perfringens isolate reproduced necrotic enteritis when inoculated into chickens [Although it is currently accepted that NetB is the main virulence factor of NE-associated his gene . In addichickens . Based ochickens .During gas gangrene, potent toxins (particularly CPA) eventually reach the circulation. At that time, they can cause a toxemia involving organ failure, circulatory collapse, and death .C. perfringens infections involve toxin production in the intestine, followed by absorption of that toxin to cause enterotoxemias that involve organs such as the brain, lungs, and so on. The foremost example of an enterotoxemia-inducing C. perfringens toxin is ETX. After production and activation in the intestine, ETX opens tight junctions [Several other unctions , which munctions . It thenunctions , where tunctions . ETX alsunctions .cpb null mutant of a type C strain lost the ability to cause those effects, implying a major role for CPB in this type C enterotoxemia [Overwhelming evidence indicates that, once produced in the intestine, CPB (and perhaps other toxins made by type C strains) are also absorbed into the circulation and then act on distant organs such as the brain and lungs. For example, neurologic and respiratory signs were observed in mice challenged intragastrically with type C strains, even though those mice showed little intestinal histologic damage . Furtherotoxemia . In furtotoxemia . Furtherotoxemia .cpb null mutant failed to cause the development of these signs in this model. Similar lesions are frequently observed in foals and calves naturally diseased with type C strains [Serosal hemorrhages, pulmonary edema, and hydropericardium have also been described in a natural host, i.e. goats, experimentally infected with a type C strain . That pa strains ,210,216. strains .C. perfringens toxin thought to induce enterotoxemia. As mentioned earlier, type F infections in psychiatric patients can become fatal; this lethality has been attributed to the absence of diarrhea due to constipation side-effects of drugs taken by these people for their preexisting psychiatric illnesses. This absence of diarrhea is thought to prolong contact between CPE and the intestine, leading to CPE absorption into the circulation, where it then affects nonintestinal organs. Studies using a mouse intestinal CPE challenge model confirmed that intestinal CPE can be absorbed into the blood and induce enterotoxemia and that this effect causes a hyperpotassemia, which likely induces death by cardiac arrest [CPE is another c arrest .C. perfringens is a multi-talented pathogen, causing histotoxic infections, enteritis/enterocolitis, and enterotoxemias. This virulence versatility is attributable, in large part, to its ability to produce a panoply of potent toxins. Histotoxic infections are usually caused by relatively “simple” type A strains producing only CPA and PFO; this association probably reflects, at least in part, type A strains being the most common C. perfringens strains in the environment so they have the most opportunity to contaminate wounds. But is this the only explanation for this association?C. perfringens typically acquires additional toxin genes. Many of these toxin genes are associated with ISs and present on conjugative plasmids, which provides considerable virulence plasticity when causing disease originating in the intestines. However, there may be a limitation to using this toxin plasmid strategy for intestinal virulence, i.e. due to plasmid incompatibility issues, only certain combinations of toxin genes can be maintained in a single C. perfringens strain. C. perfringens partially compensates for this drawback by using ISs to accumulate multiple toxin genes on a single plasmid. Eventually, such toxin gene accumulation on a single plasmid could give rise to novel hypervirulent strains of C. perfringens that do not currently exist.To cause intestinal disease, C. perfringens even produce so many toxins, particularly so many different PFTs? One insight is provided by these PFTs recognizing different receptors, which possibly allows different strains to specifically target different cell types and/or organs, particularly during enterotoxemias. Another reason for producing so many PFTs was already mentioned for type B strains, i.e. these strains produce CPB, which is sensitive to trypsin, and ETX, which is activated by GI proteases including trypsin. Thus, by producing both CPB and ETX, a single type B strain can affect a range of hosts from neonatal animals taking colostrum to adult animals with normal trypsin activity. With that said, it appears there is some evolutionary disadvantage to producing both CPB and ETX since type B strains are rare. This may be related to the necessity to maintain both CPB and ETX plasmids in a single cell, since no single plasmid has yet been identified that carries both the cpb and etx genes.Why does cpe strains and type C Darmbrand strains appear to be evolving away from other C. perfringens strains, including type F strains with a plasmid cpe gene or most other type C strains. Not only do type C Darmbrand strains and type F chromosomal cpe strains cluster apart from other C. perfringens by MLST analyses of their housekeeping gene sequences, but they also produce much more resistant spores due to their variant SASP4 and they lack the genes encoding both NanI and PFO. These strains have clearly become adapted for foodborne transmission in humans and have acquired the ability to cause acute GI infection in this host.Interestingly, there can be considerable variation even among strains belonging to the same type and producing the same toxins. For example, type F chromosomal C. perfringens toxins themselves. For example, how does CPE damage the entire small intestinal villus even though it binds only to villi tips? Why is ETX so exceptionally potent? Why does ETX commonly cause intestinal pathology in goats but not other species? What is the connection between ETX and MS? What are the specific target cells of CPE, ETX and CPB during enterotoxemia? How do toxins like CPB and CPE cross the small intestinal epithelium? Do BEC, NetF, TpeL, CPD, and CPB2 contribute to pathogenicity, when produced? When produced by a single strain, is there any synergism between NetF and CPE in causing intestinal pathology in vivo? Last, close contact of C. perfringens with host cells upregulates the production of many C. perfringens toxins involved in enteritis or enterotoxemia [C. perfringens regulatory pathways are involved in this upregulation?Many important questions remain unresolved regarding otoxemia , but whaC. perfringens virulence factors are also now coming under study. It has become apparent that, beyond toxins, factors  contribute to intestinal colonization while other factors (such as EngCP) contribute to gas gangrene. However, these studies are still in their infancy so there are also many questions remaining about non-toxin virulence factors of C. perfringens: Are sialidases important for intestinal virulence? Do they contribute to early steps in gas gangrene? Which adhesins are important for C. perfringens attachment to host tissues, particularly in the mammalian intestines? Do capsules or biofilm formation help C. perfringens evade immune responses? How does the presence of mucus impact C. perfringens growth, toxin activity and attachment to host cells? What triggers C. perfringens type F strain sporulation and CPE production in the intestines?Besides toxins, other These questions will require further study to fully understand the pathogenicity of this intriguing and important pathogen.


Gonimbrasia zambesina, Gonimbrasia krucki, Bunaea alcinoe, Gonimbrasia cocaulti, Gonimbrasia belina, Gynanisa nigra and Cirina forda. These caterpillars mostly occur twice a year during the rainy seasons and feed on specific host plants. Predictive distribution models revealed that B. alcinoe, and C. forda are mostly found in tropical and sub-tropical regions in Africa. However, climate change could cause a slight decrease in their population by the year 2050. This information will guide conservation efforts and ensure sustainable use of edible saturniid caterpillars as food.Edible insects are a traditional food source with economic benefits in sub-Saharan Africa. Caterpillars are the most popular edible insects in this region. We focus on caterpillars in the family Saturniidae. Saturniids are big colorful caterpillars with spines on their bodies, usually found in shrubs and trees. They are rich in proteins, vitamins, and minerals. Despite their economic importance, little is known about their diversity, host plants, distribution, and potential effect of climate change on edible saturniid caterpillars in Africa. The aim of this study is to identify edible saturniids, their host plants, their current distribution and to predict the possible effects of climate change on their distribution. We documented seven species of edible saturniids namely Gonimbrasia zambesina, Gonimbrasia krucki, Bunaea alcinoe, Gonimbrasia cocaulti, Gonimbrasia belina, Gynanisa nigra and Cirina forda. Two morphotypes of G. zambesina and B. alcinoe were recorded. These saturniid caterpillars occur twice a year except for G. cocaulti. Predictive models revealed that tropical and subtropical regions were potentially suitable for B. alcinoe and C. forda. The information generated from this study would be important to guide conservation efforts and their sustainable utilization as food in Africa.The promotion of edible insects, including saturniid caterpillars as potential food source is widely gaining momentum. They are adequately rich in nutrients such as proteins, amino acids, fatty acids, and micronutrients. Despite saturniids being a traditional food source with economic benefits, information on their diversity, host plants and their potential distribution in Africa are lacking, which this study seeks to address. Edible saturniids and their host plants were characterized using specific primers . Maximum entropy (MaxENT) and GARP  models were used to characterize the potential distribution of commonly consumed saturniids under current and future climate scenarios. Seven species of saturniids were recorded from 11 host plants in Kenya:  The Food and Agriculture Organization of the United Nations has termed edible insects as one of the solutions to curb food insecurity . Edible Globally, more than 2 billion people consume over 2000 species of edible insects . About 6Gonimbrasia belina (Westwood). The larvae of Go. belina is an important food commodity among rural communities that live around the mopane woodland, i.e., the mopane belt across Angola, Zambia, Zimbabwe Mozambique, South Africa and Botswana [Cirina forda (Westwood). Its larvae are considered an important food source for many rural communities in Zimbabwe, Nigeria, Togo, Ghana, Zambia, D.R. Congo, Central African Republic, and South Africa [Edible saturniids are characterized by big larval forms with spines on the surface which pupate into cocoons  that areBotswana . They haBotswana . Anotherh Africa ,25,26.Colophospermum mopane Kirk ex J. Léonard (Fabaceae) [C. forda show higher preference for the shea butter tree, Vittelaria paradoxa C.F. Gaertn (Sapotaceae) in West Africa [Edible saturniid species have been observed to inhabit different bioecological zones with considerable seasonal variability, as well as having high specificity and preference to various host plants. For example, in southern and eastern Africa, they are known to occur in large outbreaks in arid and savannah regions ,14,27. Eabaceae)  while, Ct Africa ,33.Edible saturniid caterpillars are highly nutritious, providing vital vitamins, lipids and proteins and microelements to households, especially women and children ,34,35,36The study was carried out between March 2017 and May 2019 across different agro-ecological zones ,39 and aicipe, Nairobi, Kenya, for rearing and identification. Twigs of host plants were collected and pressed in a herbarium for identification. Sampling was done for five rainy seasons between March 2017 and May 2019, both long (March to May) and short rains (October–December), in Kenya.Saturniid larvae were sampled at random along motorable roads from 15 sites  in KenyaField-collected Saturniid larvae were reared at 12 h:12 h photoperiod at 25 °C in Perspex cages (50 × 50 × 50 cm) with nets on the sides for ventilation. Cages were protected from ants and crawling insects with traps containing water placed below its metallic stand. The saturniid larvae were fed on twigs of their respective host plant from the field. The twigs were placed in a container as bouquet with stem immersed in water fastened by wet cotton wool. The twigs were changed daily to keep them fresh. The larvae fed until they reached the pre-pupal stage, when they stopped feeding, reduced movement, and moved to the floor of the cage ready to burrow and pupate. The pre-pupae were placed on moist sterile sawdust in plastic trays and allowed to burrow into the sawdust to pupate as they burrow in soil in the wild. The saw dust was kept moist by sprinkling water daily. The trays with the pupae were placed in Perspex cages (50 cm × 50 cm × 50 cm) awaiting emergence of adult moths.icipe. Host plants were identified at the Kenya Forest Research Institute (KEFRI) by an experienced plant taxonomist using available literature [The adult moths were killed by placing them in a container and freezing them at −5 °C. They were stretched out, pinned, allowed to dry, and labeled before morphological identification. Identification was carried out using published keys  and crosterature .Leaf samples of each host plant of saturniid caterpillar were carefully washed with tap water, rinsed with distilled water and dried with paper towel. They were then cut into 0.2 g of leaf sample small pieces with a sterile blade and placed in a 2 mL tube containing ceramic beads, lysis buffer PA1  and RNAse A and crushed for 3 min in a Tissue lyser II . Plant genomic DNA was extracted using Isolate II Plant DNA extraction Kit  as per the manufacturer’s instructions.A leg of each adult moth and/or a portion of larvae collected were cut with a sterile blade and placed in a 2 mL tube. Insect genomic DNA was extracted using Isolate II genomic DNA extraction kit  as per the manufacturer’s protocol. The resultant DNA was eluted in 50 µL Elution buffer  and quantified using a NanoDrop 2000/2000 c spectrophotometer . Insect and plant DNA samples were stored at −20 °C for further downstream processing.For insect identification, PCR was conducted using general insect DNA barcoding LepF1/LepR1 primers  . IsolateGeneral plant primers 3F_KIM_F/1R_KIM_R  were used to amplify a 900 bp region of the matK gene for the identification of host plants. Protocols for PCR of plant samples were similar to the PCR protocol for insect samples , except Resolution of the PCR product was done with 1% agarose gel stained with ethidium bromide (10 mg/mL) at 80 volts for 1 h . DNA bands were visualized using an ultraviolet transilluminator and photographed using the KETA GL imaging system software . The resultant PCR products for both the insects and the host plants were purified using QIAquick PCR purification kit  and quantified with a NanoDrop 2000/2000 c spectrophotometer  before being sent for bidirectional sequencing at Macrogen Inc. .https://www.ncbi.nlm.nih.gov/WebSub/) (accessed on 10 March 2020) (see Both plant and insect sequences were assembled and edited using Bioedit software v. 7.0.5.2 . A conse020) see .B. alcinoe and C. forda, because of their popularity and economic importance in sub-Saharan Africa (SSA). Cirina forda and B. alcinoe are widely consumed [Two species of edible saturniids were selected for distribution modeling, i.e., consumed ,49,50,51consumed ,52,53,54B. alcinoe, while the C. forda dataset comprised 70 points (57 from field surveys and 13 from GBIF)  for the two species was collected during field surveys in Kenya and from the Global Biodiversity Information Facility (GBIF). A total of 96 points a were acom GBIF) b.www.worldclim.org) (accessed on 15 April 2021) were considered for the study. We obtained current climate data for the period 1970–2000 at 30 arc seconds longitude/latitude degree spatial resolution (approximately 1 km at the equator). For future analysis, the downscaled and calibrated horizon 2050 IPPC (CMIP5) of climate projection bioclimatic variables were extracted from HadGEM2-ES global climate model (GCM) representing representative concentration pathways (RCP8.5) for future climate scenarios set by the intergovernmental panel on climate change (IPCC) [B. alcinoe species analysis, while seven bioclimatic variables namely, Bio2, Bio4 , Bio8 (mean temperature of wettest quarter), Bio13, Bio15, Bio18 (precipitation of warmest quarter), and Bio19 were selected for the C. forda species analysis using a cutoff of |r2| > 0.7. Aside from the spatial correlation, the ecological relevance of the variables was also considered.Nineteen bioclimatic variables downloaded from WorldClim (e (IPCC) . RCP8.5 e (IPCC) . The vare (IPCC) . Six bioC. forda and B. alcinoe was evaluated using the overall percentage contribution, area under the curve (AUC), and the Jackknife test. AUC values of 0 indicate impossible occurrence area while 1 indicates optimal occurrence area. The ROC method has shown to be effective in evaluating model performance and being independent of prevalence [Ecological niches of the two species were modeled using Maximum Entropy (MaxEnt) in the MaxEnt tool package version 3.4.1k which performs well for modeling presence only data . The ENMevalence ,58. OutpB. alcinoe, C. forda, Gonimbrasia zambesina , Go. cocaulti Darge and Terral, Go. belina Westwood, Go. krucki (Hering) and Gynanisa nigra Bouvier. Dead larval stages of Gonimbrasia belina collected from Botswana and a sample of B. alcinoe collected from Nigeria were included for comparison . However, the author expressed uncertainty and suggested that it could be a form of G. zambesina. The green forms were collected from Kilifi, Embu and Kwale, while a mixture of the green and brown forms was collected from Makuyu in Murang’a County . The green moths from Kwale and Kilifi produced larvae that were black with grey and yellow speckles and black spines  and GBMNC60687-20 , respectively. Gonimbrasia zambesina sequences were 99.09–100% similar to SAPBA773-07 ) while Go. krucki was 98.22–100% similar to SAPBA635-07 ). Cirina forda had 99.38–99.85% similarity to SATWA281-07  and STBOB620-08 . Gonimbrasia cocaulti sequences were >98% similar to SPBIS152-09 . Bunaea alcinoe sequences from samples collected in Kenya were 98.78–100% similar to LSAFR2238-12 from South Africa  (All the saturniid species showed 98.22–100% similarity to sequences in BOLD. AAA6757) .Gynanisawestwoodi and G. belina collected from Botswana were both >99% similar to STBOA580-07 (BIN-BOLD: ABY462) and SATWA003-06 (BIN-BOLD: AAB6786), respectively. Bunaea alcinoe sequences from sample collected in Nigeria was 100% similar to SATWA891-07  (AAA6756) .Gonimbrasia krucki and B. alcinoe-Nigeria species had a 100% similarity to sequences from BOLD database. Gonimbrasia zambesina had a within species pairwise distance range of 0–0.61 and a range of 0–0.91 between species and BOLD sequence SAPBA773-07 (BOLD:AAD1339)  and STBOA580-07 (BOLD:ABY4629), respectively  (AAD1339) . Gonimbrectively . GonimbrAAB6982) .Go. zambesina larvae and moths depicted different color forms morphologically, they identified as the same species using molecular characteristics. The genetic distance between all the Go. zambesina samples and the reference Go. zambesina sequence from BOLD  was 0–0.61% while the genetic distance between all the samples and Go. zambesina sequence from BOLD (SAPBA772-07) was 1.07–1.23%. The genetic distance between the samples was 0–0.61%. All the samples included in the analysis had 658 bp  was 100% similar to SATWA891-07-COI-5P  from Burkina Faso. However, the same BOLD sequence had a 3.28–3.76% genetic distance from all the other black larvae forms of B. alcinoe collected from Kenya  from South Africa. The same BOLD sequence had a genetic distance of 3.76% from the red color form collected from Nigeria. All the black forms clustered together with LSAFR2238-12  while the red form clustered with SATWA891-07-COI-5P . All the samples included in the analysis had 658 bp  showed a balance between goodness-of-fit and complexity (AUC > 0.80), for all the test and training datasets . This deB. alcinoe  P.J.H. Hurter and Mabb (Fabaceae), while B. alcinoe was feeding on Balanites aegyptiaca Linn (Zygophyllaceae) and Balanites glabra Mildbr. and Schltr. (Zygophyllaceae). Gonimbrasia cocaulti also fed on Vachellia tortilis (Forssk.) Galasso and Banfi (Fabaceae), while Go. zambesina and Go. belina on Anacardium occidentale Lin (Anacardiaceae) and Go. zambesina as well on Mangifera indica L. (Anacardiaceae). Gonimbrasia krucki was only observed feeding on Schinus terebinthifolia Raddi and Schinus molle L. (Anacardiaceae) while C. forda seemed to have a broader host range with Euclea divinorum, Acacia mearrnsii De Wild (Fabaceae) and Manilkara sulcata Engl. (Sapotaceae) (The caterpillars were observed to show very distinct host plant specificity across the various sites surveyed. We observed that otaceae) .E. divinorum, A. occidentale, M. indica, Balanites sp., V. tortilis, Manilkara sp., A. mearnsii, S. terebinthifolia and V. nilotica had a 97.06–100% similarity to GenBank sequences  [G. said [The sampled reported  and in her 2019) . The bro[G. said , but inc[G. said ,65, main[G. said  as well C. forda, with larvae occurring in April–June and October–December. In contrast, C. forda has been recorded as univoltine in Togo and Nigeria with larval occurrence between July and September [C. forda larvae coincides with the rainy seasons. For, B. alcinoe we noted a bivoltine lifecycle with larval appearance between April–May and October–December. In contrast, the same species in DR Congo is univoltine and occurs between October and May [We observed a bivoltine lifecycle in eptember ,29 and ieptember . In all  and May . UnderstB. alcinoe and C. forda demonstrates that the two species thrive well within the tropical regions of Africa. However, B alcinoe spreads slightly into the subtropics, specifically in southern Africa. The model concurs with previous reports of availability and consumption of the two edible saturniids in southern, central and western Africa [C. forda in the southern African region is not concurrent with previous reports [The model for habitat suitability of n Africa ,51,59,66 reports ,23 whichB. alcinoe feeding on B. aegyptiaca and B. glabra similar to reports in Nigeria [Sarcocephalus latifolius (JE Sm.) EA Bruce (Rubiaceae), Acacia auriculiformis A. Cunn. ex Benth. (Fabaceae), Dacryodes edulis (G. Don) H.J.Lam. (Burseraceae), Crossopteryx febrifuga (Afzel. ex G.Don) Benth. (Rubiaceae) and Anthocleista schweinfurthii Gilg (Loganiaceae) [Holarrhena floribunda (G. Don) Durand and Schinz (Apocynaceae), Ekebergia sengalensis A Juss. (Meliaceae), Fragraea fragrans Roxb. (Gentianaceae), Cleistopholis patens (Benth.) Engl. and Diels (Annonaceae) and Spondias mombin L. (Anacardiaceae) [C. forda feeding on E. divinorum, A. mearnsii and Manilkara sulcate, while in DR Congo it feeds mainly on Crossopteryx febrifuga [C. forda is confined on the shea butter tree, Vittelaria paradoxa [Burkea africana Hook (Fabaceae) and Albizia versicolor Welw. ex Oliv. (Fabaceae) [The saturniids identified fed on specific host plants and consequently their availability depends on the occurrence of these host plants. We found  Nigeria . Howeverniaceae) , and in diaceae) . In our ebrifuga . In Westparadoxa ,29,33, wabaceae) .Go. zambesina from mango and cashew nut trees in Kenya, corroborating earlier findings [Gonimbrasia zambesina is sometimes considered a pest of mango trees [Go. zambesina larvae feeding on the leaves. We collected findings . Cashew go trees . SprayinE. divinorum is utilized by the Maasai in Kenya as firewood, their stem cuttings are used as toothbrushes and their fruits are edible [E. divinorum as anti-venom [V. tortilis and V. nilotica for firewood [Balanites spp. are used to treat coughs [A. mearnsii is often planted for firewood, timber, apiculture and a source of tanning dyes, and trees are also used for shade, nitrogen fixation and controlling soil erosion [Apart from being host plants for edible insects, most of these plants have other uses in communities in Kenya and beyond. For example, e edible . Marakweti-venom  while thti-venom . Maasai firewood  while thfirewood . Balanitt coughs , and fin erosion . Such trB. alcinoe and C. forda, were mapped. This information may help in implementing conservation measures for edible saturniids and their host plants. Due to their seasonal occurrence, further research is required on prospects for mass production to ensure a continuous supply and to prevent overharvesting from the wild forest for enhance sustainability. Moreover, potential economic benefits of edible saturniids for local communities in East Africa need to be quantified and their value chains established.We successfully documented seven species of saturniids in Kenya, among which three are consumed. The identity of these species was confirmed both at molecular and morphological level. Their distribution, seasonality and host plants were also established. We emphasize the importance of combining molecular barcoding, morphological identification, phenology, and ecology studies in identification of edible saturniid species. Potential habitats under current and future climate scenarios of two edible saturniid species,


Hedysarum polybotrys var. alaschanicum is an important medicinal plant and is widely used in traditional Chinese medicine. The complete chloroplast genome of H. polybotrys var. alaschanicum was assembled from Illumina pair-end sequence reads. The whole chloroplast genome is 122,933 bp in length and encodes a total of 110 genes, including 76 protein-coding genes, 30 tRNA genes and 4 rRNA genes. The overall GC content of the cp genome is 35.3%. A maximum likelihood (ML) phylogenetic analysis revealed that H. polybotrys var. alaschanicum was close to Hedysarum semenovii. Hedysarum polybotrys Hand.-Mazz. var. alaschanicum (B. Fedtsch.) H. C. Fu et Z. Y. Chu, a perennial leguminous herb, is widely used in traditional Chinese medicine to strengthen the body  to provide genomic data for future research.H. polybotrys var. alaschanicum was collected in Chunkun Mountain, Baotou, Inner Mongolia, China . The specimen was deposited at the herbarium of Baotou Teachers’ College  under the voucher number CKS-2020-07-B06KYYHQ. Total genomic DNA was isolated from silica-dried leaf material using modified CTAB method  was used as reference genome. The complete cp genome was annotated in GENEIOUS R11 , and then manually corrected by comparing with the published complete cp genomes  of Hedysarum species using GENEIOUS R11.An individual of od Doyle , and theH. polybotrys var. alaschanicum is 122,933 bp in length. Different from typical quadripartite structure of most angiosperm cp genomes, the cp genome of this species lacks inverted repeat region. The cp genome encodes a total of 110 genes, consisting of 76 protein-coding genes, 30 tRNA genes, and 4 rRNA genes. The overall GC content of the cp genome is 35.3%.The complete cp genome of H. polybotrys var. alaschanicum was close to Hedysarum semenovii with high support . All of the 11 complete cp genome sequences were aligned using MAFFT (Katoh and Standley  support . The cp


Mastomys natalensis) as a natural reservoir of LASV, little effort has been made to characterize LASV infection in its reservoir. The natural route of infection and transmission of LASV within M. natalensis remains unknown, and the clinical impact of LASV in M. natalensis is mostly undescribed. Herein, using an outbred colony of M. natalensis, we investigate the replication and dissemination dynamics of LASV in this reservoir following various inoculation routes. Inoculation with LASV, regardless of route, resulted in a systemic infection and accumulation of abundant LASV-RNA in many tissues. LASV infection in the Natal multimammate mice was subclinical, however, clinical chemistry values were transiently altered and immune infiltrates were observed histologically in lungs, spleens and livers, indicating a minor disease with coordinated immune responses are elicited, controlling infection. Intranasal infection resulted in unique virus tissue dissemination dynamics and heightened LASV shedding, compared to subcutaneous inoculation. Our study provides important insights into LASV infection in its natural reservoir using a contemporary infection system, demonstrating that specific inoculation routes result in disparate dissemination outcomes, suggesting intranasal inoculation is important in the maintenance of LASV in the natural reservoir, and emphasizes that selection of the appropriate inoculation route is necessary to examine aspects of viral replication, transmission and responses to zoonotic viruses in their natural reservoirs.Lassa virus (LASV), a Risk Group-4 zoonotic haemorrhagic fever virus, affects sub-Saharan African countries. Lassa fever, caused by LASV, results in thousands of annual deaths. Although decades have elapsed since the identification of the Natal multimammate mouse ( Lassa fever (LF) is an often fatal haemorrhagic fever in humans, and Lassa virus (LASV) was identified as the etiologic agent of LF over 50 years ago ,2. LF isMastomys natalensis), living peri-domestically and abundant throughout much of Sub-Saharan Africa, was elucidated as a LASV natural reservoir rapidly after LASV’s initial discovery . No. NoM. na, evidenced by the presence of LASV-RNA in all sampled organs, far exceeding input RNA levels  were especially supportive of viral replication, independent of the inoculation route, while lungs and livers were secondary LASV hot-spots, even by inoculation routes not directly targeting these organs. However, viral loads in these secondary hot-spots were distinct, especially in s.c.-inoculated animals.In i.p.-inoculated animals, organs involved in excretion, residing in the abdominal cavity , displayed, expectedly, LASV-RNA by 3 dpi, while the same organs had detectible LASV-RNA in i.n.- or s.c.-inoculated animals beginning at 7 dpi. The LASV-RNA titres in the gonads, independent of sex, followed excretion organ values within respective inoculation groups. I.p.-inoculated animals uniquely had reduced clearance of LASV-RNA from the gonads.Based on the experiment’s temporal resolution, peak viral burden was at 7 dpi for all inoculation routes, after which LASV-RNA levels decreased. An exception was seen in the brains of i.n.-inoculated animals, where the measured mean LASV-RNA loads peaked at 14 dpi. Nevertheless, all inoculation routes were delayed in the clearance of LASV-RNA from lungs and lymphoid tissues up to the final 28 dpi time point.M. natalensis, oral and rectal swabs and urine of the animals were analysed for LASV-RNA and viable virus. Over the course of infection, LASV-RNA was detected at low titres (up to 10²–10³ FFU eq.) in oral and rectal swabs from several animals (r = 0.70) spleens (r = 0.66), and livers (r = 0.62), while rectal shedding correlated most closely with skin (r = 0.69) (only sampled for s.c.-inoculation), rectum (r = 0.51) and reproductive organs (r = 0.47). Interestingly, oral or rectal RNA-shedding was not correlated with viremia levels.Transmission is necessary for viral maintenance in reservoir populations. To assess whether various inoculation routes result in differential shedding of LASV in  animals . Oral swI.n.-inoculated animals consistently had the most LASV-RNA in oral swab samples at 7 and 14 dpi, while s.c.-inoculated animals had positive oral swabs only at 14 dpi. Viral RNA loads in oral swabs, when present, were of similar magnitude when compared between i.n. and i.p.-inoculated animals. Only few rectal swabs were positive for LASV from animals inoculated i.n. or i.p., while no rectal swabs of s.c.-inoculated Natal multimammate mice tested positive.0 to 104 FFU eq./mL in urine or bladder swab samples, correlating with the viral burden in kidneys (r = 0.73) and blood (r = 0.55). All successful LASV isolations originated from bladder swabs instead of urine, two from i.p.-inoculated animals at 3 dpi, the two other virus-positive bladder swabs were from animals euthanized at 7 dpi . Irrespectively, virus isolation attempts from RT–PCR-positive oral and rectal swabs were all unsuccessful. Of the few successful LASV isolations from bladder swabs, corresponding lower limits of input for viral isolation were 3×10−1, 4×101, 4×102 and 1×103 FFU eq., according to RT-qPCR, demonstrating no discernible cut-off since the overwhelming magnitude of isolations from urine, oral and rectal swabs in the RNA range of 3×101–4×102 FFU eq. were unsuccessful.Urine is reported as a putative vehicle for LASV transmission, but published virological data on this topic is incomplete. Virus isolation attempts from RT–PCR-positive bladder swabs and urine from this study upon euthanasia were mostly unsuccessful . InfectiWe observed extensive LASV replication in the Natal multimammate mouse reservoir, which was subclinical with clinical analytes exhibiting only short-lived alterations . To inveM. natalensis was identified as a primary natural reservoir of LASV, along with other Muridae species [LASV is disproportionally understudied compared to viral haemorrhagic fevers that cause fewer annual morbidities and less mortality. More than 40 years ago,  species ,36, yet M. natalensis [M. natalensis population encountered by humans [Herein, we develop a contemporary LASV infection system for this natural reservoir using a genetically heterogenous outbred breeding colony to investigate the dissemination of LASV in adult talensis . Acute iy humans . We showM. natalensis against LASV-induced disease [M. natalensis. I.n.-inoculated animals resolved infection with delayed clearance from lungs, spleens and the cLNs, while i.p.-inoculated animals struggled to clear the viral RNA from a multitude tissues as late as 28 dpi. No specific sex-dependent differences were observed; however, more study subjects likely are required to reliably identify such effects.The primary inoculation sites of the i.p. and s.c. routes were regions of early LASV-RNA accumulation, exceeding the inoculum of 10³ FFU in each animal, indicating local replication. Yet, deep lung tissue of i.n.-inoculated animals did not show this early accumulation, since early replication likely occurs in the upper respiratory tract, which was not sampled in this study. I.p. inoculation, analogous to intravenous administration, is a classical route for disease modelling to facilitate rapid and systemic virus spread and replication, often resulting in pathogenesis , making  disease ,28,34. WM. natalensis were lower than for either i.p. or i.n. inoculation, except for early brain and cLN levels, but viral clearance was similar to that for i.n. inoculation. Only i.p. inoculation of M. natalensis is published, however, the LASV-related arenaviral lymphocytic choriomeningitis virus (LCMV) also readily accumulates in lymph nodes after s.c. inoculation of its natural reservoir, Mus musculus, but with less splenic involvement when compared to intravenous LCMV inoculation [Peak LASV-RNA titres in organs of s.c.-inoculated culation .M. natalensis regarding LASV infection. Differential inoculation routes likely lead to disparate initial immune cells targets. Entry via the mucosa during i.n. inoculation involves lung epithelia and alveolar macrophages, which have evolved to facilitate immune responses against invading pathogens. I.p. inoculation circumvents this and might also target infection of immature peritoneal monocytes, for which the potent type I interferon antagonism of LASV [These differences in viral loads and dynamics highlight that a consideration for inoculation route is crucial for virological or immunological questions that remain to be addressed in  of LASV  might leM. natalensis can potentially easily be transmitted onto the mucosa of naïve adult animals within a colony. We observed oral shedding of LASV RNA in adult M. natalensis after infection, however, intradermal or s.c. inoculation of animals by biting is less likely to be a route of transmission within populations, since the required intraspecies aggression is less common in M. natalensis [M. natalensis after i.p. Morogoro virus inoculation [Similar to i.p., i.n. inoculation of Natal multimammate mice consistently caused a systemic infection, despite the necessity of LASV to transverse evolutionary-adapted immune barriers of the respiratory tract. This route likely mimics a natural mode of inter-rodent transmission in the wild, as infectious excretions of talensis . Additioculation .We hypothesize that nasal mucosal exposure could be an important entry site in nature. Oral swabs of i.n.-inoculated animals displayed high viral RNA loads similar to i.p. inoculation. Interestingly, despite the vicinity of the s.c. inoculation in the nuchal fold to salivary glands, s.c. inoculation resulted in relatively little oral viral RNA shedding late during the infection. Rectal LASV RNA shedding was observed for i.n.-inoculated animals, while s.c.-inoculated animals consistently tested negative, again showing important differences between inoculation routes, with i.n. inoculation allowing for potential virus transmission, whereas s.c. inoculation would be unlikely to allow for transmission, due to the low or absence of LASV shedding.M. natalensis were consistently negative. While urinary bladder swabs only made up 17.9% of our virus isolation attempts, they comprised all of the successful virus isolations. LASV isolation from urine of adult M. natalensis was previously demonstrated in one report from a single low-titre urine sample late in infection of an adult animal [in utero or postpartum infection [M. natalensis, infected as adults, and stress the need for comprehensive studies on this topic. The age of the animals at the time of infection should be emphasized. It has been repeatedly shown that M. natalensis and other rodents that are infected with arenaviruses in utero, perinatally or in neonatal stages postpartum, remain persistently infected and shed virus in urine for long periods [M. musculus resulted in prolonged shedding in the urine [M. natalensis and remains a potential source of rodent-to-human transmission. For the LASV-related Morogoro virus, horizontal transmission to neonates of M. natalensis from persistently infected individuals reduced the chance of persistent infection with this LASV-related arenavirus by 20–60% compared to s.c.-inoculation at day two after birth [Virus isolation attempts from urine of adult t animal , while cnfection ,43. Thes periods , which a periods . Likewishe urine . Therefoer birth . LikewisM. natalensis has been predominantly described as low to non-pathogenic due to co-evolutionary virus-host adaptations. While wild-caught individuals of M. natalensis infected with LASV displayed higher ratios of pathophysiological changes compared to LASV-free animals [M. natalensis, despite no significant influences of LASV infection on the clinical status, such as body weight changes or signs of disease. Since the experiments were conducted with the well-studied Josiah strain of LASV (a human isolate) with passaging on Vero E6 cells, an attenuation of the virus in a Natal multimammate mouse host due to a prior primate adaptation cannot be fully excluded. However, recent preliminary infection experiments with a LASV strain isolated from a Natal multimammate mouse also showed no alteration of the clinical status of the animals [LASV infection of  animals , helmint animals . Our exp animals . AdditioM. natalensis in this study show similarity to the infection course of its arenaviral cousin LCMV in its reservoir host, which induces transient disease following infection, and is cleared within 7–10 days [in utero [M. natalensis differed. S.c. inoculation resulted in less pronounced histological changes, while i.p. and i.n. inoculations of LASV displayed more widespread alterations. Future studies of additional organs and time points and staining targets will provide a detailed characterization of potential tissue pathology and resolution.The pathological changes seen in the histological examination of LASV-infected –10 days  (dependein utero . Dependein vivo models, such as mice and guinea pigs, established as lethal pathology models, results in histological changes of a similar type as the infected Natal multimammate mice in this study; with interstitial pneumonia, eosinophilic red pulp of the spleen and immune infiltrations and diffuse apoptosis in the liver, as well as serum AST and ALT peaks between 7 and 9 dpi [I.p., s.c. and aerosol inoculation with LASV in other rodent nd 9 dpi ,49–51. HM. natalensis, showing a systemic subclinical infection with histopathologic immune-related responses. Furthermore, we discovered that the inoculation route influences the clinical parameters, viral dissemination in the organs and shedding of viral RNA, leading to important implications for the study of natural infection and transmission within M. natalensis populations and to humans.The increasing emergence and re-emergence of zoonoses highlight the need for an understanding of how zoonotic viruses interact with their animal reservoirs. We, therefore, present a detailed examination of the course of LASV infection in adults of