the C. perfringens Isolated from Broiler Chickens (R. D. Eraky and W. A. Abd El-Ghany) 1 INTRODUCTION Enteric diseases are very important in the poultry industry as they lead to production loss- es, mortalities, and risk of contamination of poul- try products (Dahiya et al., 2006). Infection with Clostridium perfringens (C. perfringens) is con- sidered one of the most critical enteric problems in chickens and causes necrotic enteritis (NE) (Cooper et al., 2013). The first case of NE in fowl was reported in Australia in 1930 and was Genetic characterization, antibiogram pattern, and pathogenicity of Clostridium perfringens isolated from broiler chickens with necrotic enteritis 1 R. D. Eraky and 2* W. A. Abd El-Ghany 1 Department of Bacteriology, Animal Health Research Institute (Damietta branch), Damietta, Egypt 2 Department of Poultry Diseases, Faculty of Veterinary Medicine, Cairo University, Giza, Egypt * Corresponding E-mail: wafaa.soliman1974@gmail.com Received June 14, 2021; Accepted October 24, 2021 ABSTRACT The aims of this investigation were characterization, demonstration of the antibiogram pattern and detection of the pathogenicity of Clostridium perfringens (C. perfringens) strains isolated from broiler chickens in Damietta governorate, Egypt. A total of 357 samples representing 202 intestinal contents and 155 liver samples from freshly dead broiler chickens were collected from 18 broiler farms. Isolates of C. perfringens were identified morphologically, microscopically, and biochemically. Forty-seven C. perfringens isolates were recovered, which represented 20.3% of the intestinal contents and 3.8% of the liver samples. The toxins and virulence genes of C. perfringens were investigated using polymerase chain reaction. All of the toxigenic C. perfringens strains were type A and carried netB, tpeL, cpe, and plc genes. The in vitro antibiogram of C. perfringens strains revealed 100% sensitivity to gentamycin and levofloxacin and 100% resistance to nalidixic acid and ceftriaxone. The isolated C. perfringens strains were highly pathogenic and induced signs and lesions of necrotic enteritis as well as 43.3% mortalities in 20-day-old chicks. In conclusion, C. perfringens is an important pathogen that affects broiler chickens due to the presence of virulence genes and the pathogenicity in the inoculated birds. Keywords: Antibiotics, C. perfringens, PCR, Poultry, Toxins fully investigated in England (Parish, 1961). Lat- er, the disease spread rapidly in almost all poul- try-producing countries around the world (Finken and Wages, 1997). The disease causes severe economic problems represented by low feed conversion rate, mortalities, and increased treatment costs (Cooper and Songer, 2009). The production losses due to NE outbreaks in the global poultry industry are estimated to be US $6 billion annually (Moore, 2016). The main sources of NE infection are litter and contami- J I T A A Journal of the Indonesian Tropical Animal Agriculture Accredited by Ditjen Riset, Teknologi dan Pengabdian kepada Masyarakat No. 164/E/KPT/2021 J. Indonesian Trop. Anim. Agric. pISSN 2087-8273 eISSN 2460-6278 http://ejournal.undip.ac.id/index.php/jitaa 47(1):1-16, March 2022 DOI: 10.14710/jitaa.47.1.1-16 mailto:wafaa.soliman1974@gmail.com 2 J. Indonesian Trop.Anim.Agric. 47(1):1-16, March 2022 nated environment (Craven et al., 2003; Profeta et al., 2020), and transmission of infection oc- curs through ingestion of contaminated food and water. Husbandry practices like diet and litter types influence the incidence and severity of NE in poultry (Henry et al., 1995). Two to six-week- old broiler chickens and 12- to 24-week-old lay- ers are highly susceptible to NE (Lovland et al., 2004). Affected birds with acute NE show severe necrosis and damage of the intestinal mucosa, which lead to high mortalities (Wu et al., 2010) and poor performance in subclinical cases (Skinner et al., 2010). The causative agent of NE is C. perfringens, which is a Gram-positive, anaero- bic, and spore-forming bacillus (Timbermont et al., 2011). These bacilli are found naturally in the soil, water, sewage, food, and feces as well as in the intestinal tracts of livestock, poultry, and humans (Li et al., 2016). C. perfringens is considered a normal inhabitant of the birds’ in- testinal tract as well as a potential pathogen causing NE. Strains of C. perfringens are divid- ed into seven extracellular toxin types: A, B, C, D, E, F, and G (Rood et al., 2018; Goossens et al., 2020). However, C. perfringens type A and to a lesser extent type C have been shown to be the major cause of NE in chickens (Cooper and Songer, 2009). Moreover, alpha (α) toxin is pri- marily responsible for NE in poultry (Keyburn et al., 2010). The virulence of C. perfringens is attributed to more than 20 toxins and hydrolytic enzymes (Kiu and Hall, 2018; Gu et al., 2019), while individual strains only produce a subset of these toxins (Van Immerseel et al., 2008). Major extracellular toxins of C. perfringens are alpha (α) (cpa), beta (β) (cpb), epsilon (ε) (etx), and iota (ι) (iap). However, different strains of C. perfringens can also produce other enzymes and toxins, namely, β2, theta (θ) [perfringolysin O (PFO)], kappa (κ), delta (δ), mu (µ), sialidase, hyaluronidase, collagenase, neuraminidase, en- terotoxin (cpe), necrotic enteritis toxin B-like (netB), and toxin perfringens large (tpeL) (Lukinmaa et al., 2002; Li et al., 2013; Duff et al., 2019; Wei et al., 2020). All C. perfringens type A strains possess phospholipase C (plc) or cpa gene that produces α toxin in varying amounts (Kumar et al., 2019; Helal et al., 2019). This gene is present on the chromosome close to the origin of replication of all C. perfringens strains (Canard et al., 1989). It was found that netB and tpel toxins play a role in the virulence of some C. perfringens strains of avian origin (Rood et al., 2016; Elsharkawy et al., 2020; Thi et al., 2021). Most C. perfringens strains that produce a pore-forming toxin (netB) belong to toxin type G (Rood et al., 2018). In addition, tpel, a recently designated novel family member of large clostridial cytotox- ins, was detected in some C. perfringens type A strains isolated from NE cases (Coursodon et al., 2012; Mwangi et al., 2019). Enterotoxin gene (cpe) coding toxin of C. perfringens has been identified by Gao and McClane (2012), and it induces gastroenteritis (Lukinmaa et al., 2002). NE has become a hurdle affecting broiler production especially after the great restrictions on the application of antibiotics in ration under modern high stalking density (Van Immerseel et al., 2008). Therefore, there is an urgent need to select the drug of choice to control this critical disease. Therefore, this study aimed to characterize, investigate the antibiogram pattern and determine the pathogenicity of C. perfringens strains isolat- ed from broiler chickens in Damietta gover- norate, Egypt. MATERIALS AND METHODS Sample Collection A total of 357 samples were taken from 202 intestines and 155 livers of sacrificed diseased and freshly dead chickens (2-8-week-old) repre- senting 18 commercial broiler chicken farms at different locations in Damietta governorate, Egypt, from December 2019 to June 2020 (Table 1). Clinically suspected cases with NE showed anorexia, depression, reluctance to move, diar- rhea, and death. Sacrificed and dead chickens showed dehydration, enteritis, ballooned and fri- able intestines with hemorrhages, and yellow diphtheritic necrotic membranes on the mucosa the C. perfringens Isolated from Broiler Chickens (R. D. Eraky and W. A. Abd El-Ghany) 3 as well as liver necrosis. The samples were asep- tically collected in sterile plastic bags and quick- ly transported to the laboratory in ice-cooled containers for further microbiological examina- tion. Conventional Isolation and Identification Sample processing was done according to a routine protocol as previously described by Wil- lis (1977). For enrichment, one gram of each of the intestinal contents or liver tissue samples was inoculated into tubes of freshly prepared Robert- son cooked meat broth (Oxoid, UK) and incubat- ed for 24 h at 37°C in a Gas-Pak anaerobic jar. Aliquots of 0.1 ml were streaked onto a perfringens agar base containing 400 μg/ml of tryptose sulfite cycloserine (TSC) with egg emulsion (Oxoid, UK) and incubated anaerobi- cally. For the proliferation and detection of the hemolytic characteristics of Clostridium isolates, 5% de-fibrinated sheep blood agar with neomy- cin sulphate (200 µg/ml) was prepared. After 24- 48 h incubation at 37°C, typical black colonies were selected and cultured onto de-fibrinated 5% sheep blood agar and egg yolk agar plates and incubated anaerobically for 24 h at 37°C (Cruickshank et al., 1975). Typical colonies on blood agar or egg yolk agar were further identi- fied according to the morphological characteris- tics using Gram staining and different biochemi- cal tests, such as catalase, nitrate reduction, gelatinase, lecithinase, indole, oxidase, urease storm gas production on litmus milk medium, and fermentation of glucose, lactose, fructose, sucrose, and mannitol. Molecular Detection of the Toxins and Viru- lence Genes DNA extraction from suspected samples was performed using the QIAamp DNA Mini Kit (Qiagen, Germany, GmbH) with modifications according to the manufacturer’s recommenda- tions. Briefly, 200 µl of the sample suspension was incubated with 10 µl of proteinase K and 200 µl of lysis buffer at 56°C for 10 min. After incubation, 200 µl of 100% ethanol was added to the lysate. The sample was then washed and cen- trifuged following the manufacturer’s recommen- dations. Nucleic acid was eluted with 100 µl of elution buffer provided in the kit. Primers provid- ed by Metabion (Germany) are listed in Table (2). Multiplex polymerase chain reaction (PCR) was used for the detection of α, β, ε, and ι toxins. Primers were utilized in a 50 µl reaction contain- ing 25 µl of Emerald Amp Max PCR Master Mix (Takara, Japan), 1 µl of each primer of 20 pmol concentration, 11 µl of water, and 6 µl of DNA template. For uniplex PCR, primers were utilized in a 25 µl reaction containing 12.5 µl of Emerald Amp Max PCR Master Mix (Takara, Japan), 1 µl of each primer of 20 pmol concentration, 5.5 µl of water, and 5 µl of DNA template. All the reac- tions were performed in an Applied Biosystems 2720 thermal cycler. The products of PCR were separated by electrophoresis on 1.5% agarose gel (Applichem, Germany, GmbH) in 1x TBE buffer at room tem- perature using gradients of 5V/cm. For gel analy- Table 1. The number of the examined samples distributed in Damietta governorate, Egypt Locality Number of examined farms Number of examined samples Intestine Liver Kafer-Saad 3 18 18 Farskour 3 32 31 Om El-Reda 4 44 40 Kafer El-Batekh 2 19 9 Cinania 1 11 11 Kafer-ElGhabe 2 35 29 Zarka 3 43 17 Total 18 202 155 4 J. Indonesian Trop.Anim.Agric. 47(1):1-16, March 2022 sis, 40 µl of the multiplex PCR products and 15 µl of the uniplex PCR products were loaded in each gel slot. A gel pilot 100 bp ladder (Qiagen, Gmbh, Germany) and gene ruler 100 bp ladder (Fermentas, Thermo, Germany) were used to de- termine the fragment sizes. The gel was photo- graphed using a gel documentation system (Alpha Innotech, Biometra), and the data was analyzed through computer software. The Antibiogram Pattern The antimicrobial susceptibility testing of C. perfringens strains was done using the disc diffusion method developed by the National Committee for Clinical Laboratory Standards (NCCLS, 2007). The used chemotherapeutic agents discs (Oxoid) and the inhibition zones (susceptible, intermediate susceptibility, and re- sistant) are shown in Table (3). All C. perfringens strains were cultivated in cooked meat broth for 24 h, and then the culture broth was suspended into 0.85% NaCl to obtain an op- tical density equal to MacFarland 0.5 standards. After that, the strains were inoculated in 5% de- fibrinated sheep blood agar for 10 minutes and the antibiotic discs were dispersed in the agar plates. The plates were incubated anaerobically at 37°C overnight, and the inhibition zones were Table 2. Primers sequences, target genes, amplicon sizes and cycling conditions for C. perfringens. Target toxin and virulen ce genes Primers sequences Amplifie d segment (bp) Primary denaturation Amplification (35 cycles) Final extension Reference Secondary denaturation Annealing Extension α GTTGATAGCGCAG GACATGTTAAG 402 94˚C 5 min. 94˚C 30 sec. 55˚C 40 sec. 72˚C 45 sec. 72˚C 10 min. Yoo et al. (1997) CATGTAGTCATCT GTTCCAGCATC β ACTATACAGACAG ATCATTCAACC 236 TTAGGAGCAGTTA GAACTACAGAC ε ACTGCAACTACTA CTCATACTGTG 541 CTGGTGCCTTAAT AGAAAGACTCC ι GCGATGAAAAGCC TACACCACTAC 317 GGTATATCCTCCA CGCATATAGTC NetB GCTGGTGCTGGAA TAAATGC 560 94˚C 5 min. 94˚C 30 sec. 58˚C 40 sec. 72˚C 45 sec. 72˚C 10 min. Datta et al. (2014) TCGCCATTGAGTA GTTTCCC TpeL ATATAGAGTCAAG CAGTGGAG 466 94˚C 5 min. 94˚C 30 sec. 55˚C 40 sec. 72˚C 45 sec. 72˚C 10 min. Bailey et al. (2013) GGAATACCACTTG ATATACCTG cpe ACATCTGCAGATA GCTTAGGAAAT 247 94˚C 5 min. 94˚C 30 sec. 55˚C 30 sec. 72˚C 30 sec. 72˚C 7 min. Kaneko et al. (2011) CCAGTAGCTGTAA TTGTTAAGTGT Plc ATA GAT ACT CCA TAT CAT CCT GCT 283 94˚C 5 min. 94˚C 30 sec. 55˚C 30 sec. 72˚C 30 sec. 72˚C 7 min. Akhi et al. (2015) the C. perfringens Isolated from Broiler Chickens (R. D. Eraky and W. A. Abd El-Ghany) 5 measured as recommended by the manufacturer. The Pathogenicity Test in Broiler Chickens The experiment was done according to the National Regulations on Animal Welfare and Institutional Animal Ethical Committee (IAEC). A total of 105 day-old Cobb chicks were ob- tained from local hatcheries, and five birds were subjected on arrival to bacteriological examina- tion to confirm the absence of C. perfringens. The chicks were reared on thoroughly cleaned and disinfected semi-closed houses and vaccinat- ed using the standard protocol for vaccination. Feed and water were given ad libitum. The ration was supplemented with 12% wheat to enhance the experimental induction of infection. The chicks were divided into two equal groups, each containing 50 birds. Group (1) was the negative control non-challenged group and was inoculated with sterile phosphate buffered saline. Each bird in group (2) was orally inoculated with a field mixture of Eimeria oocysts in a dose of 1x10 3 sporulated oocysts/0.1ml of oocysts mixture at the age of 10 days. However, at the age of 20 days, each chick in group (2) was challenged orally with 1 ml of 24 hr broth culture containing 1.7×10 8 viable cells of the toxigenic strain of C. perfringens type A for four successive days (Timbermont et al., 2009). All chicken groups were kept under observation for two weeks post- challenge (PC) to monitor the clinical picture. RESULTS AND DISCUSSION C. perfringens is a widely distributed bacte- rium in the environment and is mostly found in the intestinal tracts of humans and domestic animals (Kiu and Hall, 2018). The organism is a major enteric pathogen that can lead to both clin- ical (Long and Truscott, 1976) and subclinical diseases (Lovland and Kaldhusdal, 2001). The pathogen is responsible for causing NE in poul- Table 3. The interpretation of C. perfringens antibiogram pattern Antibiotic disc (Code) Disc content/ µg Interpretation (Diameter of the zone/ mm) Susceptible ≥ Intermediate susceptibility Resistant ≤ Amoxycillin/Clavulanic acid (AMC) 20/10 18 14-17 13 Neomycin (NE) 10 17 - 16 Doxycycline (Do) 30 16 13-15 12 Erythromycin (E) 15 21 16-20 15 Nalidixic acid (NA) 30 19 14-18 13 Penicillin (P) 10 22 - 23 Ciprofloxacin (CIP) 5 21 16-20 15 Gentamycin (CN) 10 15 13-14 12 Levofloxacin (LEV) 5 17 14-16 13 Ceftriaxone (CES) 30 28 24-27 23 Table 4. The incidence rate and the type of C. perfringens in Damietta governorate, Egypt Age of chicken/Week Intestine Liver No. of samples No. of positive % positive No. of samples No. of positive % positive No. of examined farms 1-2 62 2 3.2 47 0 0 5 2-3 56 15 26.8 48 3 6.3 10 3-4 45 13 28.9 39 2 5.1 12 4-8 39 11 28.9 21 1 4.8 8 Total 202 41 20.3 155 6 3.8 18 6 J. Indonesian Trop.Anim.Agric. 47(1):1-16, March 2022 try, especially C. perfringens type A, which is the most frequently isolated clostridial type (Opengart, 2008). Based on the cultural, morphological, and biochemical characteristics of the isolates, 20.3% and 3.8% C. perfringens isolates were recovered from 202 intestine and 155 liver samples, respec- tively, from freshly dead broiler chickens in Damietta governorate (Table 4). Morphological- ly, C. perfringens isolates grew anaerobically and produced double zones of hemolysis (an in- ner zone of complete hemolysis and an outer zone of discoloration and incomplete hemolysis) on 5% sheep blood agar with neomycin sulfate (Figure 1). However, C. perfringens isolates on TSC showed black colonies due to the reduction of sulfite to sulfide, which in turn reacts with iron and forms a black iron sulfide precipitate (Figure 2). A zone of opalescence appeared around the C. perfringens colonies on egg yolk agar plates. Microscopically, C. perfringens iso- lates revealed Gram-positive, non-motile, and spore-forming large-sized bacilli. Biochemically, all C. perfringens isolates were positive for ni- trate reduction and lecithinase activity (Figure 3), but they were negative for catalase, indole production, and oxidase. The isolates produced typical stormy fermentation reaction in litmus milk medium. Manfreda et al. (2006) isolated C. perfringens from broiler farms with a rate over 90% and found C. perfringens in 87 out of 149 samples (58.40%). However, the lowest frequen- cy of isolated C. perfringens was reported by Kalender and Ertas (2005) who showed that only 5% of the intestinal contents were positive for C. perfringens. In Egypt, Hussein and Mustfa (1999) demonstrated 30 isolates of C. perfringens out of 60 intestinal samples (50%) in 4-6-week-old broiler chickens in Assiut gover- norate, while Ebtehal (2000) found that out of 470 broiler chicken samples, 231 (71.9%) strains of C. perfringens could be isolated in Assiut and El-Minia governorates. This high incidence was not surprising if the spread of the microorgan- isms in the environment, diet, water, litter, and slaughtering houses was considered. Other Egyp- tian studies reported isolation of C. perfringens from the intestines of both apparently healthy and diseased broiler chickens with high rates of 42.0% and 91.3%, respectively (El-Refaey et al., 1999); 30% and 75%, respectively (Rasha, 2009); and 35.4% and 100%, respectively (Osman et al., 2012). Moreover, C. perfringens Figure 1. Colonies of C. perfringens on 5% sheep blood agar with neomycin sulfate showing double zones of β hemolysis (Inner zone of complete hemoly- sis and outer zone partial hemolysis). Figure 2. Colonies of C. perfringens on tryptose sul- fite cycloserin (TSC) showing black colonies. Figure 3. Lecithinase activity of C. perfringens on egg yolk agar (lecithinase: α toxin phospholipase hydro- lyzes phospholipids in egg yolk agar around streaks). the C. perfringens Isolated from Broiler Chickens (R. D. Eraky and W. A. Abd El-Ghany) 7 was isolated from the intestines of chickens with NE with incidence rates of 47.70% (El-Rash, 2012) and 60% (Eman et al., 2013). Out of 120 intestine and liver samples taken from diseased broiler chickens, EI-Jakee et al. (2013) isolated 90 (75%) C. perfringens with an incidence rate of 53.8%. Multiplex PCR showed that C. perfringens strains belonged to type A as they contained the cpa gene (402 bp) that coded for α toxin and the cpb (236 bp), etx (541 bp), and iA (317 bp) genes that coded for β, ε, and ι. toxins, respectively (Figure 4). Molecular detection of the virulence genes of C. perfringens type A strains showed the presence of the netB, tpeL, cpe, and plc genes in all isolated strains (Figures 5 and 6). The PCR- based detection of α toxin is essential for the typ- ical identification of α toxigenic C. perfringens strains (Baums et al., 2004). Several Clostridia enteric diseases occur in poultry, but probably the most common and severe one is NE, caused by C. perfringens type A (Moore, 2015). In Swe- den, Engstrom et al. (2003) demonstrated that all C. perfringens strains were classified as type A without enterotoxin genes. Furthermore, in Fin- land, Heikinheimo and Korkeala (2005) showed that 118 poultry isolates of C. perfringens were classified as type A strains using multiplex PCR. In a Belgian study, five out of 63 C. perfringens isolates were β2 toxin-positive, and the authors indicated that this type of toxin is not an essential virulence factor in the development of NE in poultry (Gholamiandekhordi et al., 2006). It is well known that C. perfringens type A induces intestinal mucosal damage in chickens (Moore, 2015). The α toxin producing C. perfringens is phospholipase C sphingomyelinase that hydrolyzes lecithin into phosphorylcholine and diglyceride and as a consequence induces the production of inflammatory mediators causing blood vessel contraction, platelet aggregation, myocardial dysfunction, and finally acute death (Matsuda et al., 2019). Detection of C. perfringens toxin types and subtypes is critical for a better understanding of the epidemiology of C. perfringens infection and may be helpful in the implementation of effective preventive measures (Fancher et al., 2021). In this study, the presence of eight toxin genes (cpa, cpb, etx, iA, netB, tpeL, plc, and cpe) of C. perfringens type A isolates has been investigated. Figure 4. PCR amplification using Clostridium genus -specific primers for toxins (α, β, ε and ι), P= Positive control, L= Ladder, Lines 6-9 = C. perfringens type A strains, N= Negative control. Figure 5. PCR amplification using C. perfringens genus-specific primers (cpe and plc genes), P= Posi- tive control, L= Ladder, lines 1-4 = cpe and plc genes of C. perfringens, N= Negative control. Figure 6. PCR amplification using C. perfringens genus-specific primers (tpeL and netB genes), P= Positive control, L= Ladder, lines 1-4 = tpeL and netB genes genes of C. perfringens, N= Negative control. 8 J. Indonesian Trop.Anim.Agric. 47(1):1-16, March 2022 The results revealed the presence of netB, tpeL, plc, and cpe genes. This result confirms high production of toxins that lead to the destruction of the intestinal mucosa and consequently the development of NE (Mwangi et al., 2019). Simi- lar findings were reported by Ebtehal (2000) who indicated the role of toxigenic C. perfringens in the production of toxins that lead to NE in poultry. In addition, C. perfringens strains possess other common virulence genes (netB) producing β toxin (Yang et al., 2018). Since the discovery of this new virulence factor, the presence of the netB gene in C. perfringens strains has been in- vestigated in different regions of the world. The results indicated the existence of this gene in C. perfringens type A strains. Johansson et al. (2010) reported that more than 90% of all iso- lates from cases of NE carried the netB gene. Through the examination of 36 isolates of C. perfringens, 19 (52.8%) isolates showed pres- ence of the netB gene (Tolooe et al., 2011). A previous study of Miwa et al. (1998) demonstrat- ed that strains of C. perfringens that were netB- negative failed to cause disease in an experi- mental model, whereas all netB-positive strains produced typical lesions of NE. In addition, it has been found that netB, a pore-forming toxin, plays a role in the pathogenesis of NE in poultry as a strongly necrotizing and lethal toxin (Keyburn et al., 2010; Wade et al., 2020). Native and recombinant netB were cytotoxic for chicken hepatocytes. The netB gene is mostly found in outbreaks of NE but is relatively uncommon in healthy birds (Tolooe et al., 2011). However, several studies demonstrated the absence of the netB gene in C. perfringens isolates (Datta et al., 2014; Li et al., 2018; Zhang et al., 2019). Furthermore, all C. perfringens type A strains of avian origin possess phospholipase C (plc) or the cpa gene that produces α toxin (Abildgaard et al., 2009). This gene has also been discovered in strains of human origin (Matsuda et al., 2019). Moreover, Kimy et al. (2017) classified C. perfringens as a toxin type A based on the presence of the α toxin gene (plc). Isolates of C. perfringens that have α toxins as well as enterotoxin (cpe) are regarded as type F. Enterotoxin (cpe) is produced by about 1%- 5% of C. perfringens type A. This toxin is a sin- gle polypeptide chain of about 35 KDa and, un- like other toxins, is released upon lysis of the mother cell in the sporulation stage (Abildgaard et al., 2010). Previous studies showed that there is a relationship between C. perfringens type A isolates that carry the cpe gene and foodborne infection (Miyamoto et al., 2012) as well as non- foodborne gastrointestinal diseases (Azimirad et A A B Figure 7. A: The intestine is filled with blood (hemorrhagic enteritis) and distended with gases. B: The caecum is filled with blood (hemorrhagic typhlitis). the C. perfringens Isolated from Broiler Chickens (R. D. Eraky and W. A. Abd El-Ghany) 9 al., 2019). The findings of this study showed that C. perfringens isolates carry the cpe gene which is similar to the findings of other studies (Asaoka et al., 2004). The authors suggested that cpe plays a role in intestinal necrosis with minor in- testinal damage, allowing the multiplication of C. perfringens and consequently development of the disease. Periodic evaluation of C. perfringens antimicrobial susceptibility testing is important to avoid the losses resulting from this infection (Finken and Wages, 1997). In 47 C. perfringens strains, the in vitro sensitivity test revealed high susceptibility to levofloxacin and gentamycin (100%) as well as ciprofloxacin (85.1%). Low degree of susceptibility to doxycy- cline and erythromycin (25.5%), in addition to neomycin (23.4%), was reported. Resistance to penicillin, nalidixic acid, and ceftriaxone was 100%, while resistance to amoxycillin/clavulanic acid was 72.3% (Table 5). Nearly similar antibi- otic sensitive patterns were observed by Mehtaz et al. (2013) who found that C. perfringens iso- lates were sensitive to some fluoroquinolones, such as ciprofloxacin and ofloxacin. However, these results are inconsistent with those reported by Hussein and Mostfa (1999) who stated that neomycin was highly effective but enrofloxacin was not effective against C. perfringens. Algam- mal and Elfeil (2015) reported 100% resistance of C. perfringens to neomycin, which is com- monly used as an antimicrobial drug to treat bac- terial enteritis in poultry. In this study, C. perfringens isolates showed resistance to nalidix- ic acid and amoxicillin, similar to the results re- ported by another study (Camacho et al., 2008). Nevertheless, another study demonstrated a high level of sensitivity to penicillin (Algammal and Elfeil, 2015). Clinical signs observed among C. perfringens-challenged chicks in the challenged group were depression, ruffled feathers, de- creased appetite, and diarrhea. Mortalities were observed at 48 hr PC at a rate of 43.3%. No clini- cal signs or mortalities were observed in control birds that were inoculated with phosphate buff- ered saline. The intestines of dead and sacrificed chickens at the end of the observation period were filled with blood (hemorrhagic enteritis) and distended with gases (Figure 7 A), and the caecum was filled with blood (hemorrhagic typh- litis) (Figure 7 B). Enlargement, paleness, and necrosis of the liver were also observed (Figure 8). The pathogenesis of C. perfringens infection Figure 8. Enlarged and pale liver with necrotic foci. 10 J. Indonesian Trop.Anim.Agric. 47(1):1-16, March 2022 involves the colonization of the tissue’s host, acquisition of nutrients to allow more multiplica- tion, dodging of the immune system of the host, and finally transmission of toxins with tissue damage (Prescott et al., 2016). The presence of some risk factors associated with C. perfringens challenge enhances the development of NE clini- cal infection. Some predisposing factors, such as Eimeria species and the use of wheat and barley, are important for the induction of NE (Kocher, 2003). Moreover, C. perfringens infection was significantly higher in the presence of stress fac- tors, such as worm infestation or coccidiosis (Mateos et al., 2002). It has been found that Ei- meria species colonize the bird’s intestinal tract, causing damage and releasing plasma proteins which is the minimal requirements for growth of C. perfringens include more than 11 amino ac- ids, besides many growth factors and vitamins (Hofacre et al., 2003). Moreover, Lovland et al. (2004) reported that C. perfringens type A caus- es mucosal damage in the intestines of chickens. Regarding the pathogenicity test in broiler chicks using C. perfringens strains, the results revealed general signs with variable degrees of diarrhea, mortalities (43.3%), and intestinal and liver le- sions. Similar observations were reported in pre- vious studies (Freedman et al., 2015; Thi et al., 2021). Lovland and Kaldhusdal (2001) found that NE can present as an acute clinical disease characterized by sudden high mortality rates that can reach 50% in flocks. Moreover, Ebtehal (2000) found that C. perfringens given orally to chicks caused 80% mortality. Similar intestinal lesions were also reported in previous studies (Park et al., 2015; To et al., 2017; Abdul-Aziz and Barnes, 2018). They mentioned that infected chickens with NE showed intestinal lesions rang- ing from thin and friable walls to frank hemor- rhagic enteritis along with gas distension. In ad- dition, necrotic lesions present on the liver of chickens after C. perfringens challenge were the same as the lesions reported by Lovland and Kaldhusdal (2001), Sasaki et al. (2003), and Thi et al. (2021). CONCLUSION Continuous and periodical surveillance stud- ies should be conducted to alleviate the severe economic losses caused by C. perfringens infec- tion in broiler chicken flocks. Detection of the sensitivity of the bacterium to different antibiot- ics is a must before developing successful control and treatment strategies. Future studies on the preparation of bacterin to prevent such infection are needed. REFERENCE Abdul-Aziz, T and H.J. Barnes. 2018. Necrotic enteritis. 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