Isolation and Genotyping Study of Clostridium Perfringens From Broiler Farms Infected with Necrotic Enteritis in Sulaimania Province

ا ةيجنفربلا تايثطملا ةموثرجل ينيجلا طيمنتلا ةساردو لزع ب باصملا محللا جورف نم ةلوزعمل ةيناميلسلا ةظفاحم يف يرخنتلا ءاعملاا جمخ ةصلاخلا ةظفاحم يف محللا جورف نم ةيجنرفربلا تايثطملا ةموثرجب اهتباصا هبتشملا تلااحلا نم نافيذلا عون ةفرعمو لزعل ةساردلا هذه تيرجا ددع ناك .ةيناميلسلا ةساردلا تانيع 108 هتباصا هبتشملا تلااحلا نم ةيوافمللا تاديقعلاو ءاعملال ةيطاخملا ةقبطلا ةحسمو ءاعملاا ىوتحم نم ةنيع ا لصا نم ةصوحفملا محللا جورف يف يرخنتلا ءاعملاا جمخب 108 تناك ةصوحفم ةنيع 63 ( ةنيع 58 تايثطملا ةموثرجب ةباصمو ةبجوم )% .ةيجنرفربلا اتيبو افلأ نينافيذلا تانيجل طقف ةيباجيإ تناك تلازعلا نأ جئاتنلا تفشكو 2 . تانيجلل ينيجلا ليلحتلا ةليصحت تناكو cpa و cpb2 نيجلا نأ cpa نيجلا نيح يف .ايراغلب يف لوجعلاو ناريا يف محللا جورف نم ةلوزعملا ةموثرجلا تانيجل هباشم cpb2 .ناتسكاب يف زعاملا نم تلازع عم هباشتم ا تايثطملا ةموثرج ىلا اساسا عجري ميلقلاا يف محللا جورفل يرخنتلا ءاعملأا باهتللا ببسملا ةموثرجلا نأ جئاتنلا تنيب ةيجنرفربل A .


Introduction
Necrotic enteritis is the most common clostridial enteric disease in poultry, which typically occurs in broiler aged between two to six weeks (1) that caused by Clostridium perfringens, which is a Gram-positive, rodshaped, spore forming, anaerobic bacteria. They are widely distributed in the environment and as a part of the normal gastrointestinal tract flora of humans and animals (2). C. perfringens is a prolific toxin producer, with a high capacity for the synthesis and secretion of more than 20 different extracellular toxins (3). The C. perfringens toxins are believed to be the main virulence factors in inducing the damages by the pathogen (3). The organism has been classified traditionally into five toxinotypes (A-E) basis on the production of toxins including; alpha (α), beta (β), epsilon (ε) and iota (ɩ) (4). But this typing system was recently expanded to include two additional types, such as type F strains produce enterotoxin and alpha-toxin; type G strain produces alpha and NetB (5). C. perfringens is responsible for many histotoxic and enterotoxic Keywards: Clostridium perfringens; Characterization; Necrotic enteritis; Toxin The study was conducted to isolate and toxinotype the suspected cases of Clostridium perfringens infections of broiler farms in Sulaimania province. A total of 108 samples were collected from intestinal contents, mucosal scraping, and hemorrhagic lymphoid nodules from suspected cases of necrotic enteritis in broilers. The result of isolated and identified bacteria were revealed that 63 (58%) out of 108 samples were positive for C. perfringens. The results revealed that the isolates were only positive for alpha and beta2 toxin genes. Phylogenetic and DNA sequence analysis of cpa and cpb2 gene showed that cpa genes were highly identical to isolates from broiler in Iran, poultry stool and broiler in Brazil, and blue calves in Belgium. While cpb2 gene is closely related to the isolates of broiler in Iran, India and isolates of goat in Pakistan. The results indicated that the causative agent of necrotic enteritis in broiler farms in the region was mainly due to C. perfringens  diseases in humans and animals (6).
In poultry, C. perfringens, especially type A and type C, can cause both clinical and subclinical form of necrotic enteritis (7). The coccidial pathogen is the most important known predisposing factor that enhances the induction of necrotic enteritis by damaging the intestinal epithelium, allowing C. perfringens to penetrate and replicate rapidly to produce sufficient amount of the toxins that causes the disease (8.9). In addition, some dietary components such as fish meal, high level of indigestible-non-starch polysaccharides, dysbacteriosis although have been widely accepted as predisposing factors (10).
There was not any data about the type of the C. perfringens that caused necrotic enteritis in broiler farms in Kurdistan region. Accordingly, the aims of the study were to isolate and toxinotype the suspected cases of Clostridium perfringens in broiler farms, basis on the presence or absence of the major and the minor toxin genes, including alpha (cpa), beta (cpb), epsilon (etx), and iota (itx) toxins, beta2, necrotic enteritis toxin B (netB), using selective medium, multiplex and uniplex PCR.

Samples collection:
The samples from intestinal content, mucosal scraping, and hemorrhagic lymphoid tissue were aseptically collected from 108 diseased broiler aged between two to six weeks from 18 farms in Sulaimania province from August 2018 to May 2019. The samples were collected from farms where the birds had not been taken any antibiotic supplementation for about 72 hrs before sampling. Diseased broiler were characterized by having suspected clinical signs of necrotic enteritis, including depression, ruffled feathers, and diarrhea. The samples were taken from most affected parts of the intestines and collected in sterile plastic bags and processed immediately for isolation and identification of C. perfringens

Isolation and identification C. perfringens:
The samples directly inoculated into tubes that contained freshly prepared cooked meat broth and incubated at 37°C for 24 hrs in an anaerobic jar (BD company, U.S.A) with gaspacks (Thermo scientific, U.S.A) were used to create the anaerobic condition. A loopful of inoculated fluid medium was streaked on sheep blood agar (Himedia, India) supplied with cycloserine (400 mg/1L), at 37°C for 24 hrs under anaerobic conditions. For the purpose of bacterial purification, the single bacterial colonies were picked up and inoculated into tryptose sulfite cycloserine agar (Quelab, Canada) with and without egg yolk, which was incubated at 37°C for 24 hrs under strict anaerobic condition. The isolated colonies were further characterized by looking at the morphology of the bacteria using Gram stain.

DNA extraction:
The boiling technique was used to extract DNA from all the isolates (11). Briefly, one wellisolated colony was selected, and suspended in 100µl of (Milli Q) water in an Eppendorf tube, incubated at 100°C for 15 min. using thermoshaker (Biotech, Spain), and exposed to a pulsatile vortex (5-6 times/15 min.). Then cooled down and centrifuged (Maan lab, Sweden) at 14.000 × g for 10 min. Finally, the supernatant was kept, and the precipitate was discarded.

Multiplex and Uniplex PCR:
The multiplex PCR reaction was performed in a thermo-cycler (Techne ® Prime/ U.K) by adding 1µl of the primers mix (Table 1), 5 µl DNA into 10 µl PCR master mix cocktail (Genetbio Inc.). Then the volume was completed to the total reaction volume of 20 μl with ultrapure water. Uniplex PCR for detecting Beta2, NetB, and Alpha toxin gene was set in a total reaction volume of 20 μl by mixing 1µl of the forward and reverse primers of each gene separately (Table 2), 5 µl DNA and the volume was completed with ultrapure water. The PCR amplification was as follow: initial denaturation was 95 °C for 5 min, denaturation at 95°C/30 sec, annealing at 55°C/30 sec, and extension at 72°C/1 min. The final extension was at 72°C/10 min. DNA bands were visualized on 1.5% agarose gel (Ingenius/ U.S.A).

DNA sequence analysis and Phylogenic Tree:
Both sense and antisense strands of the amplified DNA sequences were sequenced using Sanger DNA sequencer (Macrogen Co., Korea). The obtained DNA sequences subjected to DNA analysis using Clustal Omega (Multiple Sequence Alignment) and NCBI nucleotide blast. Phylogram was created using MEGA-X software. The amino acid sequences and their corresponding codons were predicted using the ExPASy Server (https://web.expasy.org/translate/). The obtained DNA sequences of both alpha and beta-2 toxin genes recorded in the National Center for Biotechnology Information (NCBI) under different accession numbers, including MN224676, MN224677, MN224678, and MN224679 for alpha-toxin, and MN239885، MN239886 and MN239887 for beta-2 toxin genes.

Results and Discussion
Isolation and identification C. perfringens: In this study, 63 (58 %) of the isolates out of 108 samples suspected cases were positive for C. perfringens. The isolation and characterization of bacterial colonies were revealed basis on the cultural properties of the isolated bacteria on cooked meat broth, blood agar, and TSC agar with and without egg yolk.
Culturing of isolates on sheep blood agar produced small to medium-sized smooth-colonies, which had a gray and glistening appearance, surrounded by an inner zone of complete hemolysis due to theta toxin and an outer zone of partial hemolysis due to alpha toxin (Fig. 1). The bacteria produced a typical round and flat colonies, which had smooth and black color on tryptose sulfite cycloserine agar (Fig. 1). While the growth of the organism on TSC agar with egg yolk produced opalescence around the black colonies as a result of the breaking down of lecithin indicates to lecithinase activity (Fig. 1). The isolates were Gram-positive bacilli appearance (Fig. 1).

Multiplex and Uniplex PCR:
The multiplex PCR reaction showed that almost all isolates were positive for C. perfringens alpha-toxin gene and negative for other toxin-forming genes (Fig. 2). This result indicated that the isolated type was C. perfringens type A, because of the presence of alpha-toxin gene alone, which is only present in C. perfringens type A.
The uniplex PCR results reconfirmed the presence of the alpha-toxin gene; besides, the cpb2 gene (548 bp) was detected in 20 from 63 isolates of the C. perfringens type A (Fig. 3). However, all cpa gene-positive samples were negative for the NetB toxin gene.

DNA sequence analysis and Phylogenic Tree:
The DNA sequence analysis of the partially sequenced C. perfringens, which had been isolated from different broiler farms, using conventional software, including Clustal Omega (Multiple Sequence Alignment), NCBI nucleotide blast, MEGA-X software, and ExPASy Server showed that C. perfringens type A alpha gene ( Fig. 5) ( MN224677 and MN224678, and MN224676 and MN224679), had 100% to 99.22% homology respectively. The phylogenic tree analysis showed that the first two sequences were 100% similar to that of taxon L43548.1 (isolated from blue calves in Belgium), and 99.87% identical to that of taxon JQ071544.1 (isolated from poultry stool in Brazil). Still, it has a low rate identity of 84.18% with AF204209.1, which has been isolated from a diseased swan in the UK (Fig. 4). The other two sequences MN224676 and MN224679 were 100% similar to taxon X13608.1, L43547.1, and KT020614.1, which isolated from veterinary isolate, blue calves and broiler chicken in the UK, Belgium and Brazil respectively. However, the sequences were 99.68% identical to taxon GU581194 (isolated from the intestinal tract of diseased broiler chicken in Iran), and 84.31% homology with taxon AF204209.1 which was isolated from a diseased swan in the UK (Fig. 4).
The DNA sequence of the isolates showed that there were several nucleotides substitutions within the sequence of the partially sequenced alpha-toxin genes (MN224677 and MN224678, Similar to the alpha gene, C. perfringens type A beta2 toxin gene was amplified from 20 of alphatoxin gene positive isolates. Beta2 toxin gene was found to be highly conserved. The sequence analysis showed that there was only one nucleotide substitution at site 428 of the Beta2 toxin gene (MN239887) (Fig. 8). The substitution of the nucleotide Guanine (MN239886 and MN239885) with adenine that leads to the alteration of the corresponding codon (UGU to UAU) and replacement of the amino acid cysteine with tyrosine ( Fig. 9). The phylogenic analysis of cpb2 partially sequenced gene revealed that MN239886 and MN239885 were 100% identical with the sequence MF471365.1 (isolated from broiler chicken in India), AY884037.1 (from an unknown source), GU581183.1 (isolated from diseased broiler chicken in Iran) and MF191716.1 (isolated from goat in Pakistan). The sequence homology with the other taxons was ranged between 93.75% to 99.75%, respectively (Fig. 7). The MN239887 sequence of the isolate was 100% identical to that of the taxon KX924463.1 isolated from gout in Pakistan and taxon GU581182.1 and GU581185.1, which had been isolated from broiler chicken in Iran. Also, the sequence homology with other sequences were ranged between 93.50% to 99.50%, respectively ( Fig. 7).

Isolation and identification C. perfringens:
Clostridium perfringens infections are of economic concern in poultry production, resulting in gastrointestinal dysbacteriosis and necrotic enteritis (15). Proper characterization and identification of the causative agent's disease are very crucial in minimizing economic losses due to the clostridial infections in the poultry.
The characteristic of the colonial morphology, which included, appearance of inner zone of complete hemolysis due to theta toxin and an outer zone of incomplete hemolysis due to alpha toxin on sheep blood agar and blackish colonies on TSC agar which is due to reduction of sulfite to sulfide by C. perfringens which in turn react with iron and form a black iron sulfide precipitate, is concurred with observation by other authors (16,17). The growth of the isolates on medium contained egg yolk produced opalescence around the colony due to the breaking down of lecithin by alpha toxin (14). Microscopically the bacteria characterized by having gram-positive rod-shaped with blunt ends after being stained with gram stain.

Multiplex and Uniplex PCR:
Alpha and beta2 toxin genes, which have been detected in the present study, appear to be associated with the virulence of the bacteria. Alpha toxin is one of the most important lethal, hemolytic, and dermonecrotic toxins produced by C. perfringens, is considered to be the major virulence factor and lethal toxin in the pathogenesis of necrotic enteritis (15,18). The beta2 toxin was found to have in-vitro cytotoxicity and be lethal in mice (19). Although it seems to be associated with enteric diseases in piglet (20), horses (21). Although Beta2 toxin gene was isolated from sheep dysentery and an African elephant ulcerative enteritis (22,23).
NetB toxin gene is plasmid coded, pore-forming toxin plays a crucial role in the pathogenesis of necrotic enteritis in broiler (24). The isolates in the present study were found to be negative for netB gene similar to that which have been reported by Nakano et al. (25) and Merati et al. (26).

DNA sequence analysis and Phylogenic Tree:
The cpa sequence (100% to 99.22%) similarity with the recorded sequences, including isolated C. perfringens cpa gene from blue calves in Belgium (L43548.1 and L43547.1), veterinary isolate in UK (X13608.1), broiler in Iran (GU581194), poultry stool (JQ071544.1) and broiler (KT020614.1) in brazil, and cpb2 gene of C. perfringens homology with C. perfringens isolates from broiler in India (MF471365.1), Iran (GU581183.1, GU581182.1, and GU581185.1), goat in Pakistan (MF191716.1 and KX924463.1), with a minor point mutations which were observed in the present study might be related to the fact that all isolates were obtained from broiler of the same age group at a limited geographical region, they appears to be epidemiologically related (27). Although those homologies with other various isolates from different countries might be associated with a substantial border and a large trade relation with other countries, especially, regarding the sources of chicks, and litter in the farms. Eggshell fragments, chick fluff, and paper pads in commercial hatcheries were reported to be contaminated with C. perfringens (28). The wild migratory birds might have a contribution in introducing the C. perfringens strains across countries. Wild birds have been reported as a reservoir of C. perfringens and might have a role in the transmission of the bacteria (29). The ration of animal origin contained high protein, particularly in fish meal followed by meat, bone meal and dry fish, was detected to be contaminated with C. perfringens (30). Also, a high level of C. perfringens contamination was found in processed animal proteins (31). It has been revealed that C. perfringens strains of mammalian species can cause necrotic enteritis in chickens (32).