All the samples received to the laboratory, were inoculated in Robertson’s cooked meat media (RCMM). Inoculated RCMM were heated at 80°C for 10 minutes to destroy vegetative form of organisms, followed by anaerobic incubation using anaerobic jars with anaerogas pack at 37°C for 24-48 hours. After incubation, a loopful of broth cultures were further streaked onto 5% sheep blood agar for cultivation of Clostridium perfringens and for determination of hemolytic activity of the organism. The blood agar plates were incubated anaerobically for 24 hrs at 37° C (Balachandran et al, 2018).

Two to three suspected pure colonies from5% sheep blood agar plates, showing a double hemolysis pattern were suspended in 100μl of distilled water in a 1.5 ml microcentrifuge tube and further DNA was isolated by boiling method (Dar et al, 2017).

The isolates were confirmed for detection of C. perfringensusing species specific 16S rRNA gene sequences (Tonooka et al., 2005). After DNA extraction, the identity of the samples was confirmed as C. perfringens based on the amplification of specific 16S rRNA gene, using specific primers (Table 1). PCR was performed by preparing a final reaction volume of 20 μl in 0.2 ml thin walled PCR tubes. It was prepared by taking 3.3 master mix supplied with Taq DNA, MgCl2 and dNTPs; adding 2 each of forward and reverse primers, 2 μl template DNA and 10.7 μl nuclease free water. The reaction was carried out in a thermocycler as follows: initial denaturation of 94°C for 2 min, followed by 35 cycles of 94°C for 30 sec, 56°C for 30 sec, 72°C for 1 min and a final extension at 72°C for 2 min.

For typing, all the isolates of C. perfringens confirmed by species specific gene were subjected for multiplex PCR (m-PCR) using toxin genes a (cpa), β (cpb) and β2 (cpb- 2) as referred previously by Brady et al. (2010) (cpa and cpb gene) and Dar et al., 2017 for cpb2. (Table 1). The multiplex PCR assays in this study were performed in 25 μl reaction volume in Mastercycler gradient (Eppendorf AG, Germany). Three pairs of specific primers corresponding to each toxin were used to determine the presence of cpa, cpb, and cpb2 toxin genes using a multiplex PCR technique for all species specific confirmed isolates. PCR was performed by preparing a final reaction volume of 25 μl in 0.2 ml thin walled PCR tubes. It was prepared by taking 3.5 pl master mix supplied with Taq DNA, MgCl ₂and dNTPs; adding 1.5 pl each of forward and reverse primers, 2 pl each template DNA and 10.5 pl nuclease free water. Detail about primers given in table 1. Samples were subjected to 35 PCR cycles, each consisting of Initial denaturation of 15 min at 95 °C; 30 sec of denaturation at 94°C; 90 sec of annealing at 53°C, 90 sec of elongation at 72°C and final extension of 10min at 72°C.

For the detection of virulent gene netB all the positive isolates were subjected to Uniplex PCR (Ezatkhah et al,2016). Details of the primers are given in Table 1. The Uniplex PCR assays in this study were performed in 25 pl reaction volume in Mastercycler gradient (Eppendorf AG, Germany). PCR was performed by preparing a final reaction volume of 25 pl in 0.2 ml thin walled PCR tubes. It was prepared by taking 12.5 pl master mix supplied with Taq DNA, MgCl₂ and dNTPs; adding 2 pl each of forward and reverse primers, 3 pl template DNA and 5.5 pl nuclease free water. PCR conditions were 95°C for 10 min, 35 cycles of 94°C for 30 sec, 55°C for 30 s and 72°C for 1 min followed by 72°C for 10 min.

The aim of present study was toxinotyping of C. perfringens associated with necrotic enteritis (NE) in poultry. Out of 50 samples of NE suspected birds and 75 samples of healthy birds, 35 isolates from NE suspected birds and 08 isolates from healthy birds respectively (total = 43), were characterize phenotypically as C. perfringens.For phenotypic characterization all the samples were inoculated in RCM, which showed heavy turbidity along with gas production in RCMM after 24-48 hrs. of incubation,turning the meat particles pink in colour along with foul odor.

RCMM along with Brain heart infusion (BHI) broth was found to be the best medium for initial isolation of C. perfringens as described by Malmurugan et al. (2012). Das et al. (2008) and EI-Jakee et al. (2013) used RCMM for initial isolation of C. perfringens from necrotic enteritis cases of poultry.

After enrichment, samples were streaked on 5% sheep blood agar. On 5% sheep blood agar after incubation, grey, glistening, smooth and round colonies of 1-2 mm diameter surrounded by double zone of haemolysis, were visualized, which is a characteristic feature of C. perfringens. Similar types of colonies were observed by Salah-Eldin et al.(2015) and Keyburn et al. (2010) of C. perfringens on blood agar. For morphological characteristics the culture smears were prepared from the pure colonies and stained by Gram’s method which revealed Gram- positive, short and plumpy rods with blunt ends which correlated well with the reports of Hafez et al. (2011) and Khairy et al.(2013).

All the total 43 phenotypically confirmed isolates were further subjected to species specific 16S rRNA gene PCR, which showed 100 percent positive towards C. perfringensby species specific PCR. Molecular characterization revealed that all 43 isolates were positive for C. perfringensby species specific PCR showing amplicon size of 481 bp on 1.5% agarose gel electrophoresis (Fig. 1).

These present findings were in close association with the results of Nyrah et al. (2017) who recorded 66 isolates from poultry and further confirmed by 16S rRNA secies specific PCR. Similar findings were reported by Dar et al. (2017) who confirmed all the isolates of C. perfringensby species specific PCR. Nazki et al. (2017) recorded 125 (70.62%) isolates were positive for C. perfringens, by using species specific primers.

Total 43 isolates which were confirmed by 16S rRNA were further processed for toxinotyping of cpa, cpb and cpb2 toxin by multiplex PCR (Fig. 2 and 3).

Lane 1,2,3,6,7- Positive 3amples for cpa and cpb2; Lane 4,5,8Negative control; L - l00 bp DNA Ladder

Lane 1,2,3,6,7- Positive samples for cpa and cpb2; Lane 4,5,8Negative control; L -100 bp DNA Ladder

Out of total 35 isolates from NE suspected birds, 17 (48.57%) isolates showed cpa toxin, 12 (34.28%) isolates showed cpb toxin, 06 (13.95%) isolates were positive for cpb2 toxin. Among this, 06 (13.95%) isolates carried both the gene cpa and cpb2. Total 08 isolates from healthy birds were found positive for C. perfringens, out of which cpa toxin was detected in 50 percent of isolates while, cpb and cpb2 were found negative. On toxinotyping of the isolates in different types, it was seen that, out of 43 isolates, 27(62.79%) isolates belonged to C. perfringens type A and 12 (27.90%) isolates belonged to C. perfringens type C. Toxin cpa (alpha) has been indicated as a main virulence factor for the development of NE in poultry and this toxin hydrolyses phospholipids and promotes cellular membrane disorganization which leads to destruction of intestinal mucosa. Nyrah et al. (2017) detected total 66 isolates of C. perfringens and showed 100 percent positivity towards cpa toxin while 13 isolates (19.69%) showed positivity for cpb2 toxins. Recently, it was speculated that cpb2 positive C. perfringens may cause focal duodenal necrosis in egglaying chickens (França et al., 2016). The results of cpb2 in their study was in agreement with the present findings with similar agreement. Park et al. (2015) and Balchandran et al. (2018) recorded 100 percent prevalence of cpa toxin in the isolates of C. perfringens from NE cases while Rasool et al. (2017) reported 86.4 percent prevalence of cpa gene, but the prevalence of cpa gene in the present study was recorded less as compared to other studies. The possibility of the same may be due to the presence of other toxin gene which were prominently expressed and reduced the expression of the cpa gene.

Isolates carrying both the gene i.e. cpa and cpb2 were recorded in the present study, the findings of which was similar to the findings of Guran et al. (2013) and Datta et al. (2014) who found cpa and cpb2 with 2.1 percent and 33.3 percent prevalence rate, respectively. Toxin cpb (betatoxin) is a highly trypsin-sensitive protein that causes mucosal necrosis and possibly central nervous signs observed in domestic animals. It is responsible for the diseases caused by C. perfringens type C which causes necrotic enteritis in fowls.

Recently, C. perfringens strains producing netB toxin have been considered as the definitive cause of NE in chickens (To et al., 2017). In present study, total 04 (11.42%) isolates showed positivity towards netB out of 35 isolates from diseased birds (Fig. 4). None of isolates were positive for netB from healthy birds. NetB showed overall 9.30 percent prevalence from 43 isolates. These findings were in correlation with Lyhs et al. (2013) reported that the netB was identified in 6.6 percent from C. perfringens strains which were isolated from NE cases in turkeys. Tolooe et al. (2011) who reported, 52.8 percent prevalence of netB from diseased birds and none from healthy birds.

Lane 1 - Positive samples; L- 100 bp DNA Ladder

Ezatkhah and associates (2016) reported netB gene for the first time at a low incidence (7.77%) in chickens with NE in organic broiler farms and detected 17.98 percent prevalence by PCR. Keyburn et al. (2009) studied various C. perfringens isolates from NE- positive and NE- negative flocks from three continents and found 70 percent and 3.6 percent netB positive isolates, respectively. Further studies suggest that, potentially virulent toxins cpb2 along with netB could contribute to disease effects in C. perfringens isolates (Keyburn et al. 2008). Gaucher et al. (2017) reported an increase in the proportion of C. perfringens strains harboring the cpb2 gene and the netB gene. A possible explanation could be that netB is easily transferred between several C. perfringens genotypes, thus increasing their virulence (Johansson et al, 2009). NetB is involved in virulence and is a secreted protein that is readily accessible to the host immune system, and this represents a promising target for vaccine development (Keyburn et al. 2010). Confirmation of the role of netB in disease came from the finding that most necrotic enteritis outbreak strains carry the netB gene, whereas nonnecrotic enteritis derived C. perfringens isolates lack this gene (Keyburn et al., 2008; Keyburn et al, 2010). Present study focused on targeting the netB toxin which is one of the virulent gene detected in NE disease from poultry. Toxin netB is produced only when the C. perfringensconcentration is high and sufficient damage is caused to host cell (Timbermont et al. 2011).

I would like to thank my institute, Krantisinh Nana Patil College of Veterinary Science, Shirwal for their financial supports.