Propagation of monospecific culture of Eimeria maxima
20 numbers of day-old coccidian-free commercial White leghorn chicks (BV-300 strain) were procured from Sri Venkateswara Hatcheries (Hyderabad, India) and raised in a sterile brooder cage with provision of ad libitum anticoccidial drugs free water and feed. The birds were examined for Eimeria infection periodically using conventional faecal examination. Birds were challenged with pure E.maxima oocyst culture (obtained from Veterinary College and Research Institute, Namakkal, Tamil Nadu, India) orally on Day-26. The oocysts were isolated from 4 days post challenge (dpc) and propagated by subsequent passages in 3 weeks old coccidia free birds. The purity of oocyst culture was evaluated by species-specific nested PCR based on ribosomal Internal Transcribed Spacer-I (ITS-I) region as described by Bhaskaran et al. 2010.
Rna Extraction And Amplification Of Emgam56 Coding Sequence
The E.maxima oocysts purification, sporulation and excystation were done as mentioned by Wallach et al 1990. Briefly, oocysts were harvested from faecal samples of infected birds using saturated salt solution by flotation technique and purified with 4% hypochlorite solution. The oocysts were incubated with 2.5% (w/v) potassium dichromate solution for 48 hours at 25°C with constant agitation. The sporozoites were obtained by subjecting the oocyst suspension to mechanical disruption using glass beads (1 mm diameter; Sigma-Aldrich, USA), followed by incubating the suspension with Hank’s Balanced Salt Solution (Sigma-Aldrich, USA) comprised of 0.25% (w/v) trypsin and 1% (w/v) sodium taurodeoxycholate (Sigma-Aldrich, USA) at 41°C for 3 hours on shaker incubator. Total RNA was extracted from sporozoites using Trizol reagent (Invitrogen, USA). cDNA was synthesized, amplified using the primer sequences (P1: 5´-CCCAAGCTTACCATGGCCCGCCTCGGCCTCG-3´ and P2: 5´-CCCGAATTCGGTCGTT¬TAGAAGGGCATGG-3´) containing EcoR I and Hind III restriction enzyme sites and under the thermal cyclic condition as described by Xu et al. 2013. The cDNA was cloned into prokaryotic expression vector pET 28a(+) (Novogen, NJ) downstream from an NH2-terminal His-6 epitope tag. The EmGam 56 (1754 bp) clone in plasmid was verified and transformed chemically into component Escherichia coli BL21 pLysS (DE3) cell (Invitrogen, USA).
Expression And Purification Of Recombinant Emgam 56
1 mM Isopropyl β-D-thiogalactoside (IPTG, Bangalore Genei, India) was used to induce recombinant protein expression from E.coli clone using mass fermentor (New Brunswick Scientific) at OD600 = 0.6 for overnight at 25°C with constant agitation (150 rpm). The induced bacterial cell incubated for 4 hours at 37°C and then centrifuged at 1500 rpm for 20 minutes. The cleared lysate was prepared and purified using Ni-NTA chromatography under denaturing condition as per the protocol provided by manufacturer (Qiagen, Germany). The protein concentration of purified protein was measured using Bicinchoninic acid kit (Sigma-Aldrich, USA). Characterization of purified recombinant EmGam 56 has been done by 12% SDS-PAGE analysis under reducing condition (Laemmli, 1970). In western blot analysis, the antibody reactivity of specific chicken antisera (1:25 dilution-Primary antibody) against purified EmGam56 protein was confirmed, while 1:400 dilution of rabbit anti-chicken IgG HRP conjugated antibody used as a secondary antibody (Towbin et al. 1979).
Immunisation Trial
A total of 40 birds specific- pathogen free commercial broiler were purchased as day old chicks (IAEC approval letter no.1803/DFBS/IAEC/2019, dated: 31.12.2019) from the local poultry suppliers. The birds were reared under clean confinement animal house with anticoccidial drugs free diet throughout the experiment. The birds were separated into 2 groups (Group I- EmGam 56, Group II- Infected control). The Group-I were immunized with 50 µg Gam-82 emulsified in Montanide ISA 71 VG adjuvant, intramuscularly on Day-7, followed by same dose of secondary immunization, subcutaneously on Day-14. Both groups of birds were challenged with 20,000 virulent mixed oocyst culture of E. maxima, E. tenella, E. acervulina, orally on Day-21.
Assessment Of Protective Efficacy Of Emgam 56
The protective efficiency of EmGam 56 against homologous and heterologous challenges in immunized group was determined as described by Vijayashanthi et al. (2022). Briefly, the performance parameter of both groups were measured by means of body weight gain using standard weighing balance on 7, 14, 28, 42, and 56 days post immunization (dpi). The parasitological parameters were estimated by oocyst shedding from 5 days post challenge (dpc) to 12 dpc using modified Mc master technique and lesion scoring (7dpc to 12 dpc) as evaluated by Raman et al. (2011). The humoral immunity of immunized group have been estimated by indirect ELISA using pre and post immune sera (0, 14, 21 and 29 dpi) against recombinant EmGam56.