Differential Expression of Cytokine in Kadaknath and Commercial Chicken Against Coccidial Challenge

Genetic resistance to disease in chicken or any other species governed by the many genes and their interactions. This is indicated by the communication of the immune cells or membrane proteins such as molecules of Major Histocompatibility Complex (MHC), T-cells receptors (TCR) and immunoglobulins (B cell receptors and secreted proteins such as cytokines and antibodies). Genetic selections for high immune responsiveness and disease resistance can make improvement in the fitness traits of birds and also responsible for effectiveness of vaccination against diseases (Zacharias et al., 2002). Cytokines such as interleukins and interferon gamma (IFN-Y) are produced by T-helper-1 cells and are responsible for delayed hypersensitivity, macrophage activation and enhanced non-specific immunity to parasites (Hong et al., 2006 and Kim et al., 2012).
        
Cytokines are small glycoproteins (<30 kDa) molecules that mediate and orchestrate the complex events in the immune reaction, from the initiation of the acute phase reaction to the clonal expansion of effectors T and B cells. The availability of new technologies such as real-time quantitative PCR allows the estimation of expression of mRNA from cytokine genes without the need for protein or antibody. This has opened up a large area of possibilities to determine cytokine levels in disease, giving increased understanding of the mechanisms of both pathogenesis and immunity (Wigley and Kaiser, 2003).

Experimental birds
 
The present investigation was conducted on Kadaknath (n=12), Cari Vishal (n=12) and Cobb (n=12) broiler chicken under the research project funded by Madhya Pradesh Biotechnology Council, Bhopal, Department of Animal Genetics and Breeding, College of Veterinary Science and Animal Husbandry, NanajiDeshmukh Veterinary Science University (NDVSU), Jabalpur.
 
Coccidial challenge
 
Chicks from each Kadaknath, Cari Vishal and Cobb broiler chicken were randomly divided into a control (C) group and two treatment groups (T₁ and T₂) comprising 12 chicks under each group. On arrival (day 1) chicks were wing-banded, weighed and randomly allocated to the treatments. During the entire experimental periods the standard chick diet free of any anti-coccidial additives and sanitized tap water were given to the birds ad lib throughout the experiment. Group T₁ and T₂ were challenged by gavaging 10,000 and 20,000 sporulated oocyst, respectively to each bird on 21^st days of age. The control group was given 1 ml of Hanks Balanced Salt Solution (HBSS). On day 4, 7 and 14 post challenge, two birds from each group were sacrificed and lesion score, oocyst index, oocyst production (OPG) and mortality (if any) were recorded on these intervals.
 
Total RNA isolation, cDNA preparation and its quality check
 
Total RNA was extracted from intraepithelial lymphocytes using TRIzol method as per manufacturer’s instructions (Rio et al., 2010). The concentrations of total RNA as quantified by spectrophotometer (ND 2000) ranged from 130 to 165 ng/µl. The OD 260/280 of these samples was just above 1.9. The DNase treated total RNA was converted into cDNA by using cDNA synthesis kit. Integrity and quality of cDNA were checked by GAPDH primer. Amplicon of size 264 bp on 2% agarose gel clearly showed no contamination of DNA, having good integrity of total RNA. 
 
Primer selection and its specificity confirmation
 
Specific primers of cytokine genes IL-1β, IL-2, IL-6, IL-17 and IFN-Y were used for expression analysis of these cytokine genes (Table 1; Hong et al., 2006). The cDNA from 1000ng of total RNA extracted from intraepithelial lymphocytes cell of control, treatment group one (T₁) and treatment group two (T₂) on day 4,7 and 14 post challenge were used as templates for expression study IL-1β, IL-2, IL-6, IL-17 and IFN-Y genes. Amplification of all the gene fragments was confirmed by agarose gel electrophoresis (2%) of PCR product for all cDNA samples. Single band of the expected size, i.e., 244 bp for IL-1β, 256 bp for IL-2, 272 bp for IL-6, 292 bp for IL-17 and 259 for IFN-Y genes were observed on 2% agarose gel electrophoresis. The specificity of primers was also confirmed by real-time PCR dissociation curves. A primer concentration of 1.25 pmol was optimized for the amplification of all the gene fragments.
 

 
Real-time RT-PCR protocol
 
The real time RT-PCR was performed by SYBR green chemistry (ABI, USA) using cDNA as template for different genes. All PCR reactions were performed in triplicates with GAPDH as an internal control to normalize the amount of sample RNA in optical 96 well plate in final reaction volume of 20 μl. Thermal cycling condition comprised of initial denaturation at 95°C for 10 min followed by 45 cycles of denaturation at 95°C for 15 s, primer annealing and extension at 60°C for 1 min. At the end of extension step, fluorescence was captured. At the end of each run, a melt-curve analysis (95°C for 15 s, 60°C for 1 min and increase of 0.5°C/5 s until 95°C) was performed to assess the specificity of the amplification. The specificity of the amplification products was also confirmed by the appearance of predicted-size fragments after 2% agarose gel electrophoresis.
        
After quantiûcation, the data were analyzed with the Sequence Detection System (SDS) software v1.3.0 (Applied Biosystems). Comparative C_T method was used for relative quantiûcation of the target cDNA relative to GAPDH cDNA (endogenous control) as per the standard methods (Livak and Schmittgen, 2001). The specificity of real time PCR amplification was determined on the basis of specific band size of the respective gene on 2% agarose gel electrophoresis.
 
Relative expression of cytokine genes
 
The efficiency of real-time PCR and relative quantification of target genes was calculated based on the methods described by Livak and Schmittgen (2001). The relative expression of cytokine genes in treatment groups (T₁ and T₂) was compared to that of control of respective breeds /lines and fold changes in gene expression were calculated using the Sequence Detection System (SDS) software v1.3.0 (Applied Biosystems). Fold changes were subjected to analysis of variance (ANOVA) using the MSTAT-C software to determine the effect of breed, coccidial dose and interval (day) on expression of cytokine genes. 

The relative expression of IL-1β, IL-2, IL-6, IL-17 and IFN-Y in Kadaknath, Cari-Vishal and Cobb broiler chicken in control and treatment group on day 4, 7 and 14 post challenge have been depicted in Fig 1. The least squares analysis of variance (Table 2) revealed highly significant (p<0.01) effect of breed for IL-1β gene and IFN-Y gene. The effect of dose was significant (p<0.05) for IL-6 gene and non significant for remaining cytokine genes. The effect of interval (post challenge) was found to be highly significant (p<0.01) for all five cytokine genes.
 

 

        
The LSM for relative fold expression of these five cytokine genes (IL-1β, IL-2, IL-6, IL-17 and IFN-Y) have been presented in Table 3 and depicted in Fig 1. The relative mean fold expression of IL-1β gene for T₁ (dose 1) and T₂ (dose 2) ranged from 9.91±0.14 (Kadaknath) to 105.78±0.52 (Cobb) and 14.47±0.28 (Kadaknath) to 119.01±0.29 (Cari-Vishal), respectively. The highest and lowest relative mean fold expression for IL-2 gene for T₂ (dose 2) was found to be 242.19±0.40 and 6.25±0.03 in Cobb at day 4 and 14 post challenge, respectively. However, the relative mean fold expression of IL-2 gene for T₁ (dose 1) was found to be maximum in Cobb (75.84±0.58) and minimum in Kadaknath (12.77±0.38) at day 7 and day 14 post challenge, respectively.
        
For IL-6 gene, the lowest relative mean fold expression was observed in Kadaknath (8.11±0.05) for dose 1 at day 14 while the highest value was recorded in Cobb (99.04±0.92) at day 7 for dose 2. The relative mean fold expression for IL-17 gene was found to be lowest with a value of 11.04±0.29 (dose 1, day 14) in Kadaknath and maximum was seen in Cobb i.e. 197.40±0.37 (dose 2, day 4). For IFN-Y gene, the lowest relative mean fold expression was found to be 8.11±0.19 (dose 1, day 14) in Kadaknath while the highest value was recorded in Cobb (84.74±0.25) for dose 2 at day 7 post challenge. These differences in expression of cytokine genes among the three genetic groups could be attributed to the fact that expression of cytokine genes varies depending upon the genetic background of birds, level of coccidial dosage, genotype of birds and age of infection (Schat and Davis, 1999; Zhang et al., 2012). The higher relative expression of all five cytokine genes in all the three genetic groups, confirming that these cytokine genes play an important role in the immune response to coccidiosis.
        
The upregulation of IL-10 gene (Table 3; Fig 1), as observed in the present study, was also reported by Rothwell et al., (2004). They measured the expression of mRNA for chIL-10 and other signature cytokines in gut and spleen of resistant (line CB12) and susceptible (line 15I) chickens during the course of an E. maxima infection. Further their findings were in accordance with the present results that susceptible chickens showed higher levels of this cytokine gene expression after infection in the small intestine than did resistant chickens. The upregulation of IFN-Y, IL-10 and IL-12 genes have also been reported by Hong et al., (2006) in intestinal lymphocytes following E. acervulinaand E. tenellaprimary and secondary infection which is in agreement with present findings. They found that transcripts encoding the cytokines genes IFN-Y, IL-10 and IL-12 were increased following E. tenellaprimary infection. They also observed that cytokines IL-3 gene increased upto 327 fold following primary or secondary infection with both parasites. Similar findings were also reported by Gadde et al., (2011) in turkey poults. They found an increased expression of IFN-Y and IL10 genes on day 7 post challenge and IL1β and IL13 genes on day 7 post challenge following an oral infection with 12.5 × 10³ oocysts of Eimeria adenoeides. Significant up-regulation of IL-10 mRNA in the caecum was also reported by Haritova and Stanilova (2012) in poultry with experimental coccidiosis. Zhang et al., (2012) also observed significant up regulation (156.1-1117.1-fold change) of IFN-α, IFN-β, IFN-Y, IL-1β and IL-12 genes in day-old chickens at 3 hour post infection in comparison to the different peak level times and relatively low expression of cytokine genes in the 3 week old chickens. Similar findings were reported by Al-Idreesi et al., (2013) who noticed significantly higher level of IFN-Y in the serum samples when compared with those of the control negative group. They observed that the serum levels of IFN-Y increased gradually after first and second dose of vaccine but increased drastically after challenge, as compared with control negative group. In contrast to the present findings, Kim et al., (2014) reported the down regulation of the chIL-17RA gene transcript in caecum tissues from chickens infected with E. tenella. These finding commensurate that the higher level of cytokine genes as observed in Cobb and Cari-vishal can be correlated to genetic susceptibility to coccidial infection.
 

The differential expression pattern of cytokine genes in three genetic groups showed different degree of mucosal immune response to Eimeria infection. In view of the relative expression of different cytokine genes, it can be concluded that Kadaknath birds were the most resistant to coccidial challenge followed by Cari-vishal and Cobb. These differences in expression of cytokine genes among the three genetic groups could be attributed to the fact that expression of cytokine genes varies depending upon the genetic background of birds, level of coccidial dosage, genotype of birds and age of infection. The mean relative fold expression of cytokine genes increased with dose and was lowest on day 14 post challenge. The relative expression of these cytokine genes could play a crucial role by driving higher immune responses susceptible breed/line.

The authors are highly thankful to M.P. Biotechnology Council, 26-Kisan Bhawan, Bhopal for granting financial support to carry out this research work under MPBT project on coccidiosis.