تنوع ژنتیکی MHC کلاس دو بوقلمون تجاری و ارتباط آن با پاسخ ایمنی هومورال

Introduction

Newcastle disease (ND) is a highly contagious viral disease that represents a considerable threat to the poultry industry all over the world. It is caused by Newcastle disease virus (NDV) which has been designated as serotype 1 of Avian Paramyxovirus (APMV-1) (Mishra et al., 2000). NDV is able to infect almost all avian species (domestic and wild species), of which chickens are the most susceptible. Dyspnea, gasping, cyanosis of comb, wattle, crop dilatation, catarrh, foamy mucus in the pharynx, ataxia, paralysis, and torticollis are the most obvious signs of Newcastle disease that affect the respiratory tract, gastrointestinal tract, circulatory and nervous systems (Senne et al., 2004).

Different avian species show different patterns of disease resistance or susceptibility to the Newcastle. Variations in the level of immune responses among different populations can be as a result of several factors including genetic background, environmental factors and virus strain (Alexander et al., 2012; Norup et al., 2011). Major histocompatibility complex (MHC) is one of the best characterized genetic regions controlling disease resistance and susceptibility in chicken. Chicken MHC can influence the immune responses against viral diseases, including Marek disease (Briles et al., 1977; Hunt and Dunn, 2013), Rous sarcoma tumor (Suzuki et al., 2010; Taylor Jr, 2004), Infectious bursal disease (Butter et al., 2013; Juul-Madsen et al., 2006), Newcastle disease (Norup et al., 2011), and Avian Influenza (Hunt et al., 2010). Therefore, MHC could be considered as a genetic marker for identifying immune responses and their association with resistance or susceptibility to diseases.

Our previous studies have shown that polymorphisms at chicken MHC region are associated with immune response to vaccination and productive traits (Emam et al., 2013; Nikbakht and Esmailnejad, 2015). The architecture of MHC region in turkey is similar to chicken. It is divided into two genetically unlinked regions designated as MHC-B and MHC-Y loci located on the same microchromosome (Chaves et al., 2010). The MHC-B locus (BF–BL region) is generally considered as the homolog to the mammalian MHC, which contains most of the antigen processing and presenting genes. MHC-Y locus contains non-classical MHC genes, lectin-like loci, and other unknown genes with varied effects on disease susceptibility (Chaves et al., 2010). Association studies in chicken typically concentrated on the peptide-binding region (PBR) encoded in exons 2 and 3 of class I and exon 2 of the class IIB genes.

In this study, MHC genetic diversity was evaluated in commercial turkey poults (meleagris gallopavo), vaccinated by lentogenic VG/GA Newcastle vaccine. Subsequently, association of MHC haplotypes with humoral immune responses (IgY level) was also analyzed. Although the efficacy of VG/GA vaccine has been evaluated and proved in commercial broiler chickens, there is no information regarding the MHC genotypes and their association with antibody response to this vaccine in turkey.

Material and Methods

Turkey poults and Housing: A total of 92 one day old male commercial turkey poults (Meleagris gallopavo), Hybrid strain (French origin), were selected and kept for 42 days in the experimental pens. The poults were raised in concrete floor pens covered with 8 cm of clean pine wood shavings, and each pen was equipped with one tube feeder and one automatic waterer. Throughout the study, the poults were grown following standard temperature regimens, which gradually decreased from 38 ºC to 23 ºC and under 16L: 8D light schedule. Birds were allowed to consume feed and water ad libitum.

Newcastle Vaccine: One dose of vaccine containing 106.5 EID50 (50 percent Embryo Infectious Dose) of lentogenic freeze-derived live VG/GA strain of Newcastle disease vaccine (Merial Animal Health Limited, Lyon, France) was used in this study. All the turkey poults were vaccinated at age of 10 and 20 days by eye dropper according to the recommendations of the manufacturer. Blood samples were collected at 1, 9, 20, 28, 35 and 42 days of age. The blood samples pre first vaccination (days 1 and 9) were used for detection of maternal antibody level. Samples of pre second vaccination (day 20) were used to assess the IgY titers of the first vaccination, while the blood samples of days 28, 35 and 42 were utilized for evaluation of second vaccination humoral immune responses. Blood samples were immediately centrifuged to separate serum and stored at −20 °C until further analyses.

Assessment of humoral immunity: Specific antibody titer (IgY) against VG/GA Newcastle vaccine was measured using indirect enzyme-linked immunosorbent assay (ELISA) proposed by (Al-Karagoly et al., 2017) Al-Karagoly et al., (2017). Checkerboard titration was carried out to select the optimal concentration of coating antigen and antibody dilution. According to data obtained from checkerboard titration, a 96-well U-shaped polystyrene plate (Nunc, Roskilde, Denmark) was coated with 50µl of VG/GA antigen (7 µg/ml) in PBS (pH = 7.2) per well and incubated overnight at 4 °C. Plates were washed with PBS-T three times and nonspecific binding sites were blocked with 100 µl of PBS-Blotto, incubated for 1 h at room temperature. Plates were washed three times, and all tested sera were diluted in duplicate at 1:100 with PBS-T. Diluted (50 μl) sera were added into each well, at room temperature for 45 min, and then washed three times. Finally, 50 μl of diluted (1:10000) HRP-labeled goat anti-turkey IgY was added to each well and left for 30 min at 37 oC. After washing, TMB substrate was added and the mixture was incubated in dark place for 15 min at room temperature. The reaction was then stopped by the addition of 100 μl of 0.5 N sulfuric acid. The optical density (OD) was measured with an ELISA microplate reader (Stat FAX 2000, Awareness Technology, Inc., USA) at wavelengths of 450 nm. 

The Limit of detection (LOD): The limit of detect (LOD) of IgY was expressed as the analytic concentration corresponding to the sample blank value plus three standard deviation as shown in the following equations:

LOD=X_b1+3S_b1

Where X_b1 is the mean concentration of the blank and S_b1 is the standard deviation of the blank.

MHC genotyping: DNA was extracted from whole blood samples using a Genomic DNA Extraction Kit (Bioneer, Korea). Second exon of MHC class II B locus (389bp) was amplified and used for High Resolution Melting analysis (HRM). Turkey MHC sequences (Genbank DQ993255 and EU522671) were used to design primers. Amplification was performed in a final volume of 25 µL containing 20 ng template DNA, 1.5 mM MgCl2, 250 μM of each dNTP, PCR buffer (20 mM Tris–HCl pH 8.4, 50 mM KCl), 1 µl EvaGreen dye (20X), 1 U/μL of Taq DNA polymerase (CinaClon, Iran) and 20 pmol of specific primers (F: 5’-CTGCCCGCAGCGTTST-3’; R: 5’-CGAGACCCGCACCTTGG-3’). The thermal cycling profile was 1 cycle at 95 °C for 2 min, 30 cycles at 95 °C for 30 sec, 60.5 °C for 30 sec and 72 °C for 40 sec, with a terminal step of 5 min at 72 °C.

HRM curve analysis was performed in a Rotor-Gene™ 6000 thermal cycler (Corbett Life Science Pty Ltd). In order to determine the optimal melting condition for differentiation of MHC alleles, the PCR products were setup on 0.22 °C/s ramping between 75 °C and 95 °C. All specimens were tested and their melting profiles analyzed using Rotor-Gene 1.7 software and the HRM algorithm provided. Normalization regions of 80.47 - 80.97 and 89.71 - 90.21 were used for analysis. The HRM saturating fluorescent dye (Evagreen) was used in the PCR reaction to amplify the MHC class IIB exon2 (389bp). The Evagreen fluoresces strongly only when bound to dsDNA. The changes of the fluorescence during the test can be used both to measure the increase in DNA concentration during PCR amplification and, subsequently, to measure temperature- induced DNA dissociation during HRM. After PCR in the presence of the dsDNA-binding fluorescent dye (Evagreen), amplicons are briefly denatured and then rapidly reannealed. If the DNA sample is heterozygous, perfectly matched hybrids (homoduplexes) and mismatched hybrids (heteroduplexes) are formed. When the temperature is slowly increased again, the DNA begins to melt. The software HRMA system offers different methods of observing the data which assist with distinguishing genotypes even when the melting profile of each sample is similar. One of these methods explained in our study was the normalized HRM curve that was represented as the amount of fluorescence at each given temperature. The second analysis method involves plotting the fluorescence difference between normalized melting curves, called fluorescence difference curve. In this method, the genotype A is chosen as a reference and the difference between each curve and the reference is plotted against temperature. The reference curve becomes a horizontal line at zero and the other genotypes cluster along different paths for easy visual discrimination of the genotype classes.

DNA sequencing: The HRM method was used to detect genotypes and direct sequencing was applied to detect allele sequences and subtypes. Purification of PCR products and sequencing was performed by Macrogen Inc. (Seoul, Korea) on an ABI 3730 XL automatic DNA sequencer. Sequence analysis was achieved by NCBI, BLAST, EMBOSS Transeq, Chromas software version 2.5.1 (www.technelysium.com.au) and bioinformatics software BioEdit 7.0.5.3 (Hall 1999).

Repeatability of the experiment: The experiment was repeated several times to validate the results and a good repeatability as well as reproducibility was found.

Statistical analysis: The amount of gene diversity in the population was evaluated using by number of alleles (Na) and unbiased expected heterozygosity (Hexp), according to the formula proposed by (Nei, 1973). Deviation from Hardy-Weinberg equilibrium (HWE) was also estimated using likelihood ratio test in this population. Association of MHC IIB alleles with immune responses against VG/GA vaccine was analyzed using the following model:

Y_i=µ+ Σ b_jf_ij+εi

Where Y_i is a dependent variable for specific IgY response in ith turkey; µ is a general mean; f_ij is the copy number of the MHC IIB jth allele in the ith turkey; b_j is half the substitution effect for the MHC IIB jth allele; and εi is the residual effect for the ith turkey. Effects were considered to be significant when probability (P) values of ≤ 0•05 were obtained. The association study was evaluated using multivariate regression analysis and GLM procedures of SPSS software version 21.

Table 1.  MHC IIB allele frequency in commercial turkey poults.

Table 2.Significant association of MHC II B alleles with humoral immune responses in commercial turkey poults