Expression of the DRB 1 * 1101 Allele in Meat Goats Pasture Exposed to Haemonchus contortus

The Major Histocompatibility class II complex plays an extremely important role in disease resistance and susceptibility. A mutation in the DRB1*1101 gene of the MHC II has been implicated in parasite resistance in sheep. Evaluation of the DRB1*1101 allele and the role it plays in Haemonchus contortus infected goats has not been addressed. This study evaluated expression of the DRB1*1101 allele in goats naturally infected with Haemonchus contortus infection. Total RNA was isolated from whole blood and RT-PCR (Reverse-Transcriptase Polymerase Chain Reaction) using primers designed to target the conserved regions of the human and sheep DRB1*1101 allele. The expected cDNA product was observed at the 201bp of the targeted DRB1*1101 region and verified by cloning and sequencing. Alignment of the Goat DRB1*1101 sequence showed 92% homology at the transcriptional level and 86% homology at the translational level with sheep. Goats pasture exposed to Haemonchus contortus had elevated (P < 0.05) DRB1*1101 allele expression. Expression was highest (P < 0.05) in Boer goats, lower in Myotonic goats and lowest (P < 0.05) in Spanish goats. Males had higher (P < 0.05) DRB1*1101 expression than females. DRB1*1101 gene expression was higher (P < 0.05) in younger vs. older goats. Goats showing resistance to Haemonchus contortus had lower (P < 0.05) DRB1*1101 expression than susceptible goats. Based on our findings, the DRB1*1101 gene in goats can potentially be used as a genetic marker for either resistance or susceptibility to Haemonchus contortus.


Introduction
Gastrointestinal nematode (GIN) infection is a major problem in the small ruminant industry worldwide.According to southern states in Northern America, sixty-two percent of small ruminant producers have problems with GIN.The GIN that causes the most economic burden in small ruminants, are within the Order Strongylida (Keane et al., 2006).A major GIN of concern is Haemonchus contortus.Haemonchus contortus is a blood sucking nematode that causes severe blood loss which can lead to anemia, anorexia, depression, loss of condition, and eventual death (Clark, Keisel, & Goby, 1962;Le Jambre, 1995;Rowe, Nolan, de Chaneet, Teleni, & Holmes, 1988).Haemonchus contortus costs the global small ruminant industry billions of dollars a year spent on anthelmintics and other treatments of this GIN.The major histocompatibility complex (MHC) is a cell surface molecule involved in antigen presentation by immune cells (B lymphocytes, Dendritic cells, and Macrophages).The MHC class II mediates specific immunity, to an antigen.The primary function of the MHC complex is to code for histocompatibility molecules that are antigen presenting receptor glycoproteins (Dukkipati, Blair, Garrick, & Murray, 2006).In ruminants, DRB1*1101 falls in the class MHC IIa (Andersson & Rask, 1988).The DRB is the most polymorphic locus of the MHC gene complex (Andersson & Rask, 1988).The DR genes are highly polymorphic and the molecules encoded by these genes are highly expressed on the cell membranes of macrophages and B cells (Outteridge et al., 1996).Because of the association between the MHC complex and the resistance to nematodes there have been studies on the MHC complex in many mammals (Axtner & Sommer, 2012;Paterson, Wilson, & Pemberton, 1998;Sayers et al., 2005;Schwaiger et al., 1995;Stear, Innocent, & Buitkamp, 2005;Buítkamp, Filmether, Stear, & Epplen, 1996;Stear et al., 1996;Stear et al., 2007).The DRB1*1101 exon 2 encodes the β1 domain, which makes up part of the PBR (peptide binding region) of the DR molecules.It has been found that the residues found in this region have close contact with the peptides that are presented in the PBR or the TCR (T-cell receptor) region (Brown et al., 1993(Brown et al., , 2015)).Thus this region(s) is likely to have functions such as disease resistance and susceptibility (Buítkamp et al., 1996;Li et al., 2011;Sayers et al., 2005;Schwaiger et al., 1995;Untalan, Pruett, & Steelman, 2007;Valilou et al., 2015).Sheep carrying the DRB1*1101 in the second exon has shown resistance to the GIN Teladorsagia circumcincta (Hassan et al., 2011).Studies on the evaluation of the DRB1*1101 exon 2 in goats pasture exposed to Haemonchus contortus have not been conducted.Therefore the objective of this study was to isolate and characterize expression of the DRB1*1101 (Exon 2) allele in meat goats grazing Haemonchus contortus infected pastures.

Experimental Animals and Haemonchus contortus Verification
Meat goats (Boer, Spanish and Myotonic) ranging in ages from 1-5 years old, grazing pasture and supplemented with hay, cracked corn (78%) and ground soybean meal (20%) were housed at VSU Randolph farm in accordance with institutional animal care and use guidelines.Records were kept on these goats as to their dates of birth, place of origin etc.).More than 100 Goats were screened for parasite load and clinical anemia status via fecal egg counts (FEC) in eggs per gram (EPG), packed cell volume (PCV) and FAMACHA eye color charts (FAM), and divided into susceptible and resistant groups accordingly.Our previously published work describe these procedures in detail (Corley & Jarmon, 2012b).The FEC, FAM and PCV data collected were analyzed using SAS version 9.3, (Cary, North Carolina).We determined that goats with > 2000 EPG with PCV ≤ 18 were naturally susceptible and those goats with > 2000 FEC with PCV ≥ 18 were resistant.From the resistant and susceptible groups, thirty animals were sacrificed and blood samples collected and stored at -80 o C for molecular analysis.Haemonchus contortus spp.was verified via nucleotide sequencing as previously published (Corley & Jarmon, 2012a).Nucleotide sequences were analyzed using sequence analysis software (NCBI-BLAST (Altschul, Gish, Miller, Myers, & Lipman, 1990), CLC Main Workbench).

Goat Blood Collection and Preparation
Goats were adequately restrained and blood collected via jugular venipuncture.In brief, the vein was identified by palpation and visual inspection.The area was swabbed with 70% alcohol.Gentle pressure was applied at the thoracic inlet to produce distension of the vein.Blood samples (3 ml) were collected in vials without coagulant using 16-20G needles.Blood samples were subsequently stored at -80 °C for later molecular (RNA) analysis.

Total RNA Extraction from Goat Blood
Total RNA was isolated from selected goat whole blood samples previously stored at -80 °C using the modified RNA extraction protocol (Gauthier, Madison, & Michel, 1997).Approximately 500 l of each sample was mixed with 3 ml of 1X Dulbecco's phosphate buffered saline (Invitrogen; Grand Island, NY 14072) and centrifuged at 5,000 rpm for 20 minutes at 4 °C in a Heraeus MegaFuge 16R centrifuge (ThermoFisher-Scientific).After discarding the supernatant, the addition of PBS and centrifugation was repeated until cells were clean.Once the cells were adequately washed, 1 ml of Guanidium Thiocynate (4 M GTC, 25 mM Sodium citrate pH 7.0 and N-0.5% lauroylsarcosine, Sigma-Aldrich) was added and the cells were suspended by pipetting.Under the fume hood, 10 l of β-mercaptoethanol (Omni-Pur), 2M sodium acetate (pH4) at one-tenth of the volume and 1 volume of phenol:chloroform:isoamyl alcohol (25:24:1) (Fisher Scientific; Fair Lawn, New Jersey 0740) were added and mixed before placing on ice for 15 minutes.The tubes were then placed in the centrifuge for 15 minutes at 4 °C and 5,000 rpm.After centrifugation, the aqueous layer was removed and placed into a sterile tube.The samples were placed on ice for 10 minutes after the addition of 2 volumes of 95% ethanol before being placed in the centrifuge for 15 minutes at 5,000 rpm, allowing RNA to precipitate.Two additional washes with 15 minutes of centrifugation at 5,000 rpm were completed using 200 l of 70% ethanol before allowing samples to air dry.Samples were then resuspended in Diethylpyrocarbonate (DEPC) treated water and stored at -80 °C.A NanoDrop 2000 Spectrophotometer (ThermoScientific) was used to measure RNA concentration and purity.

Reverse Transcriptase PCR (RT-PCR) of Goat DRB1*1101
Oligonucleotide primers were designed from mRNA of the sheep DRB1*1101 nucleotide sequences using the bioinformatics software, CLC Main Workbench (http://www.clcbio.com).Primers and target regions used for isolation of the goat DRB1*1101 gene are given in Table 1.The RT-PCR was conducted using the recommended protocol of the Verso 1-step RT-PCR kit (Thermo Scientific).Modified thermocyling conditions for 40 cycles were as follows: 50 °C 15 minutes, 95 °C, 2 minutes (initial denaturation), 95 °C, 30 secs, 55 °C, 1 minute, 72 °C, 1 minute repeated 39 times and a final extension at 72 °C for 5 minutes.The target DRB1*1101 cDNA was visualized by 1.5% agarose gel electrophoresis and a UGenius UV gel documentation system (SynGene, Fredericksburg, MD) equipped with a high resolution CCD camera.

Nucleotide Sequencing of Goat DRB1*1101 cDNA
For DRB1*1101 nucleotide sequencing, the cDNA (201 bp) products were cut out, purified from agarose gels (Qiagen and Bio-Rad) and cloned using the TOPO TA vector (Life Technologies) and transformed in E.coli DH5 alpha.Colonies were screened (Blue, White), cDNA inserts verified via PCR (DRB1*1101 primers) and positives prepared for nucleotide sequencing per commercial instructions.Samples were sent for sequencing at GeneWiz (South Plainfield, New Jersey).Raw nucleotide sequences were analyzed using sequence analysis software (NCBI-BLAST, CLC Main Workbench).Subsequently, Gene Tools software (SynGene, Fredericksburg, MD) analysis was conducted to determine gene expression of goat DRB1*1101 cDNA.

Measurement of Goat DRB1*1101 Gene Expression
Gene expression of DRB1*1101 in goat blood was measured via UV Gel Documentation and GeneTools analytical software.Area densities of each sample were measured and standardized with automatic correction for background.An average of three replications was measured.The analytical parameters were as previously published (Corley & Jarmon, 2012a).

Statistical Analysis
Area density quantitation data were analyzed using the General Linear Model procedure of SAS and Pearson Correlation analysis.Means (n = 30) were considered significant at P < 0.05.Breed, age and gender differences were considered.

Results and Discussion
Partial codons of the Goat DRB1*1101 exon 2 were successfully isolated.The expected 201 bp product was observed, purified, cloned and sequenced (Figure 1).Sequence alignment of the goat with sheep DRB1*1101 showed 92% homology at the transcriptional level (Figure 2) and 86% homology at the translational level (Figure 3).This sequence has been submitted to the NCBI GenBank (Accession No. KM196595).Expression of the DRB1*1101 was elevated (P < 0.05) in different breeds of meat goats pasture exposed to Haemonchus contortus.Gene expression was highest (P < 0.05) in Boer goats, lower in Myotonic goats and lowest (P < 0.05) in Spanish goats (Figure 4) when compared to each other.Gender differences (P < 0.05) in DRB1*1101 expression were observed (Figure 5) where males showed higher (P < 0.05) expression of the DRB1*1101 than females.These support previous studies that show male goats are more susceptible to Haemonchus contortus infection that females (Corley & Jarmon, 2012a, 2012b).Studies also show that male sheep exhibit more susceptibility to GIN than females, which may or may not be due to a testosterone effect (Saddiqi et al., 2011;Saddiqi, Sarwar, Iqbal, Nisa, & Shahzad, 2012).Age differences influenced DRB1*1101 gene expression.The DRB1*1101 allelic expression was higher (P < 0.05) in younger (4 yrs old) than older (> 4 yrs old) (Figure 6).These data would support the theory that younger goats would be more susceptible to Haemonchus contortus and not yet undergone much gene conversion at the DRB1 gene locus.Overall, regardless of breed, DRB1*1101 expression was higher (P < 0.05) in naturally susceptible (< 18 PCV; > 2000 FEC) than in resistant (> 18 PCV; > 2000 FEC) groups of pasture infected goats (Figure 7) when compared to each other.The packed cell volume was somewhat negatively (47%) correlated (P < 0.05) with DRB1*1101 expression, indicating that the DRB1*1101 was more correlated with susceptibility to Haemonchus contortus rather than resistance (data not shown).However, in the context of resistance or susceptibility to Haemonchus contortus, expression of the DRB1*1101 allele was different within breeds.Boer and Spanish goats naturally resistant to Haemonchus contortus had elevated expression of the DRB1*1101 than those that were susceptible (Figures 8 and 9), whereas Myotonic goats naturally susceptible to Haemonchus contortus showed elevated expression of the DRB1*1101 allele (Figure 10).The DRB is the most polymorphic locus of the MHC gene complex (Andersson & Rask, 1988) therefore, results in this study are not unexpected, as the MHC is highly polymorphic and interbreed differences at the DRB1 locus is expected to be different depending on the length and time of exposure, and the gene conversion events that can occur at the MHC locus.These data also support the existence of allelic differences at the DRB1 locus in sheep with relevance to GIN infection (Polat, Aida, Takeshima, Aniwashi, & Halik, 2015;Sayers et al., 2005;Schwaiger et al., 1995).Since it has been shown that the MHC complex and nematode infection are linked (Axtner & Sommer, 2012;Paterson et al., 1998;Sayers et al., 2005;Schwaiger et al., 1995;Stear et al., 2005), these data support that a correlation between DRB1*1101 expression and Haemonchus contortus infection in goats does exist.However due to the allelic diversity, a SNP approach looking for correlations between DRB1*1101 exon 2 and Haemonchus contortus in inter and intra breed experiments in goats is necessary.Isolation and characterization of the DRB1*1101 allele allows further elucidation of the mechanism of immunity to Haemonchus contortus infection in goats and in the future can lead to support innovative technology in the fight against damage to the small ruminant industry by Haemonchus contortus.

Conclusion
The results of this study indicated that the cross species oligonucleotide primers designed from the conserved regions of the DRB1*1101 genes can successfully be used to isolate the goat DRB1*1101, from blood samples, and used as a biomarker to evaluate gene expression in response to Haemonchus contortus infection in meat goats.Based on our findings, the DRB1*1101 allele could potentially be used as a genetic marker for susceptibility or resistance to Haemonchus contortus between and within breeds.Haemonchus contortus continues to be a problem for the small ruminant industry.As GIN resistance to anthelmintics prevails, cost to the industry does.Persistent evaluation of the DBR1 locus will help us to better understand its role in disease resistance or susceptibility of goats to Haemonchus contortus.

Figure 2 .
Figure 2. Nucleotide Sequence Alignment Showing Homology of Goat and Sheep DRB1*1101 Note.Generated using CLC Bioinformatic Software.

Figure 3 .
Figure 3. Amino Acid Alignment of Translated Goat and Sheep DRB1*1101 Note.Generated using CLC Bioinformatic Software.
Table1.Primers and Target Regions used for Isolation of the goat DRB1*1101 gene from goat blood