Expression of The Transient Receptor Potential Channel 4 ( TRPC 4 ) Gene in Goats Naturally Exposed to Haemonchus contortus Infection

Expulsion of gastrointestinal nematodes (GIN) requires gut contractions and glycoprotein hyper-secretion for detachment from the gut wall. The Transient receptor potential cation channels (TRPC) facilitates contraction of smooth muscle. A mutation in the TRPC4 of mice significantly reduces contraction and motility of the intestine. Thus far, the correlation between TRPC4 and GIN infection has not been evaluated in goats or any other species. This study evaluated gene expression of TRPC4 in Haemonchus contortus exposed resistant goats. Goats that were naturally susceptible and resistant to Haemonchus contortus were sacrificed and intestinal tissues collected. From conserved regions of human, mouse, rat, and bovine TRPC4 gene alignments, oligonucleotide primers were generated using CLC Main Workbench bioinformatics software. The RT-PCR and quantitative real time pcr were performed using total RNA extracted from intestinal tissues. The expected 388bp cDNA product was amplified and sequenced. The goat TRPC4 showed 88 and 87% homology to rat and mouse and 98%, 92%, 91% and 90% homology to the bovine, horse, pig and human TRPC4 genes, respectively. The TRPC4 expression increased (P<0.05) in naturally susceptible goats. There were breed and gender effects (P<0.05) on TRPC4 expression. A strong (P<0.05) correlation was evident when the variables TRPC4 gene expression, clinical anemia, and parasite load were compared in goats. These data indicate that TRPC4 may aid in elucidation of the mechanism of action of the TRPC genes involved in gastrointestinal contraction and motility and their link to GIN infection.


Justification
The ability of the intestine to expel worms is dependent on many factors, one of which is intestinal contractility (Artis, 2006;Hasnain et al., 2011).The TRPC4 in particular has been linked to gastrointestinal contraction and motility (Kim, So, & Kim, 2006;Unno et al., 2006).Although the role of TRPC4 has been identified as crucial in intestinal contractility in mice, the TRPC4 gene has not been isolated in goats, nor evaluated in Haemonchus contortus infection.To date, the relationship between gene expression of TRPC4 and the response to GIN infection has not been assessed in goats or any other species.Moreover, the relationship between TRPC4 control over gastrointestinal motility and contractility and Haemonchus contortus infection in goats has not been investigated.Therefore the objectives of this study were to identify and characterize the TRPC4 gene of the goat and examine gene expression of TRPC4 in selected pasture exposed goats.

Experimental Animals and Haemonchus Contortus Detection
Animals used in this study were Spanish and Myotonic goats.These animals were housed at VSU Randolph farm in accordance with institutional animal care and use guidelines.More than 100 Goats were screened for parasite load and clinical anemia status via fecal egg counts (FEC), 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, 2012).The FEC, FAM and PCV data collected were analyzed using SAS version 9.1.3,(Cary,North Carolina).We determined that goats with > 2000 eggs per gram of feces (EPG) with PCV <18 were naturally susceptible and those goats with > 2000 FEC with PCV >18 were resistant.Animals were sacrificed and intestinal tissue samples collected and stored at -80 o C for nucleic analysis.Haemonchus contortus spp.was verified via nucleotide sequencing as previously published (M.Corley & A. Jarmon, 2012).Nucleotide sequences were analyzed using sequence analysis software (NCBI-BLAST (Altschul, Gish, Miller, Myers, & Lipman, 1990), CLC Main Workbench).

Goat Intestinal Tissue Collection and Preparation
Animals were sacrificed as previously published (Corley & Jarmon, 2012) in accordance with national humane euthanasia guidelines.Intestinal including jejunal tissues were collected in sterile PBS and RNAlater (Invitrogen, NY) and placed at -80 o C for further molecular analysis.Tissues were homogenized in sterile PBS, centrifuged at 10,000 g and supernatants collected for gene expression analysis.

Total RNA Extraction from Goat Intestinal Tissue
Total RNA was isolated from goat tissue samples previously stored at -80 o C using a modified RNA isolation procedure (Gauthier, Madison, & Michel, 1997).Total RNA extraction procedures were performed according to previously published methods (Corley & Jarmon, 2012).Concentration and purity of total RNA were measured using a Nanodrop ND-1000 spectrophotometer (Thermoscientific).The RNA was stored at -80 o C for later use in RT-PCR and qRT-PCR.

Reverse Transcriptase PCR (RT-PCR) of Goat TRPC4
Oligonucleotide primers were designed from mRNA of the bovine human, horse, pig, mouse and rat TRPC4 nucleotide sequences using the bioinformatics software, CLC Main Workbench (http://www.clcbio.com).Primers and target regions used for isolation of the goat TRPC4 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 o C 15 minutes, 95 o C, 2 minutes (initial denaturation), 95 o C, 30 secs, 55 o C, 1 minute, 72 o C, 1 minute repeated 39 times and a final extension at 72 o C for 5 minutes.The target TRPC4 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 TRPC4 cDNA
For TRPC4 nucleotide sequencing, the cDNA (388bp) products were cut out and purified from agarose gels (Qiagen and Bio-Rad).The purity and concentration of gel purified cDNA was measured and 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

Discuss
The TRPC4 is upregulated in more Haemonchus contortus susceptible goats.The TRPC4 was also upregulated in male goats compared to female goats.This response is logical, as it has been previously demonstrated that male goats are more susceptible to Haemonchus contortus infection than female goats (M.Corley & A. Jarmon, 2012).There was no significant difference in TRPC4 gene expression relative to age, but older goats (5-7 years old) tended to express more TRPC4 than younger goats (1-5 years old).This response could be explained by the fact that older goats have a weaker immune system than younger but a closer look would have to be made by widening the age gap in different groups of goats to see any significant effect in age groups for TRPC4 gene expression.Overall, there was a strong positive correlation between TRPC4 gene expression and FAMACHA eye color chart scores and FEC, and a strong negative correlation with PCV in goats.An increase in TRPC4 gene expression correlated with a high parasite load and anemia.On the other hand TRPC4 was down regulated in goats that were not experiencing anemia.This indicated that TRPC4 is upregulated in goats more susceptible to Haemonchus contortus infection.These data indicate thatTRPC4 gene expression correlates with susceptibility in goats pasture exposed to Haemonchus contortus.This would indicate that TRPC4 may be a potential biomarker for susceptibility rather than resistance to Haemonchus contortus infection in goats.

Conclusion
The results of this study indicated that the cross species oligonucleotide primers designed from the conserved regions of the TRPC4 genes can successfully be used to isolate a partial sequence of the goat TRPC4, from intestinal tissue samples, and used as a biomarker to evaluate gene expression in response to Haemonchus contortus infection in goats.Based on our findings, the TRPC4 gene could potentially be studied as a genetic marker for susceptibility to GIN infection, specifically Haemonchus contortus and could potentially be targeted for drug development in the treatment of GIN infection.