Candidate Gene Analysis of Genetic Resistance to Gastrointestinal Nematodes in Sheep Through Association of Single Nucleotide Polymorphism with Phenotypic Traits

Gastrointestinal nematodes (GIN) such as Haemonchus contortus impose severe constraints in small ruminant production. Conventional approaches including deworming and other medication are not effective because of the anthelmintic resistance developed by the parasites. A potential alternative measure to alleviate the problem is breeding for disease resistance. The genetic variations within and among livestock breeds against the diseases were reported, in which some breeds or individuals within a breed are more resistant to the disease than others in the same population (Jovanovic et al., 2009). Indigenous sheep breeds of India exhibit enhanced resistance to various diseases. There are well-documented shreds of evidence for within and between-breed genetic dissimilarities in resistance to GIN infection and offers the opportunity to select animals for enhanced genetic resistance to the diseases (Zvinorova et al., 2016).
       
In India, the native sheep breeds have been found to have better resistance against parasites compared to crossbreds and exotic sheep. There are reports for genetic variation between indigenous sheep and exotic crosses in resistance to gastrointestinal nematodes such as Haemonchus contortus, Trichostrongylus colubriformis and Ostertagia circumcinta (Arora and Prince, 2004). Several studies have been carried out to detect susceptible and resistant individuals based on phenotypic indicator traits like fecal egg counts (FEC), packed cell volume (PCV) (Nimbkar et al., 2003). ICAR-Central Sheep and Wool Research Institute, Avikanagar, Rajasthan developed the resistant line for Haemonchus contortus in Malpura and Avikalin sheep through regular screening for fecal egg counts at naive and exposed stage of infection in flocks and selection of progenies. The resistant line had lower fecal egg counts compared to their counterparts in susceptible line (Gowane et al., 2020). Over the last few years, various studies conducted to analyze the role of Toll-like receptor (TLR) in disease resistance against infectious diseases across the world, but studies in Indian breeds for analyzing the host genetic resistance against the parasites at molecular level are limited. This study was executed to analyze the genetic resistance to GIN infection through SNP markers within TLR genes in Kilakarsal and Vembur breeds of sheep.

A sum of 50 Kilakarsal sheep breed from Livestock Farm Complex, Veterinary College and Research Institute, Tirunelveli and District Livestock Farm, Abishegappatti, Tirunelveli and 50 Vembur sheep breed from Livestock Farm Complex, Veterinary College and Research Institute, Tirunelveli and Government Sheep Farm, Sattur, Virudhunagar, of both sexes, 6 to 18 months of age were randomly selected. They are maintained under natural free grazing. The animals were dewormed as per the standard procedure. The phenotypic indicator traits for GIN infection viz., FEC, change in PCV and change in body weight were recorded at 0, 30 and 60 days after deworming for a period of two years from 2015 to 2017. The eggs and third-stage infective larvae of GIN (Haemonchus contortus, Oesophagostomum sp., Strongyloides sp.) were examined from fecal samples of naturally infected sheep. DNA samples were extracted from the whole blood using a modified high-salt method (Miller et al., 1988). A sum of 25 SNP markers from five TLR genes viz., TLR3, TLR5, TLR6, TLR9 and TLR10 were applied for genotyping (Table 1). Competitive allele-specific PCR-based endpoint genotyping was accomplished to type the SNPs. Endpoint allelic discrimination module implemented in ILLUMINA Eco Real-Time PCR was employed to call the genotypes formed on fluorescent intensity recorded for individuals of the two alleles. The emission data of all the samples were plotted in cluster plots for respective alleles and the genotypes were called based on distinct clustering (Fig 1). Basic diversity indices namely, allele frequency and genotype frequency were assessed by PEAS -Package for Elementary Analysis of SNP data software program (Xu et al., 2010). A complete fixed effects model was used for the analysis of the association of various genotypes at each SNP with FEC, change in PCV and change in body weight. The data on FEC were converted to log transformation for applying the LSMLMW program of least squares.
 

Indicator traits for GIN infection
 
In the present study, the mean of post-deworming FEC values ranged from 0 to 4,500 in Kilakarsal sheep and from 0 to 3,150 in Vembur sheep. The post-deworming change in PCV values varied from -11 to 7% in Kilakarsal sheep and -7 to 5% in Vembur sheep. The post-deworming change in body weight varied from -4.25 to 3.70 kg in Kilakarsal sheep and -2.2 to 6.25 kg in Vembur sheep.
 
Competitive allele specific PCR based end point genotyping
 
In the sum of 25 SNPs, 22 SNPs were polymorphic, while SNPs TLR3_1081_AC and TLR9_2036_CT were monomorphic in Kilakarsal and Vembur sheep, whereas TLR10_1180_AG SNP was monomorphic in the Kilakarsal population only. The global minor allele frequency (MAF) for 22 polymorphic SNP loci ranges from 0.06 to 0.48 (mean: 0.23). However, 49.8% of SNP loci showed MAF of 0.20, 29.7% of SNP loci showed MAF of 0.30 and 18.1% of SNP loci showed MAF of 0.40.
 
Effect of non-genetic factors on phenotypic indicator traits for resistance to GIN
 
The effect of the farm had no significant influence on FEC and change in body weight, however, had a significant effect on change in PCV (P<0.05; Table 2). The sex of the sheep population also results in no significant differences with respect to the phenotypic indicator traits such as FEC, change in PCV and change in body weight (P>0.05). As reported earlier, in the present study also the indicator traits were neither influenced by the breed (Kathiravan et al., 2014) nor by other non-genetic factors (Varadhrajan and Vijayalakshmi, 2015). Since markers with MAF >0.05 were reported to be suitable for association studies (Tabangin et al., 2009), 22 polymorphic SNP loci with MAF >0.06 in this study are suitable for such studies.
 

 
Association of SNPs in TLR genes with resistance to GIN infection
 
All three possible genotypes were observed in the 22 polymorphic SNP loci except TLR10_1180_AG in which only two genotypes GG and AG were present (Table 3). In the TLR9_1769_CT locus, a significant difference (P<0.05) was observed with mean FEC of 460.36±1.16, 23.42±2.72 and 400.39±1.55 for CC, TT and CT genotypes, respectively (Table 4). The TT genotype in TLR9_1769_CT locus showed the lowest least-squares mean FEC and showed a significant difference, hence the individual with this genotype is considered resistant to GIN infection in these breeds. The remaining 22 SNP loci showed no significant difference (P>0.05) with mean FEC ranging from 184.53±1.72 to 3701±4.21 in Kilakarsal and Vembur sheep breeds taken together. The highest least-squares mean FEC was observed in AA genotype of TLR10_595_AG SNP and TT genotype of TLR10_771_CT with a mean of 3701±4.21. The association of genotypes with PCV change revealed no significant effect (P>0.05) at all the SNP loci studied. With respect to change in body weight, all the SNP loci studied showed no significant association of genotypes (P>0.05).
 

 

       
Among the 23 SNP, analyzed for the association with resistance to GIN infection the TLR9_1769_CT locus had a significant difference and the TT genotype showed a significant difference, hence, the individual with this genotype is considered resistant to GIN infection. Similarly, Kathiravan et al., (2014) reported significant variances (P<0.05) in FEC, change in body weight and PCV at SNP loci in Corriedale, Pampinta, Indonesian Thin Tail and Indonesian Fat Tailed sheep and concluded that the SNP loci detected had the high potential for imminent association studies. Among different breeds, Indonesian fat-tailed sheep showed the lowest mean FEC (mean log FEC 3.2360.16) and Corriedale sheep exhibited the highest mean logFEC 3.5860.079, but this breed variation is not significantly different (P>0.05). It is in accordance with our results, in which no breed differences were found between Kilakarsal and Vembur sheep breeds. They also stated that genotypes at NAV3_591 and GLI1_576 were found to have significant differences in their FEC, whereas genotypes at ZBTB39_51, IL20RA_422, PIK3CD_433 and TLR7_2491 showed no significant differences in their mean log-transformed FEC. Also, they reported that significant association of genotypes with change in PCV at SNP loci namely, ANKRD52_113, CSRNP2_65, ESYT1_157, NAV3_591, TIMP3_716 and IL2RA_388, however, the similar SNPs to our present study did not show any significant association with change in PCV. Also, they stated that four SNP loci viz., ACVRL1_445, GPR84_520, SMCR7L_517 and TARBP2_97 had a significant association (P<0.05) and other SNPs including TLR5_2276, TLR7_2491 and TLR8_1045 did not show any significant association with change in body weight. This is comparable with our study in which the SNPs located in TLR5, TLR7 and TLR8 genes were not significantly associated with phenotypic indicator traits.
       
Similarly, McRae et al., (2014) analyzed the genome-wide SNP data for loci associated with genetic resistance to GIN infection in Romney and Perendale sheep and revealed candidate genes for cytokine response and chitinase activity were interned within QTLs associated with resistance. Mohammad et al., (2019) identified genomic regions on OAR2, 6, 18 and 24 which were associated with GIN resistance in Australian sheep. Ahbara et al., (2021) analyzed SNP genotypes in Tunisian sheep and reported that the candidate genes IL-4, IL-13, SLC22A4 and SLC22A5 were involved in innate immune against GIN infection. Maria et al., (2021) reported the associations between candidate genes and FEC, PCV in Corriedale and Pampinta sheep infected with Haemonchus contortus and identified SNPs positioned on OARs 3, 6, 12 and 20, represented allelic variants from the MHC-Ovine Lymphocyte Antigen-DRA, IL2 receptor β, C-type lectin domain families, TLR 10 and NLR showed significant differences. Carracelas et al., (2022) identified genomic regions linked with GIN resistance in Corriedale sheep by and validated the association of SNPs in TIMP3, TLR5, TLR9 and LEPR with FEC. Thorne et al., (2023) studied the association of SNPs with GIN indicator traits as FEC and PCV in Rambouillet and Dorper lambs and concluded that the candidate genes CAPZB, GALNT6, IGF1R, PTK2B and SCUBE1 involved in immunity and cellular signaling for coagulation and wound healing following epithelial damage in GIN infection.
       
In India, Arora and Prince (2004) reported that there is significant difference in fecal egg count, hematological and biochemical parameters between the indigenous sheep breeds and exotic crosses with respect to infection with gastrointestinal nematodes and concluded that the indigenous sheep excreted fewer worm eggs in faeces and had lower morbidity and mortality rates compared to exotic breed and their crosses and Garole sheep are naturally resistant to haemonchosis. Swarnkar et al., (2017) examined the host response in Malpura lambs selected for resistance or susceptibility to Haemonchus contortus that was measured by means of fecal egg count, body weight, biochemical and hematological parameter for post challenge in which, an increase of 0.3 kg was noticed in resistance line lambs than to a decrease of 4.1 kg in susceptible line on single challenge and in most of the parameters for evaluation of infection by Haemonchus contortus was lower in resistance line than in susceptible line and It was concluded that the animals selected for resistance can tolerate parasite challenge effectively with reduced intensity of infection, higher body weight gain and reduced pathogenic effect. Gowane et al., (2020) studied the genetic structure of Haemonchus contortus resistance and susceptibility lines in Malpura and Avikalin sheep and analyzed the genetic parameters for fecal egg count. They reported that the log transformed FEC was significantly (P<.05) affected by sex, year and month of recording for all Malpura resistant, Malpura susceptible, Avikalin resistant and Avikalin susceptible and there was low permanent environment effect, low heritability for LFEC and Repeatability for LFEC were 0.05, 0.11, 0.07 and 0.06 for Malpura resistant, Malpura susceptible, Avikalin resistant and Avikalin susceptible, respectively.

In this study the genetic resistance to gastrointestinal nematodes through candidate gene analysis in Kilakarsal and Vembur breeds of sheep were assessed using the single nucleotide polymorphic markers within five toll like receptor genes TLR3, TLR5, TLR6, TLR9 and TLR10. Among 25 SNPs analyzed, 22 were found to be polymorphic in both Kilakarsal and Vembur sheep. The global minor allele frequency of the polymorphic SNP loci varied from 0.06 to 0.48 with a mean of 0.23. The effect of farm had no significant influence on FEC and change in body weight, however had significant effect on change in PCV (P<0.05). No significant difference was observed between the sexes with FEC, change in PCV and change in body weight. TLR9_1769_CT locus had significant difference (P<0.05) with mean FEC of 460.36±1.16, 23.42±2.72 and 400.39±1.55 for CC, TT and CT genotypes, respectively in Kilakarsal and Vembur sheep population. The TT genotype in TLR9_1769_CT locus showed the lowest least-squares mean FEC. The remaining 22 SNP loci showed no significant difference (P>0.05) with mean FEC ranging from 184.53 ± 1.72 to 3701±4.21.  Association of 23 TLR SNP genotypes with change in PCV and change in body weight revealed no significant effect (P>0.05). The findings of this study will aid in genetic selection against nematode infection in sheep breeds of Tamil Nadu as well as India, which will have positive effect on reduced use of anthelmintic drugs, pasture contamination, spread of anthelmintic resistance and provide a better knowledge for managing the problem in a sustainable manner.

The authors declared that there is no conflict of interest.