Retrospective Single Nucleotide Polymorphism Analysis of Host Resistance and Susceptibility to Ovine Johne's Disease Using Restored FFPE DNA.

Johne's disease (JD), also known as paratuberculosis, is a chronic, untreatable gastroenteritis of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP) infection. Evidence for host genetic resistance to disease progression exists, although it is limited due to the extended incubation period (years) and diagnostic challenges. To overcome this, previously restored formalin-fixed paraffin embedded tissue (FFPE) DNA from archived FFPE tissue cassettes was utilized for a novel retrospective case-control genome-wide association study (GWAS) on ovine JD. Samples from known MAP-infected flocks with ante- and postmortem diagnostic data were used. Cases (N = 9) had evidence of tissue infection, compared to controls (N = 25) without evidence of tissue infection despite positive antemortem diagnostics. A genome-wide efficient mixed model analysis (GEMMA) to conduct a GWAS using restored FFPE DNA SNP results from the Illumina Ovine SNP50 Bead Chip, identified 10 SNPs reaching genome-wide significance of p < 1 × 10-6 on chromosomes 1, 3, 4, 24, and 26. Pathway analysis using PANTHER and the Kyoto Encyclopedia of Genes and Genomes (KEGG) was completed on 45 genes found within 1 Mb of significant SNPs. Our work provides a framework for the novel use of archived FFPE tissues for animal genetic studies in complex diseases and further evidence for a genetic association in JD.

Transcriptome analysis reveals hepatic disordered lipid metabolism, lipotoxic injury, and abnormal development in IUGR sheep fetuses due to maternal undernutrition during late pregnancy.

Although lipid metabolism in fetal livers under intrauterine growth restriction (IUGR) conditions has been widely studied, the implications of maternal undernutrition on fetal hepatic lipid metabolism, lipotoxic injury, and abnormal development remain largely unknown. Therefore, this study investigated the effects of maternal undernutrition on disordered hepatic lipid metabolism, lipotoxic injury, and abnormal development in IUGR sheep fetuses using transcriptome analysis. Seventeen singleton ewes were randomly divided into three groups on day 90 of pregnancy: a control group (CG; 0.63 MJ metabolic energy/body weight (ME/BW)0.75/day, n = 5), maternal undernutrition group 1 (MU1; 0.33 MJ ME/BW0.75/day, n = 6), and maternal undernutrition group 2 (MU2; 0.20 MJ ME/BW0.75/day, n = 6). The fetuses were euthanized and recovered on day 130 of pregnancy. The levels of free fatty acids (FFA) in maternal blood (P < 0.01), fetal blood (P < 0.01), and fetal livers (P < 0.05) were increased in the MU1 and MU2 groups, but fetal hepatic triglyceride (TG) levels in the MU2 group (P < 0.01) and ß-hydroxybutyrate levels in the MU1 and MU2 groups (P < 0.01) were decreased compared to the CG. Severe inflammatory cell infiltration and increased non-alcoholic fatty liver disease activity scores were observed in MU1 and MU2 fetuses (P < 0.01). Progressive deposition of fetal hepatic reticular fibers and collagen fibers in the fetal livers of the MU1 and MU2 groups and significant hepatic fibrosis were observed in the MU2 fetuses (P < 0.05). Gene set enrichment analysis showed that genes involved in lipid accumulation and FFA beta oxidation were downregulated in both MU groups compared to those in the controls. The fetal liver mRNA expression of the ß-oxidation regulator, acetyl-CoA acetyltransferase 1, and the TCA regulator, isocitrate dehydrogenase were reduced in MU1 (P < 0.05) and MU2 (P < 0.01) fetuses, and downregulated mRNA expression of long chain fatty acid CoA ligase 1 (P < 0.05) and glycerol-3-phosphate acyltransferase (P < 0.01) was observed in MU2 fetuses. Differentially expressed genes (DEGs) in MU1 versus CG (360 DEGs) and MU2 versus CG (746 DEGs) were identified using RNA sequencing. Bioinformatics analyses of the 231 intersecting DEGs between MU1 versus CG and MU2 versus CG indicated that neutrophil extracellular traps (NETs) were induced and played a central role in fetal hepatic injury in IUGR sheep. Increased maternal blood myeloperoxidase (MPO) levels (P < 0.01), NE (Elane)-positive areas in fetal liver sections (P < 0.05), and fetal liver MPO protein expression (P < 0.01) were found in the MU1 and MU2 groups; however, MPO levels were reduced in the fetal membrane (P < 0.01) and fetal blood (P < 0.05) in the MU1 group, and in the maternal-fetal placenta and fetal blood in the MU2 group (P < 0.01). Analysis of gene expression trends in the intersecting DEGs between MU1 versus CG (129 DEGs) and MU2 versus CG (515 DEGs) further revealed that 30 hub genes were essential regulators of the G2/M cell cycle, all of which were associated with hepatocellular carcinoma. G0/G1 phase cells of the fetal liver were reduced in the MU1 (P < 0.05) and MU2 (P < 0.01) groups, whereas G2/M phase cells were elevated in the MU1 and MU2 groups (P < 0.01). The representatives of upregulated hub genes and fetal liver protein expression of maternal embryonic leucine zipper kinase and protein regulator of cytokinesis 1 were progressively enhanced in the MU1 and MU2 groups (P < 0.01), and topoisomerase II alpha protein expression in the MU2 group (P < 0.05), as expected. These results indicate that FFA overload, severe lipotoxic injury, and NETs were induced, and disease-promoting regulators of the G2/M cell cycle were upregulated in the fetal liver of IUGR sheep. These findings provide new insights into the pathogenesis of impaired hepatic lipid metabolism and abnormal development and the molecular origin of post-natal liver disease in IUGR due to maternal undernutrition. This information can support the development of new therapeutic strategies.

Genomic Regions Associated with Resistance to Gastrointestinal Parasites in Australian Merino Sheep.

The objective of this study was to identify genomic regions and genes associated with resistance to gastrointestinal nematodes in Australian Merino sheep in Uruguay, using the single-step GWAS methodology (ssGWAS), which is based on genomic estimated breeding values (GEBVs) obtained from a combination of pedigree, genomic, and phenotypic data. This methodology converts GEBVs into SNP effects. The analysis included 26,638 animals with fecal egg count (FEC) records obtained in two independent parasitic cycles (FEC1 and FEC2) and 1700 50K SNP genotypes. The comparison of genomic regions was based on genetic variances (gVar(%)) explained by non-overlapping regions of 20 SNPs. For FEC1 and FEC2, 18 and 22 genomic windows exceeded the significance threshold (gVar(%) ≥ 0.22%), respectively. The genomic regions with strong associations with FEC1 were located on chromosomes OAR 2, 6, 11, 21, and 25, and for FEC2 on OAR 5, 6, and 11. The proportion of genetic variance attributed to the top windows was 0.83% and 1.9% for FEC1 and FEC2, respectively. The 33 candidate genes shared between the two traits were subjected to enrichment analysis, revealing a marked enrichment in biological processes related to immune system functions. These results contribute to the understanding of the genetics underlying gastrointestinal parasite resistance and its implications for other productive and welfare traits in animal breeding programs.

Unveiling genetic signatures associated with resilience to neonatal diarrhea in lambs through two GWAS approaches.

Neonatal diarrhea presents a significant global challenge due to its multifactorial etiology, resulting in high morbidity and mortality rates, and substantial economic losses. While molecular-level studies on genetic resilience/susceptibility to neonatal diarrhea in farm animals are scarce, prior observations indicate promising research directions. Thus, the present study utilizes two genome-wide association approaches, pKWmEB and MLM, to explore potential links between genetic variations in innate immunity and neonatal diarrhea in Karacabey Merino lambs. Analyzing 707 lambs, including 180 cases and 527 controls, revealed an overall prevalence rate of 25.5%. The pKWmEB analysis identified 13 significant SNPs exceeding the threshold of ≥ LOD 3. Moreover, MLM detected one SNP (s61781.1) in the SLC22A8 gene (p-value, 1.85eE-7), which was co-detected by both methods. A McNemar's test was conducted as the final assessment to identify whether there are any major effective markers among the detected SNPs. Results indicate that four markers-oar3_OAR1_122352257, OAR17_77709936.1, oar3_OAR18_17278638, and s61781.1-have a substantial impact on neonatal diarrhea prevalence (odds ratio: 2.03 to 3.10; statistical power: 0.88 to 0.99). Therefore, we propose the annotated genes harboring three of the associated markers, TIAM1, YDJC, and SLC22A8, as candidate major genes for selective breeding against neonatal diarrhea.

Characterization of the Hepatic Transcriptome for Divergent Immune-Responding Sheep Following Natural Exposure to Gastrointestinal Nematodes.

Infections with gastrointestinal nematodes (GINs) reduce the economic efficiency of sheep operations and compromise animal welfare. Understanding the host's response to GIN infection can help producers identify animals that are naturally resistant to infection. The objective of this study was to characterize the hepatic transcriptome of sheep that had been naturally exposed to GIN parasites. The hepatic transcriptome was studied using RNA-Sequencing technology in animals characterized as high (n = 5) or medium (n = 6) based on their innate immune acute-phase (AP) response phenotype compared with uninfected controls (n = 4), and with biased antibody-mediated (AbMR, n = 5) or cell-mediated (CMR, n = 5) adaptive immune responsiveness compared to uninfected controls (n = 3). Following the assessment of sheep selected for innate responses, 0, 136, and 167 genes were differentially expressed (DE) between high- and medium-responding animals, high-responding and uninfected control animals, and medium-responding and uninfected control animals, respectively (false discovery rate (FDR) < 0.05, and fold change |FC| > 2). When adaptive immune responses were assessed, 0, 53, and 57 genes were DE between antibody- and cell-biased animals, antibody-biased and uninfected control animals, and cell-biased and uninfected control animals, respectively (FDR < 0.05, |FC| > 2). Functional analyses identified enriched gene ontology (GO) terms and metabolic pathways related to the innate immune response and energy metabolism. Six functional candidate genes were identified for further functional and validation studies to better understand the underlying biological mechanisms of host responses to GINs. These, in turn, can potentially help improve decision making and management practices to increase the overall host immune response to GIN infection.

Divergent humoral responses between males and females against 24 kDa excretory-secretory protein of Haemonchus contortus and influence of ovine ß-globin polymorphism.

Lambs harboring the Hb-AA ß-globin haplotype present improved cell-mediated responses and increased resistance against Haemonchus contortus infection. The aim of the present study was to compare the effect of sex and ß-globin haplotypes on specific humoral responses and phenotypes of resistance during H. contortus infection in Morada Nova sheep. As expected, females displayed stronger resistance during the first and second experimental challenges. Differential systemic humoral immune responses were observed comparing sex groups, in which higher levels of specific antibodies targeting 24 kDa excretory-secretory (ES24) protein of H. contortus of IgG and IgM antibodies were respectively observed as predominant isotypes in males and females. The IgM levels were significantly correlated with phenotypes of resistance, evaluated by packed cell volume and fecal egg counts. To our knowledge this is the first study reporting divergent humoral responses profiles to H. contortus infection between male and female sheep. The impact of ß-globin haplotypes was less pronounced in females compared to males. Notably, only males showed significant weight differences across haplotypes, with Hb-AA lambs being the heaviest. Additionally, Hb-AA males had significantly higher PCV (indicating better red blood cell health) and lower FEC (indicating lower parasite burden). These findings suggest a more pronounced effect of ß-globin polymorphisms on H. contortus infection in males, potentially due to their generally weaker resistance compared to females. This study highlights the importance of sex and ß-globin haplotypes in shaping immune responses to H. contortus infection. Specifically, IgM antibodies targeting the ES24 protein appear to play a crucial role in host-parasite interactions and may hold promise for therapeutic development.

Reducing fecal egg count through selective breeding alters dorper lamb response to Haemonchus contortus in an artificial challenge trial.

Infection by gastrointestinal nematodes (GIN), particularly Haemonchus contortus, can be detrimental to sheep health and performance. Genetic susceptibility to GIN varies between breeds, with those lacking high levels of natural resistance often requiring frequent anthelmintic treatment when facing parasitic challenge. Genetic technology can serve as a tool to decrease GIN susceptibility via selection for sheep with reduced fecal egg count (FEC) estimated breeding values (EBVs). However, the physiological changes that result from implementation of this strategy are not well described. Additionally, there is a need for comparison of animals from recent selective breeding against breeds with inherent GIN resistance. In this study we administered a challenge of H. contortus to Dorper x White Dorper (DWD; n = 92) lambs that have been genetically selected for either low (DWD-) or high (DWD+) FEC EBVs and Barbados Blackbelly x Mouflon (BBM; n = 19) lambs from a genetically resistant breed backgrounds. Lamb FEC, packed-cell volume (PCV) and serum IgG were measured at intermittent levels over 5 weeks. At day 21 and day 35, the selectively bred DWD- had a lower mean FEC compared to DWD+, but were higher than BBM. Reductions in both PCV and serum IgG from initial day 0 levels were observed in DWD lambs, but not in BBM. Furthermore, from a subset of lambs (n = 24) harvested at day 21, DWD- only tended (p = 0.056) to have lower mean worm counts than DWD+, with BBM having the lowest mean worm count. Differentially expressed genes (DEGs) identified via RNA-sequencing of abomasal tissue at day 21 indicate a more pronounced Th2 immune response and more rapid worm expulsion occurred in iBBM than iDWD- and iDWD+ lambs. However, gene expression in DWD- suggests an association between reduced FEC EBV and gastric acid secretion and the ability to limit worm fecundity. Ultimately, selection of Dorper sheep for low FEC EBV can reduce susceptibility to GIN, but it will likely require multiple generations with this trait as a breeding priority before presenting a similar resistance level to Caribbean breeds.

Association of ß-globin polymorphisms and tolerance to haemonchosis in ewes and lambs of different sheep breeds.

Gastrointestinal nematodes (GIN), especially Haemonchus contortus, represent a significant challenge for sheep production. Given the global concern about GIN anthelmintic resistance, alternative control methods able to reduce the dependence on these drugs are highly advisable. Since previous studies have shown that sheep carrying the Hb-A allele of ß-globin are more resistant to H. contortus, this study aimed to investigate the relationship between the different haplotypes (Hb-AA, Hb-AB and Hb-BB) and phenotypes in Santa Inês (SI), Texel (TX) and White Dorper (DO) breeds infected with H. contortus. Blood samples were collected from 180 ewes and 123 lambs of the three breeds for DNA extraction followed by qPCR using a hydrolysis probe to identify the ß-globin haplotypes. Phenotypic data, including fecal egg count (FEC), packed cell volume (PCV), FAMACHA score and body condition score for ewes and lambs, as well as weight gain for lambs, were collected. The genotypic frequencies of ß-globin for ewes and lambs were, respectively: 21.7% and 21.4% Hb-AA, 50% and 50% Hb-AB and 28.3% and 28.6% Hb-BB in SI; 0% and 0% Hb-AA, 18.6% and 9.4% Hb-AB and 81.4% and 90.6% Hb-BB in TX; and 0% and 0% Hb-AA, 13.1% and 0% Hb-AB and 86.9% and 100% Hb-BB in DO. In ewes, mean PCV differed (p<0.05) between the three haplotypes, with higher PCV in Hb-AA animals, followed by Hb-AB and Hb-BB. When considering each breed separately, SI Hb-AA ewes presented higher PCV (p<0.05), highlighting that even in a breed already considered resistant, animals with Hb-AA haplotype showed superior performance. Lambs with the Hb-AA haplotype exhibited a higher (p<0.05) mean PCV compared to those with Hb-AB and Hb-BB. The same pattern was found in SI when analyzing each breed separately. No significant association was found between ß-globin haplotypes and FEC, FAMACHA score, body condition score, or weight gain. Nevertheless, given that anemia is the major clinical sign of haemonchosis, our findings on PCV reinforce that sheep carrying the Hb-A allele of ß-globin are more tolerant to haemonchosis. This study may support the development of a valuable tool, targeting genetic selection for GIN control, reducing the dependence on anthelmintics and boosting sheep production worldwide.

Revealing the associated microflora hosted by the globally significant parasite Trichostrongylus colubriformis.

Trichostrongylus colubriformis is a parasitic helminth that primarily infects small ruminants, causing substantial economic losses in the livestock industry. Exploring the microbiome of this helminth might provide insights into the potential influence of its microbial community on the parasite's survival. We characterised the intestinal microbiome of T. colubriformis that had been collected from the duodenum of sheep, and compared the helminth microbiome with the duodenal microbiome of its host, aiming to identify contributions from the helminth's environment. At the same time, we explored the isolation of fastidious organisms from the harvested helminth. Primary alpha and beta diversity analyses of bacterial species revealed statistically significant differences between the parasite and the host, in terms of species richness and ecological composition. 16S rRNA differential abundance analysis showed that Mycoplasmoides and Stenotrophomonas were significantly present in T. colubriformis but not in the duodenal microbiome of the sheep. Furthermore, two bacteria, Aeromonas caviae and Aeromonas hydrophila, were isolated from T. colubriformis. Examinations of the genome highlight differences in genome size and profiles of antimicrobial resistance genes. Our results suggest that T. colubriformis carries a specific bacterial community that could be supporting the helminth's long-term survival in the host's digestive system.

Putative SNPs in Ovar-DRB1 and GALNTL6 Genes Conferring Susceptibility to Natural Infection of Haemonchus Contortus in Southern Indian Sheep.

AIM: To explore associations between phenotypic traits and polymorphisms in the DRB1 and GALNT6 gene in Nellore, Deccani and Kenguri sheep naturally infected with Haemonchus contortus. MATERIALS AND METHODS: Blood and faecal samples were collected to evaluate fecal worm egg counts (FEC), packed cell volume (PCV), hemoglobin (Hb), eosinophilia and for DNA isolation. RESULTS: Animals were grouped into susceptible and resistant groups based on EPG counts. FEC and circulating eosinophilia were higher in a susceptible group. Log FEC was negatively correlated (P < 0.01) with PCV, and Hb estimates. The second exon of DRB1 and intron variant of GALNTL6 genes were amplified from DNA samples of resistant and susceptible sheep. Characterization of Ovar-DRB1 amplicon by RFLP revealed two genotypes ('bb' and 'ab'). The genotype frequencies differed significantly between both groups (P < 0.05). The 'bb' genotypes had higher (P < 0.05) log FEC value than 'ab' genotypes and 'b' allele was linked with susceptibility to haemonchosis in sheep. The mean FEC of Nellore sheep was high indicating susceptibility of the breed and also in which the frequency of 'b' allele was more compared to the other two breeds. OVAR-DRB1 genotypes associated with FEC did not affect PCV and Hb. PCR-RFLP assay developed to determine the genotypes with respect to SNP rs424521894 of GALNTL6 revealed monomorphic nature at the locus in the breeds studied. CONCLUSION: MHC polymorphism could be used as a genetic marker for the selection of sheep resistant to H. contortus. However, a more intensive study, involving controlled infections and other GALNTL6 SNPs may be enforced to make any decisive assertion.

Identification of genomic regions associated with resistance to gastrointestinal parasites in an indigenous sheep by single- and multiple-locus methods.

We investigated the association between 157 SNPs located in 75 candidate genes involved in the immune system and proxy traits for resistance to gastrointestinal nematodes in sheep. A total of 211 lambs from eight flocks were sampled. Nematode eggs per gram were counted and classified as: (i) Strongyles, (ii) Nematodirus spp., (iii) Trichuris spp. and (iv) Marshallagia marshalli. Single- and multiple-locus models were used to test the marker-trait associations. Seven significant SNPs were identified on chromosomes OAR6, 15, 16, and 19. These findings provide insights for breeding nemarode-resistant traits in low-input production systems. General linear model, fixed and random model circulating probability unification, and Bayesian-information and linkage-disequilibrium iteratively nested keyway analyses identified a significant association between the eggs per gram of Strongyles nematodes and a specific variant of the PRLR gene.

MFSD2A frameshift variant in Kerry Hill sheep with microcephaly.

Microcephaly is a rare neurodevelopmental disorder characterized by reduced skull circumference and brain volume that occurs sporadically in farm animals. We investigated an early-onset neurodegenerative disorder observed in seven lambs of purebred Kerry Hill sheep. Clinical signs included inability to stand or severe ataxia, convulsions, and early death. Diagnostic imaging and brain necropsy confirmed microcephaly. The pedigree of the lambs suggested monogenic autosomal recessive inheritance. We sequenced the genome of one affected lamb, and comparison with 115 control genomes revealed a single private protein-changing variant. This frameshift variant, MFSD2A: c.285dupA, p.(Asp96fs*9), represents a 1-bp duplication predicted to truncate 80% of the open reading frame. MFSD2A is a transmembrane protein that is essential for maintaining blood-brain barrier homeostasis and plays a key role in regulating brain lipogenesis. Human MFSD2A pathogenic variants are associated with a neurodevelopmental disorder with progressive microcephaly, spasticity, and brain imaging abnormalities (NEDMISBA, OMIM 616486). Here we present evidence for the occurrence of a recessively inherited form of microcephaly in sheep due to a loss-of-function variant in MFSD2A (OMIA 002371-9940). To the best of our knowledge, this is the first report of a spontaneous MFSD2A variant in domestic animals.

Characterization and population genetics of Haemonchus contortus in Merino sheep in Lesotho.

Haemonchus contortus is the most pathogenic and economically restrictive gastrointestinal nematode in the small ruminant industry globally. Morbidity, poor cross-bodily state, and mortality of sheep in Lesotho suggest the presence of H. contortus. The present study investigated the morphological, molecular, and population genetics of H. contortus third-stage larvae infecting sheep in four ecological zones (EZ) of Lesotho. Coprocultures were prepared for larval morphological identification and PCR determination. Larvae were identified morphologically as 100% H. contortus. The Second Internal Transcribed Spacer (ITS-2) gene of the ribosomal DNA of H. contortus isolates in the present study revealed nucleotide homology ranging from 97 to 100% when compared with selected GenBank reference sequences. Pairwise evolutionary divergence among H. contortus isolates was low, with 0.01318 recorded as the highest in the present study. Five haplotypes resulted from 14 Lesotho sequences. Haplotype diversity and nucleotide diversity were 0.76923 and 0.00590, respectively. Genetic differentiation among isolates was low but not statistically significant. An analysis of molecular variance revealed that most molecular variation was distributed within topographic populations at 94.79% (FST = 0.05206, p > 0.05) and 5.21% among populations. There was high gene flow and no definite population genetic structure among Lesotho isolates.

CRISPR-Cas12a test strip (CRISPR/CAST) package: In-situ detection of Brucella from infected livestock.

BACKGROUND: Brucellosis is a common zoonotic disease caused by Brucella, which causes enormous economic losses and public burden to epidemic areas. Early and precise diagnosis and timely culling of infected animals are crucial to prevent the infection and spread of Brucella. In recent years, RNA-guided CRISPR/Cas12a(Clustered Regularly Interspaced Short Palindromic Repeats and its associated protein 12a) nucleases have shown great promise in nucleic acid detection. This research aims to develop a CRISPR/CAST (CRISPR/Cas12a Test strip) package that can rapidly detect Brucella nucleic acid during on-site screening, especially on remote family pastures. The CRISPR/Cas12a system combined with recombinase polymerase amplification (RPA), and lateral flow read-out. RESULTS: We selected the conserved gene bp26, which commonly used in Brucella infection detection and compared on Genbank with other Brucella species. The genomes of Brucella abortus 2308, Brucella suis S2, Brucella melitansis 16 M, and Brucella suis 1330, et al. were aligned, and the sequences were found to be consistent. Therefore, the experiments were only performed on B. melitensis. With the CRISPR/CAST package, the assay of Brucella nucleic acid can be completed within 30 min under isothermal temperature conditions, with a sensitivity of 10 copies/µl. Additionally, no antigen cross-reaction was observed against Yersinia enterocolitica O:9, Escherichia coli O157, Salmonella enterica serovar Urbana O:30, and Francisella tularensis. The serum samples of 398 sheep and 100 cattle were tested by the CRISPR/CAST package, of which 31 sheep and 8 cattle were Brucella DNA positive. The detection rate was consistent with the qPCR results and higher than that of the Rose Bengal Test (RBT, 19 sheep and 5 cattle were serum positive). CONCLUSIONS: The CRISPR/CAST package can accurately detect Brucella DNA in infected livestock within 30 min and exhibits several advantages, including simplicity, speed, high sensitivity, and strong specificity with no window period. In addition, no expensive equipment, standard laboratory, or professional operators are needed for the package. It is an effective tool for screening in the field and obtaining early, rapid diagnoses of Brucella infection. The package is an efficient tool for preventing and controlling epidemics.

Transgenerational epigenetic inheritance and immunity in chickens that vary in Marek's disease resistance.

Marek's disease virus (MDV), a naturally oncogenic, highly contagious alpha herpesvirus, induces a T cell lymphoma in chickens that causes severe economic loss. Marek's disease (MD) outcome in an individual is attributed to genetic and environmental factors. Further investigation of the host-virus interaction mechanisms that impact MD resistance is needed to achieve greater MD control. This study analyzed genome-wide DNA methylation patterns in 2 highly inbred parental lines 63 and 72 and 5 recombinant congenic strains (RCS) C, L, M, N, and X strains from those parents. Lines 63 and 72, are MD resistant and susceptible, respectively, whereas the RCS have different combinations of 87.5% Line 63 and 12.5% Line 72. Our DNA methylation cluster showed a strong association with MD incidence. Differentially methylated regions (DMRs) between the parental lines and the 5 RCS were captured. MD-resistant and MD-susceptible markers of DNA methylation were identified as transgenerational epigenetic inheritable. In addition, the growth of v-src DNA tumors and antibody response against sheep red blood cells differed among the 2 parental lines and the RCS. Overall, our results provide very solid evidence that DNA methylation patterns are transgenerational epigenetic inheritance (TEI) in chickens and also play a vital role in MD tumorigenesis and other immune responses; the specific methylated regions may be important modulators of general immunity.

Abomasal RNA-seq reveals a strong local cellular response in suckling lambs with resistance against Haemonchus contortus.

Santa Ines (SI) and Ile de France (IF) sheep are known to be resistant and susceptible to Haemonchus contortus infection, respectively. Several studies have shown some genes as potential biological markers for sheep resistance against gastrointestinal nematodes using molecular tools, including transcriptomic analysis. In this study, we sequenced the polyadenylated RNA of the abomasal tissue of SI and IF suckling lambs to identify mucosa-specific transcript alterations between breeds artificially infected with H. contortus. Naïve SI (n = 4) and IF (n = 4) lambs were artificially infected every other day, over a period of 52 days, from 14 to 66 days old, with a total of 5,400 H. contortus infective larvae. Fundic abomasal tissue samples were collected at 68 days old, and submitted to high-throughput RNA sequencing (RNA-seq). Differential expression analysis (P value < 0.001 and False Discovery Rate (FDR) < 0.05) between SI and IF samples identified 292 genes, most of which showed greater expression in SI lambs. To help annotate and assign possible function to differentially expressed genes (DEGs), we used previously available single-cell RNA-seq (scRNA-seq) data from ovine abomasal mucosa to putatively identify cell types and possible mechanisms involved in resistance to H. contortus. In particular, genes associated with endothelial and tuft cells showed the greatest increases in expression in SI relative to IF lambs. SI lambs had higher percentages of tuft cells than IF lambs in the fundic abomasal mucosa. Although we found innate immunity (cell-mediated in mucosa) acting as a protagonist in impairing H. contortus infection, a stronger acquired immune response was being modulated at an earlier stage by SI lambs. We suggest that the complex connection between innate and adaptive immunity is via cellular antigen processing and presentation (APP). Based on comparison with scRNA-seq data, SI lambs showed a robust APP mechanism characterized mainly by greater T cell APP, macrophage differentiation, and cytokine signalling. We identified potential mechanisms and markers to advance knowledge for selection of H. contortus resistance at a very early age, in SI as well as in other commercial sheep breeds.

Genome-Wide Analysis of Sheep Artificially or Naturally Infected with Gastrointestinal Nematodes.

The anthelmintic resistance of gastrointestinal nematodes (GINs) poses a significant threat to sheep worldwide, but genomic selection can serve as an alternative to the use of chemical treatment as a solution for parasitic infection. The objective of this study is to conduct genome-wide association studies (GWASs) to identify single nucleotide polymorphisms (SNPs) in Rambouillet (RA) and Dorper × White Dorper (DWD) lambs associated with the biological response to a GIN infection. All lambs were genotyped with a medium-density genomic panel with 40,598 markers used for analysis. Separate GWASs were conducted using fecal egg counts (FECs) from lambs (<1 year of age) that acquired their artificial infections via an oral inoculation of 10,000 Haemonchus contortus larvae (n = 145) or naturally while grazing on pasture (n = 184). A GWAS was also performed for packed cell volume (PCV) in artificially GIN-challenged lambs. A total of 26 SNPs exceeded significance and 21 SNPs were in or within 20 kb of genes such as SCUBE1, GALNT6, IGF1R, CAPZB and PTK2B. The ontology analysis of candidate genes signifies the importance of immune cell development, mucin production and cellular signaling for coagulation and wound healing following epithelial damage in the abomasal gastric pits via H. contortus during GIN infection in lambs. These results add to a growing body of the literature that promotes the use of genomic selection for increased sheep resistance to GINs.

Genotype by environment interaction and heteroscedasticity influence the expression of parasite resistance in Katahdin sheep.

Increasingly, sheep producers are choosing breeds that express resistance to gastrointestinal parasites due to reduced efficacy of anthelminthic drugs. One such breed is Katahdin. Katahdins are raised in various climates and management systems in the United States, which can be combined into eco-management clusters to describe production environments more holistically. The objectives of this study were to determine if genotype by environment interaction (G × E) and heteroscedasticity existed across these eco-management clusters for traits indicative of parasite resistance. Body weights (BW), FAMACHA scores (FAM), and fecal egg counts (FEC) were collected at around 90 d in 3,527 Katahdin lambs delineated into nine eco-management clusters. A tri-variate animal model including birth-rearing type, sex, and dam age (as a quadratic covariate) as fixed effects, and eco-management cluster, direct additive, uncorrelated maternal environmental (for BW), and residual as random effects, was fitted with ASReml. Heritability estimates for BW, FEC, and FAM were 0.36 ±â€…0.07, 0.31 ±â€…0.07, and 0.26 ±â€…0.05, respectively. The genetic (additive) correlation between BW with FEC was -0.26 ±â€…0.08 and with FAM was -0.16 ±â€…0.08, and thereby favorable. Heritabilities were also estimated univariately within eco-management clusters and ranged from 0.30 ±â€…0.05 to 0.37 ±â€…0.05 for BW, 0.18 ±â€…0.12 to 0.50 ±â€…0.13 for FEC, and 0.07 ±â€…0.06 to 0.40 ±â€…0.19 for FAM. Significant genetic and phenotypic heteroscedasticity among eco-management clusters was detected in FEC and FAM. A sire by eco-management cluster interaction term was added to the initial model fitted to evaluate G × E. This interaction defined substantial variation (P < 0.01) in all traits and explained 12% (FEC) to 20% (BW) of the phenotypic variation. Accounting for G × E and heteroscedasticity in the design and implementation of breeding programs may introduce operational challenges. Still, doing so would improve the efficacy of selection programs to improve parasite resistance.

Sheep producers increasingly rely on breeds that express resistance to gastrointestinal parasites because anthelminthic drugs are often ineffective. An example is Katahdin sheep, which are raised in various climates and management systems in the United States. These factors can be combined into eco-management clusters to describe production environments more holistically. Our objective was to determine if a genotype by eco-management cluster (environment) interaction (G × E) affected performance levels, particularly for traits indicative of parasite resistance. Body weights (BW), fecal egg counts (FEC), and FAMACHA scores (FAM) were collected at around 90 d in 3,527 Katahdin lambs delineated into nine eco-management clusters. Heritabilities of BW, FEC, and FAM were 0.36, 0.31, and 0.26, respectively. Genetic correlations of BW with FEC and FAM were low to moderate and favorable (negative). When estimated within eco-management cluster, heritabilities were 0.30 to 0.37 for BW, 0.18 to 0.50 for FEC, and 0.07 to 0.40 for FAM. For FEC and FAM, these differences corresponded with heterogenous variances (heteroscedasticity) across environments. Furthermore, G × E explained 13% (FAM) to 20% (BW) of the variation in a trait. In genetic evaluation of parasite resistance, G × E and heteroscedasticity should be incorporated to improve the efficacy of the breeding program.

Genetic polymorphism in European and African sheep breeds reared in Hungary based on 48 SNPs associated with resistance to gastrointestinal parasite infection using KASP-PCR technique.

This pilot study used an alternative and economically efficient technique, the Kompetitive Allele-Specific Polymerase Chain Reaction (KASP-PCR) to examine 48 SNPs from 11 parasite-resistance genes found on 8 chromosomes in 110 animals from five sheep breeds reared in Hungary; Hungarian Tsigai, White Dorper, Dorper, Ile de France, and Hungarian Merino. Allele and genotype frequencies, fixation index, observed heterozygosity, expected heterozygosity, F statistic, and their relationship with the Hardy-Weinberg equilibrium (WHE) and the polymorphic information content (PIC) were determined, followed by principal component analysis (PCA). As much as 32 SNPs out of the 48 initially studied were successfully genotyped. A total of 9 SNPs, 4 SNPs in TLR5, 1 SNP in TLR8, and 4 SNPs in TLR2 genes, were polymorphic. The variable genotype and allele frequency of the TLRs gene indicated genetic variability among the studied sheep breeds, with the Hungarian Merino exhibiting the most polymorphisms, while Dorper was the population with the most SNPs departing from the HWE. According to the PIC value, the rs430457884-TLR2, rs55631273-TLR2, and rs416833129-TLR5 were found to be informative in detecting polymorphisms among individuals within the populations, whereas the rs429546187-TLR5 and rs424975389-TLR5 were found to have a significant influence in clustering the population studied. This study reported a moderate level of genetic variability and that a low to moderate within-breed diversity was maintained in the studied populations.

Ovine PAPPA2 gene coding variants are linked to decreased fecal egg shedding in native Turkish sheep naturally infected with gastrointestinal nematodes.

In this study, the association between PAPPA2 coding variants and gastrointestinal (GI) nematode fecal egg count (FEC) score in adult Turkish sheep was investigated. For this purpose, the FEC score was determined in adult sheep from six breeds: Karacabey Merino (n = 137), Kivircik (n = 116), Cine capari (n = 109), Karakacan (n = 102), Imroz (n = 73), and Chios (n = 50). Sheep were classified as shedders or non-shedders within breeds and flocks. The first group was the fecal egg shedders (> 50 per gram of feces), and the second group was the no fecal egg shedders (≤ 50 per gram of feces). The exon 1, exon 2, exon 5, exon 7, and a part of 5'UTR of the ovine PAPPA2 gene were genotyped by Sanger sequencing of these two groups. Fourteen synonymous and three non-synonymous single-nucleotide polymorphisms (SNPs) were found. The non-synonymous SNPs, D109N, D391H, and L409R variants, are reported for the first time. Two haplotype blocks were constructed on exon 2 and exon 7. The specific haplotype, C391G424G449T473C515A542 on the exon 2 that carries the 391H variant, was tested against four other common haplotypes. Our results indicate that C391G424G449T473C515A542 haplotype was significantly associated with fecal egg shedding status in adult Turkish sheep (p-value, 0.044).