Single-Cell Transcriptomic Profiling Unveils Dynamic Immune Cell Responses during Haemonchus contortus Infection.

BACKGROUND: Haemonchus contortus is a parasite widely distributed in tropical, subtropical, and warm temperate regions, causing significant economic losses in the livestock industry worldwide. However, little is known about the genetics of H. contortus resistance in livestock. In this study, we monitor the dynamic immune cell responses in diverse peripheral blood mononuclear cells (PBMCs) during H. contortus infection in goats through single-cell RNA sequencing (scRNA-Seq) analysis. METHODS AND RESULTS: A total of four Boer goats, two goats with oral infection with the L3 larvae of H. contortus and two healthy goats as controls, were used in the animal test. The infection model in goats was established and validated by the fecal egg count (FEC) test and qPCR analysis of the gene expression of IL-5 and IL-6. Using scRNA-Seq, we identified seven cell types, including T cells, monocytes, natural killer cells, B cells, and dendritic cells with distinct gene expression signatures. After identifying cell subpopulations of differentially expressed genes (DEGs) in the case and control groups, we observed the upregulation of multiple inflammation-associated genes, including NFKBIA and NFKBID. Kyoto Encyclopedia of the Genome (KEGG) enrichment analysis revealed significant enrichment of NOD-like receptor pathways and Th1/Th2 cell differentiation signaling pathways in CD4 T cells DEGs. Furthermore, the analysis of ligand-receptor interaction networks showed a more active state of cellular communication in the PBMCs from the case group, and the inflammatory response associated MIF-(CD74 + CXCR4) ligand receptor complex was significantly more activated in the case group, suggesting a potential inflammatory response. CONCLUSIONS: Our study preliminarily revealed transcriptomic profiling characterizing the cell type specific mechanisms in host PBMCs at the single-cell level during H. contortus infection.

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.

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.

Breeding options for nematode resistance in Lacaune dairy sheep.

Due to progressing anthelmintic resistance of gastrointestinal nematodes (GIN), supportive measures are needed to control these parasites. In sheep, it has been shown that selection towards an increased nematode resistance is feasible and that faecal egg count (FEC) is the generally acknowledged trait for selection. However, a selection based on FEC would come with certain costs, therefore auxiliary, cheaper resistance traits would be most welcome. FAMACHA©, a colour classification of the eyelid, usually used to determine the manifestation of an infection with Haemonchus contortus, could serve as such. Therefore, we collected FAMACHA©, packed cell volume (PVC) and FEC phenotypes of approx. 1150 naturally infected Lacaune ewes on 15 commercial farms in Switzerland. The Haemonchus-proportion was determined on farm level. Phenotypic correlations of FEC and FAMACHA© as well as FAMACHA© and PCV were 0.25 (SE 0.03) and -0.35 (SE 0.08), respectively, and correspond well with the results of other studies. A multi-trait animal model was applied to estimate genetic parameters with FEC, FAMACHA©, PVC and milk yield as dependent variables. The heritabilities of FEC, FAMACHA©, PCV and milk yield were estimated to be moderate with values of 0.33 (SE 0.08), 0.30 (SE 0.08), 0.36 (SE 0.08) and 0.34 (SE 0.08), respectively. The genetic correlations between FEC and FAMACHA© and between FEC and PCV were estimated to be close to zero with values of 0.03 (SE 0.22) and 0.01 (SE 0.21), respectively. The average Haemonchus-proportion compared to other GIN was found to be 43%. The FAMACHA© classification of the Lacaune ewes seems to indicate a rather high worm challenge, with 38, 14 and 2% of observations classified to scores 3, 4 and 5, respectively. However, the worm challenge according to FEC was moderate. It has been suggested that the genetic correlation between FAMACHA© and FEC is more pronounced when FEC was high. It could therefore be that the lack of genetic correlation was due to an insufficient worm challenge, even though the Lacaune were grazing at least 70 days before phenotyping. The genetic correlation between FEC and milk yield was estimated to be 0.07 (SE 0.22, slightly unfavourable). We conclude that if FEC is used as trait, the Lacaune could be selected for lower susceptibility towards nematode infection. The use of FAMACHA© as an auxiliary trait for FEC is not feasible, due to an inexistent genetic correlation between these two traits.

Characterization of interferon gamma gene in relation to immunological responses in Haemonchus contortus resistant and susceptible Garole sheep.

Garole sheep exhibits within-breed difference in resistance to natural gastrointestinal nematode infection predominated by Haemonchus contortus. In the present study, interferon gamma gene (IFN-γ) was characterized in relation to parasitological, haematological, and immune response against H. contortus in resistant and susceptible Garole sheep. Resistant and susceptible Garole sheep were selected from the field based on consistent low faecal egg counts (FEC) for one year and single nucleotide polymorphisms (SNPs) in the IFN-γ gene. The partial amplification of IFN-γ gene (1282 bp) revealed 4 SNPs exclusively in resistant sheep and 3 SNPs were shared between resistant and susceptible Garole sheep. The selected resistant and susceptible Garole sheep were challenged with H. contortus infection. The parasitological, haematological, immunological responses, and expression of IFN-γ gene were compared between the resistant and susceptible Garole sheep. The FEC of resistant sheep was significantly (P < 0.05) lower than the susceptible sheep infected with H. contortus. There was spontaneous elimination of H. contortus from 28 to 33 days post infection (DPI) in resistant sheep. Haemoglobin and packed cell volume were significantly (P < 0.05) higher in resistant sheep than the susceptible sheep. The serum concentration of immunoglobulin (Ig)G1 and IgA and cytokine IFN-γ activity and also the expression of IFN-γ gene were significantly (P < 0.05) higher in the infected resistant sheep from 14 to 28 DPI compared to the susceptible sheep. In resistant sheep, IgA and IgG1 and cytokine IFN-γ positively correlated with expression of IFN-γ gene, and the SNPs recorded in the resistant sheep only might play an important role in conferring resistance against H. contortus. Further studies are required to elucidate the role of IFN-γ gene in H. contortus resistance in Garole sheep.

Differential responses of abomasal transcriptome to Haemonchus contortus infection between Haemonchus-selected and Trichostrongylus-selected merino sheep.

Haemonchus contortus is the most prevalent and pathogenic gastrointestinal nematode infecting sheep and goats. The two CSIRO sheep resource flocks, the Haemonchus-selected flock (HSF) and Trichostrongylus-selected flock (TSF) were developed for research on host resistance or susceptibility to gastrointestinal nematode infection. A recent study focused on the gene expression differences between resistant and susceptible sheep within each flock, with lymphatic and gastrointestinal tissues. To identify features in the host transcriptome and understand the molecular differences underlying host resistance to H. contortus between flocks with different selective breeding and genetic backgrounds, we compared the abomasal transcriptomic responses of the resistant or susceptible animals between HSF and TSF flocks. A total of 11 and 903 differentially expressed genes were identified in the innate infection treatment in HSF and TSF flocks between resistant and susceptible sheep respectively, while 52 and 485 genes were identified to be differentially expressed in the acquired infection treatment, respectively. Among them, 294 genes had significantly different gene expression levels between HSF and TSF flock animals within the susceptible sheep by both the innate and acquired infections. Moreover, similar expression patterns of the 294 genes were observed, with 273 genes more highly expressed in HSF and 21 more highly expressed in the TSF within the abomasal transcriptome of the susceptible animals. Gene ontology enrichment of the differentially expressed genes identified in this study predicted the likely differing function between the two flock's susceptible lines in response to H. contortus infection. Nineteen pathways were significantly enriched in both the innate and adaptive immune responses in susceptible animals, which indicated that these pathways likely contribute to the host resistance development to H. contortus infection in susceptible sheep. Biological networks built for the set of genes differentially abundant in susceptible animals identified hub genes of PRKG1, PRKACB, PRKACA, and ITGB1 for the innate immune response, and CALM2, MYL1, COL1A1, ITGB1 and ITGB3 for the adaptive immune response, respectively. Our results offered a quantitative snapshot of host transcriptomic changes induced by H. contortus infection between flocks with different selective breeding and genetic backgrounds and provided novel insights into molecular mechanisms of host resistance.

Single nucleotide polymorphisms from candidate genes associated with nematode resistance and resilience in Corriedale and Pampinta sheep in Argentina.

Selective breeding of genetically resistant animals is considered a promising strategy to face the problem of nematode resistance to anthelmintics and mitigate concerns about the presence of chemical residues in animal food products and the environment. Gastrointestinal nematode resistance is a complex, multifactorial trait related to host immunity. However, the mechanisms underlying host resistance and response to infection remain to be fully elucidated. In this context, the objective of this study was to provide insight into the chromosomal regions determining nematode resistance and resilience in Corriedale and resistance in Pampinta sheep breeds. A total of 170 single nucleotide polymorphisms (SNP) from 76 candidate genes for immune response were studied in 624 Corriedale and 304 Pampinta animals. Lambs underwent artificial or natural challenges with infective larvae mainly from Haemonchus contortus. Fecal egg counts, estimated breeding values for fecal egg counts, and rate of packed cell volume change and FAMACHA© score change over the challenge were used, when available, as indicators of host parasite resistance or resilience. Phenotype-genotype association studies were conducted and significance values obtained were adjusted for multiple testing errors. Eight SNPs, located on OARs 3, 6, 12, and 20, reached significance in Corriedale sheep under artificial challenge. Those SNP represent allelic variants from the MHC-Ovine Lymphocyte Antigen-DRA, two C-type lectin domain families, the Interleukin 2 receptor ß, the Toll-like receptor 10, the Mannan binding lectin serine peptidase 2, and the NLR family, CARD domain containing 4 genes. On Pampinta lambs under natural challenge, we found three significant SNPs, located in the TIMP metallopeptidase inhibitor 3, the FBJ murine osteosarcoma viral oncogene homolog, and the Interleukin 20 receptor alpha genes, on OARs 3, 7, and 8, respectively. The results obtained herein confirm genomic regions previously reported as associated with nematode resistance in other sheep breeds, reinforcing their role in host response to parasites. These findings contribute to gain knowledge on parasite resistance and resilience in Corriedale sheep and report for the first time SNPs associated with resistance to gastrointestinal parasite infections in Pampinta breed.

Selecting sheep for Haemonchus contortus resistance and susceptibility: Flock dynamics and genetic architecture.

The study was carried out in the selected strains of Malpura and Avikalin sheep for nematode resistance versus susceptibility (Malpura resistant: MR, Malpura susceptible: MS, Avikalin resistant: AR and Avikalin susceptible: AS). Strains were selected since 2004 and inter-se breeding followed since year 2007. The objective was to decipher the genetic structure of strains and to estimate genetic parameters for fecal egg count (FEC). The population size was small (107-279); however, all the four flocks had sizable effective population size (fe) for founders. The ratio for fe to effective number of ancestors (fa) was nearly 1 indicating genetic stability. Inbreeding was under control. The log transformed FEC (LFEC) was significantly (P < .05) affected by sex, year and month of recording for all 4 strains. Generation had desired genetic trend. There was low heritability for LFEC in MR(0.02), MS(0.06), AR(0.07) and AS(0.02). The estimate of the permanent environment effect (c2) was low. Repeatability (ρ) for LFEC were 0.05, 0.11, 0.07 and 0.06 for MR, MS, AR and AS, respectively. Pruning the data for repeated records in peak infection period (1: "Aug-Sep" and 2: only "Sep") resulted in considerable increase in ρ for MR (Aug-Sep: 0.07 ± 0.03 and Sep: 0.12 ± 0.07) and AR (Aug-Sep: 0.05 ± 0.04 and Sep: 0.09 ± 0.10). Results revealed genotype x environment interaction for repeated measures across months. Low flock size and low genetic variability are hurdles for improvement in R strains. It is recommended to incorporate new candidates post selection from the other breeding flocks and admit them in the R strains for selective breeding.

Four single nucleotide polymorphisms (SNPs) are associated with resistance and resilience to Haemonchus contortus in Brazilian Morada Nova sheep.

Gastrointestinal nematodes are a major constraint in sheep production. Breeding for resistance has proven to be an effective and feasible approach to address this problem. The use and investigation of genetic markers for resistance traits could accelerate genetic progress and lead to a better understanding of underlying molecular mechanisms. Thus, the aim of this study was to evaluate if five single nucleotide polymorphisms SNPs OAR2_14765360, OAR6_81718546, OAR11_62887032, OAR12_69606944 and OAR15_59871543 are associated with resistance and resilience traits in a flock of the Morada Nova sheep breed. Lambs were submitted to two consecutive parasite challenges by oral infection with 4000 infective larvae L3) of Haemonchus contortus. Fecal egg counts (FEC), packed cell volume (PVC) and body weight were measured every one or two weeks for 42 days in each trial. DNA samples from 287 lambs, 131 ewes and 4 rams were amplified by ARMS-PCR or PCR-RFLP and genotypes were determined. Analysis of variance (ANOVA) was used for association analyses between genotypes and phenotypes. In case of significant association, the allele substitution effect was calculated based on a linear model. OAR2_14765360 and OAR12_69606944 were associated with FEC, and OAR12_69606944 also had significant effects on PCV and weight gain, showing favourable associations of the CC genotype with all evaluated traits. Both OAR6_81718546 and OAR11_62887032 were associated with weight gain, and OAR6_81718546 had an additional effect on PCV. OAR15_59871543 was not polymorphic in the population. OAR6_81718546 and OAR12_69606944 presented significant allele substitution effects of -1.06 ±â€¯0.52 kg for the T allele on final body weight and 0.74 ±â€¯0.32 for the C allele in PCV of the same sampling date, respectively. This is the first report of SNPs associated with gastrointestinal nematode resistance in this sheep breed. Our findings support the existence of quantitatice trait loci (QTL) for resistance and resilience in linkage disequilibrium with the polymorphic SNPs and suggest their future use for explorations of these traits in Morada Nova sheep.

RNA-Sequencing of ovine PBMC after exposure to Haemonchus contortus larval antigen.

Mechanisms of immune activation in effector cells during Haemonchus contortus infection in sheep are currently unknown. Microarray experiments have been performed on tissues of H contortus infected sheep of varying parasite resistance during early and late points of infection, but not in immune effector cells. The purpose of this study was to compare early gene activation in peripheral blood mononuclear cells (PBMC) from primed parasite susceptible (Suffolk) and resistant (St. Croix) sheep in response to H contortus larval antigen (HcLA). Peripheral blood mononuclear cells were cultured for 6 hours with HcLA, and RNA-sequencing was performed. St. Croix PBMC upregulated 499 unique genes in response to HcLA while Suffolk PBMC upregulated 130 unique genes and 25 genes were shared between the two breeds. St. Croix PBMC had increased expression of genes associated with immune function, signal transduction, response to stress and others. In addition, while mechanisms of innate recognition of H contortus are unknown, multiple pattern recognition receptors were found to be upregulated in St. Croix PBMC cultured with HcLA and none were found to be upregulated in Suffolk PBMC. These patterns of immune gene activation may contribute to St. Croix's rapid response and ability to resist H contortus infection.

Genome-wide association study to identify genetic loci associated with gastrointestinal nematode resistance in Katahdin sheep.

Resistance to gastrointestinal nematodes has previously been shown to be a moderately heritable trait in some breeds of sheep, but the mechanisms of resistance are not well understood. Selection for resistance currently relies upon faecal egg counts (FEC), blood packed cell volumes and FAMACHA visual indicator scores of anaemia. Identifying genomic markers associated with disease resistance would potentially improve the selection process and provide a more reliable means of classifying and understanding the biology behind resistant and susceptible sheep. A GWAS was conducted to identify possible genetic loci associated with resistance to Haemonchus contortus in Katahdin sheep. Forty animals were selected from the top and bottom 10% of estimated breeding values for FEC from a total pool of 641 sires and ram lambs. Samples were genotyped using Applied Biosystems™ Axiom™ Ovine Genotyping Array (50K) consisting of 51 572 SNPs. Following quality control, 46 268 SNPs were included in subsequent analyses. Analyses were conducted using a linear regression model in plink v1.90 and a single-locus mixed model in snp and variation suite. Genome-wide significance was determined by a Bonferroni correction for multiple testing. Using linear regression, loci on chromosomes 2, 3, 16, 23 and 24 were significantly associated at the genome level with FEC estimated breeding values, and we identified a region on chromosome 2 that was significant using both statistical analyses. We suggest a potential role for the gene DIS3L2 for gastrointestinal nematode resistance in Katahdin sheep, although further research is needed to validate these findings.

Innate Immune Responses Associated with Resistance against Haemonchus contortus in Morada Nova Sheep.

The immune response against Haemonchus contortus infections is primarily associated with the Th2 profile. However, the exact mechanisms associated with increased sheep resistance against this parasite remains poorly elucidated. The present study is aimed at evaluating mediators from the innate immune response in lambs of the Morada Nova Brazilian breed with contrasting H. contortus resistance phenotypes. Briefly, 287 lambs were characterized through fecal egg counts (FEC) and packed cell volume (PCV) after two independent experimental parasitic challenges with 4,000 H. contortus L3. 20 extreme resistance phenotypes (10 most resistant and 10 most susceptible) were selected, subjected to a third artificial infection with 4,000 L3, and euthanized 7 days later. Tissue samples were collected from abomasal fundic and pyloric mucosa and abomasal lymph nodes. Blood samples were collected at days 0 and 7 of the third parasitic challenge. RNA was extracted from tissue and blood samples for relative quantification of innate immune-related genes by RT-qPCR. For the abomasal fundic mucosa, increased TNFα and IL1ß expression levels (P < 0.05) were found in the susceptible animals, while resistant animals had IL33 superiorly expressed (P < 0.05). Higher levels (P < 0.05) of TLR2 and CFI were found in the abomasal pyloric mucosa of resistant animals. TNFα was at higher levels (P < 0.05) in the blood of susceptible lambs, at day 0 of the third artificial infection. The exacerbated proinflammatory response observed in susceptible animals, at both local and systemic levels, may be a consequence of high H. contortus parasitism. This hypothesis is corroborated by the higher blood levels of TNFα before the onset of infection, which probably remained elevated from the previous parasitic challenges. On the other hand, resistant lambs had an enhanced response mediated by TLR recognition and complement activation. Nevertheless, this is the first study to directly associate sheep parasitic resistance with IL33, an innate trigger of the Th2-polarized response.

Transcriptome analysis unraveled potential mechanisms of resistance to Haemonchus contortus infection in Merino sheep populations bred for parasite resistance.

Haemonchus contortus is one of the most pathogenic gastrointestinal nematodes in small ruminants. To understand molecular mechanisms underlying host resistance to this parasite, we used RNA-sequencing technology to compare the transcriptomic response of the abomasal tissue, the site of the host-parasite interaction, of Merino sheep bred to be either genetically resistant or susceptible to H. contortus infection. Two different selection flocks, the Haemonchus selection flock (HSF) and the Trichostrongylus selection flock (TSF), and each contains a resistant and susceptible line, were studied. The TSF flock was seemingly more responsive to both primary and repeated infections than HSF. A total of 127 and 726 genes displayed a significant difference in abundance between resistant and susceptible animals in response to a primary infection in HSF and TSF, respectively. Among them, 38 genes were significantly affected by infection in both flocks. Gene ontology (GO) enrichment of the differentially expressed genes identified in this study predicted the likely involvement of extracellular exosomes in the immune response to H. contortus infection. While the resistant lines in HSF and TSF relied on different mechanisms for the development of host resistance, adhesion and diapedesis of both agranulocytes and granulocytes, coagulation and complement cascades, and multiple pathways related to tissue repair likely played critical roles in the process. Our results offered a quantitative snapshot of changes in the host transcriptome induced by H. contortus infection and provided novel insights into molecular mechanisms of host resistance.

Early and late gene expression profiles of the ovine mucosa in response to Haemonchus contortus infection employing Illumina RNA-seq technology.

We conducted herein transcriptome sequencing of the ovine abomasal tissues using the Illumina HiSeq 4000 platform to segregate early and late H. contortus-infected sheep (7 and 50days post-infected groups, respectively) from the control naive ones. A total of 548, 357 and 7 were substantially induced genes in 7days post-infection versus uninfected-control group, 50days post-infection versus 7days post-infection (7dpi), and 50days post-infection (50dpi) versus uninfected-control group, respectively. However, a total of 301, 355 and 11 were significantly repressed genes between 7dpi versus uninfected-control group, 50dpi versus 7dpi, and 50dpi versus uninfected-control group, correspondingly. This indicates that H. contortus infection induced a more potent activation of abomasal gene expression in the early stage of infection as compared to the late stage. Seven pathways were annotated by Kyoto Encyclopedia of Genes, and Genomes pathway analysis accounted for the significant percentage in early H. contortus infection. This study shows for the first time that both galectin-11 and matricellular protein osteopontin are up-regulated in abomasal tissue after chronic H. contortus infection, while galectin-4 is specifically down-regulated in the early infection. Additionally, our results showed that the induction or repression of these molecules is likely to determine the infection progression.

Phenotypic expression of parasite susceptibility to Haemonchus contortus in Pelibuey sheep.

The objective of this study was to determine if the mean faecal egg count (FEC) from a first experimental Haemonchus contortus infection could be used to classify parasite-naïve Pelibuey hair sheep as parasite-resistant high responders and parasite-susceptible low responders. Twenty 6- to 7-month-old Pelibuey male sheep raised free of gastrointestinal nematodes were challenged with 7500±1412 H. contortus L3 larvae administrated orally on day 0 of the study. Faecal samples from each lamb were obtained daily from 21 to 41days post-infection (Stage I). Lambs received a second artificial infection of 8420±1545L3 larvae on day 42, with faecal samples collected from day 65 to day 78 (Stage III). The mean FEC for each lamb in Stage I was used to classify 8 lambs with means for FEC that were more than two standard errors (SE) below the overall mean (i.e., <4764 eggs per gram of feces; epg) as high responders. The remaining 12 lambs were classified as low responders. Means for FEC in Stage I were 2449±194 epg for high responders and 14,461±1044 epg for low responders (P<0.05). High responders also had lower FEC than low responders in Stage III (actual means of 650±220 vs. 5933±1990 epg; P<0.05 following log transformation to normalize the FEC distribution). Lambs were then reclassified as high and low responders based on their mean FEC in Stage III. Fourteen lambs with means for FEC that were more than one standard error (SE) below the overall mean (i.e., below 1537 epg) were classified as high responders. The remaining six lambs were classified as low responders. Use of the Stage I responder class to predict the Stage III responder class resulted in an 83.3% sensitivity but only a 50% specificity. The positive predictive value was 41.7% and the negative predictive value was 87.5%. The poor positive predictive value was caused by 5 animals with high FEC in Stage I, but low FEC in Stage III. The first infection thus identified most high-responder lambs, but a second infection may improve accuracy by separating lambs with an intermediate level of resistance from truly susceptible lambs. This protocol now requires additional validation under more practical conditions involving natural parasite infections and larger lamb numbers.

Exploring the Genetic Resistance to Gastrointestinal Nematodes Infection in Goat Using RNA-Sequencing.

Gastrointestinal nematodes (GINs) are one of the most economically important parasites of small ruminants and a major animal health concern in many regions of the world. However, the molecular mechanisms of the host response to GIN infections in goat are still little known. In this study, two genetically distinct goat populations, one relatively resistant and the other susceptible to GIN infections, were identified in Yichang goat and then four individuals in each group were chosen to compare mRNA expression profiles using RNA-seq. Field experiment showed lower worm burden, delayed and reduced egg production in the relatively resistant group than the susceptible group. The analysis of RNA-seq showed that 2369 genes, 1407 of which were up-regulated and 962 down-regulated, were significantly (p < 0.001) differentially expressed between these two groups. Functional annotation of the 298 genes more highly expressed in the resistant group yielded a total of 46 significant (p < 0.05) functional annotation clusters including 31 genes (9 in innate immunity, 13 in immunity, and 9 in innate immune response) related to immune biosynthetic process as well as transforming growth factor (TGF)-ß, mitogen-activated protein kinase (MAPK), and cell adhesion molecules (CAMs) pathways. Our findings provide insights that are immediately relevant for the improvement of host resistance to GIN infections and which will make it possible to know the mechanisms underlying the resistance of goats to GIN infections.

Haemonchus contortus: the then and now, and where to from here?

Haemonchus contortus (Barber's pole worm or "BPW") is the nematode "nemesis" of small ruminant production systems in tropical and subtropical regions of the world. Its reputation derives from a combination of high fecundity and a short generational interval that provides an enviable developmental plasticity for adaptation or resistance to control measures. This review critically examines the historical and current literature on the host-parasite-environment interaction for H. contortus, particularly in sheep, to highlight changes in parasite distribution and ecology on pasture, changes to the seasonal inhibition of fourth stage larvae and the most appropriate models to identify protective responses and assess vaccines. The review also proposes pathways to bring host genetics to fruition and avenues where advances in the parasite genome may complement control measures.

Early IL-4 gene expression in abomasum is associated with resistance to Haemonchus contortus in hair and wool sheep breeds.

Early immune events associated with reduced larval burden remain unclear in parasite-resistant breeds of sheep. Therefore, our objective was to determine breed differences in immune-related gene expression following infection with H. contortus. Gene expression in abomasal tissue and mucosa and in abomasal lymph nodes (ALN) was measured in 24 St. Croix (hair) lambs and 24 Dorset x (Finn-Rambouillet) (wool) lambs at 0 (uninfected), 3, 5 and 7 days after infection with 10 000 L3 H. contortus larvae. Expression of IL-4 in abomasal mucosa was detected on day 3 and increased to day 7 in hair lambs, but was not detectable in wool lambs. Genes that recruit neutrophils (CXCL1) and macrophages (MCP1) were upregulated in abomasal mucosa of hair lambs. Genes associated with alternative macrophage activation (ARG-1) and eosinophil activation (Gal-14) were also upregulated in the abomasal mucosa of hair lambs. Tissue remodeling genes (MMP13, PDGF) and tumour necrosis factor alpha (TNF-α) and MCP1 were upregulated in abomasal tissue of wool lambs; these lambs also had greater expression of forkhead box P3 in ALN. These data indicate a role for early IL-4 expression locally and demonstrate potential downregulation of immunity in wool sheep that could facilitate establishment of H. contortus.

Galectins and collectinis expression are increased in Haemonchus contortus-infected corriedale sheep.

Galectins and collectins are proteins classified in the lectin family that have the ability to recognize molecular patterns associated with pathogens. Studies on cattle have demonstrated high expression of these proteins during infection with gastrointestinal nematodes. The aim of this study was to investigate whether the level of Haemonchus contortus infection would alter the expression of galectins (Gal11 and Gal14) and collectins (SPA and CGN) in sheep. Twelve Corriedale sheep exposed to natural infection with nematodes were divided into two groups: group 1 (G1, n = 7) and group 2 (G2, n = 5), with low and high parasite burdens, respectively, based on fecal egg counts and abomasal parasite counts. The fecal egg counts and abomasal parasite counts were significantly different (p < 0.05) between the groups. Galectin and collectin gene expression was observed in all sheep abomasal samples. However, animals with lower infection levels showed lower expression of the genes Gal14, SPA and CGN (p < 0.05). Expression of lectins was associated with the abomasal H. contortus burden, thus suggesting that these proteins may have a role in controlling of this infection.