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Aims: Local and systemic immune mediators of Morada Nova lambs with divergent Haemonchus contortus resistance phenotypes were evaluated., Methods and Results: Lambs were ranked through faecal egg counts (FEC) after two parasitic challenges with 4,000 H.contortus L 3 . After the second challenge, the lambs underwent a third artificial infection and were euthanized 7 days later. Immune-related genes were quantified locally in abomasal mucosa and lymph nodes (CD4, IFNγ, IL4, IL5, IL13, IL2RA and MS4A2) and systemically in the whole blood (IL4 and IL13). Anti-H. contortus IgG and IgA antibodies and eosinophils and mast cells counts were also investigated. Resistant animals presented higher systemic IgG and IgA titres, both negatively correlated with FEC. Susceptible animals had higher blood levels of IL4 transcripts. At the local level, resistant lambs had higher eosinophils counts and superior MS4A2 levels in abomasal fundic mucosa, besides higher IgA levels in abomasal mucus, while susceptible lamb had superior IL4 expression in abomasal lymph nodes., Conclusion: These data indicate that resistant lambs had an immune response mediated by antibody-mediated cytotoxicity. Also, the systemic humoral profile, particularly IgA isotype, seems to be a good resistance marker for Morada Nova sheep, as we found differences between groups even when FEC did not differ., (© 2020 John Wiley & Sons Ltd.)

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 L 3 ) 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 ± 0.52 kg for the T allele on final body weight and 0.74 ± 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., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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 L 3 . 20 extreme resistance phenotypes (10 most resistant and 10 most susceptible) were selected, subjected to a third artificial infection with 4,000 L 3 , 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 IL1β 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., Competing Interests: The authors have no conflict of interest., (Copyright © 2019 João Henrique Barbosa Toscano et al.)

Due to the great economic impact of Haemonchus contortus on sheep farming, there is an increasing number of studies addressing host resistance against this nematode, including identification of directly related immune mechanisms. In this context, relative gene expression by RT-qPCR have been largely used, due to its rapidity, high sensitivity, specificity, and reproducibility. Although, appropriate reference gene selection is crucial for accurate interpretation of results. In this study, five reference genes (GAPDH, G6PDH, YWHAZ, ACTB, and B2M) were tested for expression stability in abomasum (fundic and pyloric regions) and abomasal lymph nodes of Morada Nova sheep classified as resistant (n = 5) or susceptible (n = 5) to H. contortus infection in a flock of 151 animals. GAPDH combined with YWHAZ were selected as reference genes for abomasal fundic region and abomasal lymph nodes, whereas YWHAZ was the most stable gene for abomasal pyloric region. These genes presented the lowest intra- and inter-group variations and, consequently, highest stability. In contrast, expression of G6PDH was the least stable in all tissues. The impact of reference gene selection was demonstrated by relative quantification of a pro-inflammatory cytokine (TNFα) in abomasal fundic region. Significant differences in TNFα expression levels between resistant and susceptible groups were only observed when the most stable genes (GAPDH combined with YWHAZ) or GAPDH were used as reference genes, whereas no significant differences were observed when other tested reference genes were used. It was demonstrated that normalization of expression data using inappropriate reference genes may significantly influence interpretation results.