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 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.

Graphical abstract

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.

No abstract

Haemonchus contortus, the most pathogenic sheep parasite, is the major limiting factor for sheep farming in tropical countries, including in Brazilian territory. 1-4 As chemical control is the main tool for combating this parasite, the widespread multiple resistance of H. contortus to anthelmintics highlights the urgent need for alternative control measures, such as the selective breeding for parasite resistance or development of immune-related therapeutics. 5-8 Therefore, better understanding of the immuno-genetic mechanisms underlying this resistance is required. Immunity against helminths depends on cellular adaptive responses, through participation of T-helper lymphocytes (Th, CD4+), which are functionally grouped into two subpopulations: Th1,