Ana Carolina de Souza Chagas¹, David Miguel Ribeiro², Hugo Osório³, Ana Alexandra Pinto Abreu³, Cintia Hiromi Okino¹, Simone Cristina Méo Niciura¹, Alessandro Francisco Talamini Amarante⁴, Hornblenda Joaquina Silva Bello¹, Rafaela Tami Ikeda Kapritchkoff ⁵, Sérgio Novita Esteves¹, André Martinho de Almeida²

Haemonchus contortus is the main gastrointestinal nematode (GIN) species that parasitizes sheep and goats in tropical and subtropical countries, triggering severe anemia, submandibular edema and death. The detection of protein profiles that occur in more parasitized/susceptible animals is of high relevance for targeted selective treatment strategies (TST). This proteomic study aimed to elucidate physiological mechanisms associated to H. contortus infection in susceptible (Dorper- D) and resistant (Santa Inês - S) sheep breeds. Three naïve female lambs of each breed were infected with 10.000 H. contortus L₃ (day zero – D0). Weekly, for 28 days, feces were collected for individual fecal egg counts (FEC) and blood for packet cell volume (PCV). Proteomics studies were performed with sera from D0 (-) and D21 (+). They were prepared following the single-pot, solid-phase-enhanced sample preparation protocol (SP3) and analyzed by LC-MS/MS-FAIMS. Output results were analyzed by ClustVis (PCAs and Heatmaps) and gene ontology categorization by PANTHER. Functional enrichment analysis was conducted using ClueGo and String tools through CytoScape. The MS proteomics data was uploaded to the ProteomeXchange Consortium via PRIDE (PXD028361). FEC and PCV confirmed no infection on D0, with continuous increasing of EPG, as well as PCV decrease. On D28 there was a significant difference between the two breeds for logFEC means (D = 3774 and S = 3141, p=0.033) and PCV means (D = 16.3% and S = 24.3%, p=0.038). In total, 754 proteins were identified, connected with Ovis aires UNIPROT database, from which 68 differential abundant proteins (DAPs) were noted: 33 for S- vs D-, 9 for D+ vs D-, 8 for S+ vs S-, and 18 for S+ vs D+. PCA indicated clear clustering of the four experimental groups. S- vs D-: DAPs were associated with the innate and adaptive immune system of both breeds (which was also observed for S+ vs D+ DAPs). Focusing on the consequences of infection for each breed: D+ vs D-: the upregulated proteins (Insulin-like growth factor-binding (IGFBP1), Gasdermin B (GSDMB)) were associated with inflammation control and immune response in infected Dorper, where the downregulated ones (Acetyl-CoA acetyltransferase 2 (ACAT2), Phosphopyruvate hydratase (ENO3), Aggrecan (ACAN)) indicated a negative impact of the infection regarding tissue anabolism, compromising muscle growth and fat deposition. S+ vs S-: the upregulated proteins (ENO3, Haptoglobin, Semaphorin 6B (SEMA6B)) were related to an immune response mounted by infected Santa Inês, while the downregulated ones (Tubulin alpha chain (TUBA4A), Collagen type I alpha 2 chain (COL1A2), ACAN) are possibly linked to the Santa Inês lambs’ muscular development and growth, unimpaired by parasitemia. Taken together, our data would suggest that ENO3 plays a relevant role in the immune system of Santa Inês and appears to be differentially regulated between breeds, while THEMIS-like deeply contributes to the muscle development and carcass conformation through energy metabolism in Dorper. The abundant presence of acute phase proteins (APPs) (Alpha-1-acid glycoprotein, Haptoglobin, Serum amyloid A protein, Ceruloplasmin) reinforce their relation with infection, inflammation and stress conditions.  

Agradecimentos: This study was supported by São Paulo Research Foundation (FAPESP- Process n◦ 2021/02535–5, 2022/12536–1, 2022/07720–8, and 2022/00776-8). Authors DMR and AMA acknowledge funding from the Fundação para a Ciência e a Tecnologia via research center LEAF (UIDB/04129/2020), associate laboratory TERRA (LA/P/0092/2020) and PhD fellowship attributed to DMR (SFRH/BD/143992/2019).