Immunohistochemistry analyses of the abomasal mucosa show differences in cellular-mediated immune responses to Haemonchus contortus infection in resistant and susceptible young lambs.

Haemonchus contortus is known for its high pathogenicity in sheep, and the uncontrolled use of anthelmintics resulted in the emergence of multiple drug-resistant populations. Breeding sheep for gastrointestinal nematode resistance is a sustainable alternative to reduce dependence of anthelmintic drugs, and differences in the degree of resistance between breeds have been reported. Here we compare two sheep breeds (Santa Ines and Ile de France), concerning the differences in innate and adaptive immune response involved in the resistance against H. contortus infection. Immunohistochemical analyses of the abomasum were conducted in naïve Santa Ines (n = 14) and Ile de France (n = 12) lambs randomized into four groups: infected Santa Ines (n = 8), non-infected control Santa Ines (n = 6), infected Ile de France (n = 8), and non-infected control Ile de France (n = 4). The infected lambs were initially infected with H. contortus infective larvae at 14 days of age, and multiple infections were conducted every second day until they reached 66 days of age. There was a significant effect (P < 0.001) of the infection with increase in numbers of CD3+ T; CD79α+ B; GATA3+ Th2/ILC2; POU2F3+ tuft cells; FOXP3+ T reg; and IgE + cells in the fundus of the abomasal mucosa in both Santa Ines and Ile de France lambs. Nevertheless, the infected Santa Ines lambs presented the highest averages for CD79α+ B; GATA3+ Th2/ILC; IgE + cells; and POU2F3+ tuft cells and there was a significant association of the breed and infection status with regards to POU2F3+ tuft cells, with the highest mean in the infected Santa Ines group. The infected Santa Ines group had three lambs with high degree of resistance and five lambs that showed a moderate infection. Our results suggest a mechanism of synergistic coordination between different immune-cell types in promoting resistance of suckling lambs under H. contortus infection.

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.

Diagnosis of Oestrus ovis infestation in sheep by PCR and enzyme-linked immunosorbent assay.

The aim of this study was to assess the suitability of PCR and ELISA as diagnostic method in young sheep naturally infested by Oestrus ovis larvae. The experiment was carried out from December 2020 to April 2021 with 39 lambs divided into two groups: infested (n = 26) and control treated group (n = 13). The infested group did not receive treatment against oestrosis, and the control group was treated with closantel (10 mg/kg orally) every 28 days in order to keep the animals as free as possible of O. ovis infestation. The clinical signs varied among animals regardless of the number of recovered larvae of each lamb, however, the thick mucus and mucopurulent nasal discharge scores were less frequent in lambs from treated group. There was no correlation between the nasal discharge score and the number of O. ovis recovered larvae (R² = 0.012, P = 0.165). Three control treated animals only presented first instar larvae (L1) (1 - 4 larvae/animal) which were smaller than L1 found in the lambs of the infested group. Ninety-two percent of the lambs from infested group (24/26) were parasitized by O. ovis with number ranging from 1 to 54 larvae per animal. A gradual increase in plasma IgG (anti-antigen of O. ovis larvae) levels of animals from infested group after the third week of the trial was observed, whereas the control lambs had low levels of IgG until the end of the experiment. The PCR had low sensitivity (26 %) and high specificity (100 %), and it presented poor agreement (k = 0.177) with the larvae detection after the lamb slaughter. The oestrosis clinical signs were not related to larvae infestation intensity and ELISA showed a greater advantage over the PCR technique in identifying animals that are carrying O. ovis.

Prophylactic Effects of Ivermectin and Closantel Treatment in the Control of Oestrus ovis Infestation in Sheep.

The sheep nasal bots Oestrus ovis is parasite of the nasal cavities and sinuses of small ruminants causing oestrosis, one of the most frequent parasitic diseases in sheep and goats. The widely use of ivermectin and closantel by the sheep breeders in the treatment and prophylaxis of gastrointestinal nematodes resulted in widespread cases of anthelmintic resistance. However, there is no report about cases of O. ovis with drug-resistance. In this study, we evaluated the prophylactics and therapeutic effects of both antiparasitics in sheep with O. ovis natural infestation. The trial was carried out from early December 2019 to March 2020, with 30 crossbred males lambs allocated into three groups of 10 animals each: control (without treatment), treated with ivermectin (0.2 mg/kg subcutaneously) and treated with closantel (10 mg/kg orally). The animals were kept together grazing the same pasture area. The treatment groups were drenched in two occasions 70 days apart: on 5th December 2019 and on 13th February 2020. On 19th March 2020, all lambs were slaughtered. The lamb heads were removed and sectioned along their longitudinal and sagittal axis to search for larvae. Recovered O. ovis larvae were counted and identified according to their developmental stage (L1, L2, and L3). Seven of the control lambs were infested with O. ovis larvae ranging from six to 17 larvae (11.6 mean infestation intensity). All recovered larvae from control group were intact and active. Three animals treated with ivermectin had O. ovis larvae (1-3 larvae), however they were dead and in degeneration. The animals treated with closantel did not have any larvae. The clinical suggestive signs of oestrosis were scarce over the experimental period. The averages of daily weight gain were similar (p > 0.05) among groups. Closantel and ivermectin had high efficacy against oestrosis and O. ovis parasitism did not hinder the performance of lambs.