Scott A. Bowdridge

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.

Effects of peripheral blood mononuclear cells on Haemonchus contortus larval motility in vitro.

The objective of this experiment was to determine effects of peripheral blood mononuclear cells (PBMC), derived from parasite‐resistant St. Croix (STC) and parasite‐susceptible Suffolk (SF) sheep, on motility of Haemonchus contortus L3 stage larvae in vitro. Peripheral blood mononuclear cells were collected from 10 lambs of each breed, 5 naïve and 5 which had received a priming infection with H. contortus. Larval motility was quantified using a MIF Nikon Sweptfield microscope and NIS Elements AR software, and measurements included path length (PL) (μm), velocity (VEL) (μm/s) and acceleration (ACC) (μm/s2). After 18 h of incubation, PL and VEL were greatest in larvae cultured with SF‐derived PMBC and were significantly different from all other groups (P < 0·01). No difference was observed in PL or VEL between larvae exposed to naïve or primed STC‐derived PBMC and primed‐SF PBMC. Differences in ACC were detected between larvae cultured with primed STC‐derived PBMC (10·91 μm/s2) and naïve SF‐derived PBMC (45·7 μm/s2) (P = 0·035). These data indicate an innate ability of STC‐derived PBMC to severely inhibit larval motility.

Serum interleukin-4 (IL-4) production is associated with lower fecal egg count in parasite-resistant sheep.

The objective of this experiment was to determine serum interleukin-4 (IL-4) concentration in response to Haemonchus contortus infection in parasite-resistant and -susceptible lambs. St. Croix (STC) (resistant) and Suffolk/Hampshire crossbred (SX) (susceptible) lambs were either not infected (n=5/breed), given a primary inoculation (n=5/breed) or challenged infected with H. contortus (n=5/breed). Each inoculum given consisted of 10,000 L3 larvae. Blood was collected daily for 14 days and then weekly to day 49. Feces were collected on day 0 and weekly until day 49. Challenged STC lambs generated significantly lower fecal egg count (FEC) (1520 eggs/g SX vs. <50 eggs/gram STC; P<0.001) and had higher PCV (34% vs. 29%; P<0.001). Serum IL-4 concentrations of primary-infected STC and challenge-infected SX lambs were greater during early infection (days 0-7) than mid (days 14-28) and late (days 35-49) infection, but was significantly reduced (P<0.001) by day 49. Challenge-infected STC lambs incrementally increased serum IL-4 from early to late infection. Change in serum IL-4 concentration during early, mid and late infection indicated IL-4 concentration in challenge-infected STC lambs increased during mid and late infection. These data demonstrate that parasite-resistant St. Croix sheep generate a potent Th2-response, as measured by elevated serum IL-4 concentration, which is associated with a marked FEC reduction.

Serum-mediated Haemonchus contortus larval aggregation differs by larval stage and is enhanced by complement.

The objectives of this study were to measure Haemonchus contortus larval aggregation by complement/antibody complexes, determine effect of breed resistance and infection status and determine the effect of larval maturation on larval aggregation in vitro. Larval binding assays were performed on H. contortus L3, exsheathed L3 and L4 incubated with serum from either parasite naïve or H. contortus primed St. Croix (resistant) and Suffolk (susceptible) lambs. No differences in L3 aggregation were observed between serum from either breed or infection status. Exsheathed L3 (60%) and L4 (42%) aggregation by primed Suffolk serum was significantly reduced compared with L3 (80%, P<.001). Removal of either complement or antibody effectively eliminated L3 aggregation (P<.001). Combination of antibody‐depleted and complement‐inactivated serum restored L3 aggregation to levels consistent with unprocessed serum, supporting a role for antibody and complement in aggregation (P<.001). Use of fluorescence‐labelled anti‐sheep IgG antibody allowed documentation of IgG bound to serum complexes within L3 masses and was present only in larvae incubated with normal serum, and complement‐ and antibody‐depleted serum combination. These data indicate that complement/antibody complexes inhibit larval motility through larval aggregation which may be critical in early larval clearance of H. contortus.

The effect of ovine peripheral blood mononuclear cells on Haemonchus contortus larval morbidity in vitro

The objective of this study was to determine the effects of peripheral blood mononuclear cells (PBMC), derived from parasite‐resistant St. Croix (STC) hair sheep and parasite‐susceptible Suffolk (SUF) sheep, on Haemonchus contortus L3 stage larval death in vitro, with or without autologous serum. Larval morbidity was quantified by measuring larval ATP concentration following incubation with PBMC. Larvae exposed to either STC‐ or SUF‐derived PBMC had lower ATP than live larvae (0.12 μmol/L ATP and 0.16 μmol/L ATP vs 0.27 μmol/L ATP, respectively) (P<.001) and greater ATP of dead larvae (0.03 μmol/L ATP) (P<.001). Breed differences were observed with addition of autologous serum. Larvae exposed to SUF‐derived PBMC with autologous serum were not significantly different from live larval ATP. STC‐derived serum did not significantly reduce larval ATP compared to PBMC alone (0.11 μmol/L ATP), but was significantly reduced compared to live larvae (0.22 μmol/L ATP) and SUF‐derived PBMC with autologous serum (0.23 μmol/L ATP) (P<.001). These data indicate that a cellular response alone is capable of significantly reducing larval ATP in a breed‐independent manner. However, addition of serum to SUF‐PBMC failed to reduce larval ATP, indicating breed‐dependent humoral response to H. contortus.

Ovine vital neutrophil extracellular traps bind and impair Haemonchus contortus L3 in a breed-dependent manner

This study aimed to characterize neutrophil response to Haemonchus contortus (Hc) in vitro using cells from parasite‐resistant St. Croix (STC) and parasite‐susceptible Suffolk (SUF) sheep. Neutrophils from Hc‐primed and naive STC and SUF sheep were incubated with Hc larval antigen (HcLA), Hc worm antigen (HcWA) or complete media (CM). After HcLA exposure, neutrophils from STC and SUF formed extracellular traps composed of DNA. Stimulation with HcLA induced a 35‐fold increase in extracellular DNA compared to CM controls. However, extracellular DNA was not found when neutrophils were cultured with HcWA. The formation of neutrophil extracellular traps (NET) in response to HcLA yields a low percentage of necrotic cells indicating a form of vital NETosis. Neutrophils from primed and naïve STC bound Hc L3 greater (93% and 68%) than SUF (78% and 45%; P < 0.001). Furthermore, STC neutrophils significantly reduced larval ATP levels compared to SUF neutrophils (0.05 μmol/L vs 0.1 μmol/L ATP, P < 0.001). These data indicate that ovine neutrophils bind, form vital NET and reduce ATP to Hc L3 in a breed and infection status–dependent manner.

Impaired IL-4 signaling promotes establishment of Haemonchus contortus in sheep

Reduced worm burden in St. Croix (STC) sheep during Haemonchus contortus (Hc) infection is predicated on rapid interleukin‐4 (IL‐4) signalling and T helper type 2 immune (Th2) response. The aim of these studies was to further elucidate differences in Th2 responses by STC and Suffolk (SUF) sheep. Ten days after challenge Hc infection, peripheral blood mononuclear cells (PBMC) were collected and cultured with larval (HcLA) and adult worm (HcWA) antigen. STC PBMC produced nearly twice as much IL‐4 as SUF (823.57 pg/mL vs 454.28 pg/mL) at 6 hours of HcLA culture despite no difference in IL‐4 gene expression and the IL‐4 receptor (IL4Rα) was upregulated in STC PBMC but was undetectable in SUF. Expression of other Th2‐type genes were increased in STC PBMC including IL13, IL5 and MRC1. IL‐4 supplementation to HcLA culture was insufficient to achieve upregulation of Th2 genes in SUF PBMC. Production of IL‐4 did not occur in SUF PBMC until 24 hours after culture with HcLA, and expression of IL4 in the abomasum was similarly delayed until 10 days after challenge infection, which was associated with significantly higher larval burden (530 vs 16). These data demonstrate delayed upregulation of Th2 genes in SUF sheep contributes to susceptibility to Hc.

Effects of grazing birdsfoot trefoil-enriched pasture on managing Haemonchus contortus infection in Suffolk crossbred lambs

High-tannin forages can be used to help mitigate the serious limitations associated with gastrointestinal nematode (GIN) infections on efficient small ruminant production. The objective of this experiment was to determine how grazing a GIN-free, established stand of a high-tannin cultivar of birdsfoot trefoil (Lotus corniculatus L.) influenced the prevention or treatment of Haemonchus contortus (Hc) infection in lambs. A birdsfoot trefoil-enriched pasture was established on an area that was previously row cropped and not grazed for at least 15 yr. Treatments included preventative (PREV) with parasite-naïve lambs transitioned onto pasture 1 wk prior to receiving an infection of 10,000 Hc larvae, therapy (THER) with parasite-naïve lambs infected with 10,000 Hc larvae 4 wk prior to the start of grazing, and control (CONT) with naïve, uninfected lambs to verify that natural infection did not occur on pasture. Each treatment group of 12 Suffolk crossbred lambs was divided into 3 replicates per treatment, and all were supplemented with a grain mix to provide 16% CP. Fecal egg count (FEC, eggs/g wet feces) in THER lambs peaked 1 wk after the start of grazing (9,404) and after 4 wk fell to 1,068, equivalent to a FEC reduction of 88.6%. Lambs in PREV had a peak FEC of 4872 at 4 wk after infection where peak FEC was 48% less in PREV than THER lambs. Lambs in CONT did not have measurable FEC for the duration of this study. Packed cell volume (PCV, %) reflected infection status of the lambs in each group, where CONT (32%) had the highest (P < 0.05) PCV followed by THER (29%) and PREV (26%). Total weight gained in CONT lambs was greatest at 5.51 kg (P < 0.01), whereas THER and PREV (2.68 and 2.97 kg, respectively) did not differ. Grazing birdsfoot trefoil-enriched pasture can have both therapeutic and preventative effects on Hc infection in lambs and can be used in a systems approach to control GIN parasites in grazing sheep.