Reducing fecal egg count through selective breeding alters dorper lamb response to Haemonchus contortus in an artificial challenge trial.

Infection by gastrointestinal nematodes (GIN), particularly Haemonchus contortus, can be detrimental to sheep health and performance. Genetic susceptibility to GIN varies between breeds, with those lacking high levels of natural resistance often requiring frequent anthelmintic treatment when facing parasitic challenge. Genetic technology can serve as a tool to decrease GIN susceptibility via selection for sheep with reduced fecal egg count (FEC) estimated breeding values (EBVs). However, the physiological changes that result from implementation of this strategy are not well described. Additionally, there is a need for comparison of animals from recent selective breeding against breeds with inherent GIN resistance. In this study we administered a challenge of H. contortus to Dorper x White Dorper (DWD; n = 92) lambs that have been genetically selected for either low (DWD-) or high (DWD+) FEC EBVs and Barbados Blackbelly x Mouflon (BBM; n = 19) lambs from a genetically resistant breed backgrounds. Lamb FEC, packed-cell volume (PCV) and serum IgG were measured at intermittent levels over 5 weeks. At day 21 and day 35, the selectively bred DWD- had a lower mean FEC compared to DWD+, but were higher than BBM. Reductions in both PCV and serum IgG from initial day 0 levels were observed in DWD lambs, but not in BBM. Furthermore, from a subset of lambs (n = 24) harvested at day 21, DWD- only tended (p = 0.056) to have lower mean worm counts than DWD+, with BBM having the lowest mean worm count. Differentially expressed genes (DEGs) identified via RNA-sequencing of abomasal tissue at day 21 indicate a more pronounced Th2 immune response and more rapid worm expulsion occurred in iBBM than iDWD- and iDWD+ lambs. However, gene expression in DWD- suggests an association between reduced FEC EBV and gastric acid secretion and the ability to limit worm fecundity. Ultimately, selection of Dorper sheep for low FEC EBV can reduce susceptibility to GIN, but it will likely require multiple generations with this trait as a breeding priority before presenting a similar resistance level to Caribbean breeds.

Genome-Wide Analysis of Sheep Artificially or Naturally Infected with Gastrointestinal Nematodes.

The anthelmintic resistance of gastrointestinal nematodes (GINs) poses a significant threat to sheep worldwide, but genomic selection can serve as an alternative to the use of chemical treatment as a solution for parasitic infection. The objective of this study is to conduct genome-wide association studies (GWASs) to identify single nucleotide polymorphisms (SNPs) in Rambouillet (RA) and Dorper × White Dorper (DWD) lambs associated with the biological response to a GIN infection. All lambs were genotyped with a medium-density genomic panel with 40,598 markers used for analysis. Separate GWASs were conducted using fecal egg counts (FECs) from lambs (<1 year of age) that acquired their artificial infections via an oral inoculation of 10,000 Haemonchus contortus larvae (n = 145) or naturally while grazing on pasture (n = 184). A GWAS was also performed for packed cell volume (PCV) in artificially GIN-challenged lambs. A total of 26 SNPs exceeded significance and 21 SNPs were in or within 20 kb of genes such as SCUBE1, GALNT6, IGF1R, CAPZB and PTK2B. The ontology analysis of candidate genes signifies the importance of immune cell development, mucin production and cellular signaling for coagulation and wound healing following epithelial damage in the abomasal gastric pits via H. contortus during GIN infection in lambs. These results add to a growing body of the literature that promotes the use of genomic selection for increased sheep resistance to GINs.

Effects of a multicomponent microbial feed additive containing prebiotics and probiotics on health, immune status, metabolism, and performance of newly weaned beef steers during a 35-d receiving period.

We examined the effects of dietary supplementation of a multicomponent blend of prebiotics and probiotics on health, immune status, metabolism, and performance of newly weaned beef steers during a 35-d receiving period. Eighty newly weaned crossbred steers (12-hour postweaning; 206 ± 12 kg of body weight [BW]) from a single source were stratified by BW into four pens (20 steers per pen) such that each pen had similar BW at the beginning of the experiment. The pens were randomly assigned to receive a corn silage-based diet with no additive (CON; two pens; n = 40 steers) or a basal diet supplemented with SYNB feed additive at an average of 28 g/steer/d (SYNB; two pens; n = 40 steers). The SYNB additive is a blend of live Saccharomyces cerevisiae and the fermentation products of S. cerevisiae, Enterococcus lactis, Bacillus licheniformis, and Bacillus subtilis and was supplemented for the first 21 d only. Percentage of steers treated for bovine respiratory disease (BRD) was calculated for each dietary treatment. Daily dry matter intake (DMI) and meal events (meal frequency and duration) were measured. Weekly BWs were measured to calculate average daily gain (ADG). Blood samples collected on days 0, 14, 21, 28, and 35 were used for ex-vivo tumor necrosis factor alpha (TNF-α) release assay following lipopolysaccharides (LPS) stimulation, plasma metabolome analysis, and mRNA expression analysis of 84 innate and adaptive immune-related genes. Compared with CON, supplemental SYNB increased (P ≤ 0.05) ADG, DMI, and meal events during the first 7 d. At d 21, there was no treatment effect (P > 0.05) on final BW, DMI, ADG, and meal events; however, beef steers fed supplemental SYNB had greater (P = 0.02) meal duration. Over the entire 35-d receiving period, beef steers fed supplemental SYNB had greater (P = 0.01) ADG and feed efficiency, tended to have greater (P = 0.08) meal duration, and had lower percentage (35 vs. 50%) of animals treated for BRD and lower percentage of sick animals treated for BRD more than once (7.15 vs. 45%). Whole blood expression of pro-inflammatory genes was downregulated while that of anti-inflammatory genes was upregulated in beef steers fed supplemental SYNB. Beef steers fed supplemental SYNB had lower (P = 0.03) plasma concentration of TNF-α after LPS stimulation. Six nutrient metabolic pathways associated with health benefits were enriched (false discovery rate ≤ 0.05) in beef steers fed supplemental SYNB. This study demonstrated that dietary supplementation of SYNB during the first 21 d of arrival reduced BRD morbidity, improved the performance, immune, and metabolic status of beef steers over a 35-d receiving period thereby extending the SYNB effect by a further 14 days post supplementation.

Response of Rambouillet Lambs to an Artificial Gastrointestinal Nematode Infection.

Gastrointestinal nematodes (GIN) threaten the productivity and health of sheep worldwide, prompting the need for genetic selection to reduce GIN susceptibility. Fecal egg count (FEC), packed-cell volume (PCV), and various production traits were examined in parasitized Rambouillet sheep and compared to sire FEC estimated breeding value (EBV). Rambouillet lambs (n = 77) were inoculated with 10,000 H. contortus L3 larvae. Subsequently, FEC, PCV, and body weight (BW) were captured at seven-day intervals for six weeks. Lambs were sired by one of two rams with post-weaning FEC EBV of −9% or +9%. Mean FEC differed (p = 0.0132) with lambs from the lower EBV sire ("Sire L") being reduced, versus those from the higher EBV sire ("Sire H"), being 2135 ± 211 vs. 2912 ± 207 eggs per gram, respectively. Males and females did not differ for FEC, but females exhibited a higher mean PCV than males, (33.74 vs. 29.65%, p < 0.0001). Lambs were shorn ~120 d post artificial infection and wool measurements were captured. A negative correlation between FEC and grease fleece weight was observed. Our results describe the response of Rambouillet lambs to artificial H. contortus infection and suggest FEC EBV can reduce susceptibility to GIN in this breed.

Immune mechanisms of Texel sheep to adult and egg stages of Haemonchus contortus.

Experimental Haemonchus contortus (Hc) infections revealed that Texel sheep have faecal egg counts (FEC) comparable to parasite-resistant St. Croix but adult worm burden comparable to parasite-susceptible Suffolk sheep. The aim of these studies was to further investigate the mechanism causing this disparity. Cellular and humoral immune responses to adult- and egg stage Hc were compared amongst St. Croix, Texel and Suffolk sheep. Adult worms and eggs were exposed to peripheral blood mononuclear cells (PBMC) and serum from the three breeds in vitro. There was a greater IgA binding to eggs when treated with St. Croix and Texel serum (p < .05), and these eggs exhibited a lower hatch rate (p < .05) when exposed to serum and PBMC. Adult worms exposed to St. Croix and Texel-derived PBMC, and serum had greater binding around the head and reproductive structures than worms exposed to Suffolk-derived cells and serum. Consequently, egg release tended to be affected by breed (p = .09). To further examine differences, Suffolk and Texel lambs (n = 5/breed) were infected with 10,000 Hc L3 for 30 days. Suffolk sheep had greater egg release per worm than Texel (3.8 vs. 2.1 eggs/worm). Taken together, these data would indicate one of the mechanisms in Texel's targeting adult-stage Hc reducing worm fecundity.

Characterization of ovine monocyte activity when cultured with Haemonchus contortus larvae in vitro.

AIMS: The objective of this study was to identify and characterize cell populations within ovine peripheral blood mononuclear cells (PBMCs) associated with Haemonchus contortus (Hc) larval morbidity and impairment in vitro. METHODS AND RESULTS: Monocytes and lymphocytes were separated from PBMC from parasite-resistant St. Croix (STC) sheep and parasite-susceptible Suffolk (SUF) sheep. Cells were cultured with Hc third stage larvae (L3) for 9 h. Larval morbidity was assessed using ATP concentration. Activation status was determined through gene expression analysis and enzyme inhibition. Enzymes arginase-1 (Arg1) and inducible nitric oxide synthase (iNOS) were inhibited using BEC (S-(2-boronoethyl)-I-cysteine) and 1400W (N-(3-(aminomethyl)benzyl)acetamidine), respectively. Larval ATP was lower when cultured with STC-derived monocytes (0.015 µmol/L ATP) compared to SUF-derived monocytes (0.067 µmol/L ATP) (P < .001), or lymphocytes from either breed (STC: 0.085 µmol/L, SUF: 0.112 µmol/L ATP) (P < .001). SUF-derived monocytes displayed higher expression of M1 genes, whereas STC-derived monocytes displayed M2 genes continuously. Inhibition of Arg1 decreased monocyte function in both breeds, whereas iNOS inhibition restored SUF-derived monocyte function. CONCLUSIONS: Together, these data indicate STC-derived monocytes favour M2 phenotype when exposed to L3, where SUF-derived monocyte function resembled M1 phenotype and described potential for improving Suffolk sheep through modulating inflammatory responses.

Interleukin-13 induces paralysis of Haemonchus contortus larvae in vitro.

AIMS: Interleukin-13 (IL-13) is a Th2-associated cytokine that typically induces gut contractility and mucus secretion to eliminate helminth parasites from the digestive tract. Little evidence exists of IL-13's direct effect on Haemonchus contortus larvae (L3) and thus was the objective of this study. METHODS: To test effects of IL-13 on H contortus, L3 were treated with ovine recombinant (r) IL-13 (1 µg/mL); motility and morbidity were assessed. Monocytes isolated from H contortus-resistant St. Croix (STC) and susceptible Suffolk (SUF) sheep were treated with anti-IL-13 blocking antibody to elucidate differences in host immune response. RESULTS: rIL-13 treatment reduced L3 speed (27 µm/s) and distance (7.5 µm) compared to untreated L3 (speed: 94 µm/s; distance: 27 µm) (P < .001). Comparison of larval speed to known paralytic levamisole (LEV) revealed no difference between treatments (rIL13: 23 µm/s; LEV 27 µm/s). Additionally, rIL-13 had no effect on larval morbidity. Blocking IL-13 reduced monocyte-driven larval morbidity (0.13 µmol/L ATP) and increased larval motility (88 µm/s; 27 µm) compared to larvae treated with STC-monocytes alone (0.07 µM ATP; 34 µm/s; 8 µm) (P < .05). CONCLUSIONS: These data indicate IL-13 has a dual capability paralysing L3 and contributing to monocyte-driven larval morbidity, and also indicate breed differences.

RNA-Sequencing of ovine PBMC after exposure to Haemonchus contortus larval antigen.

Mechanisms of immune activation in effector cells during Haemonchus contortus infection in sheep are currently unknown. Microarray experiments have been performed on tissues of H contortus infected sheep of varying parasite resistance during early and late points of infection, but not in immune effector cells. The purpose of this study was to compare early gene activation in peripheral blood mononuclear cells (PBMC) from primed parasite susceptible (Suffolk) and resistant (St. Croix) sheep in response to H contortus larval antigen (HcLA). Peripheral blood mononuclear cells were cultured for 6 hours with HcLA, and RNA-sequencing was performed. St. Croix PBMC upregulated 499 unique genes in response to HcLA while Suffolk PBMC upregulated 130 unique genes and 25 genes were shared between the two breeds. St. Croix PBMC had increased expression of genes associated with immune function, signal transduction, response to stress and others. In addition, while mechanisms of innate recognition of H contortus are unknown, multiple pattern recognition receptors were found to be upregulated in St. Croix PBMC cultured with HcLA and none were found to be upregulated in Suffolk PBMC. These patterns of immune gene activation may contribute to St. Croix's rapid response and ability to resist H contortus infection.

Neutrophils rapidly produce Th2 cytokines in response to larval but not adult helminth antigen.

Host protective immunity to Haemonchus contortus (Hc) infection in parasite-resistant St. Croix (STC) sheep is initiated early and characterized by an influx of innate cells and robust interleukin-4 (IL-4) production, resulting in T-helper type 2 immune (Th2) responses. The purpose of these studies was to elucidate the source of early IL-4 production. Neutrophils were isolated from whole blood, and populations >98% purity were cultured with larval or adult antigen to access cytokine production. Interleukin-4 and IL-13 were measured in sample supernatant using an ovine-specific enzyme-linked immunosorbent assay (ELISA). Neutrophils exposed to HcLA peaked in IL-4 production at 30 minutes (STC, 3153.65 pg/mL and SUF, 4665.22 pg/mL). A similar trend was observed in IL-13 production by 6 hours (STC, 391.02 pg/mL and SUF, 419.6 pg/mL). Adult antigen stimulation resulted in low cytokine production when compared to HcLA stimulation (STC IL-4, 6.04 pg/mL and SUF, 8.05 pg/mL, respectively; STC IL-13, 10 pg/mL and 12.5 pg/mL; P < .001), and no breed differences were observed. Mixed immune cell assays revealed an ability of neutrophils to induce IL-4 production in peripheral blood mononuclear cell (PBMC). Taken together, these data implicate neutrophils as a potential effector cell responsible for Th2 initiation.

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.

Impaired interleukin-4 signalling 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.

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.

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.

St. Croix sheep produce a rapid and greater cellular immune response contributing to reduced establishment of Haemonchus contortus.

The objective of this study was to determine breed differences in immune response shortly following Haemonchus contortus infection. Peripheral and local cellular and humoral immune responses were evaluated in 24 St. Croix hair lambs and 24 Dorset×(Finn-Rambouillet) wool lambs at 0, 3, 5 and 7 days after infection with 10,000 L3 H. contortus larvae. Blood samples taken immediately before harvest revealed no differences in circulating effector cell populations, yet there were significant differences in levels of circulating neutrophils. Across all time points, hair lambs had a higher average circulating neutrophil concentration (3018 cells/µl) than wool lambs (1818 cells/µl; P<0.05). Infected hair lambs also had greater serum total-IgA compared to wool lambs (1.8 vs 0.9 mg/ml; P=0.006). Breeds did not differ in eosinophil or globule leukocyte (GL) counts in abomasal tissue, but infiltration of these cell populations increased with time. Globule leukocyte counts peaked at day 3 after infection whereas eosinophil numbers continued to increase to day 7 after infection. When averaged across all time points, abomasal neutrophil counts were higher in hair lambs (831 cells/mm(2)), than wool lambs (561 cells/mm(2); P<0.0001). Total abomasal lymph node (ALN) weight increased exponentially from 2.60 g at day 0 to 6.57 g by day 7 in hair lambs whereas ALN weight only marginally increased in wool lambs and was significantly lower than hair lambs by day 7 (P=0.0003). This result suggests a greater expansion of lymphocytes in the ALN promoting early development of antigen-specific acquired immune responses in hair lambs. Greater IgA production and infiltration of immune cells to the abomasal mucosa at an earlier stage of infection may limit establishment of adult parasites and thereby shorten the duration and severity of infection.