Variants of NLRP3 Protein in Haemonchus contortus Infected Sheep: Impact on Immune Cell Responsiveness to LPS In Vitro.

Pathogen recognition is an essential component to achieve the desired outcome of host protection. Nod-like receptor pyrin containing domain 3 (NLRP3) is a cytoplasmic pattern recognition receptor (PRR) with a wide array of agonists, such as PAMPs, DAMPs, ATP, bacterial product and viral products. Stimulation of the NLRP3 inflammasome results in proteolytic activation of IL-1ß and IL-18, cell pyroptosis and classically, the induction of proinflammatory responses. St. Croix (STC) sheep have resistance traits exhibiting the appropriate T-helper type 2 immune response ensuing protection during helminth parasitic infection whereas parasite-susceptible Suffolk (SUF) sheep have an impaired response resulting in parasite establishment and adverse symptoms. The objective of these experiments was to determine if NLRP3 protein in H. contortus-infected SUF sheep was defective using the classical activation pathway of NLRP3 inflammasome. Peripheral blood mononuclear cells (PBMCs) derived from H. contortus-infected STC and SUF sheep were isolated from whole blood and treated (MCC950 treatment for 2 h followed by LPS treatment for 3 h, 1400 W treatment for 2 h followed by LPS treatment for 3 h, LPS treatment for 3 h or culture media for 3 h). qPCR analysis of LPS-stimulated PBMC revealed an upregulation in inflammatory associated genes IL-1ß, TLR4, TNFα and NFκB (p < 0.0001) in STC PBMC and downregulation in IFNγ, IL-6 and iNOS for SUF PBMC. Pharmacological inhibition of iNOS in SUF PBMC resulted in an upregulation in the expression of IFNγ. These preliminary data begin to discover a relationship between NLRP3 activation and TLR4 signalling in PBMC of STC and SUF sheep.

Determining muscle plasticity and meat quality development of low-input extended fed market-ready steers.

In March 2020, the World Health Organization declared COVID-19 a pandemic, which ultimately led to many meat processors temporarily shutting down or reducing processing capacity. This backlog in processing capacity forced many feedlots to retain cattle for longer periods of time and assume the risk of major market fluctuations. The aim of this study was to understand how a dietary insult affects meat quality and muscle metabolism in market-ready steers (590 kg). Sixteen market-ready (590 kg) commercial Angus crossbred steers were subjected to a maintenance diet of either forage or grain for 60 d. Longissimus lumborum (LL) muscle samples were collected immediately postmortem and processed for characteristics reflecting the underlying muscle fiber type and energy state of the tissue. Despite cattle being subjected to a 60-d feeding period, there were no detectable differences (P > 0.05) in carcass characteristics, color of lean, or ultimate pH (pHu). Moreover, our data show that muscle plasticity is rather resilient, as reflected by lack of significance (P > 0.05) in oxidative and glycolytic enzymes, myosin heavy chain isoforms (MyHC), myoglobin, and mitochondrial DNA (mtDNA) contents. These data show that market-ready steers are capable of withstanding a low-input feeding strategy up to 60 d without dramatically impacting underlying muscle characteristics and meat quality development.

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.

Evaluating the relationship between fecal egg count, FAMACHA score, and weight in dewormed and non-dewormed Katahdin rams during a parasite challenge.

The objective of this study was to evaluate and to estimate the relationship between fecal egg counts (FECs) and FAMACHA score and the body weight of growing Katahdin rams during a parasite challenge. One of the largest factors negatively influencing reproduction and economics in the sheep industry is gastrointestinal nematode (GIN) parasites. Due to anthelmintic resistance of these parasites, animals are selected for parasite resistance using FEC and FAMACHA scores. Data were used from the Virginia Tech Southwest Agricultural Research and Extension Center Ram Test in Glade Spring, VA, from the year 2012 to 2018 in which animals were tested in 14-d intervals for 70 d. Mixed models for repeated weight measurements were made from backward stepwise selection to evaluate the relationships between weight and GIN FEC. A total of 576 animals within 23 contemporary groups derived from test year and pasture group were analyzed. Ram, contemporary group, and consignor were considered random effects, and fixed effects were birth type, test day, age, age squared, starting weight, FEC, and FAMACHA score. Pairwise contrasts were used in the statistical analysis of parameters and their interactions. Weight and age were found to have a quadratic relationship. Increased FEC was associated with weight loss at a rate of 0.00030 kg/FEC (P < 0.0001). Animals dewormed at any point during the trial weighed less than those that were not and increased with test day to a maximum difference of 4.66 kg (P < 0.001). FAMACHA score was found to be significant (P < 0.05), but a direct relationship with weight was not conclusive. Overall, rams with severe enough parasite load to require deworming had lesser weights, which could impact the profitability of sheep production and reinforced the need to select animals that had greater innate parasite resistance.

Lamb performance in hardwood silvopastures, II: animal behavior in summer.

Integrating trees into pastures, a practice known as silvopasture, may benefit livestock in the summertime through the provision of shade. The purpose of this project was to compare the behavioral patterns of sheep grazing in silvopastures and open pastures. Black walnut (Juglans nigra L.) and honeylocust (Gleditisia triacanothose L.) based silvopasture systems were compared with open pastures in a randomized complete block design with three blocks over two summers. Behavior measures were recorded within a replicate within a week, and these measures were taken sequentially within three experimental periods. Ewe lambs (n = 3) within each experimental unit were equipped with a wideband audio-recording device to detect prehension events. Time-lapse cameras documented sheep behavior every 60 s. In the silvopastures, the lambs spent over 90% of daylight hours within shade from trees. Lambs in silvopastures spent more time lying down than animals in the open pastures (P ≤ 0.01), while lambs in the open pastures spent more than 2 h longer each day standing (P < 0.0001). Lambs in the black walnut silvopastures spent more time grazing (488 ± 14 min · d-1) than lambs in the honeylocust silvopastures (438 ± 14 min · d-1; P = 0.0493) and lambs in the open pastures (417 ± 14 min · d-1; P = 0.0026). There was no difference in grazing time for lambs in the latter two systems (P = 0.5597). Spectral analysis of the imagery revealed that the lambs in the black walnut silvopastures grazed more frequently than the lambs in the other systems for both years. The acoustic analysis, though limited by recorder durability to 47 complete recordings, revealed no difference in total bites taken per day (P ≥ 0.7222) or in the morning (P ≥ 0.2069), afternoon (P ≥ 0.5816), and evening periods (P ≥ 0.9337). Silvopastures provide an opportunity to improve lamb comfort in the summer.

Lamb performance in hardwood silvopastures, I: animal gains and forage measures in summer.

The integration of trees into pasture systems can have variable effects on forage and animal growth. Some reports of these systems have indicated that animal gains are similar or better even when tree presence lowers forage yield. Forage production and animal performance were compared in black walnut (Juglans nigra L.)-based and honeylocust (Gleditisia triacanthose L.)-based silvopasture systems and open pastures in a randomized complete block design with three blocks over three summers. Cool season-based, mixed grass pastures were rotationally stocked with four to seven lambs depending on available forage. A rising plate meter was used to estimate pre- and post-graze forage mass. Forage samples of the mixed sward were collected and analyzed for nitrogen (N) and neutral detergent fiber (NDF) concentrations. Species percent cover was estimated using a modified Daubenmire approach at the same 12 points within each experimental unit every 4 wk during the study. Pre-graze herbage mass was similar (P = 0.0717) in honeylocust silvopastures (5020 ± 30 kg·ha-1) and open pastures (4930 ± 30 kg·ha-1) and lowest (P < 0.0001) in the black walnut silvopastures (3560 ± 30 kg·ha-1). Forages in the black walnut and honeylocust silvopastures had similar (P = 0.4867) N concentrations (23.3 ± 0.4 and 23.9 ± 0.4 g·kg-1, respectively), which was greater (P ≤ 0.0003) than that of the forages in the open pastures (21.0 ± 0.4 g·kg-1). Forages in the honeylocust silvopasture had lower (P ≤ 0.0042) NDF concentrations (507 ± 3 g·kg-1) than forages in the black walnut silvopasture and open pastures (mean = 525 ± 3 g·kg-1). Forage species present in the black walnut silvopastures differed from those present in the open and honeylocust systems, which had similar composition. Despite differences in stocking rates, total lamb weight gains per system did not differ (P ≥ 0.7592) among black walnut, honeylocust, and open pasture systems (10 ± 2, 12 ± 2, and 10 ± 2 kg·d-1, respectively). Silvopasture practices can improve land productivity when incorporated into cool season forage pastures.

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.

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.