BACKGROUND:Peripheral immune cells participate in the pathogenesis and progression of central nervous system diseases including relapsing-remitting multiple sclerosis (RRMS), which is an immune-mediated demyelinating disorder. The association between IL-6 and RRMS pathogenesis is clear but there is some uncertainty about the role of IL-6 and IL-6 pathway components in blood and the molecular mechanisms through which T regulatorys (Tregs) contribute to MS pathogenesis. The purpose of this study was to identify markers of IL-6 pathways in serum and regulatory CD8+ and CD4+ T cells in the blood of RRMS patients and to analyze their associations with multiple sclerosis, with each other, and with age. METHODS:Peripheral blood was collected from female RRMS patients (16), and healthy controls (18) recruited between December, 2019 and July, 2022. The serum levels of IL-6, TGF-β1, IL-6Rα, IL-6/IL-6Rα complex, and soluble glycoprotein-130 (sgp-130) were measured by ELISA. Flow cytometry was used to quantify the surface expression of IL-6R (CD126), membrane glycoprotein 130 (gp130, CD130), and phospho-STAT3 (pSTAT3) and pSTAT5 in CD4+CD25+FoxP3+, CD8+CD25+FoxP3+, and CD8+CD122+ Tregs. Differences were compared using the Student's t-test or Welch's t-test. Pearson product-moment correlations were used to detect correlations. A p-value ≤ 0.05 was considered statistically significant. RESULTS:The CD8+CD122+ Treg subset in RRMS patients exhibited an increased level of surface CD126 and CD130 associated with classical IL-6R signaling without STAT3 phosphorylation. For CD4+CD25+FoxP3+ and CD8+CD25+FoxP3+ Tregs, no changes in classical IL-6R surface markers were observed in RRMS, but there was an increased percentage of pSTAT3 in these cells. Age-related changes in pSTAT5 expression across Treg subsets in healthy controls were absent in RRMS patients. CONCLUSIONS:Our findings underscore the complex interplay between IL-6 signaling and Tregs as well as age-related immune regulation. The observed alterations in the expression of receptors and in signaling activity may contribute to the dysregulation of CD8+CD122+ Treg function activated via the classic IL-6 pathway and suggests IL-6 trans-signaling in CD25-positive Tregs. RRMS may interfere with normal immune aging patterns, possibly by promoting a sustained inflammatory state that overrides the senescence of Tregs.

Parasitic nematodes produce a variety of molecules that modulate the immune system of their hosts. Over 30 molecules have been identified from more than a dozen nematode species. Some molecules are present in many species; immunomodulation has been demonstrated in some species and is assumed to exist in the remainder. Other immunomodulators appear to exist in only one or a few closely related species. The well studied nematodes produce multiple molecules to modulate the immune response and there is considerable synergy among these molecules. It is not clear why immunomodulation is so complex; possible explanations include more precise control of the host immune response or evasion of host responses against individual molecules.

Pituitary pars intermedia dysfunction (PPID) is the most common endocrine disorder of older horses. Immune dysfunction in horses with PPID could increase susceptibility to infectious diseases, including strongyle infections; however, few data are available. The aim of this study was to determine if horses with PPID had increased strongyle faecal egg counts (FEC) compared with control horses, over a fourteen-week period in Victoria, Australia. Clinical signs and plasma adrenocorticotropic hormone (ACTH) concentrations were used to categorise horses into PPID (n=14) or control (n=31) groups. Faecal samples were collected for FEC determination prior to anthelmintic treatment, and fortnightly post-treatment for each horse. Generalised linear mixed modelling using a gamma distribution was used to compare differences between groups in the repeated measures study. The confounding variable of age was controlled for as a fixed effect. Following anthelmintic treatment, mean FEC was greater for the PPID group compared to the control group on day 56 (405 ± 756 eggs per gram [EPG] vs 40 ± 85 EPG, p=0.05) and day 70 (753 ±1598 EPG vs 82 ±141 EPG, p=0.04). There were no differences in mean FEC between groups on days 84 and 98. Cumulative FEC (day 14 to day 98) was significantly greater for the PPID horses than control horses (2118 ± 4016 EPG vs 798 ± 768 EPG, p<0.0001). Group egg reappearance period was shorter for PPID horses (day 56 post-anthelmintic treatment) compared to control horses (day 70) and 30% of the PPID horses reached a FEC threshold of >200 EPG on day 42, compared to 0% of control horses (p=0.02). These results suggest that the rate of a re-established patent infection between groups could be different due to a comprised immune response in PPID horses or differences in the host-parasite relationship regarding encysted stage larvae. However, despite differences between groups, some horses with PPID consistently had no detectable or low FEC (<200 EPG) during the study period. These findings highlight the importance of individual FEC monitoring to determine if anthelmintic treatment is required in line with sustainable parasite management practices.

The control of parasitic nematode infection in sheep and other animals is threatened by the evolution of drug resistance in parasite populations. One recommendation to delay the onset of drug resistance is to estimate the flock mean faecal egg counts by sampling a subpopulation and to treat sheep only when egg counts are high. However, there is little research on the accuracy and precision of estimates of the flock mean obtained from samples. In silico sampling was used to quantify the influence of flock variation, sample size, flock size and mean egg count on the accuracy and precision of the estimated mean egg count. Commonly used and recommended sampling schemes gave alarmingly imprecise estimates of the true flock means. Simply providing a point estimate of the flock egg count can be seriously misleading. Therefore, quantiles were provided for the proportion of estimates in a plausible scenario that is likely to require treatment. It may be more informative to use these quantiles to predict the probability that the true flock mean is sufficiently high to consider treatment.

Mast cells are essential immune cells involved in the host’s defence against gastrointestinal nematodes. To evade the immune response, parasitic nematodes produce a variety of molecules. Galectin 1, produced by Teladorsagia circumcincta (Tci-gal-1), reduces mast cell degranulation and selectively regulates mediator production and release in an IgE-dependent manner. To uncover the activity of Tci-gal-1, we have examined the effect of the protein on gene expression, protein production, and apoptosis in activated basophilic leukaemia RBL-2H3 cells. Rat RBL-2H3 cells were activated with anti-DNP IgE and DNP-HSA, and then treated with Tci-gal-1. Microarray analysis was used to examine gene expression. The levels of several apoptosis-related molecules and cytokines were determined using antibody arrays and ELISA. Early and late apoptosis was evaluated cytometrically. Degranulation of cells was determined by a β-hexosaminidase release assay. Treatment of activated RBL-2H3 cells with Tci-gal-1 resulted in inhibited apoptosis and decreased degranulation, although we did not detect significant changes in gene expression. The production of pro-apoptotic molecules, receptor for advanced glycation end products (RAGE) and Fas ligand (FasL), and the cytokines IL-9, IL-10, IL-13, TNF-α, and IL-2 was strongly inhibited. Tci-gal-1 modulates apoptosis, degranulation, and production of cytokines by activated RBL-2H3 cells without detectable influence on gene transcription. This parasite protein is crucial for modulation of the protective immune response and the inhibition of chronic inflammation driven by mast cell activity.

Gastrointestinal nematodes (GINs) are a concern for sheep production and are associated with significant economic losses globally, driving the need for effective management strategies. The aims of this research were first to determine the influence of GIN infections on sheep grazing behaviours, specifically grazing time (GT) and grazing event length (GL), and then to estimate the genetic and phenotypic correlation between faecal egg counts (FEC) and salivary specific IgA activity vs GT and GL. Data were collected from 146 Merino sheep on a commercial farm in Victoria, Australia. Faecal, saliva and body weight (BW) were collected at four time points (A: Oct 2019, B: early May 2020, C: late May 2020, and D: Jul 2020), while tri-axial accelerometer sensors recorded GT and GL for the length of 26 days between B and C. Univariate linear regression and Bayesian Generalised Linear Mixed Model, estimated using Markov Chain Monte Carlo, were used to assess the relationships between the behaviours and infection indicators. We found an increase in the logarithm of FEC was associated with a significant rise in grazing time (p < 0.05), suggesting compensatory behaviour in infected sheep. GT also accounted for 6% of the FEC variation. In contrast, the effect of IgA on GT was not significant (p > 0.05). There was a positive genetic and phenotypic correlation estimated between GT and FEC, however, the High Posterior Density interval for the genetic and phenotypic correlations between GT and FEC indicates some uncertainty in these estimates. These results imply that sheep may modify their grazing strategies as an adaptive response to parasitic infections to ease the impact on their nutritional intake. This study improves our understanding of how natural GIN infections affect sheep behaviour and highlights the potential to use animal behaviours as a precision livestock farming tool and in breeding for resilience against parasites.

Nematode infection is a major threat to the health of humans, domestic animals and wildlife. Nematodes vary in their effect on the host and in the mechanisms underlying immunity but the general features are becoming clear. There is considerable variation among individuals in resistance to infection and much of this variation is due to genetic variation in the immune response. The major histocompatibility complex has a strong influence on resistance to infection but other genes are collectively more important. Resistant individuals produce more IgA, eosinophils, IgE and mast cells than susceptible individuals and this is a consequence of stronger type 2 (Th2) immune responses. A variety of factors promote Th2 responses including genetic background, diet, molecules produced by the parasite and the location of the infection. A variety of cells and molecules including proteins, glycolipids and RNA act in concert to promote responses and to regulate the response. Nematodes themselves also modulate the host response and over 20 parasite-derived immunomodulatory molecules have been identified. Different species of nematodes modulate the immune response in different ways and probably use multiple molecules. The reasons for this are unclear and the interactions among immunomodulators have still to be investigated.

Parasitic nematodes and their products are promising candidates for therapeutics against inflammatory bowel diseases (IBD). Two species of nematodes, the hookworm Necator americanus and the whipworm Trichuis suis, are being used in clinical treatment trials of IBD referred to as "helminth therapy". Heligmosomoides polygyrus is a well-known model for human hookworm infections. Excretory-secretory (ES) products of H. polygyrus L4 stage that developed during colitis show a different immunomodulatory effect compared to the ES of H. polgyrus from healthy mice. The aim of the study was to evaluate excretory-secretory proteins produced by H. polygyrus L4 stage males and females that developed in the colitic milieu. Mass spectrometry was used to identify proteins. Blast2GO was used to investigate the functions of the discovered proteins. A total of 387 proteins were identified in the ES of H. polygyrus L4 males (HpC males), and 330 proteins were identified in the ES of L4 females that developed in the colitic milieu (HpC females). In contrast, only 200 proteins were identified in the ES of L4 males (Hp males) and 218 in the ES of L4 females (Hp females) that developed in control conditions. Most of the proteins (123) were detected in all groups. Unique proteins identified in the ES of HpC females included annexin, lysozyme-2, apyrase, and galectin. Venom allergen/Ancylostoma-secreted protein-like, transthyretin-like family proteins, and galectins were found in the secretome of HpC males but not in the secretome of control males. These molecules may be responsible for the therapeutic effects of nematodes in DSS-induced colitis.

Introduction The automated collection of phenotypic measurements in livestock is of interest to both researchers and farmers. Real-time, low-cost, and accurate phenotyping can enhance precision livestock management and could lead to the optimized utilization of pasture and breeding of efficient animals. Wearable sensors provide the tools for researchers to develop novel phenotypes across all production systems, which is especially valuable for grazing conditions. The objectives of this study were to estimate the repeatability and heritability of traits related to grazing and rumination activities and their correlations with other traits. Methods This study was conducted on a commercial Merino farm in the west of Victoria, Australia, from 4 May 2020 to 29 May 2020. A total of 160 ActiGraph sensors embedded in halters were attached to the left side of the muzzles of Merino sheep (M = 74, F = 86) aged 10–11 months while the sheep were grazing on pasture. Support vector machine (SVM) algorithms classified the sensor output into the categories of grazing, rumination, walking, idle, and other activities. These activities were further classified into daily grazing time (GT), number of grazing events (NGE), grazing length (GL), rumination time (RT), number of rumination events (NRE), rumination length (RL), walking time (WT), and idle time (IT). The data were analyzed using univariate and bivariate models in ASReml-SA to estimate the repeatability, heritability, and phenotypic correlations among traits. Results The heritability of GT was estimated to be 0.44 ± 0.23, whereas the other traits had heritability estimates close to zero. The estimated repeatability for all traits was moderate to high, with the highest estimate being for GT (0.70 ± 0.03) and the lowest for RT (0.44 ± 0.03). The intraclass correlation or repeatability at a 1-day interval (i.e., 2 consecutive days) was high for all traits, and steadily reduced when the interval between measurements was longer than 1 week. Discussion The estimated repeatability for the grazing traits showed that wearable sensors and SVM methods are reliable methods for recording sheep activities on pasture, and have a potential application in the ranking of animals for selective breeding.

Accumulating data suggest an important role of growth factors in autoimmune diseases and parasitic nematode infections. Nematodes are used in clinical studies of autoimmune diseases and parasite-derived molecules are widely studied for their therapeutic potential in various types of disorders. However, the effect of nematode infection on growth factors in autoimmune disorders has not been studied. The objective of this study was to evaluate the influence of infection with the intestinal nematode Heligmosomoides polygyrus in murine autoimmune models on the production of growth factors. Here, the level of a variety of growth factors related mainly to angiogenesis was evaluated by protein array in the intestinal mucosa of C57BL/6 dextran sodium sulfate-induced colitic mice and in cerebral spinal fluid of experimental autoimmune encephalomyelitis (EAE) mice infected with nematodes. In addition, vessel formation was evaluated in the brains of EAE mice infected with H. polygyrus. A significant influence of nematode infection on the level of angiogenic factors was observed. Parasitic infection of colitic mice resulted in upregulation of mucosal AREG, EGF, FGF-2, and IGFBP-3 in the intestine of the host and better adaptation (infectivity). In EAE mice, infection increased the level of FGF-2 and FGF-7 in CSF. In addition, remodeling of brain vessels was observed, with a higher density of long vessels. Nematode-derived factors are promising tools to fight autoimmune diseases and to study angiogenesis.

Context Monitoring the behaviour of animals can provide early warning signs of disease or indicate loss of appetite. Also, an understanding of the variation in behaviours among animals and their distributions is essential for meaningful statistical inference. Therefore, quantifying the variation of behaviours is of both biological and statistical interest. Aim The objectives of this study were to determine the distributions and quantify the variation among animals with respect to the times spent grazing, ruminating, idling, walking, and licking. Methods The activities of 147 (male = 67, female = 80) Merino lambs at 10–11 months of age on a commercial farm in Edenhope, Victoria, Australia were recorded for 26 days, using ActiGraph accelerometer sensors attached to the left side of the sheep’s muzzle. The male and female sheep were kept in separate paddocks. A Support Vector Machine algorithm was used to differentiate sheep behaviour into six categories: grazing, ruminating, idling, walking, licking, and other activities. The distributions of behaviours were analysed using energy statistics-based tests and Generalised Additive Models for Location, Scale, and Shape (GAMLSS). Different distributions were compared using Akaike Information Criterion (AIC) values. Key results Among the distributions that were considered, we found that times spent ruminating in both male and female sheep populations as well as idling in male sheep were best described by the skew exponential type 2 distribution. Grazing, walking and licking behaviours were best described by the Box–Cox t distribution. The distribution of time spent grazing was symmetrical and unimodal in males, and adequately modelled by a normal distribution, but the distribution in females had a prominent left skew. Also, we found that females typically grazed for a longer time than males. However, males spent more time ruminating than grazing. Conclusions The time spent by the animal in each activity varied during the day. Within each population, the variation among animals in the time spent grazing was best described by a Box–Cox t distribution. Implications This study has enhanced our understanding of grazing behaviour and will facilitate more appropriate analyses of the causes of variation among animals in grazing behaviour.

Galectin-11 (LGALS-11) and galectin-14 (LGALS-14) are ruminant specific galectins, first reported in sheep. Although their roles in parasite immunity are still being elucidated, it appears that they influence protection against parasites. In gastrointestinal infections with the nematode Haemonchus contortus, both galectin-11 and galectin-14 appear to be protective. However, in a chronic infection of liver fluke, Fasciola hepatica, these galectins may aid parasite survival. To unravel the structural, functional, and ligand profile of galectin-11 and galectin-14, recombinant production of these proteins is vital. Here we present the recombinant production of soluble galectin-11 and galectin-14 from domestic sheep for in vitro and structural biology studies. These methods include parasite cultivation and infection, galectin staining of host and parasite tissue, surface staining of parasites with recombinant galectins, pull-down assays to identify endogenous galectin binding proteins, and in vitro assays to monitor the effect of galectins on parasite development.

Altered regulatory T cell (Treg) function could contribute to MS. The expression of activating and inhibitory receptors influences the activity of Tregs. Our aim was to investigate T cell phenotypes in relapsing–remitting MS (RRMS) patients at an early phase of the disease. We examined the influence of demographic parameters on the distribution of CD4+ and CD8+ T cell subclasses by generalized linear modeling. We also studied the expression of the following markers—CTLA-4, GITR, PD-1, FoxP3, Helios, CD28, CD62L, CD103—on T cell subsets from peripheral blood with a 14-color flow cytometry panel. We used an antibody array to define the profiles of 34 Th1/Th2/Th17 cytokines in the serum. Expression of PD-1 and GITR on CD4+ and CD8+ Tregs was decreased in RRMS patients. The proinflammatory factors IFN-γ, IL-17, IL-17F, TGFβ-1, TGFβ-3, IL-1SRII, IL-12 p40, sgp130, IL-6sR were significantly increased in RRMS patients. Therefore, a deficiency of PD-1 and GITR immune checkpoints on CD4+ and CD8+ Tregs is a feature of RRMS and might underlie impaired T cell control.

Mast cell degranulation is the major mechanism influencing establishment and survival of the abomasal nematode Teladorsagia circumcincta and probably many other gastrointestinal nematodes. Host galectins-3 and -9 have been shown to bind IgE and positively and negatively influence mast cell degranulation. As incoming nematodes produce large amounts of galectin, we hypothesised that nematode galectin competes with host galectin and inhibits mast cell degranulation. ELISA was used to show that nematode galectin reduced total IgE activity. Galectin also reduced the binding of sheep IgE to the surface of a mast cell line and decreased the release of LCT-4 and Beta hexosaminidase but not MMP-9. These results indicate that nematode galectin influences mast cell degranulation and identify a potential immunomodulatory mechanism used by nematodes to enhance their establishment and survival.

Historically, there has been little interaction between parasitologists and oncologists, although some helminth infections predispose to the development of tumours. In addition, both parasites and tumours need to survive immune attack. Recent research suggests that both tumours and parasites suppress the immune response to increase their chances of survival. They both co-opt the transforming growth factor beta (TGFβ) signalling pathway to modulate the immune response to their benefit. In particular, there is concern that suppression of the immune response by nematodes and their products could enhance susceptibility to tumours in both natural and artificial infections.

Accelerometer sensors were attached to 147 Merino sheep for 26 days on a commercial farm in Victoria, Australia. Using a Support Vector Machine learning algorithm, 6 behaviours were defined, including grazing and rumination. The overall repeatability of grazing and rumination were 0.44±0.03 and 0.49±0.03, respectively. When repeatability was estimated on 2 consecutive days, it was approximately 0.7 for each trait and by increasing the interval between days (over 22) dropped to below 0.1. The heritability estimates for all grazing behaviour traits were close to zero, which is likely to be associated with the small sample size in our study and weak genetic connections between individuals. Nevertheless, the repeatability results were promising and suggested that animals maintain their ranking at least over short periods of time. These results were preliminary and from a small sample size. Hence, we recommend a larger sample size for estimating heritability of grazing and rumination traits.

Although Nematodirus battus is a serious threat to the health and survival of young lambs, there are few options to control this parasite. Bayesian Monte Carlo Markov Chain modelling with a zero-inflated Poisson distribution was used to estimate the heritability of egg counts in both June and July for each of five consecutive cohorts of 200 Scottish Blackface lambs. In one of the 10 analyses, the results failed the diagnostic tests. In seven of the analyses, there was no convincing evidence that the variation in egg counts was heritable. In the 2 years of high infection, the heritability was approximately 0.4 in June but the estimates lacked precision and the 95% highest posterior density credible intervals ranged from just above zero to 0.7. Selective breeding for resistance to N. battus will be difficult because genetically resistant or susceptible lambs cannot be consistently identified by phenotypic markers.

Context Lamb loss and dyctocia are two major challenges in extensive farming systems. While visual observation can be impractical due to the large sizes of paddocks, number of animals and high labour cost, wearable sensors can be used to monitor the behaviour of ewes as there might be changes in their activities prior to lambing. This provides sufficient time for the farm manager to nurse those ewes that are at risk of dyctocia. Aim The objective of this study was to determine whether the behaviour of a pregnant ewe could predict the time of parturition. Methods Two separate trials were conducted: the first trial (T1), with 32 ewes, included human/video observations, and the second trial (T2), with 165 ewes, conducted with no humans present, to emulate real extensive farming settings. The ewes were fitted with tri-axial accelerometer sensors by means of halters. Three-dimensional movement data were collected for a period of at least 7 and 14 days in T1 and T2 respectively. The sensor units were retrieved, and their data downloaded using ActiGraph software. Ewe behaviour was determined through support vector machine learning (SVM) algorithm, including licking, grazing, rumination, walking, and idling. The behaviours of ewes predicted by analysis of sensor data were compared with behaviours determined using visual observation (video recordings), with time synchronisation to validate the results. Deep learning and neural-network algorithms were used to predict lambing time. Key results The concordance percentages between visual observation and sensor data were 90 ± 11, 81 ± 15, 95 ± 10, 96 ± 6, and 93 ± 8% ± s.d. for grazing, licking, rumination, idling, and walking respectively. The deep-learning model predicted the time of lambing with 90% confidence via a quantile regression method, which can be interpereted as 90% prediction intervals, and shows that the time of lambing can be predicted with reasonable confidence approximately 240 h before the actual lambing events. Conclusion It was possible to predict the time of parturition up to 10 days before lambing. Implications The behaviour of ewes around lambing time has a direct effect on the survival of the lambs and therefore plays an important part in animal management. This knowledge could improve the productivity of sheep and considerably decrease lamb mortality rates.

Galectins are a family of proteins that bind β-galactosides and play key roles in a variety of cellular processes including host defence. They have been well studied in hosts but less so in gastrointestinal nematodes. Both host and parasite galectins are present in the gastrointestinal tract following infection. Parasite galectins can both bind antibody, especially highly glycosylated IgE and be bound by antibody. Parasite galectins may act as molecular sponges that soak up antibody. Host galectins promote mast cell degranulation while parasite galectins inhibit degranulation. Host and parasite galectins can also bind mucins and influence mucus viscosity. As the protective response against gastrointestinal nematode infection is partly dependent on IgE mediated mast cell degranulation and mucus, the interactions between host and parasite galectins play key roles in determining the outcome of infection.

Eosinophils play a key role in defence against gastrointestinal nematodes. There is considerable variation among animals in the intensity of eosinophilia following nematode infection. However, the statistical distribution of eosinophils among animals has still to be determined. A better description of the variation among animals could provide biological insight and determine the most appropriate way to analyse the effect of eosinophils. We estimated blood eosinophil numbers in a flock of Scottish Blackface sheep that were naturally exposed to mixed, predominantly Teladorsagia circumcincta infection. Three of the four eosinophil counts were better described by a gamma distribution than by a lognormal distribution. The scale and shape parameters of the gamma distribution varied over time. Eosinophil counts differed among animals kept on separate fields before weaning and between singletons and twins but were not significantly different between years and genders. Eosinophil counts also differed among offspring from different sires and dams. The parameters of the gamma distribution were used to enable a power analysis. Large numbers of animals were required to reliably detect even large differences between two groups. These results indicate that methods appropriate for gamma distributions, such as generalized linear mixed models, will provide more reliable inferences than traditional methods of analysis and experimental design.