Comparative evaluation of different molecular methods for DNA extraction from individual Teladorsagia circumcincta nematodes.

BACKGROUND: The purpose of this study was to develop a reliable DNA extraction protocol to use on individual Teladorsagia circumcincta nematode specimens to produce high quality DNA for genome sequencing and phylogenetic analysis. Pooled samples have been critical in providing the groundwork for T. circumcincta genome construction, but there is currently no standard method for extracting high-quality DNA from individual nematodes. 11 extraction kits were compared based on DNA quality, yield, and processing time. RESULTS: 11 extraction protocols were compared, and the concentration and purity of the extracted DNA was quantified. Median DNA concentration among all methods measured on NanoDrop 2000™ ranged between 0.45-11.5 ng/µL, and on Qubit™ ranged between undetectable - 0.962 ng/µL. Median A260/280 ranged between 0.505-3.925, and median A260/230 ranged - 0.005 - 1.545. Larval exsheathment to remove the nematode cuticle negatively impacted DNA concentration and purity. CONCLUSIONS: A Schistosoma sp. DNA extraction method was determined as most suitable for individual T. circumcincta nematode specimens due to its resulting DNA concentration, purity, and relatively fast processing time.

Immunoglobulins as Biomarkers for Gastrointestinal Nematodes Resistance in Small Ruminants: A systematic review.

The rise of anthelmintic resistance worldwide has led to the development of alternative control strategies for gastrointestinal nematodes (GIN) infections, which are one of the main constraints on the health of grazing small ruminants. Presently, breeding schemes rely mainly on fecal egg count (FEC) measurements on infected animals which are time-consuming and requires expertise in parasitology. Identifying and understanding the role of immunoglobulins in the mechanisms of resistance could provide a more efficient and sustainable method of identifying nematode-resistant animals for selection. In this study we review the findings on immunoglobulin response to GIN in the literature published to date (june 2019) and discuss the potential to use immunoglobulins as biomarkers. The literature review revealed 41 studies which measured at least one immunoglobulin: 35 focused on lamb immune response (18 used non-naïve lambs) and 7 on yearlings. In this review we propose a conceptual model summarizing the role of immunoglobulins in resistance to GIN. We highlight the need for more carefully designed and documented studies to allow comparisons across different populations on the immunoglobulin response to GIN infection.

Bioinformatic analysis of eosinophil activity and its implications for model and target species.

Eosinophils are important immune cells that have been implicated in resistance to gastrointestinal nematode (GIN) infections in both naturally and experimentally infected sheep. Proteins of particular importance appear to be IgA-Fc alpha receptor (FcαRI), C-C chemokine receptor type 3 (CCR3), proteoglycan 3 (PRG3, major basic protein 2) and EPX (eosinophil peroxidase). We used known human nucleotide sequences to search the ruminant genomes, followed by translation to protein and sequence alignments to visualize differences between sequences and species. Where a sequence was retrieved for cow, but not for sheep and goat, this was used additionally as a reference sequence. In this review, we show that eosinophil function varies among host species. Consequently, investigations into the mechanisms of ruminant immune responses to GIN should be conducted using the natural host. Specifically, we address differences in protein sequence and structure for eosinophil proteins.

Analysis of pooled genome sequences from Djallonke and Sahelian sheep of Ghana reveals co-localisation of regions of reduced heterozygosity with candidate genes for disease resistance and adaptation to a tropical environment.

BACKGROUND: The Djallonke sheep is well adapted to harsh environmental conditions, and is relatively resistant to Haemonchosis and resilient to animal trypanosomiasis. The larger Sahelian sheep, which cohabit the same region, is less well adapted to these disease challenges. Haemonchosis and Trypanosomiasis collectively cost the worldwide animal industry billions of dollars in production losses annually. RESULTS: Here, we separately sequenced and then pooled according to breed the genomes from five unrelated individuals from each of the Djallonke and Sahelian sheep breeds (sourced from Ghana), at greater than 22-fold combined coverage for each breed. A total of approximately 404 million (97%) and 343 million (97%) sequence reads from the Djallonke and Sahelian breeds respectively, were successfully mapped to the sheep reference genome Oar v3.1. We identified approximately 11.1 million and 10.9 million single nucleotide polymorphisms (SNPs) in the Djallonke and Sahelian breeds, with approximately 15 and 16% respectively of these not previously reported in sheep. Multiple regions of reduced heterozygosity were also found; 70 co-localised within genomic regions harbouring genes that mediate disease resistance, immune response and adaptation in sheep or cattle. Thirty- three of the regions of reduced heterozygosity co-localised with previously reported genes for resistance to haemonchosis and trypanosomiasis. CONCLUSIONS: Our analyses suggest that these regions of reduced heterozygosity may be signatures of selection for these economically important diseases.

Targeted anthelmintic treatment of parasitic gastroenteritis in first grazing season dairy calves using daily live weight gain as an indicator.

Control of parasitic gastroenteritis in cattle is typically based on group treatments with anthelmintics, complemented by grazing management, where feasible. However, the almost inevitable evolution of resistance in parasitic nematodes to anthelmintics over time necessitates a reappraisal of their use in order to reduce selection pressure. One such approach is targeted selective treatment (TST), in which only individual animals that will most benefit are treated, rather than whole groups of at-risk cattle. This study was designed to assess the feasibility of implementing TST on three commercial farms, two of which were organic. A total of 104 first-grazing season (FGS), weaned dairy calves were enrolled in the study; each was weighed at monthly intervals from the start of the grazing season using scales or weigh-bands. At the same time dung and blood samples were collected in order to measure faecal egg counts (FEC) and plasma pepsinogen, respectively. A pre-determined threshhold weight gain of 0.75kg/day was used to determine those animals that would be treated; the anthelmintic used was eprinomectin. No individual animal received more than one treatment during the grazing season and all treatments were given in July or August; five animals were not treated at all because their growth rates consistently exceeded the threshold. Mean daily live weight gain over the entire grazing season ranged between 0.69 and 0.82kg/day on the three farms. Neither FEC nor pepsinogen values were significantly associated with live weight gain. Implementation of TST at farm level requires regular (monthly) handling of the animals and the use of weigh scales or tape, but can be integrated into farm management practices. This study has shown that acceptable growth rates can be achieved in FGS cattle with modest levels of treatment and correspondingly less exposure of their nematode populations to anthelmintics, which should mitigate selection pressure for resistance by increasing the size of the refugia in both hosts and pasture.

Combatting African Animal Trypanosomiasis (AAT) in livestock: The potential role of trypanotolerance.

African Animal Trypanosomiasis (AAT) is endemic in at least 37 of the 54 countries in Africa. It is estimated to cause direct and indirect losses to the livestock production industry in excess of US$ 4.5 billion per annum. A century of intervention has yielded limited success, owing largely to the extraordinary complexity of the host-parasite interaction. Trypanotolerance, which refers to the inherent ability of some African livestock breeds, notably Djallonke sheep, N'Dama cattle and West African Dwarf goats, to withstand a trypanosomiasis challenge and still remain productive without any form of therapy, is an economically sustainable option for combatting this disease. Yet trypanotolerance has not been adequately exploited in the fight against AAT. In this review, we describe new insights into the genetic basis of trypanotolerance and discuss the potential of exploring this phenomenon as an integral part of the solution for AAT, particularly, in the context of African animal production systems.

Implementation of an extended ZINB model in the study of low levels of natural gastrointestinal nematode infections in adult sheep.

BACKGROUND: In this study, two traits related with resistance to gastrointestinal nematodes (GIN) were measured in 529 adult sheep: faecal egg count (FEC) and activity of immunoglobulin A in plasma (IgA). In dry years, FEC can be very low in semi-extensive systems, such as the one studied here, which makes identifying animals that are resistant or susceptible to infection a difficult task. A zero inflated negative binomial model (ZINB) model was used to calculate the extent of zero inflation for FEC; the model was extended to include information from the IgA responses. RESULTS: In this dataset, 64% of animals had zero FEC while the ZINB model suggested that 38% of sheep had not been recently infected with GIN. Therefore 26% of sheep were predicted to be infected animals with egg counts that were zero or below the detection limit and likely to be relatively resistant to nematode infection. IgA activities of all animals were then used to decide which of the sheep with zero egg counts had been exposed and which sheep had not been recently exposed. Animals with zero FEC and high IgA activity were considered resistant while animals with zero FEC and low IgA activity were considered as not recently infected. For the animals considered as exposed to the infection, the correlations among the studied traits were estimated, and the influence of these traits on the discrimination between unexposed and infected animals was assessed. CONCLUSIONS: The model presented here improved the detection of infected animals with zero FEC. The correlations calculated here will be useful in the development of a reliable index of GIN resistance that could be of assistance for the study of host resistance in studies based on natural infection, especially in adult sheep, and also the design of breeding programs aimed at increasing resistance to parasites.

Potential role for mucosal IgA in modulating Haemonchus contortus adult worm infection in sheep.

Haemonchus contortus (H. contortus) is a haematophagous parasite which causes important economic losses in small ruminants. On the island of Gran Canaria, two sheep breeds coexist which differ in their susceptibility to the infection with H. contortus; the resistant Canaria Hair Breed (CHB) sheep and the susceptible Canaria Sheep (CS) breed. The major target of resistance mechanisms in CHB sheep are directed to the adult parasite stage, reducing the worm burden, and decreased length and fecundity of surviving worms. Mucosal IgA (mIgA) has been shown to be an important regulator of immunity in Haemonchus and Teladorsagia infections; through correlations with larval stages where such mechanisms as antibody-dependent cell cytotoxicity and enzyme inhibition may mediate resistance. Here for the first time, we demonstrate a significant negative correlation between mIgA and adult worm length and fecundity only in the resistant CHB sheep. In contrast, and as reported in other sheep breeds, mIgA was only negatively correlated against the larval stage in the more susceptible CS breed. This study suggests mIgA may play a role in resistance to both larval and adult stages.

The host immune response to gastrointestinal nematode infection in sheep.

Gastrointestinal nematode infection represents a major threat to the health, welfare and productivity of sheep populations worldwide. Infected lambs have a reduced ability to absorb nutrients from the gastrointestinal tract, resulting in morbidity and occasional mortality. The current chemo-dominant approach to nematode control is considered unsustainable due to the increasing incidence of anthelmintic resistance. In addition, there is growing consumer demand for food products from animals not subjected to chemical treatment. Future mechanisms of nematode control must rely on alternative, sustainable strategies such as vaccination or selective breeding of resistant animals. Such strategies take advantage of the host's natural immune response to nematodes. The ability to resist gastrointestinal nematode infection is considered to be dependent on the development of a protective acquired immune response, although the precise immune mechanisms involved in initiating this process remain to be fully elucidated. In this study, current knowledge on the innate and acquired host immune response to gastrointestinal nematode infection in sheep and the development of immunity is reviewed.

A comprehensive mapping of the structure and gene organisation in the sheep MHC class I region.

BACKGROUND: The major histocompatibility complex (MHC) is a chromosomal region that regulates immune responsiveness in vertebrates. This region is one of the most important for disease resistance because it has been associated with resistance or susceptibility to a wide variety of diseases and because the MHC often accounts for more of the variance than other loci. Selective breeding for disease resistance is becoming increasingly common in livestock industries, and it is important to determine how this will influence MHC polymorphism and resistance to diseases that are not targeted for selection. However, in sheep the order and sequence of the protein coding genes is controversial. Yet this information is needed to determine precisely how the MHC influences resistance and susceptibility to disease. METHODS: CHORI bacterial artificial chromosomes (BACs) known to contain sequences from the sheep MHC class I region were sub-cloned, and the clones partially sequenced. The resulting sequences were analysed and re-assembled to identify gene content and organisation within each BAC. The low resolution MHC class I physical map was then compared to the cattle reference genome, the Chinese Merino sheep MHC map published by Gao, et al. (2010) and the recently available sheep reference genome. RESULTS: Immune related class I genes are clustered into 3 blocks; beta, kappa and a novel block not previously identified in other organisms. The revised map is more similar to Bovidae maps than the previous sheep maps and also includes several genes previously not annotated in the Chinese Merino BAC assembly and others not currently annotated in the sheep reference chromosome 20. In particular, the organisation of nonclassical MHC class I genes is similar to that present in the cattle MHC. Sequence analysis and prediction of amino acid sequences of MHC class I classical and nonclassical genes was performed and it was observed that the map contained one classical and eight nonclassical genes together with three possible pseudogenes. CONCLUSIONS: The comprehensive physical map of the sheep MHC class I region enhances our understanding of the genetic architecture of the class I MHC region in sheep and will facilitate future studies of MHC function.

Efficacy of treatment of cattle for liver fluke at housing: influence of differences in flukicidal activity against juvenile Fasciola hepatica.

Flukicides are commonly administered at housing to cattle that have grazed fluke-infected pastures or that have been purchased from endemic areas. The choice of product is determined by numerous factors, one of which is the stages of Fasciola hepatica that are killed. Flukicides can be categorised into three main groups: (A) those that kill all juvenile stages and adults; (B) those that kill juveniles from six to eight weeks of age and adults and (C) those that kill adults only. This study was conducted on a commercial beef farm in Scotland and was designed to compare the efficacy of flukicides from each of these three classes in terms of their effects on faecal egg output, coproantigen and liveweight gain. The majority of animals in the untreated control group were positive for coproantigen, fluke eggs or both throughout the study duration of 16 weeks. Egg reappearance interval following housing treatment was eight weeks for clorsulon and 13 weeks for nitroxynil, though patent infections in both groups developed in only a small minority of animals; no fluke eggs were recovered from cattle treated with triclabendazole. Coproantigen was detected four weeks before the reappearance of fluke eggs in the dung. Animals treated with flukicides had significantly fewer faecal samples positive for eggs (P<0.006) and coproantigen (P<0.05) following treatment compared with the controls. Despite differences in the efficacy profiles among the flukicide-treated groups, there were no significant differences (P>0.05) in growth rates among any of the four treatment groups. There was, however, a significant negative association (P<0.001) between fluke positivity at housing and subsequent growth performance, irrespective of treatment group.

Differences between female and castrated male lambs in susceptibility to natural, predominantly Teladorsagia circumcincta infection.

The difference between castrated male lambs and entire female lambs in susceptibility to gastrointestinal nematode infection was investigated in a flock of Scottish Blackface sheep. Castrated male lambs had higher faecal egg counts, higher body weights, lower parasite-specific plasma IgA activity and more adult nematodes. There were no detectable differences in the length of adult Teladorsagia circumcincta. Heavier egg shedding by males was associated with greater worm burdens. The difference in plasma IgA activity may have been a consequence of larger numbers of adult nematodes inhibiting the transfer of mucosal IgA to the plasma. At least part of the sex difference in egg production could be a consequence of males ingesting larger amounts of grass and hence numbers of infective larvae because of their larger size. The differences between castrated males and females contribute to the observed variation in nematode egg production in naturally infected lambs.

Genome-wide association and regional heritability mapping to identify loci underlying variation in nematode resistance and body weight in Scottish Blackface lambs.

The genetic architecture underlying nematode resistance and body weight in Blackface lambs was evaluated comparing genome-wide association (GWA) and regional heritability mapping (RHM) approaches. The traits analysed were faecal egg count (FEC) and immunoglobulin A activity against third-stage larvae from Teladorsagia circumcincta, as indicators of nematode resistance, and body weight in a population of 752 Scottish Blackface lambs, genotyped with the 50k single-nucleotide polymorphism (SNP) chip. FEC for both Nematodirus and Strongyles nematodes (excluding Nematodirus), as well as body weight were collected at approximately 16, 20 and 24 weeks of age. In addition, a weighted average animal effect was estimated for both FEC and body weight traits. After quality control, 44 388 SNPs were available for the GWA analysis and 42 841 for the RHM, which utilises only mapped SNPs. The same fixed effects were used in both analyses: sex, year, management group, litter size and age of dam, with day of birth as covariate. Some genomic regions of interest for both nematode resistance and body weight traits were identified, using both GWA and RHM approaches. For both methods, strong evidence for association was found on chromosome 14 for Nematodirus average animal effect, chromosome 6 for Strongyles FEC at 16 weeks and chromosome 6 for body weight at 16 weeks. Across the entire data set, RHM identified more regions reaching the suggestive level than GWA, suggesting that RHM is capable of capturing some of the variation not detected by GWA analyses.

Conserved haplotype blocks within the sheep MHC and low SNP heterozygosity in the Class IIa subregion.

This report describes single-nucleotide polymorphisms (SNPs) in the sheep major histocompatibility complex (MHC) class II and class III regions and provides insights into the internal structure of this important genomic complex. MHC haplotypes were deduced from sheep family trios based on genotypes from 20 novel SNPs representative of the class II region and 10 previously described SNPs spanning the class III region. All 30 SNPs exhibited Hardy-Weinberg proportions in the sheep population studied. Recombination within an extended sire haplotype was observed within the class II region for 4 of 20 sheep chromosomes, thereby supporting the presence of separated IIa and IIb subregions similar to those present in cattle. SNP heterozygosity varied across the class II and III regions. One segment of the class IIa subregion manifested very low heterozygosity for several SNPs spanning approximately 120 Kbp. This feature corresponds to a subregion within the human MHC class II region previously described as a 'SNP desert' because of its paucity of SNPs. Linkage disequilibrium (LD) was reduced at the junction separating the putative class IIb and IIa subregions and also between the class IIa and the class III subregions. The latter observation is consistent with either an unmapped physical separation at this location or more likely a boundary characterized by more frequent recombination between two conserved subregions, each manifesting high within-block LD. These results identify internal blocks of loci in the sheep MHC, within which recombination is relatively rare.

Selective forces shaping diversity in the class I region of the major histocompatibility complex in dairy cattle.

The major histocompatibility complex (MHC) is one of the most diverse regions of the mammalian genome. Diversity in MHC genes is integral to their function in the immune system, and while pathogens play a key role in shaping this diversity, the contribution of other selective forces remains unclear. The controlled breeding of cattle offers an excellent model for the identification and exploration of these forces. We characterized the MHC class I genes present in a sample of Canadian Holstein A.I. bulls and compared the results with those obtained in an earlier study. No evidence for a reduction in MHC diversity over 20 years was observed, but the relative frequency of some haplotypes had changed: the formerly rare A12 (w12B) haplotype had become the most common, together with A15, while A19, which dominated the earlier sample, had significantly reduced in frequency. Only 7% of bulls in the current study were MHC homozygous compared with the 14% expected under Hardy-Weinberg. To identify the selective forces at work, a gene substitution model was used to calculate the effects of MHC on selection traits using estimated breeding values for each bull. Significant associations between MHC and production, disease and fertility traits were identified, suggesting that MHC diversity is not merely shaped by disease in this controlled breeding system. The decrease in a common haplotype, the reduced number of homozygous bulls and the associations with disease and production traits together indicate that MHC diversity in dairy cattle is maintained by heterozygote advantage.

The influence of MHC and immunoglobulins a and e on host resistance to gastrointestinal nematodes in sheep.

Gastrointestinal nematode parasites in farmed animals are of particular importance due to their effects on production. In Australia, it is estimated that the direct and indirect effects of parasite infestation cost the animal production industries hundreds of millions of dollars each year. The main factors considered by immunologists when studying gastrointestinal nematode infections are the effects the host's response has on the parasite, which immunological components are responsible for these effects, genetic factors involved in controlling immunological responses, and the interactions between these forming an interconnecting multilevel relationship. In this paper, we describe the roles of immunoglobulins, in particular IgA and IgE, and the major histocompatibility complex in resistance to gastrointestinal parasites in sheep. We also draw evidence from other animal models to support the involvement of these immune components. Finally, we examine how IgA and IgE exert their influence and how methods may be developed to manage susceptible animals.

A single nomenclature and associated database for alleles at the major histocompatibility complex class II DRB1 locus of sheep.

The development of standardised nomenclatures with associated databases containing reference sequences for alleles at polymorphic loci within the major histocompatibility complex (MHC) has been facilitated by the development of the immuno polymorphism database (IPD). Recently, included within IPD-MHC is information on allelic diversity within sheep species (IPD-MHC-OLA). Here, we present the first report of progress in populating the sheep IPD-MHC database with alleles at the class II MHC DRB1 locus. The sequence of 63 Ovar-DRB1 alleles within 24 allelic families is now held within the database, each meeting the minimum requirement of a complete second exon. These sequences are derived from a combination of genomic and cDNA-based approaches and represent the most extensive collection of validated alleles at the sheep DRB1 locus yet described. Although these 63 alleles probably represent only a fraction of the DRB1 allelic diversity in sheep species worldwide, we encourage the research community to use the official allelic nomenclature and to contribute allelic sequences to the database via its web-based submission tool. In time, the IPD-MHC-OLA resource will underpin population-based MHC genotyping studies and help to simplify meta-analyses of multi-source data from wild and domestic sheep populations.

A mechanistic model of developing immunity to Teladorsagia circumcincta infection in lambs.

Acquired immunity influences the severity of parasitic disease, but modelling the effects of acquired immunity in helminth infections has proved challenging. This may be due to a lack of suitable immunological data, or to the perceived complexity of modelling the immune response. We have developed a model of T. circumcincta infection in domestic sheep that incorporates the effects of acquired immunity on parasite establishment and fecundity. A large data set from commercially managed populations of Scottish Blackface sheep was used, which included relationships between IgA activity and worm length, and between worm length and fecundity. Use was also made of a recently published meta-analysis of parasite establishment rates. This realistic but simple model of nematode infection emulates observed patterns of faecal egg counts. The end-of-season faecal egg counts are remarkably robust to perturbations in the majority of the parameters, possibly because of priming of the immune system early in the season, reducing parasite establishment and growth and, therefore, faecal egg counts. Lowering the amount of early infection leads to higher end-of-season egg counts. The periparturient rise in egg counts in ewes appears to have an important role in supplying infection for the priming of the immune response. This feedback in the immune priming suggests that nematode infections may be difficult to eliminate.

An evolutionary perspective on gastrointestinal nematodes of sheep.

The purpose of this paper was to discuss from an evolutionary perspective the interaction between domestic sheep (Ovis aries) and their gastrointestinal nematodes. Although evolution is the central theme of biology, there has been little attempt to consider how evolutionary forces have shaped and continue to shape the relationships between domestic animals and their parasite community. Mathematical modelling of the host-parasite relationship indicated that the system is remarkably robust to perturbations in its parameters. This robustness may be a consequence of the long coevolution of host and parasites. Although nematodes can potentially evolve faster than the host, coevolution is not dominated by the parasite and there are several examples where breeds of cattle or sheep have evolved high levels of resistance to disease. Coevolution is a more equal partnership between host and nematode than is commonly assumed. Coevolution between parasites and the host immune system is often described as an arms race where both host immune response genes and parasite proteins evolve rapidly in response to each other. However, initial results indicate that nematode antigens are not evolving rapidly; the arms race between the immune system and nematodes, if it exists, is happening very slowly. Fisher's fundamental theorem of natural selection states that genes with positive effects on fitness will be fixed by natural selection. Consequently, heritable variation in fitness traits is expected to be low. Contrary to this argument, there is considerable genetic variation in resistance to nematode infection. In particular, the heritabilities of nematode-specific IgA and IgE activity are moderate to high. The reasons for this apparent violation of the fundamental theorem of natural selection are not clear but several possible explanations are explored. Faecal nematode egg counts increase at the beginning of the grazing season - a phenomenon known as the periparturient rise. This increase benefits host and parasite and appears to be a consequence of coevolution. In conclusion, an evolutionary perspective can shed light on many aspects of the host-parasite relationship in domestic animals.

Genetic variation among lambs in peripheral IgE activity against the larval stages of Teladorsagia circumcincta.

IgA and IgE activity against Teladorsagia circumcincta was investigated in a flock of Texel lambs following natural, mixed nematode infection among lambs. The distribution of IgA activity was similar to a gamma distribution whereas IgE activity was different. Box-Cox analysis demonstrated that X0.25 was a suitable transformation to normalise IgE responses. The transformed IgE activity was under moderate to strong genetic control. Nine different allergens were identified by proteomic analysis. Tropomyosin was selected for further analysis. IgE activity against tropomyosin was moderately heritable and associated with decreased egg counts and with reduced body weight at the time of sampling.