Genetic profile of adaptive immune traits and relationships with parasite resistance and productivity in Scottish Blackface sheep.

Gastrointestinal (GI) parasites cause significant production losses in grazing ruminants which can be mitigated by breeding animals resistant to disease. Lymphocyte cytokine production and parasite-specific Immunoglobulin A (IgA) are adaptive immune traits associated with immunity to GI parasites. To explore the utility of these traits for selective breeding purposes, this study estimated the genetic parameters of the immune traits in sheep and assessed their relationship with disease and productivity traits. Whole blood stimulation assays were performed on 1 040 Scottish Blackface lambs at two months of age in 2016-2017. Blood was stimulated with either pokeweed mitogen (PWM), a non-specific activator of lymphocytes, and Teladorsagia circumcincta (T-ci) larval antigen to activate parasite-specific T lymphocytes. The type of adaptive immune response was determined by quantifying production of cytokines interferon-gamma (IFN-γ), interleukin (IL)-4, and IL-10, which relate to T-helper type (Th) 1, Th2 and regulatory T cell responses, respectively. Serum T-ci specific IgA was also quantified. Heritabilities were estimated for each immune trait by univariate analyses. Genetic and phenotypic correlations were estimated between different immune traits, and between immune traits vs. disease and productivity traits that were recorded at three months of age. Disease phenotypes were expressed as faecal egg counts (FEC) of nematode parasites (Strongyles and Nematodirus), faecal oocyst counts (FOC) of coccidian parasites, and faecal soiling score; production was measured as lamb live weight. Significant genetic variation was observed in all immune response traits. Heritabilities of cytokine production varied from low (0.14 ± 0.06) to very high (0.77 ± 0.09) and were always significantly greater than zero (P < 0.05). IgA heritability was found to be moderate (0.41 ± 0.09). Negative associations previously identified between IFN-γ production and FOC, and IL-4 production and strongyle FEC, were not evident in this study, potentially due to the time-lag between immune and parasitology measures. Instead, a positive genetic correlation was found between FOC and PWM-induced IFN-γ production, while a negative genetic correlation was found between FOC and T-ci induced IL-10. Live weight was negatively genetically correlated with IFN-γ responses. Overall, IFN-γ and IL-4 responses were positively correlated, providing little evidence of cross-regulation of Th1 and Th2 immunity within individual sheep. Furthermore, T-ci specific IgA was highly positively correlated with PWM-induced IL-10, indicating a possible role for this cytokine in IgA production. Our results suggest that while genetic selection for adaptive immune response traits is possible and may be beneficial for parasite control, selection of high IFN-γ responsiveness may negatively affect productivity.

Antagonism between co-infecting gastrointestinal nematodes: A meta-analysis of experimental infections in Sheep.

Gastrointestinal nematodes (GIN) have enormous global impacts in humans, wildlife and grazing livestock. Within grazing livestock, sheep are of particular global importance and the economics and sustainability of sheep production are greatly constrained by GIN infections. Natural infections are composed of co-infections with multiple species, and while some past work suggests species can interact negatively with one another within the same host, there is wide variation in reported patterns. Here, we undertook a systematic literature search and meta-analysis of experimental GIN co-infections of sheep to determine whether these experimental studies support the hypothesis of antagonistic interactions between different co-infecting GIN, and test whether aspects of parasite biology or experimental design influence the observed effects. A systematic search of the literature yielded 4848 studies, within which, we identified 19 experimental sheep studies comparing post-mortem worm counts across two co-infecting GIN species. Meta-analysis of 67 effects obtained from these studies provides strong evidence for interactions between GIN species. There was wide variation in the strength and direction of these interactions, but the global effect was significantly antagonistic. On average, there was a decrease in the number of worms of one species when a co-infecting species was also present, relative to a mono-infection with that species alone. This effect was dependent on the infectious dose and was rapidly lost after anthelmintic treatment, suggesting that live worms are required for the effect to occur. Individual parasite species varied in the extent to which they both exerted, and were subject to, these interspecies interactions, and these differences are more complex than simply co-localisation within the gastrointestinal tract. Antagonistic interactions between co-infecting GIN may feedback into their epidemiology as well as potentially affecting the clinical impacts of infection. Furthermore, the consequences of these interactions may be heightened when clinical interventions affect only one species within the co-infecting network. Whilst it was not possible to identify the causes of variation between GIN species in the impact of co-infection, these findings point to new avenues for epidemiological, clinical and mechanistic research on GIN co-infections.

Cloning and expression analysis of two pro-inflammatory cytokines, IL-1 beta and IL-8, in haddock (Melanogrammus aeglefinus).

This paper reports the cloning and sequencing of two pro-inflammatory cytokines, interleukin (IL)-1beta and IL-8, in haddock (Melanogrammus aeglefinus) by homology cloning. The complete transcript of the haddock IL-1beta was sequenced and contained 1043 bp, including a 762 bp open reading frame. The 3' end of the gene includes a polyadenylation signal 13 bp upstream of the poly(A) tail, along with 10 instability motifs. The predicted protein of 253aa revealed the presence of the IL-1 family signature and the absence of an ICE cut site. The cDNA of the chemokine IL-8 was sequenced in haddock and contained 903 bp of which 306 bp are the open reading frame. Interestingly, the predicted protein sequence of 101aa, contains an ELR motif preceding the CXC signature, common in all vertebrate IL-8 molecules but absent in all teleost genes sequenced to date. The expression of both haddock cytokines was studied in four different tissues: head kidney, spleen, liver and gill. Tissues were obtained from both healthy fish and fish stimulated in vivo with four commercial serotypes of LPS, namely Escherichia coli 026:B6, 055:B5, 0111:B4 and 0127:B8 and PMA. Haddock IL-1beta was not constitutively expressed and expression was only observed following stimulation. However, this expression was stimulant dependent and only PMA and LPS 026:B6 induced high levels of expression in the head kidney. The haddock IL-8 gene on the other hand, showed a constitutive expression, that could be up or down-regulated depending on the immunostimulant used, although to a lesser extent than IL-1beta.

Vaccination experiments in the gadoid haddock, Melanogrammus aeglefinus L., against the bacterial pathogen Vibrio anguillarum.

Vibrio anguillarum is one of the primary pathogens responsible for high levels of fish mortality in the aquaculture industry, and among gadoids O2a and b are the most common pathogenic serotypes. In this paper a variety of studies were performed to assess the optimal route by which to challenge haddock against this pathogen, and an optimal regime to vaccinate haddock. The most efficient method to challenge haddock with V. anguillarum in this study was immersion in a bath containing 10(7)cfu/ml, where 60% mortality was seen. Subsequent experiments showed that juvenile haddock could be protected against bacterial challenge with V. anguillarum, with a significant reduction in mortalities observed amongst the vaccination treatments when compared to the unvaccinated controls. However, as seen previously in cod studies, vaccination did not induce a specific antibody response.

Analysis of the cathelicidin 1 gene locus in Atlantic cod (Gadus morhua).

Cathelicidins are one of the major families of antimicrobial peptides produced as part of the innate immune responses. We have studied the gene locus of the Atlantic cod cathelicidin gene (gmCath1) by sequencing of a BAC clone. Results have revealed the genes upstream and downstream of gmCath1 in a sequence of >92.6 kb and these include APG-9, obscurin, guanylate kinase and MMTA-2. There appears to be no pattern of synteny observed when the loci of cathelicidin genes in other species was analysed. Analysis of the promoter region of 1 kb upstream of the translation start codon led to the identification of numerous different putative transcription factor binding sites (TFBS) that are believed to be important for the control of immune gene expression. Further studies into the quantitative expression of gmCath1 found that it is constitutively expressed in the head kidney and spleen and significant increases in expression are observed at 24 h post injection with the immunostimulant ergosan. By inserting a 311 bp fragment of the gmCath1 promoter into a luciferase reporter vector we found that stimulation with Poly I:C results in significant up-regulation of gene expression. This report detailing the promoter region of a cathelicidin gene in teleost fish, and the varied putative TFBS identified, suggests that the gmCath1 gene is highly likely to be induced in response to a wide range of different immune stimuli including bacteria and viruses.

RAG-1 and IgM genes, markers for early development of the immune system in the gadoid haddock, Melanogrammus aeglefinus, L.

The full sequence of the heavy chain of Immunoglobulin M (IgM) and a partial fragment of the recombination activating gene-1 (RAG-1) gene were sequenced in haddock, Melanogrammus aeglefinus, L. The complete transcript of haddock IgM consisted of 1865 bp and translated into a 572-aa peptide. The RAG-1 fragment was 1776 bp and was identified as the core region of RAG-1. These two immune genes were used in expression studies as markers of early development in haddock larvae. A DIG labelled oligoprobe of the RAG-1 gene was used in whole-mount in situ hybridisation (WISH). A hybridisation signal for RAG-1 was first detected in larvae at 25 days post-hatching (dph) in two bilateral symmetric regions of the head identified as the thymus. Further expression studies were carried out by RT-PCR analysis of RAG-1 and IgM on larval samples obtained during early development, i.e. from fertilisation to weaning. Haddock RAG-1 expression was detected after 21 days post-fertilisation (dpf) whilst IgM transcripts were not detected until 40 dpf, equivalent to day 29 post-hatching. These results suggest that the immune system in haddock starts to develop in larvae of 6-7 mm in length (25-29 dph).