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The aim of the study was to purify and characterise recombinant proteins with the potential as an anti-parasite vaccine. Full-length cDNAs encoding seryl-tRNA synthetase (srs-2) were cloned from Haemonchus contortus (HcSRS-2) and Teladorsagia circumcincta (TcSRS-2). TcSRS-2 and HcSRS-2 cDNA (1458bp) encoded proteins of 486 amino acids, each of which was present as a single band of about 55 kDa on SDS-PAGE. Multiple alignments of the protein sequences showed homology of 94% between TcSRS-2 and HcSRS-2, 76-93% with SRS-2s of eight nematodes and 68% with Mus musculus SRS-2. The predicted three-dimensional structures revealed an overall structural homology of TcSRS-2 and HcSRS-2, highly conserved binding and catalytic sites, and minor differences in the tautomerase binding site residues in other nematode SRS-2 homologues. A phylogenetic tree was constructed using helminth and mammalian SRS-2 sequences. Soluble C-terminal SRS-2 proteins were expressed in Escherichia coli strain AY2.4 and purified. Recombinant HcSRS-2 assay shows that the recombinant enzyme was active and stable. The K m and V max for ATP were 3.9 ± 1.0 μM and 2.7 ± 0.1 μmol min -1 mg -1 protein, respectively. Antibodies in serum and saliva from field-immune, but not nematode-naïve, sheep recognised recombinant HcSRS-2 and TcSRS-2 in enzyme-linked immunosorbent assays. Recognition of the recombinant proteins by antibodies generated by exposure of sheep to the native enzyme indicates similar antigenicity of the two proteins., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

An effective immune response is expected to confer fitness benefits through improved resistance to parasites but also incur energetic costs that negatively impact fitness-related traits, such as reproduction. The fitness costs and benefits of an immune response are likely to depend on host age, sex, and levels of parasite exposure. Few studies have examined the full extent to which patterns of natural selection on immune phenotypes vary across demographic groups and environments in the wild. Here, we assessed natural selection on plasma levels of three functionally distinct isotypes (IgA, IgE, and IgG) of antibodies against a prevalent nematode parasite measured in a wild Soay sheep population over 26 years. We found little support for environment-dependent selection or reproductive costs. However, antibody levels were negatively associated with parasite egg counts and positively associated with subsequent survival, albeit in a highly age- and isotype-dependent manner. Raised levels of antiparasite IgA best predicted reduced egg counts, but this did not predict survival in lambs. In adults increased antiparasite IgG predicted reduced egg counts, and in adult females IgG levels also positively predicted overwinter survival. Our results highlight the potential importance of age- and sex-dependent selection on immune phenotypes in nature and show that patterns of selection can vary even among functionally related immune markers.

A 1299 bp full length cDNA encoding Teladorsagia circumcincta enolase (TeciENO) was cloned, expressed in Escherichia coli and the recombinant protein purified and its kinetic properties determined. Helminth enolase sequences were used to construct a phylogenetic tree. The predicted protein consisted of 433 amino acids and was present as a single band of about 50 kDa on SDS-PAGE. Multiple alignments of the protein sequence of TeciENO with homologues from other helminths showed 98% similarity with Haemonchus contortus enolase, 78-95% similarity to other nematode sequences and 72-75% similarity to cestode and trematode enolases. Substrate binding sites and conserved regions were identified and were completely conserved in other homologues. The optimum pH for TeciENO activity at 25 °C was pH 7, the K m for 2-phophoglycerate 0.09 ± 0.04 mM and the V max was 604 ± 6 nmol min -1  mg -1 protein (both mean ± SD, n = 2). TeciENO activity was inhibited by 11.5% by 1 mM citrate (p < 0.001). Antibodies in both serum and saliva from field-immune, but not nematode-naïve, sheep recognised recombinant TeciENO in enzyme-linked immunosorbent assays. The recognition of the recombinant protein by antibodies generated by exposure of sheep to native enolase indicates similar antigenicity of the two proteins., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The objective of this study was to identify Scottish Blackface lambs that were at the extremes of the spectrum of resistance to gastrointestinal nematodes and characterise their response to an experimental nematode challenge. Lambs (n = 90) were monitored for faecal egg count (FEC) (2 samples from each of 2 independent natural infections). The most resistant (n = 10) and susceptible (n = 10) individuals were selected and challenged with 30,000 Teladorsagia circumcincta larvae (L3) at 9 months of age. Response to infection was monitored by measuring FEC, plasma pepsinogen, serum antibodies against nematode larval antigens and haematology profile, until necropsy at 71 days post infection. Worm burden, worm fecundity and the level of anti-nematode antibodies in abomasal mucosa were determined at necropsy. FEC was consistently higher in susceptible animals (P < 0.05), validating the selection method. Worm fecundity was significantly reduced in resistant animals (P = 0.03). There was also a significant correlation (r = 0.88; P < 0.001) between the number of adult worms and FEC at slaughter. There was no effect of phenotype (resistance/susceptibility) on plasma pepsinogen or on haematology profile. Phenotype had a significant effect on the level of anti-nematode IgA antibodies in serum (P < 0.01), reflecting a higher peak in resistant animals at day 7 post infection. It is concluded that significant variation in the response to gastrointestinal nematode challenge exists within the Scottish Blackface population with resistant animals displaying significantly lower FEC, lower worm fecundity and higher concentration of anti-nematode IgA antibodies in serum., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Teladorsagia circumcincta is among the most important gastrointestinal parasites in small ruminants and the predominant species in Southern European goats. Parasite control is largely based on metaphylactic/preventative treatments, which is often seen as non-sustainable anymore. The reasons are increased consumer demand to reduce chemicals in livestock production and anthelmintic resistance against the common drugs. This study aimed at the development of a T. circumcincta-enzyme-linked immunosorbent assay (ELISA) specifically for goats. Samples were obtained from goats raised parasite-free or infected experimentally. Sampling continued during the following pasture season and housing period. The sensitivity for the use in bulk milk samples as an indicator of T. circumcincta infection levels in grazing goats was examined. The ELISA enables clear differentiation of negative and positive animals. With a specificity of 100% negative cut-off values for serum and milk were 0.294 and 0.228 (sensitivity, 95%). Positive cut-off values (sensitivity, 90%) were 0.606 (serum) and 0.419 (milk), while a sensitivity of 95% resulted in 0.509 and 0.363, respectively. The grey-zone between negative/positive cut-offs was introduced to deal with animals in pre-patency and decreasing antibody levels after infection. There was no cross reactivity for Trichostrongylus colubriformis and Cooperia oncophora while for Haemonchus contortus and Fasciola hepatica it cannot be fully excluded currently. In bulk milk samples, 5% of the milk had to be contributed from animals infected with T. circumcincta to be detected as positive. The results derived from experimentally and naturally infected as well as parasite naïve animals indicate the potential of the ELISA to be used in targeted anthelmintic treatment regimes in goats.

The aim of this study was to evaluate the IgG activity in serum and milk samples of experimentally infected lactating ewes with the nematode Teladorsagia circumcincta as well as its relationship to the degree of infection. In a previous study 28 pregnant ewes were divided into two homogeneous groups, with high (H) and low (L) level of parasitism, respectively. Blood and milk samples were taken weekly after lambing until the end of the lactation, 126 days post-partum (pp). IgG against T. circumcincta were measured by means of an indirect ELISA. The kinetic of the immunoglubulins in serum samples showed a very low activity at the beginning but gradually increased throughout the lactation; H group showed higher values most of the sampled days than L group. Contrary, IgG in milk samples remained high during the first month pp, then decreased around 38% and by the end of the study rose again. Antibodies in both samples were correlated and especially during the second month of lactation (r=0.3; p<0.001). With the aim to correlate the immune response and the degree of infection we found an inverse relationship (r=-0.2; p<0.05) during the second month of lactation between eggs and IgG in serum. However the correlation with immunoglobulins in milk was positive, mainly, on the last third of lactation (r=0.2; p<0.01). As a conclusion, the individual detection of total IgG antibodies against T. circumcincta in lactating ewes is highly dependent on the stage of lactation. Therefore, these associations should be confirmed under field conditions., (Copyright © 2012 Elsevier B.V. All rights reserved.)

A functional immune system is important for survival in natural environments, where individuals are frequently exposed to parasites. Yet strong immune responses may have fitness costs if they deplete limited energetic resources or cause autoimmune disease. We have found associations between fitness and heritable self-reactive antibody responsiveness in a wild population of Soay sheep. The occurrence of self-reactive antibodies correlated with overall antibody responsiveness and was associated with reduced reproduction in adults of both sexes. However, in females, the presence of self-reactive antibodies was positively associated with adult survival during harsh winters. Our results highlight the complex effects of natural selection on immune responsiveness and suggest that fitness trade-offs may maintain immunoheterogeneity, including genetic variation in autoimmune susceptibility.

Sheep that are highly resistant to parasitic nematodes can suffer bad diarrhoea due to the inflammation associated with rejection of ingested larvae from pasture. We hypothesised that challenging parasite-resistant sheep indoors with nematode larvae would result in reduced faecal dry matter, and that faecal dry matter would be lowest in sheep challenged with a mixture of Trichostrongylus colubriformis and Teladorsagia circumcincta compared to those challenged with either species alone. We also hypothesised that inflammatory cells and serum antibodies and interleukin-5 would be highest in those sheep that received the mixed larval challenge. We found that faecal dry matter was reduced (P<0.05) in challenged sheep compared to unchallenged sheep, with the fastest reduction being in those sheep challenged with only Tric. colubriformis. At 14 and 23 days after challenge began, there were no differences in faecal dry matter between the three challenged groups. Within the abomasum, there were no differences in inflammatory cell numbers between unchallenged sheep and those challenged only with Tric. colubriformis. Cell numbers in sheep challenged with Tela. circumcincta were higher (P<0.05) than those in unchallenged sheep, but there were no differences between sheep challenged only with Tela. circumcincta or as a mixed challenge. In the small intestine, inflammatory cell numbers were higher (P<0.05) in sheep that received the mixed challenge compared to controls. Cell numbers in sheep challenged with either Tela. circumcincta or Tric. colubriformis were also slightly higher than those in controls. Larval challenge increased (P<0.05) levels of IgA and IgE in serum, but there were no differences between the three challenged groups. Larval challenge also increased (P<0.05) levels of IL-5, with the greatest increase being in those sheep challenged with both species. We concluded that both Tela. circumcincta and Tric. colubriformis can cause immune-mediated diarrhoea in sheep, and that a mixed challenge will not necessarily lead to worse diarrhoea or higher concentrations of antibodies in serum. We also concluded that challenge with Tric. colubriformis leads to no inflammation in the abomasum, but challenge with Tela. circumcincta may lead to some inflammation in the small intestine., ((c) 2010 Elsevier B.V. All rights reserved.)

Sheep were immunized by multiple truncated infections with the gastrointestinal nematodes Trichostrongylus colubriformis, Haemonchus contortus and Teladorsagia circumcincta. Three infections with T. colubriformis of 14 days plus five booster doses of L3 stimulated highly effective protection against challenge (99%). Three infections of 14 days plus three booster doses with H. contortus also resulted in significant protection against challenge infection (87%), but the same procedure was not effective for T. circumcincta. Antibodies derived from gastrointestinal mucus of these immunized sheep were tested for their ability to reduce worm burden following injection of antibody-coated exsheathed larvae into the abomasum (H. contortus and T. circumcincta) or duodenum (T. colubriformis) of nematode-naïve sheep in a passive immunity test. The IgG fraction from the mucus of immunized sheep reduced worm burdens by 62%, 76% and 91% in three tests with T. colubriformis but was not effective for either of the abomasal dwelling nematodes H. contortus and T. circumcincta. Antibodies in immune mucus predominantly recognized two larval surface antigens on immunoblots of L3 extract, a high MW surface glycoprotein and the carbohydrate larval antigen (CarLA). Antibodies raised against purified T. colubriformis glycoprotein Tc-120 and CarLA were tested in the passive immunity model and it was found that only the antibody against CarLA resulted in a significant reduction of infection (87%). The protective anti-CarLA antibodies strongly recognized the surface of living T. colubriformis L3. Antibodies from abomasal mucus of sheep immunized by H. contortus and T. circumcincta infections reacted weakly with CarLA and the larval surface and did not reduce worm counts in a passive immunity test. The results provide further evidence that the larval surface carbohydrate antigen CarLA has potential as a mucosal immunogen for a strongylid nematode vaccine.

Groups of yearling sheep, which had been trickle infected with Teladorsagia circumcincta for 8 weeks and then drenched, were challenged with 50 000 T. circumcincta larvae together with groups of worm-free controls. Fewer parasites and a greater proportion of early fourth stage larvae were recovered from previously infected sheep compared to controls. Worm loss and arrested development were evident by 5 days after challenge whereas growth retardation of developing worms was observed by day 10. In the previously infected sheep a secondary IgA response was observed in the efferent gastric lymph from 5 days post-infection. Western blot analysis showed the lymph IgA to be predominantly dimeric and nonsecretory in nature and that the somatic antigens recognized were predominantly in the 100-250 kDa range. The concentration of IgA in lymph was always higher than in blood and in the previously infected sheep increased fivefold 8 days post-challenge in contrast to blood where IgA levels were unchanged. The timing of the response suggested that it occurred too late to have been the cause of worm loss or arrested development, though it may have retarded the growth of developing parasites.

SUMMARY Previous experiments have shown that genetic resistance to infection by Teladorsagia circumcincta in sheep can be measured by the level of IgA in gastric mucus, jointly with other parameters. The aim of this study has been to observe the influence of IgA on adult worms. The experiment was carried out with Churra sheep experimentally infected with T. circumcincta. At slaughter, gastric content, gastric mucus, blood samples and faeces were recovered to determine the number of eggs in utero, length of adult females, worm burden, number of L4, titre of serum pepsinogen, peripheral eosinophilia and eggs per gram (epg). IgA activity in gastric mucus, serum, nasal secretions and saliva were tested against somatic antigen from fourth-stage larvae (L4), somatic antigen from the adult stage and excretory-secretory (E/S) antigen from the adult stage. The results showed a significant correlation between serum IgA and gastric mucus (P<0.01) as well as in nasal secretions (P<0.01). We found negative correlations between IgA activity in gastric mucus with the eggs in utero and with adult female length. Furthermore there were also strong relationships between the peripheral eosinophilia with serum (P<0.01) and gastric mucus IgA activity (P<0.01). Moreover serum pepsinogen and the number of L4 at slaughter were related (P<0.01).

Studies on the immunoglobulin (Ig)E immune responses to the gastric nematode, Teladorsagia circumcincta, have demonstrated a major high molecular weight allergen (HMWTc). Cross reactive allergens of similar MW were demonstrated for Trichostrongylus colubriformis and Cooperia curticei, but not for Haemonchus contortus. Purification of HMWTc was achieved by gel-filtration chromatography, and nonreducing SDS-PAGE and Western blot analysis revealed two closely associated bands with a molecular weight of approximately 140-150 kDa. Reduction showed four IgE reactive bands of 120, 50, 45 and 30 kDa, and deglycosylation abrogated the immunoreactivity of the 120 and 30 kDa bands. Ultrastructural immunolocalization by electron microscopy revealed that the IgE reactivity was confined to the cuticular surface of the infective (L3) larvae. ELISA studies to determine the IgE anti-HMWTc responses in lambs during their first grazing season, demonstrated significantly higher IgE antibody in lambs with low accumulative faecal egg count (FEC) compared to animals with high accumulative FEC. These studies provide evidence for a protective function of IgE antibody in Teladorsagia infections in lambs.

The dynamics of the humoral immune response of calves were analysed after primary infection and re-infection with the intestinal nematode Cooperia punctata. 12 male 5 month-old Holstein-Friesian calves were randomly divided into two groups A and B. At the beginning of the experiment Group A animals were each infected experimentally with a single oral dose of 130,000 infective third stage larvae (L3) of C. punctata. The animals of Group B were kept as non-infected controls. The two calves from Group A with the highest infections died of cooperiosis at 32 and 44 days after infection (DAI), respectively. On DAI 100 the calves were treated with the recommended dose of oxfendazole. On DAI 180 the remaining four calves of Group A and three animals of Group B (B1) were infected with 260,000 L3 of C. punctata, while the other three calves of Group B (B2) served as non-infected controls. Monitoring of the humoral immune response predominantly demonstrated an IgG1 response against both adult and L3 antigen of C. punctata. Moreover, re-infections increased the levels of these immunoglobulins. IgA levels were less increased than IgG1 and no significant increase was observed in IgG2 and IgM levels. Immunoblotting analysis showed that total IgG present in the serum of the primary infected animals mainly reacted against adult proteins of 12-14 and 17-20 kDa and against L3 proteins of 33 and 43 kDa. After re-infection total IgG reacted with the same adult proteins but also with an adult 29 kDa protein.

A monoclonal antibody to ovine IgE was employed in an ELISA to investigate the IgE antibody responses in serum and gastric lymph to a primary infection of Teladorsagia circumcincta, and following challenge in previously infected sheep. During a primary response, IgE antibody to antigens derived from the infective third stage (L3) and adult (L5) worms were negligible, with low levels of IgE antibody detected in serum and lymph. In contrast, there was a pronounced IgE antibody response in 2/4 sheep to L3 antigens during 2-8 days after challenge of previously infected animals but low levels of IgE antibody to L5 antigens. This response was confirmed in a second but similar experiment, where relatively high levels of IgE antibody was detected to antigens from L3. Antibody levels were higher in lymph than in serum from the same animals, and Western blots of L3 antigen following SDS-PAGE under reducing conditions revealed several bands of MW26-96KD which reacted with the IgE antibody from gastric lymph. Immunohistochemical staining indicated that these IgE antibodies may be reacting with allergens associated with the surface cuticle of the worms.

The recognition of low molecular weight proteins by sera obtained during a single oral (primary) infection with 100,000 3rd-stage Cooperia oncophora larvae was studied in calves. Three groups of 6 or 7 calves were selected based on different egg excretion patterns. SDS-gel electrophoresis of adult Cooperia antigen under reducing conditions, followed by Western blotting, revealed that resistance of individual calves to C. oncophora might be related with antibody responses (42 days post infection) against at least 2 protein fragments (14-16 kDa and 27 kDa). The 14-16-kDa protein complex was bound, to some extent, by individual sera from all calves. The intensity of staining was negatively correlated with egg excretion on Day 42 p.i. Calves with high egg counts on Day 21 p.i. either did not or only weakly recognized the 27-kDa band. It has to be established whether the 14-16 kDa (or recombinant 14.2 kDa) provides a tool for immunodiagnostics and whether the 27-kDa fragment can help further unravel immune-mediated resistance to Cooperia.

Young lambs (6-8 weeks of age) were infected with 50000 infective stage larvae of the trichostrongyle nematode Nematodirus battus. Initial mean adult worm burdens of 23884 +/- 1678 (S.E.M.) at day 14 post-infection (p.i.) were reduced significantly (P < 0.01) to a mean burden of 5383 +/- 1397 by day 21 p.i. Blood samples were taken and serum analysed using ELISA and Western blotting techniques for IgG antibodies specific for N. battus. Pre-infection serum from young naive animals showed IgG recognition of adult and larval N. battus whole worm homogenate. Recognition of adult antigens became reduced during the first 14 days after infection, followed by a subsequent increase beginning between days 14 and 18 p.i. and peaking at day 28 p.i. IgM recognition of N. battus antigens showed a single response peak, starting between days 14 and 18 p.i., without the pre-infection elevated titre as observed with IgG. These results show that young lambs can mount a significant serum response to a primary infection with N. battus during rejection of adult worms and that lambs receive from their dams IgG antibodies that are capable of recognizing adult and larval N. battus during the first few weeks of life.

The total and IgG1 antibody responses to the intestinal nematode parasites Haemonchus contortus and Trichostrongylus colubriformis were measured in the serum of 160 lambs, 4 months of age. These antibodies had developed as the result of natural exposure to the parasites on pasture. Three sires were examined and strong sire effects on half-sib progeny were found. Plotting of ELISA antibody results in two dimensions revealed clustering of responses within sire groups. Bimodal antibody distributions were also observed within sire groups and the whole population for T. colubriformis. A bimodal distribution of antibodies to H. contortus was found for one sire group but not for the whole population. The injection of blowfly larvae (Lucilia cuprina) extract into 42/160 lambs at a later age (12 months) was followed by increased antibodies to L. cuprina and an apparent increase in antibodies to T. colubriformis. A bimodal distribution for antibodies to L. cuprina was found in one sire group and in the whole population. These bimodal distributions of antibodies to L. cuprina did not coincide with the distribution of antibodies to T. colubriformis or H. contortus, measured on the same serum samples. It was concluded that high and low responder sire groups could be differentiated in lamb populations for all three parasites. These effects persisted during lamb maturation and appeared to be genetic effects. Finally, cross-reacting antibodies between L. cuprina and T. colubriformis appear to be stimulated by injection of L. cuprina antigens.

Antibodies against Ostertagia spp., Cooperia spp. and Dictyocaulus viviparus were assessed in serum and individual milk samples of cows and in bulk milk samples from the refrigerating tank. Titres in milk samples were related to serum titers, but also influenced by milk yield, lactation stage and age of the cow. Correlation coefficients between serum and individual milk samples were highly significant, and varied from 0.42 to 0.56. Correlations between herd means of serum titres and herd means of individual milk titres varied from 0.41 to 0.61, and those between herd means of serum titres and means of bulk milk samples from 0.43 to 0.67. Finally, the correlations between herd means of individual milk samples and means of bulk milk samples varied from 0.52 to 0.82. This variation was dependent upon worm species and whether or not correction for milk yield was applied. The discrimination of herds was evaluated by the between/within herd ratio of variances. F-values for all types of samples were highly significant, and at least as high for milk samples as for serum samples. If, as has been shown for serum samples, a positive relationship exists between the milk yield response to anthelmintic treatment and the mean herd titre of milk samples or bulk milk samples, assessment of these titres could offer a rational basis for treatment of dairy cows.

The excretory-secretory products of exsheathed third-stage larvae of Trichostrongylus colubriformis conferred some protection to guinea pigs against homologous challenge. A glycoprotein with an apparent molecular mass of approximately 94 kDa was the dominant immunogen in post-exsheathment products. Immunoblots revealed IgG antibodies to this glycoprotein in sera from multiply-infected guinea pigs and some sheep, and in sera of guinea pigs after three truncated infections which had been restricted by anthelmintic treatments to development of the third parasitic stage. IgA antibodies to this protein were also found in intestinal lymph of a naturally infected sheep. Fluorescent antibody studies indicated that this 94 kDa component was associated with cells in the central body cavity of third-stage larvae, but was absent from fourth-stage larvae or adult worms. Fractionation and protection assays in guinea pigs revealed that while the native and aggregated 94 kDa protein conferred some host protection, it was not the only protective component of the excretory-secretory products of exsheathed third-stage larvae of T. colubriformis.

125I-radiolabelled surface proteins of various developmental stages of Obeliscoides cuniculi were used to analyze serum and gastrointestinal immunoglobulin responses of the host to primary infection with 10,000 larvae of the nematode, using a radioimmunoprecipitation method. Serum IgM, IgA, and IgG antibodies reacted mostly with infective larval and adult nematodal surface proteins. The highest binding activity was shown by IgG against the surface of infective larvae on days 56 and 86 after infection. Local (gastric mucosal) antibodies were also predominantly directed against surface antigens of infective larvae and adults, although considerable binding of the fourth-stage larval surface proteins by gastric mucosal IgG occurred 35 days after infection. Physicochemical analysis of the radiolabelled surface antigens revealed that the strongest antigenicity was found in proteins with molecular weights of 40,000 and 145,000. Nonspecific binding demonstrated in our tests by infective larval surface proteins was associated with particles with a molecular weight of 550,000.