Immune development and performance characteristics of Romney sheep selected for either resistance or resilience to gastrointestinal nematodes.

Immunological and performance characteristics were explored in Romney sheep from lines selected for either resistance or resilience to parasite infection. At a mean 78 days-of-age, twin lambs from a line selected for resistance (RT) and lambs from a line selected for resilience (RL) were infected with the intestinal nematode Trichostrongylus colubriformis for 100 days (I) while their twin remained as an uninfected control (C). Compared with RL, RT animals had lower levels of circulating CD4+ T-cells (P = 0.003) but a greater proportion of these were activated (CD4+CD25+) in response to infection (P = 0.007). Differences between the lines in humoral immune responses to nematode infection varied with higher levels of T. colubriformis specific immunoglobulin (Ig) E in RT-I than RL-I (P = 0.002) but similar levels of both IgG (P = 0.926) and IgA (P = 0.321) responses. Temporal differences in the immune response also existed between the lines with RT-I animals displaying an earlier peak and more rapid reduction in FEC and an earlier peak in T. colubriformis specific IgA. In addition, compared with their RT-C and RL-C counterparts, infection caused a 22% reduction in feed intake from day 56 (P = 0.001) with total feed intake reduced by 15% and 9% for RT-I and RL-I, respectively. Cumulative liveweight gain was greatest for RL animals (P = 0.026) and relative to RT-C and RL-C was reduced by 5.8 kg and 4.9 kg for RT-I and RL-I, respectively. Overall, the selection lines appear to have differences in immunological characteristics that are both dependent on, and independent of parasite infection. Further, the difference in growth in the uninfected animals coupled with the similar cost of infection suggests the lower liveweight gain of RT-I compared with RL-I may be due to inherent differences between the lines in their growth potential, rather than a greater cost of infection in animals selected for resistance.

Direct anthelmintic and immunostimulatory effects of oral dosing semi-purified phytohaemagglutinin lectin in sheep infected with Teladorsagia circumcincta and Trichostrongylus colubriformis.

Lectins are plant secondary compounds that can have anthelmintic properties in vitro. In particular, the phytohaemagglutinin lectin extracted from Phaseolus vulgaris has been shown to inhibit the feeding of Trichostrongylus colubriformis and Teladorsagia circumcincta L1 larvae. However, little is known about the potential anthelmintic properties of this lectin in vivo and its suitability to control gastrointestinal parasite infections in lambs. In a 2 × 2 study, lambs were either orally dosed, or not, with 2.3mg semi-purified PHA lectin per kg live weight (LW) per day, whilst concurrently infected, or not, with 1000 T. circumcincta and 1000 T. colubriformis L3 infective larvae per day for 42 days. There were no adverse clinical effects observed with this dose of PHA lectin. Although worm burdens were similar, animals dosed with PHA lectin had reduced concentration of nematode eggs in the faeces compared with their non-lectin dosed counterparts (P=0.026), suggesting that there may be a direct effect of PHA lectin on parasite fecundity. Irrespective of infection, PHA lectin had immune-stimulatory properties with increased eosinophillia in both abomasal and small intestine tissue sections taken at slaughter on day 42 (P<0.02 for both) and a tendency for decreased ability of Teladorsagia larvae to penetrate abomasal tissue explants (P=0.06). Compared with infection alone, concurrent PHA lectin dosing and infection further increased the number of eosinophils (P<0.01), PAS-positive (mucin-producing cells) (P=0.03) and tended to increase the number of T helper cells (P=0.06). No interactions were observed for cell populations in small intestine tissue sections. These results suggest PHA lectin could have two possible modes of action against T. circumcincta and T. colubriformis, a direct anthelmintic effect on nematode fecundity and an indirect effect through enhancing local immune responses in the host.

In vitro screening of plant lectins and tropical plant extracts for anthelmintic properties.

Lectins are plant secondary metabolites (PSM) found in many forages and which may confer anthelmintic properties to gastrointestinal parasites through disrupting the development of parasitic larvae throughout its life cycle. In experiment 1, the ability of the plant lectins jacalin (JAC), concanavalin A (Con A), phytohemagglutinin E2L2 (PHA-E2L2), phytohemagglutinin L4 (PHA-L4), phytohemagglutinin E3L (PHA-E3L), kidney bean albumin (KBA), Robinia pseudoacacia agglutinin (RPA), Maackia amurensis lectin (MAA), Maclura pomifera agglutinin (MAA), Dolichos biflorus agglutinin (DBA), wheat germ agglutinin (WGA) and Galanthus nivalis agglutinin (GNA) to disrupt the feeding of the first stage larvae (L(1)) of the sheep gastro-intestinal nematodes (GIN) Teladorsagia circumcincta, Haemonchus contortus and Trichostrongylus colubriformis was investigated using a larval feeding inhibition test (LFIT). Only PHA-E3L, WGA and Con A had a potent effect on disrupting larval feeding of all of the three species of GIN investigated. The lectin concentration required to inhibit feeding in 50% of L(1) (IC50) was 7.3±1.2, 8.3±1.4 and 4.3±1.7 µg/ml for PHA-E3L; 59.1±32.4, 58.7±11.9 and 8.1±7.0 µg/ml for Con A and 78.9±11.2, 69.4±8.1 and 28.0±14.1 µg/ml for WGA for T. circumcincta, H. contortus and T. colubriformis larvae, respectively (P=0.006). The addition of the lectin inhibitors fetuin, glucose/mannose or N-acetylglucosamine for PHA-E3L, Con A and WGA, respectively, caused an increase in the proportion of larvae that had fed at all concentrations for PHA-E3L only. In experiment 2, the effect of extracts from the tropical plants Azadiractha indica, Trichanthera gigantea, Morus alba, Gliricidia sepium and Leucaena leucocephala on the feeding behaviour of H. contortus L(1,) was examined. A. indica, T. gigantea and M. alba failed to inhibit 50% of larvae from feeding at concentrations up to 10mg plant extract per ml. In contrast, both G. sepium and L. leucocephala demonstrated a dose-dependent effect on larval feeding with respective IC50 estimates (mean±s.e.) of 0.015 mg/ml ±0.001 and 3.465 mg/ml ±0.144, effects which were partly reversed by the inclusion of either the tannin inhibitor polyethylene glycol or the lectin inhibitor Fetuin. These studies demonstrate that plant lectins can have an inhibitory effect on the feeding behaviour of first stage larvae of ovine GIN in vitro. Moreover they also provide novel evidence that lectins may contribute to the anthelmintic properties of some tropical forage plant extracts, such as G. sepium and L. leucocephala.

Development and evaluation of an antibody ELISA for sarcoptic mange in sheep and a comparison with the skin-scraping method.

In this work an indirect ELISA for detecting serum-specific IgG antibodies in sheep was developed using a crude saline extract from Sarcoptes scabiei var. ovis mites and then the repeatability of the ELISA outcomes was estimated. Subsequently, its diagnostic accuracy was evaluated by Receiver Operating Characteristics (ROC) analysis using a sample collected from the entire sheep population of western Castile and Leon region in Spain, and then compared with that of the skin-scraping method. The reference method used was a combination of clinical examination, skin-scraping analysis and epidemiological surveys, but it introduced selection and probably information biases. Furthermore, we attempted to identify biological factors useful to predict the sensitivity or specificity of the ELISA as determined by comparison with the reference method. Additionally, conventional latent-class analysis [Hui, S.L., Walter, S.D., 1980. Estimating the error rates of diagnostic tests. Biometrics 36, 167-171] was also used to estimate accuracy parameters. The between-run coefficient of variation (CV) for a standard serum was 8.8% and the within-run CV 4.3%. No significant deviation between the OD% means and strength positive correlation between the OD% values (r=0.98) were found for the results from two different batches of antigen. When compared to the reference method, the Area Under the ROC curve (AUC) for the reference population was 0.967 (95% CI: 0.949-0.985) for the ELISA and 0.915 (95% CI: 0.863-0.968) for the skin-scraping method. By logistic regression analysis, one explanatory biological factor-result to the skin-scraping method-and four explanatory biological factors-Tyroglyphidae individual status, Trichophyton verrucosum individual status, Oestrus ovis status of the flock and presence of adjacent animals with a clinical disease neighbour to S. scabiei infection-were found for diagnostic sensitivity and specificity of the ELISA, respectively, although this depended on the OD% cut-off value used. Latent-class analysis, carried out for the ELISA at 17.8 OD% cut-off value (mean plus 3 SDs of sheep considered negative to anti-S. scabiei antibodies), showed a marked difference between the estimated diagnostic sensitivity of the ELISA (87.6%) and the skin-scraping method (62.8%), but closer diagnostic specificities (95.9% vs. 100%, respectively). These results demonstrate that the developed ELISA is valid for different applications in clinical as well as in epidemiological contexts.

Infestation of sheep with Psoroptes ovis, the sheep scab mite, results in recruitment of Foxp3(+) T cells into the dermis.

Regulatory T cells (Tregs) play a central role in maintenance of immune homeostasis by controlling harmful immune responses to inappropriate antigens and are thought to play a key role in modulating hypersensitivity reactions. Infestation of sheep with Psoroptes ovis results in a pronounced cutaneous hypersensitivity-type response, which appears to be crucial for mite survival. We hypothesize that (i) Tregs are involved in sheep scab lesions and (ii) Treg responses may crucially affect lesion development and subsequent mite survival. Foxp3 is a key transcription factor required for generation and maintenance of Tregs in rodents and humans, and is the most widely used marker for Tregs in these species. In this study, we sequence ovine foxp3 and show that it exhibits a high degree of homology with foxp3 from other species. Using a validated immunohistochemical staining technique, we demonstrate that infestation of sheep with P. ovis results in an influx of Foxp3(+) T cells into the skin. Future work will investigate the regulatory function of ovine Foxp3(+) T cells and determine whether the quality of the Treg response to P. ovis plays a role in individual susceptibility to the mite.

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.

Gene expression profiling of ovine keratinocytes stimulated with Psoroptes ovis mite antigen--a preliminary study.

Sheep scab is caused by the noninvasive mite, Psoroptes ovis, which initiates a profound pro-inflammatory skin response leading to lesion development. To investigate these early events between the skin and the parasite, primary ovine epidermal keratinocyte cultures were generated and challenged with mite derived antigens. The kinetics of the mRNA response of these cells were monitored by microarray. The results indicated that the cells responded within 1 h of challenge, with a significant increase in the pro-inflammatory cytokine IL-8. This result was confirmed by real-time RT-PCR, and showed that IL-8 up-regulation was maximal at 1 h but declined to pre-stimulation levels at 24 and 48 h. The IL-8 mRNA response to mite wash antigens containing secretory and/or excretory proteins was also investigated and compared to the response to whole mite antigen. These studies revealed that the mite wash antigen, at a challenge dose of 10 microg/mL, was markedly more potent and induced significantly higher levels of IL-8 mRNA than the same concentration of whole mite antigen. These results are discussed in relation to mite establishment and survival on the ovine host.

The effect of dietary sainfoin ( Onobrychis viciifolia) on local cellular responses to Trichostrongylus colubriformis in sheep.

The effect of sainfoin (Onobrychis viciifolia) hay consumption on the pathophysiology and local cellular responses of growing lambs during infection with Trichostrongylus colubriformis was investigated. Thirty-two lambs, 16 weeks of age, were allocated to 1 of 4 treatment groups (n=8) that were offered either grass (G) or sainfoin (S) hay while concurrently either infected (+), or not (-) with 12,000 L3 T. colubriformis larvae per week for 6 weeks. Liveweight gains were affected by diet (P=0.002) and reduced by infection (P<0.05). Faecal egg count was reduced in S+compared to G+ from days 35 to 42 (P=0.001); however, total egg output, worm burdens at day 42 and worm fecundity were similar between diets (P>0.05).Feeding sainfoin appeared to enhance immune cell development with tissue eosinophils, mast cells and pan T cells present in greater concentrations in S+ than in G+ animals. However, further studies are required to determine if the enhanced immune cell development is a consequence of a greater nutrient supply or a direct influence of sainfoin metabolites on local inflammatory responses to the gastrointestinal nematode T. colubriformis.

The effect of corticosteroid treatment on local immune responses, intake and performance in lambs infected with Teladorsagia circumcincta.

The nutritional cost of, and the sequential cellular changes associated with the developing immune response to the abomasal parasite Teladorsagia circumcincta were investigated using corticosteroid-induced immune-suppression. Six-month-old lambs with minimal nematode experience were either infected with 4000 L3 T. circumcincta per day (group IF), similarly infected and concurrently immune-suppressed with methylprednisolone acetate (group ISIF), immune-suppressed only (group IS) or remained as controls (group C). Food intake, faecal egg count (FEC) and antibody titres in plasma were recorded weekly, worm burden at necropsy on day 63 p.i. and body composition by X-ray computed tomography on days -2 and 62 p.i. Furthermore, sequential immunological changes at the site of parasite infestation in the abomasal mucosa were measured from serial biopsy tissue samples taken from additional animals that were fitted with an abomasal cannula and either infected with the same regime as IF animals above (group CnIF) or concurrently infected and immune-suppressed as above (group CnISIF). Corticosteroid treatment resulted in greater FECs (P<0.01) and worm burdens (P<0.01) in both ISIF and CnISIF compared with IF and CnIF sheep, respectively. Infection reduced feed intake by 17% between 14 and 28 days p.i. (P<0.05) and efficiency of energy utilisation by 20% (P=0.07) in IF animals but not in ISIF animals. Mast cells, globule leukocytes and IgA in tissue biopsy samples were elevated in CnIF from 42 days p.i., all of which were abrogated by corticosteroid treatment. The ability to regulate the worm population appeared to be associated with a rise in tissue IgA concentration and numbers of globule leucocytes (GL). The results support the hypothesis that a majority of the production losses that occur during infection of lambs with T. circumcincta in lambs are a consequence of the host immune response. These findings may have implications for regimes that promote the development of a strong host immune reaction to gastrointestinal parasites in lambs.

Emerging technologies and their applications in interactions between nutrition and immunity to gastrointestinal parasites in sheep.

Despite the plethora of evidence on the consequences of host nutrition on their immune responses to gastrointestinal parasites, the identity of molecules and mechanisms that drive the manifestations of immunity under nutrient abundance are not yet known. This is partly due to limitations of the methodologies employed to date that have failed to give comprehensive answers. The great advancements in the technological front over the past few years have opened a window of opportunity to identify these effector molecules, to explore both the mechanisms and cellular pathways and to evaluate their importance in the immune response to parasites. The aim of this paper is to present some of the novel, high-throughput technologies that are currently available in immunology and to explore their use in advancing our knowledge in interactions between nutrition and immunity to nematode parasites, with special reference to sheep. In the first part, we introduce the technologies and we discuss advantages and pitfalls of their use. We bring in successful examples of how their employment advanced knowledge and improved our understanding of the mechanisms that regulate immune responses to pathogens (both micro- and macroparasites). In the second part, we focus on the impact of nutrition on the immune response to parasites, and explore how these technologies can be used to advance our knowledge of immunonutritional interactions. We use as our starting point well-established models that have been successfully used to investigate the consequences of nutrition on the manifestations of immunity to parasites, which we further consider in the context of the novel technologies. We conclude by emphasizing the great potential of the described methodologies in unravelling the complex interactions between nutrition and immunity, but we also recommend caution when interpreting the outcomes.

The effect of vaccinating infection during pregnancy and dietary protein supply on the peri-parturient immune response of sheep to infection with Teladorsagia circumcincta and Trichostrongylus colubriformis larvae.

It is well established that dietary protein supply can influence the peri-parturient breakdown of immunity to nematode parasites but there is no information on the importance of exposure to nematode larvae during pregnancy for this response. We investigated this by exposing housed pregnant sheep, scanned as carrying two lambs, to a vaccinating infection with a trickle mixed infection of Teladorsagia circumcincta and Trichostrongylus colubriformis larvae (L3) or to no infection during weeks - 9 to - 4 relative to parturition. At the beginning of week - 3 all sheep were treated with anthelmintic to remove any vaccinating worm burden and from week - 2 to week +6 received a trickle challenge infection with the same nematodes. Within each vaccinating treatment there were two nutritional treatments (no. = 20 per subgroup) designed to provide 1.5 or 1.0 and 1.3 or 0.8 of metabolisable protein (MP) requirement during pregnancy and lactation, respectively. Five ewes were necropsied during weeks +1 and +3 to measure worm burdens and mucosal inflammatory cells and the remainder maintained until week +6. Serum levels of total, IgA and IgE antibodies against L3 antigen of each nematode were measured.Scanning errors and lamb losses resulted in some ewes carrying and/or rearing only one lamb. Numbers of lambs reared was therefore introduced as a treatment effect. Vaccinating infection delayed the peri-parturient rise in faecal egg count (FEC) by an average of 2 weeks but its effect on FEC during the first 6 weeks of lactation was smaller and less persistent than that of dietary MP supply and single- v. twin-suckling.Populations of both nematodes were lower in association with high MP supply, vaccination and single suckling. These changes were associated with increases in numbers of mucosal mast cells (MMC) as a result of both increased MP supply and vaccination. Evidence for a more rapid return of host ability to limit populations of the abdominal nematode T. circumcincta than of the intestinal nematode T. colubriformis was associated with fewer eosinophils and more globule leucocytes (GL) in abomasal than in intestinal tissue.None of the serum antibody isotypes was affected by dietary protein supply. Total and IgA antibodies were maintained by a current larval (vaccinating) intake. IgA titres, however, increased progressively during pregnancy, especially in twin-bearing ewes. IgE titres appeared to be sensitive primarily to the reproductive cycle itself, peaking around parturition.This work supports the conclusion that availability of MP supply influences the recruitment and activity of cells of the immune armoury of the gastro-intestinal tract to nematode parasites. The precise outcome may differ with site and/or nematode species.

Eukaryotic expression of recombinant Pso o 1, an allergen from Psoroptes ovis, and its localization in the mite.

A cDNA encoding the immunogen Pso o 1 from Psoroptes ovis was obtained by polymerase chain reaction (PCR) amplification. The amplicon contained the entire coding sequence for the prepro-enzyme in an open reading frame (ORF) of 966 bp. This gene encoded a predicted protein of 322 amino acids (aa) with 64% aa identity (80% similarity) to the major house dust mite faecal allergen Der f 1. The pro-enzyme form of Pso o 1 was expressed as a recombinant protein in the Pichia pastoris-eukaryotic expression system. Maturation of the recombinant pro-enzyme by autocatalytic activation was not observed, and such maturation could not be achieved using a number of techniques known to activate recombinant Der p 1 and Der f 1 expressed in the same system. Serum raised against recombinant Pso o 1 cross-reacted with mature Der p 1 and allowed Pso o 1 to be immunolocalized to the gut of P. ovis.

A house dust mite allergen homologue from poultry red mite Dermanyssus gallinae (De Geer).

Tropomyosin is an allergenic, actin-binding protein and a proposed vaccine candidate from several species of parasite. Tropomyosin cDNA, obtained by polymerase chain reaction (PCR) amplification from Dermanyssus gallinae RNA, encoded a predicted protein with 89% and 88% identity to tropomyosins from the ticks Boophilus microplus and Haemaphysalis longicornis, respectively, and 85% identity to the house dust mite (HDM) tropomyosin Der p 10. Mouse antibodies raised against HDM tropomyosin reacted with a band of 38 kDa on Western blots of D. gallinae extract, consistent with the molecular masses of acarine tropomyosins and the putative product of the cDNA encoding D. gallinae tropomyosin. When the same preparation of D. gallinae proteins was used in Western blots with serum from infested hens, the IgY component of the serum bound to a number of mite proteins, but not to tropomyosin, indicating that hens are not directly exposed to this allergen during a natural infestation. Immunolocalization of tropomyosin in mites indicated a ubiquitous distribution of the molecule in mite tissues. Immunolocalization and Western blotting also indicated that poultry red mites ingest host IgY.

Molecular characterization, expression and localization of tropomyosin and paramyosin immunodominant allergens from sheep scab mites (Psoroptes ovis).

cDNAs encoding the immunodominant allergens tropomyosin and paramyosin were amplified from RNA extracted from the sheep scab mite Psoroptes ovis. The tropomyosin cDNA contained an open reading frame (ORF) of 852 bp which encoded a predicted protein with 98% and 97% identity to the house dust mite allergens Der f 10 and Der p 10 respectively. The complete paramyosin ORF generated by RT-PCR was 2625 bp in length and encoded an 875aa predicted protein of 102.6 kDa with 97%, 95% and 89% identity to the paramyosins of Dermatophagoides pteronyssinus (Der p 11), Sarcoptes scabiei and Blomia tropicalis (Blo t 11) respectively. Full length tropomyosin and truncated and full-length paramyosin were expressed as recombinant proteins. IgG and IgE in sera from sheep with a 6-week duration primary infestation of P. ovis did not detect either full-length or truncated recombinant paramyosin. IgG in both infested and naïve sheep sera detected recombinant tropomyosin, suggesting cross-reactivity to tropomyosin and to other invertebrate species to which the sheep may have been exposed. Staining with antibodies directed against tropomyosin and paramyosin was observed throughout sections of P. ovis. Staining was especially prevalent in the anterior sections of the mites, possibly associated with locomotory muscles in this region.

Immune responses to Staphylococcus aureus and Psoroptes ovis in sheep infected with P. ovis--the sheep scab mite.

In sheep, lesions caused by Psoroptes ovis, the sheep scab mite, may become colonized by Staphylococcus aureus. The present study compares clinical signs, lesional area and the immune response to P. ovis and S. aureus in P. ovis-infested sheep with and without secondary S. aureus infection. No differences were detected in the clinical signs or lesional areas in the S. aureus-positive and -negative sheep. However, 6 weeks after infestation an IgG but not IgE isotype antibody response to S. aureus was detected in the S. aureus-positive but not the S. aureus-negative group of sheep. This response targeted S. aureus antigens with molecular weights of approximately 36, 38, 50 and 65 kDa. In addition, 6 weeks after infestation an IgE response to P. ovis was detected in the S. aureus-positive but not the S. aureus-negative group of sheep.

Ability of a proteinase inhibitor mixture to kill poultry red mite, Dermanyssus gallinae in an in vitro feeding system.

The development of a reliable in vitro feeding system has enabled the rapid testing of presumptive anti-mite factors, a mixture of protease inhibitors (PI), by incorporating these into the blood feed. Mites were collected from a caged-hen facility and underwent a regime of starvation under varying conditions of temperature and darkness to determine the optimum conditions that encouraged mites to feed in the in vitro system. The efficacy of two anti-coagulants, heparin (HS) and acid citrate glucose (ACD), on mite feeding rates and mortality was evaluated. The ability of a mixture of PI to kill mites was also evaluated. The rate of feeding was greater in mites that were starved and cooled for between 7 and 30 days compared with mites that were not starved or cooled. The percentage of mites that fed when presented with HS treated blood (70%) was greater when compared with ACD treated blood (48%). The mortality of mites fed blood treated with HS + PI increased to 45% compared with a mortality level of 5% in mites fed on blood treated with HS alone. A reliable in vitro method for feeding D. gallinae which has the potential to be used to rapidly screen blood-borne products for their efficacy in reducing mite numbers has been developed.

Progress and opportunities in the development of vaccines against mites, fleas and myiasis-causing flies of veterinary importance.

Despite the potential benefits offered by vaccination against ectoparasites, there have been few commercial successes with this strategy, in spite of sustained efforts using increasingly sophisticated techniques. This review outlines the progress and challenges offered by recent research into vaccination against some of the major ectoparasites of veterinary importance, and provides an insight into the opportunities arising from our increased understanding of the immunology of host-parasite relationships and the potential for exploitation of this knowledge and that arising from new genomic data provided by expressed sequence tag projects.

The effect of chicory ( Cichorium intybus ) and sulla ( Hedysarum coronarium ) on larval development and mucosal cell responses of growing lambs challenged with Teladorsagia circumcincta.

The aim of this study was to investigate the effects of grazing different bioactive forages on acquired immunity against Teladorsagia circumcinta infection. The development of immunity was assessed by following the response of trickle-infected lambs grazing chicory (Cichorium intybus; IC), sulla (Hedysarum coronarium; IS) or grass/clover (Lolium perenne/Trifolium repens; IGC), to a single challenge infection. Parasite-naive lambs, grazing grass/clover, were also challenged with the single infection dose providing the uninfected control (UGC) group. Trickle infection significantly reduced worm establishment, inhibited larval development and increased mucosal mast cell (MMC) and globule leucocyte (GL) cells. Grazing treatment (chicory, sulla or grass/clover) significantly affected adult worm (P<0.05), late-L4 (P<0.01) and mid-L4 (P<0.01) larval-stage recoveries of the trickle-infected lambs, with IGC group always carrying higher worm burdens than either IC or IS lambs. MMC and GL cells of trickle-infected lambs were positively correlated with the proportion of early-L4 worms recovered and negatively correlated with both the proportion of adult worms recovered and the total worm establishment, suggesting that the observed effects were due to an enhanced immune response. The results suggest elevated immune responses against T. circumcincta infections in growing lambs grazing on either sulla or chicory compared to those grazing on grass/clover, probably due to differences in forage nutritional values.

Cloning and expression of the extra-cellular part of the alpha chain of the equine high-affinity IgE receptor and its use in the detection of IgE.

The high-affinity receptor for IgE (FcepsilonRI) plays a central role in IgE-mediated allergic reactions. Cross-linking of FcepsilonRI by IgE-antigen complexes results in the activation of mast cells and basophils and is thought to contribute to the immunopathology of Heaves, a chronic obstructive pulmonary disease of horses. Recombinant protein corresponding to the extra-cellular portion of the FcepsilonRI alpha subunit, cloned and sequenced previously, was expressed using both mammalian cells and insect cells. The yield of expressed protein was considerably greater using insect cells and the baculovirus expression system. The recombinant proteins differed in size between the two systems, presumably due to differences in the extent of glycosylation. However, recombinant protein from both cell systems bound equine IgE present in bronchoalveolar lavage fluid from horses with Heaves. These results suggest that the recombinant extra-cellular part of FcepsilonRI should be a useful tool with which to study equine IgE responses.

Expression library immunization confers protection against Mycobacterium avium subsp. paratuberculosis infection.

Currently, paratuberculosis vaccines are comprised of crude whole-cell preparations of Mycobacterium avium subsp. paratuberculosis. Although effective in reducing clinical disease and fecal shedding, these vaccines have severe disadvantages as well, including seroconversion of vaccinated animals and granulomatous lesions at the site of vaccination. DNA vaccines can offer an alternative approach that may be safer and elicit more protective responses. In an effort to identify protective M. avium subsp. paratuberculosis sequences, a genomic DNA expression library was generated and subdivided into pools of clones (approximately 1,500 clones/pool). The clone pools were evaluated to determine DNA vaccine efficacy by immunizing mice via gene gun delivery and challenging them with live, virulent M. avium subsp. paratuberculosis. Four clone pools resulted in a significant reduction in the amount of M. avium subsp. paratuberculosis recovered from mouse tissues compared to mice immunized with other clone pools and nonvaccinated, infected control mice. One of the protective clone pools was further partitioned into 10 clone arrays of 108 clones each, and four clone arrays provided significant protection from both spleen and mesenteric lymph node colonization by M. avium subsp. paratuberculosis. The nucleotide sequence of each clone present in the protective pools was determined, and coding region functions were predicted by computer analysis. Comparison of the protective clone array sequences implicated 26 antigens that may be responsible for protection in mice. This study is the first study to demonstrate protection against M. avium subsp. paratuberculosis infection with expression library immunization.