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J F Huntley

Publications and source records attributed to J F Huntley.

At least 37 records · Page 2Linked to original sources

Temporal pattern of isotype-specific antibody responses in primary and challenge infestations of sheep with Psoroptes ovis--the sheep scab mite.

In sheep Psoroptes ovis provokes an allergic dermatitis with significant P. ovis antigen-specific IgE responses. The kinetics of the IgE response to primary and challenge infestations of P. ovis were reported earlier [Parasite Immunol. 22 (2000) 407]. The present study examines IgG, IgM and IgA responses to primary and challenge infestations of P. ovis and the profile of antigens/allergens reacting with IgG and IgE antibodies. Antigen-specific enzyme-linked immunosorbent assays (ELISAs) demonstrate that primary infestations elicited significant increases in levels of IgG and IgM but not IgA antibodies. IgG and IgM responses to primary and challenge infestations were not significantly different. Western blots of reduced P. ovis proteins indicate that IgG antibodies reacted with five major antigens following primary infestation and only three of these after challenge infestation. IgE antibodies bound to three major and five minor allergens after primary infestation and two additional minor allergens after challenge infestation. Immunodominant antigens >100 and <15 kDa and allergens >100 kDa were most consistent in stimulating substantial IgG and IgE antibody responses, respectively. These antigens/allergens may be exploited in immunodiagnosis and modulation of the host immune response.

Allergens↗

Cutaneous hypersensitivity reactions to Psoroptes ovis and Der p 1 in sheep previously infested with P. ovis--the sheep scab mite.

We have previously shown that infestation with Psoroptes ovis induces an IgE response and intense tissue eosinophilia, typical of a Type I hypersensitivity response [Parasite Immunol. 22 (2000) 407]. Intradermal tests (IDSTs) suggest that there are also delayed and Arthus-type responses to this parasite. In order to study the nature of ovine cutaneous reactions to P. ovis, naïve controls and experimentally infested sheep (n = 5) were challenged intradermally with mite antigen. Challenge elicited immediate (P < 0.001) and delayed (P < 0.005) wheal reactions in sensitised sheep. At 6 (P < 0.02) and 30 h (P < 0.001) the predominant infiltrating cells were eosinophils. To explore the role of circulating antibodies, naïve sheep (n = 5) were subjected to Prausnitz-Kustner (PK) tests. These elicited immediate (P < 0.02) but not delayed wheal reactions. At 6 h eosinophils (P < 0.001) dominated the infiltrate. These results suggest that P. ovis allergens provoke an IgE-dependent immediate and late phase response and a cell-mediated eosinophil-rich delayed-type hypersensitivity response (ER-DTH).

Animals↗

Is the allocation of metabolisable protein prioritised to milk production rather than to immune functions in Teladorsagia circumcincta-infected lactating ewes?

It has been suggested that the periparturient breakdown of immunity to parasites has a nutritional basis. Our overall hypothesis is that it results from a prioritised scarce nutrient allocation to reproductive functions (e.g. milk production) rather than to immune functions. We tested this hypothesis by offering five levels of dietary metabolisable protein, ranging from 0.65 to 1.25 times their assumed requirements, for 4 weeks post-parturition to twin-rearing Greyface ewes, experimentally infected with Teladorsagia circumcincta. We hypothesised that the initial increments of metabolisable protein supply would increase milk production without affecting the degree of breakdown of immunity whilst later increments would reduce the degree of breakdown of immunity. The first two increments of metabolisable protein supply indeed increased milk production and did not affect final worm burdens, but in contrast to the expectation, reduced faecal egg counts and total egg output. The last two increments of metabolisable protein supply did not further affect milk production and egg output, but resulted in reduced final worm burdens. Metabolisable protein supply did not affect plasma IgG and IgE antibody against somatic L(3) antigen but the first three increments reduced plasma pepsinogen and plasma IgA antibody. The last increment did not further reduce plasma pepsinogen but increased plasma IgA. Metabolisable protein supply did not systematically affect abomasal mucosal mast cell, globule leukocyte and eosinophil counts. Our results support the view that the priority of scarce metabolisable protein allocation to milk production over immune functions may be gradual rather than absolute. The contrast between effects of metabolisable protein supply on faecal egg count and final worm burden points towards the possibility that if different effector responses regulate fecundity and worm expulsion, then they would differ in their sensitivity towards changes in the degree of nutrient scarcity.

Animals↗

Protection studies with recombinant excretory/secretory proteins of Haemonchus contortus.

The efficacy of two recombinant proteins of Haemonchus contortus was studied in both adult sheep and young lambs. These 15 and 24 kDa excretory/secretory proteins were given combined, either supplemented or not with a glycan-rich insect cell extract. In 9-month-old sheep (trial 1), faecal egg output and worm burden were reduced by 49% and 55%, respectively, after vaccination with rec15/24, and by 46% and 65% after vaccination with rec15/24 and glycan extract. No reduction in egg output or number of worms was found in young lambs using the above recombinant proteins plus glycan-rich extract (trial 2). When trial 1 was repeated (trial 3), the protection could not be reproduced, possibly due to differences in batches of recombinant proteins. In all sheep, independent of their age, rec15/24-specific immunoglobulin (Ig)G1 and IgA titres were present, but 9-month-old protected sheep had significantly higher IgA titres than the lambs. Addition of glycans resulted in lower rec15/24-specific IgG1 and IgA in 9-month-old sheep after challenge. This did not affect the level of protection. A significant negative correlation was found between IgA and worm numbers in protected sheep immunized with rec15/24 supplemented with glycans. Total IgE and rec15/24 specific IgE titres were low. The number of eosinophils, mast cells, sheep mast cell protease (SMCP)+ cells and IgA+ cells did not differ between the protected and unprotected sheep, but the lambs had significantly fewer mast cells independent of their immunization.

Animals↗

Identification of an antigen from the sheep scab mite, Psoroptes ovis, homologous with house dust mite group I allergens.

Infestation of sheep with the ectoparasitic mite Psoroptes ovis, results in a severe allergic dermatitis. Currently, little is known about the allergens/antigens that stimulate the allergic response. We have isolated an 836-bp cDNA from a P. ovis cDNA library which displays strong homology to cysteine proteases and, in particular, to the group I house dust mite allergens Der p 1, Der f 1 and Eur m 1. The cDNA was expressed in Escherchia coli, fused to a hexahistidine tag and the recombinant protein (Pso o 1) purified using a nickel-affinity column. The recombinant Pso o 1 was tested for recognition by immunoglobulin (Ig)G and IgE in serum from P. ovis naïve and P. ovis infested sheep. Using Western blots, both classes of antibody to Pso o 1 were detected in postinfestation serum. In enzyme-linked immunosorbent assays, a pronounced IgG-antibody response to Pso o 1 was detected in five of five sheep 3 weeks postinfestation. The IgE-antibody response to whole mite extract was poor in four of five animals. However, a marked IgE response occurred in the fifth animal, and IgE anti Pso o 1 was detected in the serum.

Allergens↗

Studies on the immunoglobulin E responses to Teladorsagia circumcincta in sheep: purification of a major high molecular weight allergen.

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.

Allergens↗

Age-related protective immunity after vaccination with Haemonchus contortus excretory/secretory proteins.

Protection against an experimental challenge infection by immunization with excretory/secretory products (ES) from Haemonchus contortus, containing predominantly proteins of 15 and 24 kDa, depends on the age of the sheep. Vaccinated sheep 9 and 6 months of age had reduced final worm burdens of 82 and 77, respectively. No reduction in worm burden was found in 3-month-old lambs. Nine-month-old sheep had significantly higher ES-specific serum immunoglobulin (Ig)G1 and IgA during immunizations and after challenge infection than 3-month-old lambs. There was no correlation within the 9-month-old sheep between ES-specific IgA or IgG1 levels and protection, measured as worm burden. However, when the different age groups were combined, negative correlations between percentage protection and ES-specific IgA and IgG1 levels after challenge were found. At the end of the experiment, peripheral blood eosinophils and mast cell counts in abomasal tissue were also significantly higher in the vaccinated and challenged 9-month-old sheep than in the vaccinated and challenged 3-month-old or than in the 9-month-old sheep with challenge, but without vaccination. The responses measured in young lambs were similar to the responses in sheep, but the height of these responses was in general of a lower magnitude.

Age Factors↗

Characterization of ovine nasal-associated lymphoid tissue and identification of M cells in the overlying follicle-associated epithelium.

The distribution of mucosal lymphoid nodules in the ovine nasopharyngeal tract was studied by an acetic acid fixation technique. Nodules, which were concentrated just posterior to the opening of the Eustachian tube, were excised and examined by light microscopy, and scanning and transmission electron microscopy. Immunohistochemical examination revealed that each lymphoid structure consisted of follicles containing discrete B- and T-cell areas, characteristic of a mucosal inductive site of the mucosa-associated lymphoid tissue (MALT). Electron microscopy revealed that specialized epithelial cells, displaying features characteristic of M cells, were present in the follicle-associated epithelium (FAE) that covered the lymphoid nodules. These cells had sparse irregular microvilli and were closely associated with lymphocytes in the underlying tissue. These findings suggest that targeting the nasopharyngeal region may provide a practical and effective route for the stimulation of protective mucosal immune responses.

Acetic Acid↗

Can an increased intake of metabolizable protein affect the periparturient relaxation in immunity against teladorsagia circumcincta in sheep?

Within the overall hypothesis that the periparturient relaxation of immunity (PPRI) to gastrointestinal parasites has a nutritional basis, we studied whether an increased intake of metabolizable protein (MP) could affect the expression of immunity to Teladorsagia (Ostertagia) circumcincta of twin-bearing and -rearing Greyface ewes. Two sets of iso-energetic diets were formulated to provide either 85% (L-) or 130% (H-) of the MP requirements during the last 6 weeks of pregnancy (-P) and the first 6 weeks of lactation (-L). Thirty ewes were allocated to LP or HP. Six ewes of each group were slaughtered after lambing (S(0)); the remaining 24 ewes of each group were allocated to either LL or HL, thus creating four treatment combinations during lactation (LP/LL, LP/HL, HP/LL and HP/HL; n=12). Six ewes of each treatment combination were slaughtered at Week 3 (S(3)) or at Week 6 (S(6)) in lactation. The HP ewes were heavier at lambing, produced heavier lambs, had lower faecal egg counts (FECs), a lower concentration of plasma pepsinogen, a higher concentration of plasma albumin during late pregnancy and more abomasal globule leukocytes (GLs) but similar worm burdens at S(0) compared to the LP ewes. The LP/HL and HP/HL ewes had a higher calculated milk production than the LP/LL and HP/LL ewes. FECs increased for each treatment combination during the first 3 weeks of lactation. This increase concurred with fewer abomasal GLs at S(3) compared to S(0). The LP/HL and HP/HL ewes had a higher proportion of immature worms and more abomasal GLs than the LP/LL and HP/LL ewes at S(6). The FECs continued to increase during the second 3 weeks of lactation for the LP/LL ewes only, which concurred with an elevated plasma pepsinogen concentration and an absence of abomasal GLs at S(6). The plasma pepsinogen concentration decreased rapidly for the LP/HL ewes during lactation. We conclude that an increased intake of MP could enhance the expression of immunity to T. circumcincta of twin-bearing and -rearing Greyface ewes.

Animal Feed↗

Drug-abbreviated infections of Trichostrongylus colubriformis and development of immunity in jirds (Meriones unguiculatus).

The objective of this study was to examine the development and the duration of immunity achieved with drug-abbreviated infections of Trichostrongylus colubriformis in jirds (Meriones unguiculatus). Jirds were primarily infected either by trickle infection with 6 x 100 infective larvae (L3) of T. colubriformis at 3-day intervals or by a single infection with 600 L3. On day 35 post-infection, one batch of jirds from each group was autopsied; the others were treated with oxfendazole at a dose of 5 mg/kg and were challenged with 1,000 L3 on either day 7 or day 42 post-treatment. All jirds were autopsied at 17 days post-challenge. Trickle infection resulted in lower levels of egg production during the primary infection period. The systemic IgM and IgG antibody response was significantly stronger in trickle- and single-infected groups as compared with the negative control group (P < 0.01-P < 0.05). Significantly higher levels of intestinal IgA were demonstrated in trickle- and single-infected groups than in the negative control group (P < 0.01). Numbers of mucosal mast cells increased following infection, but this was not dependent on the type of immunisation. After challenge the extent of worm reduction was greater in trickle-infected than in single-infected subgroups. The IgM and IgG response was significantly stronger in challenged subgroups as compared with negative control subgroups (P < 0.01). However, the IgG response was weaker in control challenged subgroups than in challenged subgroups (P < 0.01). There was a negative correlation between the IgG response and the worm burden after the second challenge (r = -0.73). The acquired immunity to T. colubriformis infection in jirds developed within 5 weeks of primary infection. The level of immunity was higher after trickle infection than after single infection. Furthermore, the immunity persisted for at least 6 weeks after oxfendazole treatment in the absence of a worm burden and larval intake, which is very similar to the situation in domestic ruminant hosts.

Animals↗

cDNA sequence of two sheep mast cell tryptases and the differential expression of tryptase and sheep mast cell proteinase-1 in lung, dermis and gastrointestinal tract.

BACKGROUND: Mast cell tryptases are a family of serine proteinases which are implicated in the proliferation of smooth muscle cells and fibroblasts, upregulation of interleukin-8 synthesis by endothelial cells, and recruitment of neutrophils and eosinophils. Trials in sheep showed that administration of a specific tryptase inhibitor reduced the late-phase response to inhaled allergen. OBJECTIVES: The aim of this study was to characterize the sequence and distribution of sheep tryptase(s), to validate the sheep model of allergic lung disease. METHODS: Reverse transcriptase PCR cloning was used to obtain cDNA sequences for two sheep tryptases. Lung and gut extracts were used as a source of tryptase for partial purification and characterization of the protein. The distribution of tryptase in skin, lung and gut was determined by immunohistochemistry, and compared with the distribution of sheep mast cell proteinase-1 (sMCP-1). RESULTS: Two highly similar cDNA sequences encoding sheep tryptase were found, indicating the presence of a 28 amino acid leader sequence, and a mature peptide of 245 amino acids. Partial purification of a putative sheep tryptase from lung and gut extracts was achieved using heparin-Sepharose affinity chromatography. Rabbit antihuman skin tryptase antiserum recognized the putative sheep tryptase on Western blot (approximate Mr 32-34 000) and paraformaldehyde-fixed tissue sections. Tryptase was detected in all lung, skin and gut mast cells by this antibody, and transcripts for tryptase were detected in all three tissues by RT PCR. Sheep mast cell proteinase-1, detected by a specific monoclonal antibody, was present in all intestinal and gastric mucosal mast cells, but was not found in mast cells of the muscularis, thus defining at least two mast cell phenotypes in the gut. Whereas all dermal and pulmonary mast cells were tryptase positive, only a low proportion in the lung, and almost none in the dermis, were positive for sMCP-1. CONCLUSION: In view of the structural and functional similarities of sheep and human tryptases, and their similarity in tissue distribution in normal sheep, the sheep lung appears to be a good model for in vivo studies relating to human tryptase.

Amino Acid Sequence↗

Cutaneous and systemic responses during primary and challenge infestations of sheep with the sheep scab mite, Psoroptes ovis.

Lesional area was estimated and lesional mast cells and eosinophils were counted during primary and challenge infestations of sheep with Psoroptes ovis. In addition, circulating basophils and eosinophils and serum P. ovis-specific immunoglobulin IgE antibody were quantified. Expansion of lesional area was significantly less and serum IgE titres significantly greater in challenge than primary infestations. Lesional mast cell hyperplasia and massive eosinophil infiltration accompanied by raised titres of P. ovis antigen-specific IgE antibody were compatible with an IgE-mediated Type-1 allergic reaction, while detection of lesional basophils was suggestive of cutaneous basophil hypersensitivity. The temporal pattern of lesional mastocytosis and eosinophil infiltration and the role of these cells and serum IgE in the immune response is discussed.

Animals↗

Sheep mast-cell proteinases-1 and -3: cDNA cloning, primary structure and molecular modelling of the enzymes and further studies on substrate specificity.

Sheep mast-cell proteinase-1 (sMCP-1) is a serine proteinase expressed predominantly by mucosal mast cells, with specificity for cleavage C-terminal to basic and hydrophobic amino acid residues. A cDNA encoding sMCP-1 has been cloned using reverse transcriptase (RT)-PCR. It appears to be translated as a pre-proenzyme with a 17-amino-acid signal peptide, a basic 2-amino-acid propeptide and a 226-amino-acid catalytic domain. A second cDNA, encoding a serine proteinase 90% identical with sMCP-1, was also cloned and named sMCP-3. Molecular models were constructed for both enzymes using coordinates for the refined X-ray structures of human cathepsin G, chymase and rat mast-cell proteinase-2. The model for sMCP-1 suggests that the acidic Asp-226 side chain extends into the substrate-binding pocket, hydrogen-bonding with Ser-190 on the opposite side and bisecting the pocket. The location of an acidic moiety in this position would favour interaction with basic substrate residues and binding of aromatic residues is rationalized by interaction of the positively charged equatorial plane with Asp-226. The balance between chymotryptic and tryptic activities of sMCP-1 was found to be sensitive to salt concentration, with increasing univalent cation concentration favouring chymotryptic activity relative to the tryptic. Using a peptide substrate representing residues 36-59 of the human thrombin receptor, increasing salt concentration favoured cleavage at Phe-43 rather than at Arg-41.

Amino Acid Sequence↗

Pathogenicity and immunogenicity of different isolates of Teladorsagia circumcincta.

The effects on liveweight gain and development of immunity were studied in lambs trickle infected for 8 weeks with either a benzimidazole-resistant isolate (Moredun ovine resistant isolate, MORI), a multiple benzimidazole + ivermectin-resistant isolate (Moredun caprine resistant isolate, MCRI) or an unselected susceptible isolate (Moredun ovine susceptible isolate, MOSI) of Teladorsagia circumcincta. Plasma pepsinogen concentrations of infected groups were significantly elevated compared to an uninfected control group (P < 0.001) by day 14. The liveweight gains varied markedly but there were no statistical differences between the infected and uninfected control groups at any point in time during the study. Lambs infected with the MORI had significantly lower faecal consistency scores than the other challenged groups on days 7 and 14 (P < 0.05) but from day 21 onwards, faecal consistencies were similar in all of the groups. There was a notable difference in the pre-patent periods of the different isolates with the MOSI producing positive faecal egg counts (FECs) by day 14 of the study. The FECs remained reasonably low once infections had reached patency and there were no further differences between the groups. Following administration of anthelmintic to remove residual worms from the trickle infection, no differences between the infected groups in terms of worm burden or mucosal mast cell numbers were evident as a consequence of a single challenge infection. The changes in genetic code associated with enhanced resistance against anthelmintics do not appear to have resulted in any fundamental alteration of the pathogenicity and immunogenicity of these three isolates of Teladorsagia.

Animals↗

Differential inhibition of mast cell chymases by secretory leukocyte protease inhibitor.

The major physiological role of human secretory leukocyte protease inhibitor (SLPI), a low molecular weight inhibitor present in mucus, is the rapid formation of a tight-binding inhibitory complex with neutrophil elastase. It is also the most effective known inhibitor of human mast cell chymase. The inhibitory efficacy of recombinant SLPI towards three other mast cell chymases was therefore investigated. Rat mast cell proteinases-1 and -2 (rMCP-1 and -2, respectively) and sheep mast cell proteinase-1 (sMCP-1), a chymase with additional tryptase-like properties, were treated with the inhibitor. SLPI inhibited rMCP-1 very efficiently in the absence of heparin, with a low dissociation constant, Ki = 3 x 10(-10) M and high second order association constant, kass = 8.0 x 10(6) M(-1) s(-1), and inhibition was enhanced when heparin was present. rMCP-2 was not inhibited by SLPI in the presence or absence of heparin, and did not degrade SLPI on prolonged incubation. SLPI inhibited sMCP-1 very poorly in the absence of heparin (Ki = 9 X 10(-6) M). However, in the presence of heparin, the Ki for inhibition of sMCP-1 by SLPI was reduced to the nanomolar range. sMCP-1 was observed to cleave SLPI with chymase-like specificity at Leu72-Met73 on prolonged incubation in the absence of heparin, but increasing concentrations of heparin reduced the extent of cleavage.

Amino Acid Sequence↗

IgE responses in the serum and gastric lymph of sheep infected with Teladorsagia circumcincta.

The IgE response of naive or previously infected sheep to 50,000 infective larvae of Teladorsagia circumcincta was monitored in serum and gastric lymph using a monoclonal antibody generated to recombinant ovine IgE in a dot blot assay. In 4/5 naive sheep, lymph and serum IgE concentrations increased from days 8 and 14 after infection, respectively. In most previously infected sheep, the IgE response to challenge was more rapid, although not necessarily greater than that following a primary infection. IgE concentrations in lymph were some 4-fold higher than in serum indicating that its source was the mucosa or draining nodes.

Animals↗

IgE antibody during infection with the ovine abomasal nematode, Teladorsagia circumcincta: primary and secondary responses in serum and gastric lymph of sheep.

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.

Animals↗

The effects of recombinant ovine interleukin-3 and recombinant ovine stem cell factor on the growth and mediator expression of caprine and ovine bone marrow-derived mast cells.

The growth of ovine and caprine mast cells in bone marrow cultures has been achieved using recombinant ovine interleukin-3 (rOvIL-3) and recombinant ovine stem cell factor (rOvSCF). After approximately 2-3 weeks' growth in optimal concentrations of either rOvIL-3 alone or a combination of rOvIL-3 and rOvSCF, the majority of the cells produced in bone marrow culture from both species were mast cells. The significant increase in the total numbers of cells and survival times of the cultures when both cytokines were present compared to either alone, indicated synergy between rOvIL-3 and rOvSCF on mast cell growth. Ovine and caprine cells cultured in rOvIL-3 alone produced a four-fold increase in cell numbers compared with medium only controls. The resulting cultures contained up to 52% mast cells by day 18 and had a lifespan of 3-4 weeks. In contrast, cells from both species grown in both rOvIL-3 and rOvSCF produced up to six times more cells than the equivalent rOvIL-3 stimulated cultures, contained up to 69% mast cells by day 21 and could be maintained for at least 6 weeks. Ovine cells grown in rOvIL-3 alone or rOvIL-3 and rOvSCF contained significantly more aryl-sulfatase and serine protease but similar amounts of beta-hexosaminidase compared with caprine cells during the second week of culture. There were no significant differences in the granule-associated mediator content of cells from either individual species grown in rOvIL-3 alone compared with those grown in rOvIL-3 and rOvSCF during the first 21 days of culture.

Animals↗