Influence of environmental and genetic factors on allergen-specific immunoglobulin-E levels in sera from Lipizzan horses.

To investigate whether allergen-specific IgE production is influenced by environmental and genetic factors, IgE levels against 2 mould extracts (Alternaria alternata [Alt a] and Aspergillus fumigatus [Asp f]) and against recombinant (r) rAlt a 1, rAsp f 7 and rAsp f 8 were determined by ELISA in sera from 448 Lipizzan horses living in 6 studfarms. Statistical evaluation showed a significant effect of studfarm-specific environment on IgE levels against the different allergens, but genetic factors also influenced allergen-specific IgE production: an heritability of 0.33 was found for IgE levels against the 2 mould extracts and of 0.21 for rAsp f 8-specific IgE. Heritability estimates for rAlt a 1- and rAsp f 7-specific IgE were negligible. Investigations for a possible association between Major Histocompatibility Complex (MHC) class I antigens and specific IgE levels were carried out. The most consistent significant association was found between the equine leucocyte antigen (ELA) A8 and undetectable IgE titres against rAsp f 7 and rAsp f 8. Significant ELA associations were also demonstrated between ELA A1 and higher specific IgE levels, between ELA A14 and lower IgE levels against the mould extracts and in one studfarm between ELA Be27 and lower Aspergillus-specific IgE levels.

Allergen-specific IgE levels against crude mould and storage mite extracts and recombinant mould allergens in sera from horses affected with chronic bronchitis.

Immunoglobulin E antibody (IgE) levels against four recombinant (r) mould allergens (r-Aspergillus fumigatus [rAsp f] 7, 8 and 9; r-Alternaria alternata 1 [rAlta1]) and crude mould (Aspergillus fumigatus, Alternaria alternata, Penicillium notatum) and storage mite extracts were determined by ELISA in sera from 24 pulmonary sound control horses and 26 horses suffering from chronic bronchitis/bronchiolitis (CB), also called chronic obstructive pulmonary disease (COPD). Serum IgG and IgA titres were also determined against Aspergillus fumigatus extract and rAsp f 8.IgE against the crude extracts could be measured in all sera, but there was no significant difference between CB-affected and control horses. In contrast, only 8-30% of the horses, depending on the r-allergen tested, had detectable IgE levels in serum against the r-allergens. Horses with CB had significantly more often detectable IgE levels than controls against rAlt a 1 (10/26 and 3/24, respectively, p=0. 054), rAsp f 7 (13/26 and 2/24, respectively, p<0.01) and rAsp f 8 (11/26 and 1/24, respectively, p<0.01). Only four horses (three CB-affected and one healthy, p0.05) had detectable IgE levels against rAsp f 9. Furthermore, CB-affected horses were often sensitised against two or more r-allergens (13/26 of the CB-affected horses) while only one of the 24 healthy horses had positive IgE levels against more than one r-allergens. Similarly to IgE levels, no significant differences between CB-affected and healthy horses were found for IgG titres against the Aspergillus fumigatus extract. However, horses with CB had significantly higher serum IgG titres against rAsp f 8 than healthy controls (median=28 versus 10 relative ELISA units [REU], p<0.01). Additionally, horses with detectable IgE titres against rAsp f 8 had significantly higher IgG titres against this r-allergen than horses with undetectable IgE titres (median IgG titres=46 and 13 REU, respectively; p<0.01). For serum IgA titres, neither differences between healthy and CB-affected animals nor correlations between IgA and IgG or IgE titres could be found. These results show that horses suffering from CB are more often sensitised to some Aspergillus fumigatus and Alternaria alternata allergens than control horses and that they are partly sensitised to the same fungal proteins as mould-allergic human patients. Furthermore, this study shows that r-allergens allow a much more sensitive determination of specific serum antibody levels by ELISA than crude mould extracts.

Sulfidoleukotriene generation from peripheral blood leukocytes of horses affected with insect bite dermal hypersensitivity.

Sulfidoleukotrienes (sLT) generated in vitro after incubation of equine peripheral blood leukocytes (PBL) with different inducing agents were determined in 18 healthy and 16 insect bite dermal hypersensitivity (IDH)-affected horses. PBL from these 32 horses were stimulated with Concanavalin A, Parascaris equorum, Culicoides nubeculosus and Simulium extracts, and with a six-Grass mix. The cells of all but four horses generated sLT after incubation with Concanavalin A; these four horses did also not produce sLT with the other inducing agents. Of the 28 remaining horses (12 affected with IDH and 16 healthy), all but three generated sLT with the P. equorum extract. The six-Grass mix did not induce sLT production in any of the tested horses. sLT generation with Concanavalin A and Parascaris was statistically not different between IDH-affected and healthy horses. PBL of the diseased horses, however, produced significantly more sLT with the Culicoides (p < 0.01) and Simulium (p < 0.05) extracts than those of the healthy animals. Additionally, sLT generation with the Culicoides extract was measured at different times of the year in one IDH-affected animal and remained high even in winter, when the horse was asymptomatic. sLT and histamine release were determined in 10 horses in parallel. Positive correlations of 0.81 and 0.82 for Concanavalin A and Parascaris (p < 0.01 and p < 0.05, respectively), and of 0.95 and 0.94 for Culicoides and Simulium (p < 0.01) were found between sLT and histamine release. These results indicate that, alike in humans, sLT are released in vitro from equine basophils along with histamine in response to various stimuli and that immediate type hypersensitivity reactions to Culicoides and Simulium are often involved in the pathogenesis of IDH. Thus, sLT generation from equine basophils offers an in vitro diagnostic tool for IDH even in sensitised but asymptomatic horses.

Domain mapping and comparative binding features of eight dog IgE-specific reagents in ELISA, immunoblots, and immunohistochemistry.

Eight dog IgE-specific reagents including monoclonal and polyclonal antibodies (Ab) and a cross-reactive alpha chain of the human high affinity IgE receptor were mapped to recombinant fragments of the second (IgEf2) and third/fourth (IgEf3/4) domains of the dog IgE heavy chain. In ELISA, five out of eight reagents reacted to solid-phase bound IgEf2, of which two polyclonal Ab bound in addition to IgEf3/4. All Ab which recognized at least one recombinant IgE fragment, also bound to IgE in ELISA, immunoblots, and immunohistochemistry. In contrast, only one monoclonal Ab, that did not bind to the recombinant IgE fragments, reacted with immunoblots of serum and immunohistochemistry. The alpha chain could only be applied to ELISA with serum IgE. Furthermore, there was a wide range of heat-lability of binding reactions. Comparative analysis of available dog IgE-specific reagents enables more in-depth functional studies on IgE-mediated phenomena in dogs, and helps to further establish the dog as an animal model for allergy research.

Influence of sex and age on serum total immunoglobulin E concentration in Beagles.

OBJECTIVE: To establish an ELISA for detection of serum total IgE concentration in dogs and to analyze IgE values in a dog colony. ANIMALS: 147 healthy Beagles (31 males and 116 females). PROCEDURE: 2 canine IgE-specific polyclonal antibodies elicited by 2 recombinant fragments of the epsilon chain in hens were used to develop a capture ELISA specific for serum total IgE concentration. The IgE values were calculated by comparing serum dose-response curves (1:50 to 1:6,400) with a reference serum pool assigned 100 relative ELISA units (REU). Results-Mean IgE concentration in female Beagles was 51.2 REU (range, 0 to 337.8 REU; median, 31.4 REU), whereas mean IgE concentration in male dogs was only 7.5 REU (range, 0 to 32.6 REU; mean, 3.6 REU). Distribution of IgE values was skewed; approximately 80% of dogs had IgE values < 50 REU. Analysis of natural logarithmically transformed IgE values indicated that sex and age significantly (P < 0.05) influenced IgE values; mean serum IgE values increased until the age of 4 years. Heritability estimates of IgE concentration indicated a trend toward a genetic influence. CONCLUSION: A reliable capture ELISA specific for canine IgE was developed. Serum total IgE values vary with age and sex in the sample population. CLINICAL RELEVANCE: Serum total IgE concentration can now be evaluated in various dog breeds and, subsequently, in dogs with IgE-mediated diseases provided that these significant influences are accounted for. Serum total IgE values may then prove to be of diagnostic value, similar to their use in human beings.

Polymorphism of Old Kladruber horses, a surviving but endangered baroque breed.

Analysis of MHC class I and class II polymorphism, as well as data from other polymorphic systems (non-MHC lymphocyte alloantigen, blood groups systems, biochemical polymorphisms and microsatellite loci), was used to characterize the extent and distribution of the genic polymorphism of Kladruber horses. A breed-characteristic distribution of the MHC polymorphism was found. The repertoire of defined MHC class I specificities was restricted, especially in the grey subpopulation and in stallions, but a high frequency of blanks suggests the possible existence of undetected specificities. Despite the small population size and a relatively high degree of inbreeding, high heterozygosity in MHC haplotypes has been conserved. The extent of polymorphism and the degree of heterozygosity in other loci were also relatively high. A comparison of the two existing subpopulations, grey and black, at all the loci tested, including RAPD markers, characterized them as genetically distinct, although clearly related. The genetic distances between them were of the same order of magnitude as between distinct breeds. The results may be useful in defining short-term and long-term breeding policy within the breed and for further studies of associations with disease and other traits.

Caprine T-cell receptor variable beta-chain (TCRV beta) repertoire analysis and potential applications in cowdriosis immune response studies.

Anchor polymerase chain reaction has been applied to the study of caprine TCR V beta-chain repertoire at the mRNA level in peripheral blood of a Saanen goat. Single stranded, g-tailed cDNA synthesized from total RNA was PCR-amplified using a sheep V beta constant region primer (3') and a poly(dC) anchor primer (5') at the variable region end of the TCR V beta-chain. The obtained amplicon was subsequently cloned into Bluescript plasmid vector. A total of 72 recombinant clones whereof 61 contained an insert of appropriate size were harvested. Up to now, the full length sequences of a total of 55 clones have been obtained. Nine clones were rearranged but not functional due to stop codons. Forty-five sequences were functionally rearranged and further analyzed. They were classified into 15 different V beta families on the basis of V-region sequence homology with their human counterpart. V beta families corresponding to the 9 published bovine families were represented in our library. This complexity enables us to develop V beta family-specific primers in order to study the TCR V beta repertoire of other goat breeds of interest, i.e., the Creole goat. There the TCR V beta repertoire analysis and kinetic study of the cowdriosis model will provide insight into the type of the immune response and the status of protection during the immunization process and challenge.

Characterization of two dog IgE-specific antibodies elicited by different recombinant fragments of the epsilon chain in hens.

Two recombinant [His]6-tagged fragments of the canine immunoglobulin E (IgE) heavy chain (second domain: IgEf2 and third and fourth domains: IgEf3/4) were cloned, expressed in Escherichia coli (E. coli) as [His]6-tagged proteins, and affinity-purified over nickel-nitrilotriacetic acid columns. The recombinant proteins were used to immunize hens. The raised and affinity-purified chicken antibodies (Ab) isolated from egg yolk exhibited specific binding to the respective recombinant canine IgE fragment (IgEf) on immunoblots and displayed high titers against the IgEf in ELISA. Immunoblotting of canine serum separated by PAGE under native conditions with the IgEf2- and IgEf3/4-specific Ab resulted in staining of a protein of approximately 180 kilodaltons (kD). The IgEf3/4-specific Ab further recognized an 80 kD protein in IgEf3/4-specific Ab affinity-enriched dog serum separated under denaturing conditions. In an ELISA for the detection of antigen-specific IgE in dog serum, reduced binding of the IgEf-specific Ab was observed after heat treatment of the dog serum. The reactivity of both of the raised chicken Ab was only present in postimmune reagents and could only be inhibited by preincubation with the IgEf used for immunization and not with dog immunoglobulin G, E. coli extract, or with a nonrelevant recombinant [His]6-tagged protein. In immunohistochemistry, the IgEf3/4-specific Ab specifically recognized cells in paraffin-embedded tissue sections of lymph nodes. Furthermore, both of the IgEf-specific Ab elicited positive immediate type 1 skin reactions in dogs. Semiquantitative assessment of total serum IgE in dogs was developed using IgEf2-specific Ab as coating reagent and the biotinylated IgEf3/4-specific Ab as developing Ab in ELISA. In conclusion, both IgEf-specific Ab recognize native dog IgE with the advantages that they are directed against different and known constant domains of the IgE molecule, and that they can be used for immunohistochemistry on paraffin-embedded tissue. The two dog IgE-specific Ab could initiate clinical research on the involvement of immediate-type hypersensitivity reactions in dogs.

[The therapy of equine sarcoid with a non-specific immunostimulator--the epidemiology and spontaneous regression of sarcoids]. / Zur Therapie des Equinen Sarkoids mit einem unspezifischen Immunstimulator-Beitrag zur Epidemiologie und zur spontanen Regression des Sarkoids.

20 sarcoid-affected horses from a practice in the northern Jura were used in this experiment. The mean age of the 20 horses was 3.9 years at the time of the first observation of sarcoid tumors. On the average, 4.4 tumours were noted per horse. 10 of the horses were treated in a double-blind study with an unspecific immunostimulant (Baypamun P), 10 others received a placebo. One single tumour only was treated per horse. The injections were given under and around the sarcoid. In eight out of the 20 horses all tumours regressed totally or for more than 50% of their initial size. Five of these had received placebo, three the immunostimulant. Four animals showed a modest, but measurable reduction in tumour size (3 immuno-stimulant, 1 placebo) and in the remaining eight horses (4:4) no reduction or even an increase in tumour size was observed. The phenomenon of tumour regression was very probably due to spontaneous regression and horses which had been observed to develop sarcoid within the last three months had significantly more regressions than animals with older tumours (p < 0.05). The haplotype of the equine leucocyte antigens was thought to predispose 12 of the 20 horses for the sarcoid. However, the ELA-type did not measurably influence the phenomenon of tumour regression.

Chicken antibodies to a recombinant fragment of the equine immunoglobulin epsilon heavy-chain recognising native horse IgE.

An equine immunoglobulin E (IgE) heavy-chain cDNA fragment (CH3-CH4, nucleotides 1132 to 1592) was cloned, expressed in Escherichia coli as a fusion protein with a [His]6-tag and purified over a Ni-NTA column. The recombinant protein was used to immunise hens. Testing of the raised egg yolk immunoglobulin G (IgG) in Western-blot and ELISA revealed high titres against the recombinant equine IgE fragment (reqIgEf). The reqIgEf-specific IgG was successfully affinity-purified on an unconventional affinity matrix: the [His]6-tagged recombinant IgE fragment was bound to Ni-NTA agarose and used to adsorb specific immunoglobulins. In Western-blot of ammonium sulphate precipitated horse serum and bronchoalveolar lavage fluid, separated by SDS-PAGE under denaturing-reducing conditions, the raised antibodies reacted with a protein of approximately 80 kDa. A reaction of the reqIgEf-specific IgG was seen with a 190-200 kDa band when the same horse serum or bronchoalveolar fluid (BALF) was separated under non-reducing conditions. These reactions could be inhibited by preincubation of the immune IgG with reqIgEf, while preincubation with horse IgG did not inhibit the reaction. Antibody-affinity chromatography of horse serum with the reqIgEf-specific chicken IgG resulted in an enrichment of the 80 kDa protein in denaturing Western-blot. Determination of the amino acid composition of this protein and comparison with the equine IgE heavy- chain sequence strongly indicates that the 80 kDa band corresponds to the heavy chain of the horse IgE. The reqIgEf-specific chicken IgG was further characterised in an ELISA for the detection of allergen-specific horse IgE. It was demonstrated that heating IgE positive horse sera at 54 degrees C for 10 min drastically diminished the recognition by the reqIgEf-specific chicken IgG. The reaction is inhibitable by preincubation with reqIgEf in a concentration dependent manner. In addition, preincubation with horse IgG, a nonrelevant [His]6-tagged protein or 2% equine colostrum had no influence on the reqIgEf-specific chicken IgG binding characteristic. This antibody recognising horse IgE will be useful for further studies on the pathogenesis of equine allergic diseases.

Lack of association of bovine MHC class I alleles with carcass and reproductive traits.

The present study was carried out to examine whether a relationship between bovine major histocompatibility complex (BoLA) class I alleles and carcass traits or reproductive performance exists in Braunvieh and Fleckvieh AI (artificial insemination) bulls. The influence of BoLA class I (BoLA-A) alleles on deregressed breeding values for net growth rate, carcass index and thigh volume was assessed in Braunvieh crosses and Fleckvieh bulls with a gene substitution model. The reproductive traits: non-return rate and interval between first and last insemination of daughters (female fertility), as well as non-return rate of inseminated cows (male fertility), were only investigated in Fleckvieh animals. No influence of the BoLA-A region on the traits evaluated could be demonstrated. An improper, i.e. less restrictive analysis would have led to spurious results.

Tumour suppressor gene p53 in the horse: identification, cloning, sequencing and a possible role in the pathogenesis of equine sarcoid.

The tumour suppressor protein p53 enhances the genetic stability of the cell and plays a critical role in tumour suppression. Equine p53 was analysed by sequencing exons 5 to 9, a region which includes most known mutations and all the mutational hotspots in the species that have been investigated. The fragment was amplified, cloned and sequenced from genomic and complementary DNA. A comparison of the predicted amino acid sequences between the horse and other species resulted in identities between 66 per cent with the clawed frog and 92 per cent with the cat. Using the single strand conformation polymorphism technique, exons 5 to 8 amplified from sarcoid tissue and peripheral leucocytes of 28 sarcoid-affected and 11 healthy horses were screened for mutations. No mutations were identified, suggesting that the frequency of p53 mutations in equine sarcoid might be low. However, the high incidence of bovine papillomavirus (BPV) infection in equine sarcoid may indicate the functional inactivation of p53 by BPV-encoded E6 protein.

Partial sequence of the equine immunoglobulin epsilon heavy chain cDNA.

In order to isolate a part of the immunoglobulin E (IgE) heavy chain cDNA of the horse, primers have been designed based upon well conserved sequences in humans, sheep and rats. The PCR resulted in a 500 bp fragment which hybridised with a human IgE constant region probe. The fragment was cloned and sequenced and its derived protein sequence compared with the corresponding sequences in humans, sheep and mice. Most amino acids common to these three species are also shared by the horse.

Experimental treatment of equine sarcoid using a xanthate compound and recombinant human tumour necrosis factor alpha.

A xanthate compound with antiviral and antitumoural activities, tricyclodecan-9-yl-xanthogenate (D609) in combination with the potassium salt of the lauric acid (KC12) and, in a further investigation, the above-mentioned substances together with recombinant human TNF alpha (rh-TNF alpha), were tested on equine sarcoid tumours for therapeutic efficacy. A pilot investigation on 5 healthy horses showed that the compounds were well-tolerated; apart from a local, temporary oedema at the injection site, no other clinical symptoms were observed after subcutaneous administration of volumes from 0.1 to 10 ml per injection. The tested concentrations of D609 and KC12 (5 mg/ml solution) and of rh-TNF alpha (50 micrograms/ml) were used for the treatment experiments. The repeated injections of the compounds to 11 sarcoid affected horses were also well-tolerated, except by one horse. In this case the treatment had to be interrupted after two injections because of severe reaction, i.e. fever and lameness due to oedemas. Five horses (n = 6 sarcoids) were treated by local, subcutaneous injection of D609 and KC12 under the tumour at intervals of 3 weeks. On one periocular sarcoid the compounds were applied as an ointment. After a follow-up period of 18 months, 5 tumours did completely regress and one remained unchanged. The periocular tumour showed a reduction in size. Five horses (n = 9 sarcoids) were then treated with a combination of D609, KC12 and rh-TNF alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on the frequency and associations of equine leucocyte antigens in sarcoid and summer dermatitis.

The equine leucocyte antigen (ELA) types and the clinical diagnosis for equine sarcoid and summer dermatitis were evaluated in 2026 horses representing five breeds. Data were analysed in unrelated animals and in family material. In the case of equine sarcoid, a strong association was observed between the ELA class II DW13 antigen and its effect on Swiss (cP < 0.001), French (cP < 0.0001) and Irish (cP < 0.01) Warmblood horses. The class I antigen A3 occurred more frequently in sarcoid-affected French horses (cP < 0.001). These results confirm our earlier findings (Gerber et al. 1988). Among Freiberger horses, which lack the ELA DW13 and A3 specificities, a breed-specific class I antigen, ABe108, displayed an increased frequency (cP < 0.05) in the affected group. Among Arabian horses, a tendency for increased frequency of the A1 antigen was observed in the affected animals, but the number of affected horses is too small for statistical significance. The Mendelian segregation in diseased half-siblings by ELA DW13 heterozygous stallions showed a strong association (P < 0.0001) between the inherited DW13 antigen and susceptibility to the sarcoid effect. In the case of summer dermatitis, previously published data (Marti et al. 1992) have been extended. The ELA types in four multiple-case families, founded by the same stallion, were analysed for an association with the effect of sarcoid. Eight out of nine ELA-typed affected offspring inherited the paternal haplotype A15, DW23 in contrast to nonaffected offspring where three out of 12 displayed these antigens (P < 0.005).(ABSTRACT TRUNCATED AT 250 WORDS)

Possible association between a serologically detected haplotype of the bovine Major Histocompatibility Complex and subclinical mastitis.

SUMMARY: The incidence of subclinical mastitis in Simmental and Simmental-Red Holstein cattle in relation to the bovine major histocompatibility complex (BoLA) was investigated. Quarter milk samples of 166 cows consisting of fifteen halfsib groups of different ages and lactation stages were analysed for somatic cell counts (SCC) and bacteriological infection. From these data the udder health status (UHS) of the animal was determined. Each cow was typed serologically for BoLA class I and class II specificities. The statistical evaluation for UHS was performed using a logistic regression model. The effect of BoLA on SCC was estimated by least square analysis. Animals carrying the BoLA class II haplotype "b" were significantly more affected by subclinical mastitis than those without this haplotype. Breed group showed a significant influence on both UHS and corrected mean logSCC (P < 0.0001 resp. P < 0.05). Lactation stage had a significant effect on SCC but only a weak influence on UHS (P < 0.0001 resp. P < 0.13). ZUSAMMENFASSUNG: Mögliche Assoziation zwischen dem bovinen Haupthistokompatibilitätskomplex und subklinischer Mastitis In dieser Studie wurde die Beziehung zwischen dem bovinen Histokompatibilitätskomplex (BoLA) und der Prävalenz subklinischer Mastitis bei Simmentaler und mit Red Holstein eingekreuzten Simmentaler Kühen untersucht. Bei 166 Kühen, aus 15 Halbgeschwistergruppen, unterschiedlichen Alters und in verschiedenen Laktationsstadien, wurden Milchproben entnommen und auf Zellzahl (SCC) und bakterielle Infektion untersucht. Aus diesen Daten wurde ein Eutergesundheitsstatus (UHS) definiert. Jede Kuh wurde serologisch für BoLA Klasse I und Klasse II Spezifitäten typisiert. Die statistische Auswertung für den UHS erfolgte mit einem logistischen Regressionsmodel. Der Einfluß von BoLA-Haplotypen auf SCC wurde mit der Least Square Analyse ermittelt. Tiere mit dem Klasse II Allel "b" zeigten mehr Euterprobleme als Kühe ohne dieses Allel. Die Rassegruppe übte sowohl auf den UHS wie auch auf den korrigierten Mittelwert der Zellzahlen einen signifikanten Einfluß aus (P < 0.0001 resp. P < 0.05). Der Effekt des Laktationsstadiums auf die Zellzahl war signifikant, aber für den UHS wurde nur ein schwacher Einfluß des Laktationsstadiums festgestellt (P < 0.0001 resp. P < 0.13).

Serological and biochemical detection of B-cell antigens in goats.

The production of B-cell specific alloantisera by lymphocyte transfusion between class I matched, MLR positive goats is described. Furthermore, the usefulness of preceding IEF typing is demonstrated in the selection of immunization pairs. After suitable absorptions and cluster analysis the detected B-cell specific antigens were designated BeD1-BeD6. The specificity BeD3 could also be detected by two class II-specific murine anti-H2 monoclonal antibodies. IEF class II typing of 34 animals gave concordant results between the two techniques. One additional allelic variant could be detected by IEF typing. The detected products segregated in close linkage to the earlier described caprine class I antigens. One recombinant has been found in 78 offspring. The reactivity of cells in mixed lymphocyte cultures (MLC) was correlated with the compatibility of the test cells for their B-cell specific antigens. Three of the B-cell specific sera were characterized by immunoprecipitation. The precipitated antigens corresponded in molecular weight to MHC class II products as described in other species indicating that the here described caprine products are of class II nature.