IgE-bearing cells in bronchoalveolar lavage fluid and allergen-specific IgE levels in sera from RAO-affected horses

Recurrent airway obstruction (RAO) is a common condition in stabled horses characterized by small airway inflammation, airway neutrophilia and obstruction following exposure of susceptible horses to mouldy hay and straw and is thus regarded as a hypersensitivity reaction to mould spores. However, the role of immunoglobulin E antibodies (IgE) in the pathogenesis of RAO is unclear. We hypothesized that the number of cells with receptor-bound IgE in bronchoalveolar lavage fluid (BALF) and IgE levels in serum would be higher in RAO-affected than in healthy horses living in the same environment. Therefore, IgE-positive (+) cells were identified by immunocytochemistry on cytospins from BALF and counted. IgE levels against the mould extracts Aspergillus fumigatus (Asp. f.) and Alternaria alternata (Alt. a.) and the recombinant mould allergen Aspergillus fumigatus 8 (rAsp f 8) were measured by enzyme-linked immunosorbent assay (ELISA) in the sera of seven RAO-affected and 22 clinically healthy mature horses housed in the same conventional stable environment. After correcting for the number of neutrophils, there were no significant differences in IgE+ cells on cytospins from BALF between both groups of horses (5% versus 7%, P > 0.1). Serum IgE levels against the mould extracts were significantly higher in RAO-affected than in clinically healthy horses [median = 119 versus 66 relative ELISA units (REU), P < 0.05]. Furthermore, significantly more RAO-affected than healthy horses had detectable serum IgE against the recombinant allergen rAsp f 8 (4/7 and 3/22, respectively, P < 0.05). Age had no significant effect on BALF cell ratios or on specific serum IgE levels. These results show that high IgE levels against mould antigens are associated with RAO under controlled environmental conditions but ranges of mould-specific serum IgE levels overlapped too much between diseased and clinically healthy animals to be of any diagnostic value. Further studies are needed to assess whether IgE-mediated reactions contribute to the pathogenesis of RAO.     

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.     

In vitro allergy tests compared to intradermal testing in horses with recurrent airway obstruction

Recurrent airway obstruction (RAO) is a common condition in stabled horses characterised by small airway inflammation, airway neutrophilia and obstruction following exposure of susceptible horses to mouldy hay and straw and is thus regarded as a hypersensitivity reaction to mould spores. However, the role of IgE-mediated reactions in RAO remains unclear.The aim of the study was to investigate with a serological IgE ELISA test (Allercept?), an in vitro sulfidoleukotriene (sLT) release assay (CAST^®) and with intradermal testing (IDT) whether serum IgE and IgE-mediated reactions against various mould, mite and pollen extracts are associated with RAO. IDT reactions were evaluated at different times in order to detect IgE-mediated immediate type reactions (type I hypersensitivity reactions, 0.5–1 h), immune complex-mediated late type reactions (type III reactions, 4–10 h) and cell-mediated delayed type reactions (type IV hypersensitivity reactions 24–48 h).In the serological test, overall the control horses displayed more positive reactions than the RAO-affected horses but the difference was not significant. Comparison of the measured IgE levels showed that the RAO-affected horses had slightly higher IgE levels against Aspergillus fumigatus than controls (35 and 16 AU, respectively, p < 0.05), but all values were below the cut off (150 AU) of the test. In the sLT release assay, seven positive reactions were observed in the RAO-affected horses and four in the controls but this difference was not significant.A significantly higher proportion of late type IDT reactions was observed in RAO-affected horses compared to controls (25 of 238 possible reactions versus 12 of 238 possible reactions, respectively, p < 0.05). Interestingly, four RAO-affected but none of the control horses reacted with the recombinant mould allergen A. fumigatus 8 (rAsp f 8, p < 0.05), but only late phase and delayed type reactions were observed.In all three tests the majority of the positive reactions was observed with the mite extracts (64%, 74% and 88% of all positive reactions, respectively) but none of the tests showed a significant difference between RAO-affected and control animals. Our findings do not support that IgE-mediated reactions are important in the pathogenesis of RAO. Further studies are needed to investigate whether sensitisation to mite allergens is of clinical relevance in the horse and to understand the role of immune reactions against rAsp f 8.     

Equine insect bite hypersensitivity: immunoblot analysis of IgE and IgG subclass responses to Culicoides nubeculosus salivary gland extract

Insect bite hypersensitivity (IBH) is an allergic dermatitis of horses caused by IgE-mediated reactions to bites of Culicoides and sometimes Simulium spp. The allergens causing IBH are probably salivary gland proteins from these insects, but they have not yet been identified. The aim of our study was to identify the number and molecular weight of salivary gland extract (SGE) proteins derived from Culicoides nubeculosus which are able to bind IgE antibodies (ab) from the sera of IBH-affected horses. Additionally, we sought to investigate the IgG subclass (IgGa, IgGb and IgGT) reactivity to these proteins. Individual IgE and IgG subclass responses to proteins of C. nubeculosus SGE were evaluated by immunoblot in 42 IBH-affected and 26 healthy horses belonging to different groups (Icelandic horses born in Iceland, Icelandic horses and horses from different breeds born in mainland Europe). Additionally, the specific antibody response was studied before exposure to bites of Culicoides spp. and over a period of 3 years in a cohort of 10 Icelandic horses born in Iceland and imported to Switzerland. Ten IgE-binding protein bands with approximate molecular weights of 75, 66, 52, 48, 47, 32, 22/21, 19, 15, 13/12 kDa were found in the SGE. Five of these bands bound IgE from 50% or more of the horse sera. Thirty-nine of the 42 IBH-affected horses but only 2 of the 26 healthy horses showed IgE-binding to the SGE (p<0.000001). Similarly, more IBH-affected than healthy horses had IgGa ab binding to the Culicoides SGE (19/22 and 9/22, respectively, p<0.01). Sera of IBH-affected horses contained IgE, IgGa and IgGT but not IgGb ab against significantly more protein bands than the sera of the healthy horses. The cohort of 10 Icelandic horses confirmed these results and showed that Culicoides SGE specific IgE correlates with onset of IBH. IBH-affected horses that were born in Iceland had IgGa and IgGT ab (p< or =0.01) as well as IgE ab (p=0.06) against a significantly higher number of SGE proteins than IBH-affected horses born in mainland Europe. The present study shows that Culicoides SGE contains at least 10 potential allergens for IBH and that IBH-affected horses show a large variety of IgE-binding patterns in immunoblots. These findings are important for the future development of a specific immunotherapy with recombinant salivary gland allergens.     

Cloning and sequencing of a cDNA expressing a ribosomal P0 peptide from Culicoides nubeculosus (Diptera)

Insect bite dermal hypersensitivity (IBH) is an allergic dermatitis of horses caused by bites of Culicoides spp. and sometimes Simulium spp. The aim of the investigation presented here was to identify allergens causing IBH. A cDNA library expressing recombinant Culicoides nubeculosus proteins was screened using affinity-purified serum from an IBH-affected horse. Screening of the library resulted in identification of one immunoreactive clone. The sequence of the cDNA insert was determined and revealed a 600 bp insert with an open reading frame coding for a 78 amino acid long protein, called rCul n 1. Analysis of the deduced amino acid sequence revealed an identity of 67-78% to the C-terminal part of the 318 amino acid long ribosomal P0 protein from other Diptera. Furthermore, the 38 C-terminal amino acids displayed an identity of 57% with the C-terminal part of the acidic ribosomal protein P2 from Aspergillus fumigatus. The cDNA insert was subcloned and expressed as a [His]6-tagged protein in Escherichia coli and purified using Ni2(+)-chelate affinity chromatography. The 10kDa recombinant Cul n 1 protein bound the affinity-purified antibody fraction used for screening the expression library. Determination of IgE and IgG levels against rCul n 1 by ELISA in sera from 19 IBH-affected and 18 Swiss control horses and in sera from eight control horses living in Iceland showed no significant differences between the three groups of horses (median IgE levels = 60, 49 and 44 relative ELISA units, respectively). rCul n 1 did not induce sulfidoleukotriene (sLT) release from peripheral blood leukocytes of IBH-affected horses (N = 5), although sLT release was induced with the Culicoides whole body extract.     

Keratinopathogenic mould fungi and dermatophytes in healthy and diseased hooves of horses

Specimens of hoof horn from 187 horses were examined for a possible relationship between clinically affected hooves and the occurrence of pathogenic fungi. Specimens were taken from the coronary band and from the stratum externum and medium of the coronary horn and transferred on to Sabouraud dextrose agar, with and without cycloheximide, and incubated at 28 degrees C. Dermatophytes and mould fungi were identified by their macroscopic and microscopic characteristics. The 732 isolates could be assigned to 26 species of moulds, two different species of the dermatophyte Microsporum and three different species of the dermatophyte Trichophyton. Depending on their pathogenic potential they were assigned to three groups: (i) fungi known to be keratinopathogenic (Acremonium blochii, Alternaria alternata, Alternaria chlamydospora, Geotrichum candidum, Microsporum ferrugineum, Microsporum gypseum, Scopulariopsis brevicaulis, Trichophyton species, Trichophyton mentagrophytes, Trichophyton sch?nleinii, 57 isolates), (ii) a group of uncertain pathogenicity (223 isolates), and (iii) a group of non-pathogenic species (452 isolates). Eighty per cent of the samples from horses with hoof horn lesions and 66.7 per cent of the samples from horses with slightly affected hoof horn contained fungi of the keratinopathogenic group, whereas only 8.9 per cent of the samples from horses with healthy hoof horn contained fungi of this group. There were no significant correlations between the clinical data and the age, sex or breed of the horses or their bedding and hygiene. Twelve species of fungi were isolated from the air in the horses' stables, but none of them belonged to the keratinopathogenic group.     

"Haysickness" in Icelandic horses: precipitin tests and other studies

Blood samples were taken from 18 healthy horses (Group A), 15 horses clinically diagnosed to have "haysickness" ("farmer's lung") (Group B), 10 closely related horses (Group C) and 14 inbred horses (Group D). Precipitins in sera were measured by double gel diffusion test against Micropolyspora faeni, Thermoactinomyces vulgaris, Aspergillus fumigatus, Alternaria, Penicillium and Rhizopus species. In Group A, all the horses were precipitin negative except one with a faint reaction to Rhizopus species. In Group B all had precipitin against M faeni. One horse also had precipitins against Rhizopus species and another against A fumigatus. In Group C, seven of the 10 horses had precipitins against M faeni. Of these, five had a history of respiratory signs, but two horses with a faint reaction had no such history. In Group D, four out of 14 horses had positive precipitin tests against M faeni. Of these four horses, three also had a faint reaction to A fumigatus and one a faint reaction to Alternaria species. All were asymptomatic. These results indicate that "farmer's lung" in man and "haysickness" in horses are of the same origin. However, further studies are necessary to substantiate the diagnostic or prognostic value of these precipitin tests in equine practice. The question of whether hereditary factors play a role in causing this disease also warrants further studies.     

IgE and IgG antibodies in skin allergy of the horse

In horses, allergies have been characterized by clinical signs and/or intradermal (i.d.) allergen testing. Our aim was to find the first direct evidence that immunoglobulin E (IgE) mediates equine allergy. In addition, we tested the hypothesis that immediate skin reactions in horses can also be mediated by IgG. Anti-IgE affinity columns were used to purify IgE from serum of one healthy horse and three horses affected with summer eczema, an allergic dermatitis which is believed to be induced by Culicoides midges. A modified Prausnitz-Küstner experiment was performed in four clinical healthy horses by i.d. injection of the purified serum IgE antibodies. The following day, Culicoides allergen was injected at the same sites. Skin reactions were not observed in response to allergen alone, and in two horses after stimulation at any previous IgE injection site. However, the other two horses showed an immediate skin reaction at the previous injection sites of IgE obtained from allergic horses. In addition, purified monoclonal antibodies to various equine immunoglobulin isotypes were injected i.d. into six healthy horses. Immediate skin reactions were observed in response to anti-IgE (6/6 horses) and anti-IgG(T) injections (5/6 horses). The specificities of both antibodies for IgE and IgG(T), respectively, were confirmed by enzyme linked immunosorbent assays. The results provide the first direct evidence that IgE mediates classical Type-I allergy in horses and plays a major role in the pathogenesis of summer eczema. The data also suggest that IgG(T) can bind to skin mast cells and might contribute to clinical allergy.     

Humoral immune response in calves to single-dose, trickle and challenge infections with Fasciola hepatica

In cattle experimentally infected with Fasciola hepatica, parasite specific IgG1 and IgG2 responses were studied. Additionally parasite specific IgE production was assessed by the Passive Cutaneous Anaphylaxis reaction. The primary infection was administered either as a single-dose or as a trickle infection over a 4-week period. Animals were challenged 4 months later. Titres of IgG1 and IgG2 against excretory-secretory parasite products (FhESAg), and against a whole-worm extract (FhSomAg) were measured by enzyme-linked immunosorbent assay (ELISA) in relation to weight gain, serum hepatic enzyme levels, and fluke infection rate. At necropsy, the mean number of flukes recovered was similar in both infected groups. The two ELISAs specific for bovine IgG1 showed analogous sensitivity and specificity (92% and 94%). Cross-reactivity was observed towards Echinococcus granulosus, Cysticercus tenuicollis, and C. ovis but not towards C. bovis, Cooperia spp., and Ostertagia spp. FhESAg gave rise to apparently more stable specific IgG1 titres as compared to FhSomAg. Mean IgG1 titres were significantly higher in the single-dose-infected group than in the trickle-infected group during the early migratory phase of the infection (week 2 to week 4 (FhSomAg) or week 6 (FhESAg)). IgG2 values were consistently lower than IgG1 levels. The kinetic response of both isotypes yielded a similar pattern. Specific IgE antibodies were detected in cattle of both infected groups from week 2 post-primary infection (PPI) onwards. The mean serum glutamate dehydrogenase (GLDH) and gamma-glutamyl transferase (gammaGT) activities were significantly higher in the single-dose-infected group for 3 weeks around peak levels (12-14 weeks PPI and 14-16 weeks PPI for GLDH and gammaGT respectively). Western blotting revealed a major antigenic fraction in FhESAg (26-30 kDa) recognized specifically by sera from F. hepatica infected calves as early as 6-8 weeks PPI. Experimental challenge caused no statistically significant modification of any parameter (IgG1 and IgG2 titres, enzymatic activities, immunoblotting) used to monitor the course of the infection. No correlation was found between fluke size and number, and antibody titres, suggesting that IgG1 production has little protective effect against F. hepatica infection.     

Allergen-specific IgE in Icelandic horses with insect bite hypersensitivity and healthy controls, assessed by FcepsilonR1alpha-based serology

Insect bite hypersensitivity (IBH) and atopy can both be causes of pruritus in horses and are associated with allergen-specific IgE to biting insects and environmental allergens respectively. Information with respect to differences in IgE levels in diseased and healthy animals is crucial in enabling an understanding of the clinical relevance of results of allergen-specific IgE tests. The aim of this study was (i) to evaluate and compare levels of allergen-specific IgE, using an ELISA method, in Icelandic horses, with and without IBH, from Iceland and Sweden respectively; (ii) to investigate patterns of allergen-specific IgE to insects, pollens, moulds and mites in those groups of horses; and (iii) to investigate the clinical significance of employing two different cut-off levels for the ELISA. The study compromised a total number of 99 horses from Iceland and Sweden, with and without IBH, divided in 5 groups. Sera from the horses were analysed blindly with the use of Allercept , a non-competitive, solid-phase ELISA-test, designed to detect the presence of allergen-specific IgE in sera using the recombinant alpha chain of the high-affinity IgE receptor (FcepsilonR1alpha). The distribution of the ELISA values was shown for each insect, mould, mite and pollen allergen, in the different groups using 10th, 50th and 90th percentiles. The use of two cut-off levels, 150 EA and 300 EA, did not eliminate the false positives. Horses with IBH had a higher number of positive reactions, counting all the 29 allergens, than healthy controls and this was borderline significant (P=0.053). In this study it was shown that serological testing with an ELISA that uses the high-affinity IgE receptor (FcepsilonR1alpha) is presently not suitable as a tool for establishing a diagnosis of IBH or equine atopy. The importance of establishing a correct cut-off level for the ELISA for the different allergens is emphasised.     

Comparison of cellular and humoral immunoassays for the assessment of summer eczema in horses

The objective of this study was to compare and analyze three common diagnostic methods for summer eczema (SE) in horses, an allergic dermatitis caused by bites of Culicoides spp. Nine horses with a medical history of SE and nine control animals were intradermally challenged with whole body extracts (WBE) and the saliva of a native (C. nubeculosus) and exotic (C. sonorensis) Culicoides species. Blood and serum samples of the horses were examined for basophil reactivity by a histamine release test (HRT) and for Culicoides-specific serum immunoglobulin E (IgE) and G (IgG) by enzyme-linked immunosorbent assay (ELISA). The results of intradermal testing (IDT) at 30min (immediate reactivity) and 4h (late-phase reactivity) post challenge with most insect preparations revealed significant differences between horses with and without SE. Overall, the HRT showed the most accurate results with a sensitivity of 1.00 for all Culicoides preparations and specificities of 0.78 (WBE) and 1.00 (saliva). By contrast, delayed reactions of the IDT (24h), and levels of Culicoides-specific IgE and IgG in the native serum showed little or no distinction between allergic and non-allergic horses. However, the use of purified serum IgE and IgG indicated the possibility for elevated titers of insect-specific serum immunoglobulins in horses with SE. The IDT and HRT did not reveal obvious differences in onset and intensity of positive reactions for the native verses exotic Culicoides species, whereas the ELISA showed slightly higher numbers of positive reactions for serum IgG with the indigenous species. Saliva, as compared to WBE, was found to have improved sensitivity and/or specificity for the HRT and for the late-phase immune reactions as measured by the IDT. Overall, the results indicate that allergy tests utilizing effector cells (mast cells, basophils) are more accurate in diagnosing SE in horses than serological analysis by ELISA.     

The humoral immune response to recombinant nucleocapsid antigen of canine distemper virus in dogs vaccinated with attenuated distemper virus or DNA encoding the nucleocapsid of wild-type virus

This study compared the humoral immune response against the nucleocapsid-(N) protein of canine distemper virus (CDV) of dogs vaccinated with a multivalent vaccine against parvo-, adeno-, and parainfluenza virus and leptospira combined with either the attenuated CDV Onderstepoort strain (n = 15) or an expression plasmid containing the N-gene of CDV (n = 30). The vaccinations were applied intramuscularly three times at 2-week intervals beginning at the age of 6 weeks. None of the pre-immune sera recognized the recombinant N-protein, confirming the lack of maternal antibodies at this age. Immunization with DNA vaccine for CDV resulted in positive serum N-specific IgG response. However, their IgG (and IgA) titres were lower than those of CDV-vaccinated dogs. Likewise, DNA-vaccinated dogs did not show an IgM peak. There was no increase in N-specific serum IgE titres in either group. Serum titres to the other multivalent vaccine components were similar in both groups.     

Age-related quantitative alterations in lymphocyte subsets and immunoglobulin isotypes in healthy horses

OBJECTIVE: To characterize age-associated changes in lymphocyte population subsets and immunoglobulin isotypes. ANIMALS: 30 healthy young light-breed horses (5 to 12 years old) and 30 healthy aged light-breed horses (> 20 years old). PROCEDURE: Lymphocyte subset populations were identified, using monoclonal antibodies to cell surface markers CD5, CD4, CD8, and IgG. Subset populations were quantitated by use of flow cytometric analysis of antibody-stained cells. Serum immunoglobulin concentration was determined using single radial immunodiffusion. RESULTS: Absolute cell counts of total lymphocytes, T cells, CD4+ and CD8+ T cells, and B cells were decreased in aged horses, compared with young horses. There was a significant decrease in the percentage of CD8+ cells and an increase in the CD4+-to-CD8+ cell ratio in the aged population, compared with young horses. However, serum concentration of IgG, IgG(T), IgM, or IgA did not differ with age. CONCLUSIONS AND CLINICAL RELEVANCE: In horses, total lymphocyte count and lymphocyte subset cell counts decrease with age. Age-matched control values are necessary for optimal evaluation of hematologic variables in aged horses. The decrease in lymphocyte subset cell counts in healthy aged horses mimics that seen in other species and may contribute to an age-associated decrease in immunocompetency.     

Fungal isolation and identification in 21 cases of guttural pouch mycosis in horses (1998-2002)

This aetiological study of guttural pouch mycosis (GPM) in the horse was based on the retrospective study of 21 horses brought into the National Veterinary School of Lyon (France) between 1998 and 2002. Biopsies were taken from the lesions caused by GPM during endoscopic examination. In 87% of the cases, direct examination gave positive results, whereas 43% of the cultures were found to be negative. The main fungi observed were Aspergillus fumigatus (in three cases), A. versicolor (in two cases, together with other fungi), and A. nidulans and A. niger (one case each). In six cases, the Aspergillus species could not be identified. In two cases, cleistothecia and/or Hulle cells were observed. In three cases, fungi other than Aspergillus were seen, mixed or not with Aspergillus. These results underline the importance of Aspergillus fumigatus in the development of GPM in horses.     

Chronic obstructive pulmonary disease (COPD) in horses: aetiological studies: responses to intradermal and inhalation antigenic challenge.

Micropolyspora faeni and Aspergillus fumigatus were identified as common causes of respiratory hypersensitivity in horses affected with chronic obstructive pulmonary disease (COPD). Rye grass pollen and an Actinomycete evoked respiratory allergy in a few horses. Not infrequently, individual horses were found to have respiratory hypersensitivity to two or more antigens. The methods used to examine for allergy were intradermal testing and inhalation challenge with environmental antigens. An intradermal test using an M faeni extract was demonstrated to be suitable for diagnostic use in horses previously accurately diagnosed as suffering from COPD. In contrast, the A fumigatus antigen used proved unsatisfactory for such a purpose. Skin reaction to M faeni and A fumigatus extracts by horses affected with COPD indicated that the hypersensitivity was a dual one--a weak response shortly after injection followed by an Arthus-like response 4 to 8 hours later. As a parameter for monitoring responses to inhalation challenge, maximum intrathoracic pressure change (max delta Ppl) proved satisfactory, whereas changes in partial pressure of arterial oxygen (PaO2) did not.     

Serosurvey of horses with evidence of equine monocytic ehrlichiosis

In August 1986, an extensive serosurvey for prevalence of IgG and IgM antibodies against Ehrlichia risticii, the causative agent of equine monocytic ehrlichiosis (EME), was performed at 2 Ohio racetracks, River Downs (RD) and Beulah Park (BP). Of 840 horses at RD and 574 at BP, 13 and 20%, respectively, were IgG antibody-positive (by indirect fluorescent antibody test results), with antibody titer ranging from 1:20 to 1:10,240. The titer observed at highest frequency at both racetracks was 1:80. A higher proportion of horses was ill at RD (operating during the summer months) than at BP (winter track). Of ill horses, 41% (24/58) at RD and 58% (11/19) at BP were seropositive. At RD, 70% (589/840) of all horses and 95% (102/107) of IgG seropositive horses had been stabled only at RD during the month prior to testing. Analysis of these sera by use of an ELISA to detect IgM antibody against E risticii antigen indicated that at RD, 42% (57/137) of the seropositive horses were IgM seropositive. At BP, 17% (20/120) of seropositive horses were IgM seropositive. The larger number of IgM seropositive horses at RD indicates that more horses were recently infected at RD than at BP (P = 0.0001). Therefore, at least half the seropositive horses at RD seemed to have acquired the infection at RD. These serosurvey data also indicate that at BP and RD, 78% (85/109) and 91% (111/122) of IgG seropositive horses, respectively, had subclinical infection. At less than or equal to 1:40 titer, there was no difference in seropositive rates between healthy and ill horses.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative pharmacokinetics of antifungal drugs in domestic turkeys, red-tailed hawks, broad-winged hawks, and great-horned owls

The present research was to test in vitro activity of thiabendazole, 5-fluorocytosine, and amphotericin B against 11 isolates of Aspergillus fumigatus from avian species. Additionally, the plasma concentrations of these drugs were determined in four avian species given a range of dosages by oral, intravenous, and intratracheal routes. Thiabendazole inhibited most isolates in vitro at concentrations between 25 and 50 micrograms/ml; however, there were no detectable inhibitory concentrations in the plasma of any species at any of the doses. The arithmetic mean minimum inhibitory in vitro concentration for 5-fluorocytosine against the 11 Aspergillus isolates was 2.73 micrograms/ml. Inhibitory concentrations of 5-fluorocytosine were found 2 and 6 hours post-administration in all species when given oral doses of 30 or 60 mg/kg as a single dose or when given three divided doses a day totaling 120 mg/kg. No inhibitory concentrations were found 24 hours post-administration. Inhibitory concentrations of amphotericin B were found only 2 and 6 hours post-administration in birds receiving three doses of 1.5 mg/kg at 2-hour intervals. The arithmetic mean minimum inhibitory in vitro concentration for amphotericin B against 11 isolates of A. fumigatus was 0.81 micrograms/ml.     

Serum levels of the immunoglobulins IgG and IgG(T) in horses

Levels of the immunoglobulins IgG and IgG(T) in serum in Norwegian horses of the breeds “Døle” and “Fjord” were determined by the quantitative radial immunodiffusion test.No significant differences were apparent between the 2 Norwegian breeds. The immunoglobulin levels were approximately in the same range as previously reported for Shetland ponies.Immunoglobulins could not be detected in the newborn foal. As early as 24 hrs. after birth the mean immunoglobulin level was within the adult range. After a drop during the first month of life, the immunoglobulins increased. IgG(T) rose more rapidly and to a higher level than IgG.In 2 year old horses, IgG(T) was significantly higher than in adults, while IgG was significantly lower. IgG(T) seems to be a very important immunoglobulin in foals and young horses.     

Monoclonal anti-equine IgE antibodies with specificity for different epitopes on the immunoglobulin heavy chain of native IgE

In this study we describe the generation of monoclonal antibodies (mAbs), which recognize different epitopes of the equine IgE constant heavy chain. Equi-murine recombinant IgE (rIgE), composed of the murine V(H)186.2 heavy chain variable region, linked to the equine IgE constant heavy chain and expressed together with the murine lambda(1) chain in J558L cells was used to immunize BALB/C mice. A total of 17 different mAbs were obtained, which recognized the rIgE heavy chain constant region. None of the mAbs reacted with monoclonal equine isotypes IgM, IgG1 (IgGa), IgG3 (IgG(T)), IgG4 (IgGb) or isolated equine light chains, IgGc and IgA from horse serum, or the native mAb B1-8delta, expressing the same heavy chain variable regions and light chains. One of the mAbs (alphaIgE-132) recognized the recombinant equine IgE, but did not recognize any protein in equine serum, i.e. native IgE. A total of 16 mAbs detected a serum protein of approximately 210,000Da on Western blots, corresponding to the expected MW of native IgE. In addition, one of the mAbs (alphaIgE-176) detected a protein of 76,000Da under reducing conditions, most likely the equine IgE heavy chain. According to binding inhibition studies, the equine IgE specific mAbs recognize at least two different epitopes of the equine IgE. In an ELISA using two anti-IgE mAbs which recognized different epitopes, no significant differences in the concentration of total serum IgE could be detected between adult Icelandic horses with IgE-mediated type I allergy (summer eczema) and healthy control animals. In Icelandic horse foals, no serum IgE could be measured 6 months post partum. All anti-IgE mAbs recognized a small population (1.3+/-0.5%) of leukocytes from adult Icelandic horses by surface immunofluorescence, but no cells could be detected in foal blood. The stained leukocytes from adult horses could be enriched by magnetic cell sorting and contained 32% basophils, 53% monocytes and/or large lymphocytes, 13% small lymphocytes and 2% eosinophils.