Pharmacokinetics and effects of cetirizine in horses with insect bite hypersensitivity

Horses with insect bite hypersensitivity (IBH) have difficulty in completely avoiding allergens, so effective treatment options are required. A randomised, placebo controlled and double blinded field study was conducted to determine the pharmacokinetics and efficacy in reducing dermatitis of the antihistamine cetirizine given orally at 0.4 mg/kg twice daily for 3 weeks. The influence of protection blankets and stabling were also investigated.The estimated maximum plasma concentration (C_max) and trough plasma concentration of cetirizine were 135 ng/mL and 18 ng/mL, respectively. There was no difference in dermatitis reduction between the treatment and placebo groups (P = 0.77). The findings indicated that cetirizine was of no apparent benefit in treating IBH at the dose rate tested. The use of blankets and stabling were shown to have favourable influence on the dermatitis (P < 0.05) and may be the preferred options to prevent this condition.     

Culicoides species attracted to horses with and without insect hypersensitivity

The aims of this study were to determine (1) which species of Culicoides is most commonly attracted to horses, (2) whether horses suffering insect hypersensitivity attract more Culicoides spp. than unaffected horses, and (3) the times when Culicoides spp. are most active. Horses affected by insect hypersensitivity and unaffected horses were placed inside mosquito netting tents for 30 min at different times of the day. All Culicoides spp. trapped inside the tents were collected and identified. C. obsoletus was the most common species found, followed by C. pulicaris. Healthy horses attracted slightly more midges than horses that were affected with insect hypersensitivity. All of the Culicoides species were most active at sunset, less so at sunrise and very few or no midges were trapped in the afternoon or at night.     

Immunoglobulin-E-bearing cells in skin biopsies of horses with insect bite hypersensitivity.

The aim of the present study was to investigate, with immunohistochemistry and in situ hybridisation, if immunoglobulin-E (IgE) and mast cells are involved in the pathogenesis of insect bite hypersensitivity (IBH), an allergic dermatitis of horses. In tissue sections fixed in paraformaldehyde (PFA) for <24 h, significantly more IgE protein-bearing cells were found in the dermis and epidermis of acute and chronic IBH lesions than in skin biopsies from healthy horses (medians = 466, 236 and 110 cells/mm2, respectively; P < or = 0.01). More IgE-mRNA positive (+) cells were observed in the dermis of acute IBH lesions than in the dermis of healthy skin (median = 2.8 vs. 0.0 cells/mm2; P < or = 0.01). Significantly, more mast cells were detected with metachromatic (median = 160 vs. 62 cells/mm2; P < or = 0.001) and tryptase-specific stainings (median = 120 vs. 69 cells/mm2; P < or = 0.001) in the dermis of acute IBH biopsies compared to healthy skin. No chymase+ mast cells were found in any skin biopsy. IBH lesions fixed in PFA for >24 h were compared to dermatomycosis (DM) lesions; IBH biopsies contained a similar number of IgE-protein+ cells to DM biopsies (median = 249 vs. 192 cells/mm2; P = 0.08) but had significantly more IgE-mRNA+, metachromatic and tryptase+ mast cells than DM biopsies. This study suggests an involvement of IgE-mediated immune reactions in the pathogenesis of IBH as well as, sometimes, in dermatomycosis. Using double labelling, cells which expressed IgE protein and contained mast cell enzymes were detected.     

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.     

Identification of factors associated with the development of insect bite hypersensitivity in horses in The Netherlands

An Internet-based questionnaire among horse owners was carried out to identify factors affecting the incidence of insect bite hypersensitivity (IBHI) among horses in the Netherlands. Information was obtained for 794 horses of various breeds, but the breed distribution was not representative for the Dutch horse population. Of the horses for which information was available, 56% suffered from IBH and 44% did not. The most common clinical symptoms were pruritus, scaling, and hair loss, occurring mainly at the base of the tail and along the mane. Breed, age, region (and local habitat), stabling, type of bedding in the stable, use of the horse, deworming frequency, and season were associated with significant differences in IBH incidence. Knowledge of the factors influencing the incidence of IBH may make it possible to reduce the number of animals affected and help alleviate symptoms in affected horses.     

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.     

Determination of 'irritant' threshold concentrations for intradermal testing with allergenic insect extracts in normal horses

Abstract Sixteen healthy horses with no history of skin or respiratory disease were used for an intradermal testing (IDT) threshold study, in order to determine the concentrations of 13 commercial allergenic insect extracts most appropriate for IDT. Five dilutions of each extract were used, which included the manufacturer's recommended concentrations for equine IDT, plus one dilution higher and three lower than these standard concentrations. Allergens tested included caddisfly ( Trichoptera spp.), mayfly ( Ephemeroptera spp.), horsefly ( Tabanus spp.), deerfly ( Chrysops spp.), fire ant ( Solenopsis invicta ), black ant ( Camponotus pennsylvanicus ), cockroach mix ( Periplaneta americana and Blattella germanica ), mosquito ( Aedes aegypti ), house fly ( Musca domestica ), moth ( Heterocera spp.), flea ( Ctenocephalides canis / C. felis ), Culicoides variipennis and Culicoides nubeculosis. Two separate methods were used to calculate the allergen concentration for each insect extract where the normal horses, as a group, ceased to show false-positive ('irritant') reactions. 'Irritant' threshold concentrations were determined for 9/13 of these allergens, whereas the other 4 were undetermined due to either insufficient reactivity (flea, C. variipennis ) or excessive reactivity (black ant, moth) to the concentrations tested. Recommended concentrations for future use in equine patients with suspected insect hypersensitivity include: 125 pnu mL⁻¹ (mayfly); 250 pnu mL⁻¹ (caddisfly, horsefly, deerfly, fire ant, house fly); 500 pnu mL⁻¹ (cockroach); 1000 pnu mL⁻¹ (mosquito); and 1:10 000 w/v ( C. nubeculosis ).     

Risk factors for insect bite hypersensitivity in Friesian horses and Shetland ponies in The Netherlands

Insect bite hypersensitivity (IBH) is an equine skin allergy caused by bites of Culicoides spp. and impacts on the welfare of affected horses. The aim of this study was to identify and quantify risk factors for IBH. Data from 3453 Friesian horse mares and 7074 Shetland pony mares scored for IBH by inspectors during obligatory foal inspections were analysed using breed-specific multivariable logistic regression models. The combined effect of month and year of scoring, Province and inspector were significantly associated with IBH in both breeds. In Shetland pony mares, withers height and coat colour were also significantly associated with IBH, while body condition had a nearly significant effect. The outcomes from this study on risk factors might contribute to the development of more efficient measures to reduce the prevalence of IBH.     

Serologic evidence of Legionella infection in horses

The indirect fluorescent antibody test was used to examine 109 samples of equine sera randomly selected from serum pools. Results were compared with titers obtained by the microagglutination (MA) test. A high correlation (r = 0.89) was found between titers measured by the 2 tests. Blood samples were obtained serially from a total of 156 horses at a research farm and the sera were tested against Legionella pneumophila serogroups 1 through 4 using the MA test; 29 horses (19%) seroconverted to at least 1 serogroup of L pneumophila. The indirect fluorescent antibody test substantiated the results obtained by the MA test. Seroconversions in horses provided additional evidence that horses become naturally exposed to legionellae.     

Serologic evidence of vesivirus-specific antibodies associated with abortion in horses

OBJECTIVE: To test horses for serologic evidence of an association between vesiviral antibodies and abortion. SAMPLE POPULATION: Sera from 141 horses. PROCEDURES: 2 experiments were conducted. Experiment 1 comprised sera obtained in 2001 and 2002 from 3 groups of horses (58 mares from farms with a history of abortion problems, 25 mares between 3 and 13 years of age with unknown reproductive histories that were sold at auction [breeding-age control mares], and 29 mixed-age males and yearling females sold at auction [negative control population]). Experiment 2 comprised sera from 3 groups of pregnant mares (10 pregnant mares fed Eastern tent caterpillars [ETCs], 9 pregnant mares fed ETC frass only, and 10 pregnant control mares). Sera were analyzed for antibodies against vesivirus by use of a validated recombinant vesivirusspecific peptide antigen in an indirect ELISA. RESULTS: For experiment 1, 37 of 58 (63.8%) mares from farms with abortion problems were seropositive for vesivirus antibodies, whereas 10 of 25 (40%) breeding-age control mares were seropositive. All 29 mixed-age males and yearling females were seronegative for vesivirus antibodies. For experiment 2, 17 of 29 mares aborted (some from each group). Seropositive status for vesivirus antibodies increased from 47.1% (8/17) to 88.2% (15/17) for the pregnant mares that aborted during the experiment. CONCLUSION AND CLINICAL RELEVANCE: Significant association was detected between seropositive status for vesivirus and abortion in mares; consequently, vesivirus appears to be a pathogenic virus associated with abortion in mares. These data support adding vesivirus antibody testing into diagnostic screening to determine the cause for abortion in mares.     

The healing effects of a topical phytogenic ointment on insect bite hypersensitivity lesions in horses

Insect bite hypersensitivity (IBH) is the most common cause of pruritus in horses and is a serious welfare issue for affected animals. In this study, the effect of a topical phytogenic ointment on the healing of cutaneous lesions was investigated in a double-blind trial involving 26 horses with I B H. The number of lesions and their total surface area were recorded on days 0, 7, and 21 in horses treated for 3 weeks with either verum or placebo ointment. After unblinding of treatment assignment, the horses that had been treated with the placebo ointment received the verum preparation for an additional 3 weeks and the number of lesions and their total surface area were again recorded. This part of the study was not blinded. The number of lesions and the total surface area decreased in both treatment groups (no significant difference). Owners also scored the degree of discomfort suffered by their horses as a result of IBH lesions, and at the end of the 3-week period this score was significantly lower in the verum than in the placebo group (P = 0.04). When placebo-treated horses subsequently received the verum ointment, their wound severity score also decreased significantly (P < 0.01). Daily application of an ointment (verum or placebo) does not cure IBH, but use of the phytogenic ointment led to a decrease in the owner-assessed discomfort suffered by horses.     

Evaluation of an in vitro sulphidoleukotriene release test for diagnosis of insect bite hypersensitivity in horses

REASONS FOR PERFORMING STUDY: Insect bite hypersensitivity (IBH) is an IgE-mediated allergic dermatitis caused by bites of Culicoides and Simulium species, and improved means of diagnosis are required. OBJECTIVES: The cellular antigen simulation test (CAST) with C. nubeculosus and S. vittatum extracts was assessed in a population of IBH-affected and healthy horses. Variations in test results over a one year period and possible cross-reactivity between different insect extracts was studied. METHODS: A total of 314 mature horses were studied using the CAST. Influence of severity of clinical signs, gender and age were evaluated, and 32 horses were tested repeatedly over one year. The kappa reliability test was used to assess agreement of the test results with different insect extracts. RESULTS: Horses with IBH had significantly higher sLT release than controls with C. nubeculosus and S. vittatum. The highest diagnostic sensitivity and specificity levels were attained when using adult C. nubeculosus extracts with the CAST (78% and 97%, respectively), suggesting that most horses with IBH are sensitised against Culicoides allergens. A proportion of IBH-affected horses was found to be sensitised to allergens of Simulium spp. in addition to those of C. nubeculosus. The CAST with C. nubeculosus had positive and negative predictive values > or = 80% for a true prevalence of IBH of 12-52%. In the follow-up study, the proportion of IBH-affected horses with a positive test result ranged from 90% in November to 68% in March. Severity of clinical signs or age did not influence test results significantly. However, IBH-affected males achieved significantly more positive test results than IBH-affected females. CONCLUSIONS: The CAST with adult C. nubeculosus has high specificity and good sensitivity for diagnosis of IBH. Horses with IBH are mainly sensitised to Culicoides allergens, and some horses are additionally also sensitised to allergens in Simulium spp. POTENTIAL RELEVANCE: The CAST is likely to be a useful test for diagnosis of IBH, even allowing the identification of IBH-affected but asymptomatic horses. This test may also help in further characterisation of allergens involved in this condition.     

CD4+CD25+ T cells expressing FoxP3 in Icelandic horses affected with insect bite hypersensitivity

Insect bite hypersensitivity (IBH) is an IgE-mediated dermatitis caused by bites of midges from the genus Culicoides. We have shown previously that peripheral blood mononuclear cells (PBMC) from IBH-affected horses produce higher levels of IL-4 and lower levels of IL-10 and TGF-β1 than those from healthy horses, suggesting that IBH is associated with a reduced regulatory immune response. FoxP3 is a crucial marker of regulatory T cells (Tregs). Here we have determined the proportion of CD4(+)CD25(+)FoxP3(+) T cells by flow cytometry in PBMC directly after isolation or after stimulation with Culicoides extract or a control antigen (Tetanus Toxoid). There were no differences between healthy and IBH horses either in the proportion of FoxP3(+)CD4(+)CD25(+) cells in freshly isolated PBMC or in the following stimulation with Tetanus Toxoid. However, upon stimulation of PBMC with the allergen, expression of FoxP3 by CD4(+)CD25(+high) and CD4(+)CD25(+dim) cells was significantly higher in healthy than in IBH horses. Addition of recombinant IL-4 to PBMC from healthy horses stimulated with the allergen significantly decreased the proportion of FoxP3 expressing cells within CD4(+)CD25(+high). These results suggest that IBH is associated with a decreased number of allergen-induced Tregs. This could be a consequence of the increased IL-4 production by PBMC of IBH-affected horses.     

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.     

Serologic testing of horses for granulocytic ehrlichiosis, using indirect fluorescent antibody staining and immunoblot analysis.

OBJECTIVE: To diagnose granulocytic ehrlichiosis in horses, compare results of indirect fluorescent antibody (IFA) staining procedures with those of immunoblot analysis, and compare serologic test findings with polymerase chain reaction (PCR) results. ANIMALS: 69 horses with high rectal temperatures (> or = 39 C) and lethargy, anorexia, or limb edema. PROCEDURE: 43 convalescent serum samples obtained from 38 horses 2 to 18 weeks after onset of illness were analyzed by use of immunoblot procedures and IFA staining methods, using the NCH-1 or BDS ehrlichial strains. Blood samples from 69 acutely ill horses were tested by PCR to detect ehrlichial DNA. RESULTS: Antibodies to Erlichial equi were detected in serum samples obtained during all seasons; seropositivity rates ranged from 50 to 93%. In IFA assays using the BDS or NCH-1 strain, seropositivity rates were 70 and 79%, respectively, whereas in immunoblot analyses using the NCH-1 strain, a seropositivity value of 79% was recorded. By immunoblot analysis, all serum samples of all seropositive horses were reactive to a protein having molecular mass of about 44 kd. Blood samples from 29 of 69 (42%) acutely ill horses contained ehrlichial DNA. CONCLUSION AND CLINICAL RELEVANCE: Results of the various serologic testing procedures were in close agreement with each other. All serologic testing methods are suitable for laboratory diagnosis of equine granulocytic ehrlichiosis.     

Purpura haemorrhagica in 53 horses

The medical records of 53 horses with purpura haemorrhagica were reviewed. Seventeen of them had been exposed to or infected with Streptococcus equi, nine had been infected with Corynebacterium pseudotuberculosis, five had been vaccinated with S. equi M protein, five had had a respiratory infection of unknown aetiology, and two had open wounds; the other 15 cases had no history of recent viral or bacterial infection. The horses were between six months and 19 years of age (mean 8.4 years). The predominant clinical signs were well demarcated subcutaneous oedema of all four limbs and haemorrhages on the visible mucous membranes; other signs included depression, anorexia, fever, tachycardia, tachypnoea, reluctance to move, drainage from lymph nodes, exudation of serum from the skin, colic, epistaxis and weight loss. Haematological and biochemical abnormalities commonly detected were anaemia, neutrophilia, hyperproteinaemia, hyperfibrinogenaemia, hyperglobulinaemia and high activities of muscle enzymes. All of the horses were treated with corticosteroids; 42 also received non-steroidal anti-inflammatory drugs and 26 received antimicrobial drugs. Selected cases received special nursing care, including hydrotherapy and bandaging of the limbs. Most of the horses were treated for more than seven days and none of them relapsed. Forty-nine of the horses survived, one died and three were euthanased, either because their severe clinical disease failed to respond to treatment or because they developed secondary complications. Two of the four non-survivors had been vaccinated against S. equi with a product containing the M protein, one had a S. equi infection and the other had a respiratory infection of undetermined aetiology.