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.     

A comparison of intradermal testing and detection of allergen-specific immunoglobulin E in serum by enzyme-linked immunosorbent assay in horses affected with skin hypersensitivity

Skin hypersensitivities (allergies) in horses are often diagnosed using clinical signs only. Intradermal testing or serological assays are diagnostic options to confirm the allergic nature of the disease and to identify the allergen(s). Our objective was to develop an allergen-specific enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody specific for horse IgE and to examine its potential for allergen detection in serum in comparison to intradermal testing. Intradermal testing with 61 allergen extracts was performed on 10 horses affected with skin hypersensitivity. Their sera were analyzed by ELISA for IgE antibodies to the same allergens. The kappa test of concordance was used for comparison of the results of both tests. Out of 61 allergen extracts, only two (Timothy and Quack) had kappa values greater than 0.60, suggesting a substantial agreement between skin testing and IgE ELISA. The statistical comparison of the remaining 59 allergens showed little or no concordance between the tests beyond chance. To identify parameters that may influence the sensitivity of the ELISA, the assay was modified to detect allergen-specific IgGb and IgG(T) in serum, and the protein content in all allergen extracts was determined by SDS-PAGE. The commercial allergen extracts revealed a high variation in detectable protein. High concentrations of allergen-specific IgG in horse serum were found to compete with IgE for binding to the plates. In conclusion, an ELISA using whole serum and crude allergen preparations provides limited diagnostic information in horses. The reliable diagnosis of allergens in equine skin hypersensitivity is essential to improve allergen-specific treatments, such as hyposensitization, or the development of allergy vaccines.     

Incidence of Insect Bite Hypersensitivity in a Small Population of Warmblood Horse Breed in the Czech Republic

Insect bite hypersensitivity (IBH) is a serious equine disease with a complex etiology, affecting many breeds. The availability of a small closed population with a relatively high incidence of IBH (28%) offered the chance to contribute to a better understanding of the etiology of IBH. All 736 Warmblood horses, kept in lowland floodplain meadows in the Czech Republic, were analyzed in the period 1998-2010. The morbidity rate in the offsprings of particular stallions was significantly different; the lowest was 10% and the highest 75%. The assumption that the highest occurrence of healthy offspring would be in healthy parents and vice versa was confirmed. The sex of the offsprings was not significant. To decline the disorder prevalence in this population, stud stallions should be IBH negative; afflicted mares can be tolerated to a certain extent, but their male offspring must never be used as stud. Heredity control is necessary, and stallions with IBH+ offspring should be culled. There is to date no reliable treatment available, and therefore, the development of preventive measures is particularly important. The incidence of IBH could be lowered by prudent breeding, and thus immunotherapy and complex herd management would not be necessary. However, this would be a long-term and complex process.     

Malassezia species isolated from the intermammary and preputial fossa areas of horses

BACKGROUND: Malassezia-type yeasts previously have been observed on cytologic examination of the intermammary region of mares that presented with tail-head pruritus; topical antiyeast treatment resolved the pruritus. Further, Malassezia dermatitis has been observed in horses in intertriginous areas such as the udder and prepuce; the species of yeast was not confirmed. It is not known whether healthy mares or male horses can be carriers of this yeast in these body areas. HYPOTHESIS: Malassezia spp. are present in the intermammary region in healthy mares and the preputial fossa in healthy geldings. ANIMALS: Eleven healthy horses (5 mares and 6 geldings). METHODS: Samples of surface material were taken digitally from the intermammary area of 5 mares and the preputial fossa region of 6 geldings. The samples were examined cytologically and were cultured on modified Sabouraud's dextrose agar. The DNA from yeast colonies grown on the agar was extracted, and samples were assayed using fungal generic polymerase chain reaction (PCR) analysis. Amplicons with positive PCR results were sequenced and compared with sequences in the BLAST database search program. RESULTS: Of 44 attempts at culture, 5 yielded a species identified as Malassezia equi, and 2 yielded M slooffiae. In contrast, of 44 cytologic examinations, yeasts with the morphology of Malassezia spp. were seen in 40 samples. CONCLUSIONS AND CLINICAL IMPORTANCE: Due to its presence in healthy horses, finding of Malassezia-type yeast on cytologic examination may not incriminate it as a pathogen. Despite difficulty in culturing, cytologic examination was an effective tool to rapidly demonstrate the organism.     

Characterisation of lymphocyte subpopulations in the skin and circulation of horses with sweet itch (Culicoides hypersensitivity)

Circulating lymphocyte numbers are elevated in horses with the allergic skin disease sweet itch and skin lesions are typified by an infiltrate of eosinophils and mononuclear cells, the latter of which have not been fully characterised. The aim of the present study was to characterise the lymphocyte subpopulations in the circulation and skin of ponies with sweet itch by flow cytometry and a newly developed modified alkaline phosphatase immunohistochemical technique. Sweet itch ponies were found to have significantly greater numbers of circulating CD5+ and CD4+ T-lymphocytes than normal animals. Increased numbers of CD3+ T-lymphocytes, most of which were CD4+, and eosinophils were present in the skin of these animals following intradermal injection of a Culicoides antigen extract (97 +/- 21 vs. 449 +/- 49 CD3+ T-lymphocytes/mm2 in deep dermis of vehicle vs. antigen injected sites; 83 +/- 8% CD4+ T-lymphocytes at antigen injected site). T-lymphocytes, which are thought to be important in the pathogenesis of human allergic skin disease, may therefore contribute to the development of sweet itch lesions via the release of cytokines which can cause eosinophil accumulation and activation. An understanding of the pathology of this disease may lead to a more rational approach to therapy.     

Serologic and molecular survey of horses to Coxiella burnetii in East of Iran a highly endemic area

There are few reports about Q fever in horse populations worldwide. This study aimed to detect the C. burnetii infection by serologic and molecular confirmation using commercial ELISA kit and real-time PCR in the East of Iran a region highly endemic. A total of 177 blood samples and 115 vaginal swabs were randomly collected from horses in East of Iran. The sera samples were analyzed for anti C.burnetii Ig G antibodies by a commercial ELISA kit and nucleic acid extraxted from vaginal samples were used to determine the C. burnetii DNA by real-time PCR assay. Antibodies were detected in 5.64 % (10/177) of sera samples and C. burnetii DNA was detected in 7.82 % (9/115) of horse vaginal samples. There was no significant difference in seroprevalence in different sex, age and breed groups. Our study showed that horses could be considered as a mild potential reservoir of C. burnetii which may be effective on horse health status. However, additional studies are needed to assess whether the horse could be considered as a relevant transmission risk indicator for Q fever.     

Detection of bacterial DNA in synovial fluid from horses with infectious synovitis

Standard culturing techniques are often unrewarding in confirming diagnosis of synovial infection in the equine patient. Several human studies report the use of sensitive polymerase chain reaction (PCR) techniques for the detection of bacterial involvement in acute synovitis. However, successful extraction of bacterial DNA directly from clinical samples from horses without prior culture has not been reported yet. The goal of this study was to develop a sensitive and reliable method for molecular detection and identification of bacterial species in synovial fluid from horses with infectious synovitis. Synovial fluid samples from 6 horses with culture confirmed synovial infection were used for broad range 16S rRNA gene PCR. Synovial aspirates of 2 healthy horses were used as negative controls. Following extraction and purification of synovial fluid DNA, all samples were processed by touchdown PCR. Amplicons were detected by reverse line blot hybridisation and visualised with chemiluminescence. Pathogen-specific detection of 16S rRNA gene sequences was successful in all 6 synovial fluid samples. No bacterial DNA was detected in the aspirates from the negative control horses using touchdown PCR followed by 25 additional cycles of amplification. The identity of the pathogens was confirmed by DNA sequencing of the amplicons. It can be concluded that broad range 16S rRNA gene PCR followed by reverse line blot hybridisation is a promising technique for detection of bacterial DNA in synovial fluid samples. Further research should aim at the detection of bacterial DNA in synovial fluid samples suspected of infection but having negative culture results. When the 16S PCR proves to be reliable and more sensitive than standard culturing techniques, it may become a powerful tool in the diagnosis of synovial infection.     

Immunoglobulin E-bearing cells and mast cells in skin biopsies of horses with urticaria

The pathogenesis of equine urticaria is not well understood. In man, urticaria has been associated with immunological and nonimmunological mechanisms leading to the release of various mediators by mast cells. Skin biopsies of 32 horses with a history of urticaria were stained with toluidine blue, a double-labelling method for chymase and tryptase, and immunohistochemistry for immunoglobulin (Ig)E. These horses were compared with horses with pemphigus foliaceus, insect bite hypersensitivity and control horses with healthy skin. Neither formalin fixation time nor biopsy site influenced the staining methods. No chymase-positive cells were found. In all groups of horses, cells staining with toluidine blue and for tryptase and IgE were found in the epidermis and hair follicle papilla and significantly more positively staining cells were observed in the subepidermal dermis compared with the deep dermis. Horses with urticaria had significantly more IgE-bearing cells in the subepidermal dermis than control horses. However, horses with urticaria had significantly fewer toluidine-blue-stained mast cells in both subepidermal and deep dermis compared with the insect bite hypersensitivity and pemphigus foliaceus groups. This study suggests that IgE-mediated reactions play a role in the pathogenesis of urticaria.     

MCT1, MCT4 and CD147 gene polymorphisms in healthy horses and horses with myopathy

Polymorphisms in human lactate transporter proteins (monocarboxylate transporters; MCTs), especially the MCT1 isoform, can affect lactate transport activity and cause signs of exercise-induced myopathy. Muscles express MCT1, MCT4 and CD147, an ancillary protein, indispensable for the activity of MCT1 and MCT4. We sequenced the coding sequence (cDNA) of horse MCT4 for the first time and examined polymorphisms in the cDNA of MCT1, MCT4 and CD147 of 16 healthy horses. To study whether signs of myopathy are linked to the polymorphisms, biopsy samples were taken from 26 horses with exercise-induced recurrent myopathy. Two polymorphisms that cause a change in amino acid sequence were found in MCT1 (Val₄₃₂Ile and Lys₄₅₇Gln) and one in CD147 (Met₁₂₅Val). All polymorphisms in MCT4 were silent. Mutations in MCT1 or CD147 in equine muscle were not associated with myopathy. In the future, a functional study design is needed to evaluate the physiological role of the polymorphisms found.     

Risk factors for fecal shedding of Salmonella from horses in a veterinary teaching hospital

Identification of risk factors for horses shedding Salmonella in their feces helps identify patients at-risk of infection and protect the overall population through heightened biosecurity. Fecal samples from 230 hospitalized horses were cultured for Salmonella spp. Historical data were collected on 21 putative risk factors and assessed for association with the risk of a horse being culture positive using forwards stepwise logistic regression. Salmonella was isolated from 13 horses—most commonly from either the first (n=5) or second (n=4) sample collected. Only presenting complaint (confounded by age, breed and gender) was significantly (P≤0.05) associated with positive Salmonella-culture results. Analysis of residuals showed that the model was robust, but individual risk-factor estimates were changed by removal of outliers. Overall, presenting complaint (for example, lower-respiratory-tract disease) was the most important indicator of culture status.     

Serum vitamin D,calcium, and phosphorus concentrations in ponies,horses and foals from the United States and Thailand

Vitamin D is essential in calcium and phosphorus regulation, bone physiology, cell proliferation and epithelial integrity. Literature on vitamin D in growing horses is sparse, and the effect of age on vitamin D has not been evaluated in equids in the United States or in tropical countries. The goal of this study was to determine if there was an effect of age on serum 25(OH)D₃ concentrations in equids in the US (Ohio/Kentucky) and Thailand (Chiang Rai and Kanchanaburi) during the same time of the year. Blood samples were collected from healthy ponies (n = 21) and Thoroughbred foals (n = 13), yearlings (n = 10), and horses (n = 20) in Thailand and from Thoroughbred foals (n = 10) and horses (n = 17) in the US. Serum concentrations of 25(OH)D₃, calcium and phosphorus were measured.In both countries, serum 25(OH)D₃ concentrations were lower in foals than in yearlings and adult horses. Serum 25(OH)D₃ concentrations were higher in horses than in ponies in Thailand, but were not different between horses from either country. Calcium concentrations were not different between groups or location. In both countries, phosphorus concentrations were higher in foals than in older groups; however, were not different between ponies and horses. This study shows that independent of geography there are age-related differences in 25(OH)D₃ concentrations in horses and further confirms that 25(OH)D₃ concentrations are lower in horses compared to other species. The information will serve as the basis for future clinical studies and to help understand better the pathophysiology of equine disorders associated with calcium and phosphorus dysregulation.     

In Vivo and In Vitro Evidence of the Involvement of CXCL1, a Keratinocyte-Derived Chemokine, in Equine Laminitis

Background: C-X-C motif ligand 1 (CXCL1) is an important chemokine of epithelial origin in rodents and humans.
Objectives: To assess in vivo and in vitro the regulation of CXCL1 in equine laminitis.
Animals: Twenty adult horses.
Methods: Real-time quantitative polymerase chain reaction (PCR) was used to assess expression of CXCL1 in samples of laminae, liver, skin, and lung from the black walnut extract (BWE) model of laminitis, and in cultured equine epithelial cells (EpCs). Tissue was obtained from control animals (CON, n = 5), and at 1.5 hours (early time point [ETP] group, n = 5), at the onset of leukopenia (developmental time point [DTP] group, n = 5), and at the onset of lameness (LAM group, n = 5) after BWE administration. EpCs were exposed to Toll-like/Nod receptor ligands, oxidative stress agents, and reduced atmospheric oxygen (3%). In situ PCR was used to localize the laminar cell types undergoing CXCL1 mRNA expression.
Results: Increases in laminar CXCL1 mRNA concentrations occurred in the ETP (163-fold [ P = .0001]) and DTP groups (21-fold [ P = .005]). Smaller increases in CXCL1 expression occurred in other tissues and organs. In cultured EpCs, increases ( P < .05) in CXCL1 mRNA concentration occurred after exposure to lipopolysaccharide (LPS [28-fold]), xanthine/xanthine oxidase (3.5-fold), and H₂O₂ (2-fold). Hypoxia enhanced the LPS-induced increase in CXCL1 mRNA ( P = .007). CXCL1 gene expression was localized to laminar EpCs, endothelial cells, and emigrating leukocytes.
Conclusion and Clinical Importance: These findings indicate that CXCL1 plays an early and possibly initiating role in neutrophil accumulation in the BWE laminitis model, and that laminar keratinocytes are an important source of this chemokine. New therapies using chemokine receptor antagonists may be indicated.     

Detection of IgG and IgE serum antibodies to Culicoides salivary gland antigens in horses with insect dermal hypersensitivity (sweet itch).

We postulated that all horses exposed to the bites of Culcoides (midges) would have an antibody response to the antigen secreted in Culcoides saliva, but that IgE antibody would be restricted to allergic individuals. Using immunohistology on sections of fixed Culicoides, we have demonstrated the presence of antibodies in horse serum which recognise Culicoides salivary glands. Antibodies were detected in the serum of horses with insect dermal hypersensitivity and in the serum of normal horses exposed to Culicoides bites. In contrast, no antibodies were detected in serum from native Icelandic ponies which had not been exposed to Culicoides. Anti-salivary gland IgG antibodies were detected in serum from both allergic and healthy horses exposed to Culicoides. IgE antibodies were only detected in horses with signs of insect dermal hypersensitivity, they were not found in serum of healthy controls nor in the serum of horses with a history of hypersensitivity but in remission at the time of sampling. Using western blotting we confirmed the presence of antibodies to Culicoides antigens and demonstrated that individual horses react to different numbers of antigens. This paper demonstrates the ability of serum from allergic horses to detect Culcoides antigens and will enable further studies to isolate and characterise the allergens.     

Alveolar macrophage graded hemosiderin score from bronchoalveolar lavage in horses with exercise-induced pulmonary hemorrhage and controls

The objective of this study was to determine if a quantitative scoring system for evaluation of hemosiderin content of alveolar macrophages obtained by bronchoalevolar lavage provides a more sensitive test for the detection of exercise-induced pulmonary hemorrhage (EIPH) in horses than does endoscopy of the lower airways. A sample population composed of 74 Standardbred racehorses aged 2-5 years was used. Horses were grouped as either control (EIPH-negative) or EIPH-positive based on history and repeated postexertional endoscopic evaluation of the bronchial airways. Bronchoalveolar lavage was performed and cytocentrifuge slides were stained with Perl's Prussian blue. Alveolar macrophages were scored for hemosiderin content by a method described by Golde and associates to obtain the total hemosiderin score (THS). Test performance criteria were determined with a contingency table. All subjects had some degree of hemosiderin in the alveolar macrophages, regardless of group. The distribution of cells among the different grades followed a significantly different pattern for the control group versus horses with EIPH (P < .05). When using a THS of 75 as a cutoff point, the THS test was found to have a sensitivity of 94% and a specificity of 88%. The level of agreement beyond chance, between the EIPH status and the THS test result was very good (Cohen's kappa = 74%). The conclusion was made that careful assessment and scoring of alveolar macrophages for hemosiderin by means of the Golde scoring system shows promise as a more sensitive approach than repeated postexertional endoscopy alone to detect EIPH.     

Clinical, histopathological and immunophenotypical findings in five horses with cutaneous malignant lymphoma

This study documents the clinical, histopathological, immunohistochemical and flow-cytometric findings in five horses with cutaneous non-epidermotropic malignant lymphoma (ML). The median survival time after discovery of the first subcutaneous nodules was 3.8 years (range 2-5 years: n=4). Histologically, the cutaneous ML had a pleiomorphic structure and contained a mixture of large reticulo-endothelial cells, medium-large sized lymphoid cells with a rounded nucleus and small nucleoli, many medium sized lymphoid cells with irregular nuclei, and some small lymphoid cells. Immunohistochemically (IHC) the lymphoid cells were positive for the pan-T-lymphocyte marker CD3 but negative for the B-lymphocyte markers CD21 and kappa and lambda immunoglobulin light chains. Although routine haematological examination revealed no abnormalities in the horses with cutaneous ML, changes in the peripheral blood lymphocyte population were apparent flow-cytometrically. Compared to clinically healthy horses, a decreased total percentage of cells was recorded in the lymphocyte gate. In three horses with cutaneous ML, an increase in CD4 positive cells was noticed in the monocyte gate. Flow-cytometric analysis of tumour cells collected by fine needle aspiration (FNA) suggested that the cutaneous MLs consisted primarily of CD4 and CD8 positive T-lymphocytes. The results were compared to those of a monomorphic multicentric T- and a monomorphic multicentric B-cell lymphoma. The results of immunohistochemistry and flow-cytometry were largely but not completely in accordance. In conclusion, the results of this study suggest that cutaneous non-epitheliotropic malignant lymphomas in the horse are of T-cell origin and that, after improvement of its accuracy, flow cytometric analysis of FNAs might become a useful aid to rapid tumour identification.     

The effect of a topical insecticide containing permethrin on the number of Culicoides midges caught near horses with and without insect bite hypersensitivity in the Netherlands

Insect bite hypersensitivity (IBH) in horses is most likely caused by Culicoides species, although other insects may also play a role. Until now no effective cure has been found for this condition, although numerous therapeutic and preventive measures have been used to control insect hypersensitivity. One such method is to apply a topical insecticide to horses. In this study, the effect of a topical insecticide containing permethrin (3.6%) was examined in seven pairs of horses. The horses were placed inside a tent trap to collect Culicoides spp. and other insects attracted to the horses on two subsequent evenings. On the first evening, both horses were untreated. After the end of this session, one horse of each pair was treated with the pour-on insecticide; treated horses were kept separate from untreated horses. The next evening the pairs of horses were again placed inside the tent trap and insects were collected. Similar percentages of Culicoides were trapped as in earlier studies (C. obsoletus 95.34% and C. pulicaris 4.54%), with healthy horses attracting more Culicoides than horses affected by IBH. The number of Culicoides, the percentage of blood-fed Culicoides obsoletus, and the total number of insects attracted to horses 24 hours after treatment with permethrin were reduced but the reduction was not statistically significant. No negative side effects of permethrin administration were observed.     

Exercise-induced alterations in plasma concentrations of ghrelin, adiponectin, leptin, glucose, insulin, and cortisol in horses

Six Standardbred (STB) mares (11+/-2 years, 521+/-77 kg; means+/-SD) performed an exercise trial (EX) where they underwent an incremental exercise test (GXT) as well as a parallel control trial (CON) to test the hypothesis that short-term, high intensity exercise would alter plasma concentrations of glucose, leptin, adiponectin, ghrelin, insulin and cortisol. Plasma samples were taken before (0 min), during (last 10s at 6, 8m/s, and the velocity eliciting VO(2max)), and after exercise (2, 10, 30, 60 min; 12 and 24h post-GXT). A second set of blood samples was collected before and after an afternoon meal given at 1515 h (at 1500, 1514, 1530, and 1545 h). Data were analyzed using ANOVA for repeated measures and Tukey's test. During the GXT, there were no changes (P>0.05) in the plasma concentrations of glucose, leptin, adiponectin or ghrelin. However, there was a 29% increase (P<0.05) in mean plasma cortisol concentration and a 35% decrease (P<0.05) in mean plasma insulin concentration. Substantial increases (P<0.05) in the mean plasma concentrations of glucose and cortisol of 36% and 102%, respectively, were seen in the EX trial during the first 60 min post-GXT. Plasma leptin concentration, measured at the 24h post-GXT time point, was 20% lower (P<0.05) during the EX trial compared with the parallel time point in the standing control (CON) trial. Plasma ghrelin concentration was 37% lower (P<0.05) in the EX trial compared with CON before and after the afternoon meal, but was 43% higher (P<0.05) 12h post-GXT. There were no differences between EX and CON for plasma concentrations of insulin or adiponectin during recovery. It was concluded that short-term high intensity exercise alters plasma leptin and ghrelin concentrations in STB mares post-exercise, which may signal the exercised animals to alter energy intake.     

Sucrose concentration in blood: a new method for assessment of gastric permeability in horses with gastric ulceration

A urine sucrose test has recently been reported to be a reliable method of detecting gastric ulcers in horses; however, technical difficulties associated with urine collection have limited the practical value of the test. The objective of this pilot study was to determine whether gastric sucrose permeability, as evaluated by serum sucrose concentration, could be used to detect gastric mucosal injury in horses. Twelve adult horses with naturally acquired gastric ulceration were studied. After a 20-hour nonfeeding period, each horse was dosed with 250 g of sucrose via nasogastric intubation. Blood samples were collected at 0, 15, 30, 45, 60, and 90 minutes, and horses underwent gastroscopy 4 hours later. The severity of gastric ulceration in each horse was defined by means of a 4-point ulcer-scoring system, and the relationship with serum sucrose concentration was analyzed by means of a linear mixed-effects model. Serum sucrose concentration was measured by liquid chromatography operating in tandem with electrospray mass spectrometry. After nasogastric administration of table sugar, horses with moderate to severe gastric ulceration had significant increase in serum sucrose concentration at 30, 45, 60, and 90 minutes, relative to earlier times (P < .05). Peak sucrose concentration was observed at 45 minutes, and was correlated with ulcer severity (Spearman's rank correlation coefficient = 0.898, P < .05). These data indicate that determination of sucrose concentration in equine serum may be a useful test for identifying horses with endoscopically visible gastric ulceration and has potential use as a noninvasive method for screening and monitoring horses engaged in racing training and other performance-related disciplines.     

Lymphoid nodules in skin biopsies from dogs, cats, and horses with nonneoplastic dermatoses

In a retrospective histopathologic study of nonneoplastic dermatoses, lymphoid nodules were found in 0.3% of 3,408 canine, 5.1% of 469 feline, and 4.5% of 325 equine skin biopsies. In all 3 species, the majority of cases wherein lymphoid nodules were found were diseases of presumed immune-mediated nature. In cats and horses, the majority of cases were also diseases characterized by tissue eosinophilia.