Pilot study to determine the concordance between skin prick and intradermal test (IDT) reactivity to environmental allergens in atopic dogs using IDT as the gold standard.

BACKGROUND: Prick testing is widely used as the first-line in vivo test for environmental allergens in people owing to its noninvasive nature and speed of performance. OBJECTIVES: To determine concordance between skin prick testing (SPT) and intradermal testing (IDT) reactivity to environmental allergen mixes in dogs with atopic dermatitis (cAD). ANIMALS: Forty client-owned dogs with cAD. MATERIALS AND METHODS: Skin prick testing (GREER Pick System; Stallergenes Greer) and IDT were performed on 40 dogs using seven glycerinated and aqueous environmental allergen mixes, respectively (tree, grass and weed pollens, house dust mites and three mould mixes). Reactions for IDT and SPT were evaluated both subjectively and objectively (mean wheal diameter; MWD) and compared to saline and histamine controls. RESULTS: Using IDT as the gold standard, with subjective scoring, SPT was 47.0% sensitive [95% confidence interval (CI) 36.0%-58.7%], 92.1% specific (95% CI 87.6%-95.3%) and agreement was moderate (79%, Cohen's kappa = 0.424). The positive predictive value of SPT was 36% and negative predictive value was 95%. Objective and subjective scores had only fair agreement. CONCLUSIONS AND CLINICAL RELEVANCE: Skin prick testing with allergen mixes was specific yet poorly sensitive as compared to IDT. For both IDT and SPT, 95% (38 of 40) dogs failed to react to an allergen mix, despite showing a positive reaction to at least one component. Future studies comparing SPT and IDT should test individual allergens rather than mixes to prevent the dilution of individual components, which may have resulted in false negatives.

Investigation of the diagnostic performance of prick test with two environmental allergens in dogs with atopic dermatitis: A study using intradermal test as the gold standard.

BACKGROUND: There are no studies investigating the correlation between prick test (PT) and intradermal test (IDT) with environmental allergens in dogs with atopic dermatitis (AD). OBJECTIVES: To investigate the correlation between PT and IDT for two environmental allergens, and to calculate the sensitivity, specificity and Youden index of PT, using IDT as the gold standard. MATERIALS AND METHODS: Twenty-two dogs with AD were selected. PT was performed with glycerinated allergen extracts, along with negative and positive controls, using the Greer Prick System. Reactions were interpreted (positive/negative) subjectively and by using seven objective criteria, by an examiner blinded to the IDT results. IDT reactions to the same allergens were interpreted, subjectively and objectively, by another masked investigator. The agreement between PT and IDT, the sensitivity, specificity and Youden index of PT, using IDT as gold standard, were calculated. RESULTS: On subjective evaluation, the correlation between PT and IDT was poor and sensitivity of PT was 0%. Of the seven criteria for the objective evaluation of PT, the best diagnostic performance was attained when allergen-induced wheals were considered positive if their longest diameter was ≥8.5 mm. However, even then, the correlation with IDT was moderate, and the sensitivity of PT, albeit based on few positive IDT reactions, was low. CONCLUSION AND CLINICAL RELEVANCE: At least as performed herein, PT has a poor-to-moderate correlation with IDT, mainly as a consequence of the lack of positive PT reactions. Further studies are needed to improve PT technique, yet, meanwhile, it cannot be recommended as a substitute for IDT.

Grass pollen sensitization in dogs in Paraná, Brazil: comparison between percutaneous and intradermal testing / [Sensibilização a polens em cães no Paraná, Brasil: comparação entre o teste percutâneo e o teste intradérmico]

Canine atopic dermatitis is an inflammatory, genetic, pruritic and chronic dermatosis that affects between 10 and 30% of dogs and one of the most important allergens is grass pollen. The objective of this study was to evaluate the sensitization to grass pollen allergens in dogs with canine atopic dermatitis and to compare intradermal skin test (IDT) with percutaneous test (PT). For this study, ten healthy dogs and 39 dogs with atopic dermatitis were tested. Dogs were submitted to IDT and PT for Lolium multiflorum, Cynodon dactylon and Paspalum notatum. The IDT and PT tests were compared using the Proportion Test. All healthy dogs were negative to both tests. Ten atopic dogs (25.6%) responded positively to the PT and none were positive in IDT. C. dactylon, L. multiflorum and P. notatum were responsible for positive reactions in 70%, 70% and 30% of positive dogs, respectively. The number of positive reactions in PT were statistically higher than IDT (P<0.05). In conclusion, grass pollen can be important source of allergens for dogs in Paraná state (Brazil) and the PT showed higher sensitization to grass pollen in dogs with atopic dermatitis than IDT.(AU)

A dermatite atópica canina é uma dermatose inflamatória, genética, prurítica e crônica que afeta entre 10% e 30% dos cães, e um dos alérgenos mais importantes são os polens de gramíneas. O objetivo deste estudo é avaliar a sensibilização a alérgenos de polens de gramíneas em cães com dermatite atópica e comparar o teste intradérmico (TID) com o teste percutâneo (TP). Para o estudo, 10 cães hígidos e 39 cães com dermatite atópica foram testados. Estes foram submetidos ao TID e ao TP para Lolium multiflorum, Cynodon dactylon e Paspalum notatum. TID e TP foram comparados usando-se o teste de proporção. Todos os cães hígidos foram negativos em ambos os testes. Dez cães atópicos (25,6%) responderam positivamente ao TP e nenhum ao TID. C. dactylon, L. multiflorum e P. notatum foram responsáveis por reações positivas de 70%, 70% e 30% dos cães positivos, respectivamente. O número de reações positivas no TP foi estatisticamente maior que no TID (P<0,05). Foi concluído que os polens de gramíneas podem ser importantes fontes de alérgenos para cães no estado do Paraná (Brasil) e que o TP mostrou maior sensibilização a polens em cães com dermatite atópica que o TID.(AU)

Immunoglobulin G1 subclass responses can be used to detect specific allergy to the house dust mites Dermatophagoides farinae and Dermatophagoides pteronyssinus in atopic dogs.

BACKGROUND: In dogs with atopic dermatitis, intradermal testing (IDT) or allergen specific IgE serological testing are routinely employed to identify causative allergens. These allergens can then be used for allergen-specific immunotherapy and allergy management. The clinical relevance of this testing is affected by the source of allergen, and other biomarkers that are more related to specific allergens still need to be identified. The aim of this study was to investigate levels of specific IgE, total IgG, and IgG1 and IgG2 subclasses against the local house dust mites (HDM) Dermatophagoides farinae (DF) and D. pteronyssinus (DP) as biomarkers by using in-house ELISAs in healthy (n = 33) and atopic dogs (AD) (n = 44) that were either positive or negative by IDT to HDM. RESULTS: Being over 3 years of age was a risk factor for AD (Odds Ratio (OD) = 4.10, 95% Confidence interval (CI) 1.57-10.75, p = 0.0049), but there was no relation to IDT outcomes (OR = 0.9091, 95% CI 0.22-3.74, p = 1.00). High levels of all antibody isotypes (IgE, IgG, IgG1 and IgG2) against HDM were found in aged healthy dogs (> 3 years old). In AD, HDM-IgE and IgG1 levels were higher in dogs that were IDT positive to HDM than in IDT negative animals. Levels of IgE and IgG1 could be used to distinguish the specific allergens, whereas total IgG and IgG2 levels were not different between IDT-positive and IDT-negative AD. By the receiver operating characteristic curve at a false-positive rate = 0.10, both IgE and IgG1 showed better sensitivity than IgG and IgG2. Similar to IgE, serum IgG1 concentration was also relevant to IDT outcomes. CONCLUSIONS: Our in-house ELISAs coated with local HDM were useful for evaluating antibody levels, and we propose use of the HDM-specific IgG1 subclass as a biomarker to detect HDM specific allergens in AD, potentially together with an IgE based platform.

Dynamics and risk of transmission of bovine tuberculosis in the emerging dairy regions of Ethiopia.

The Ethiopian government has several initiatives to expand and intensify the dairy industry; however, the risk of bovine tuberculosis (bTB) spread is a challenge. To assess the rate of expansion and risk factors for transmission of bTB within-herds, we carried out a repeated cross-sectional survey at two time points, 2016/17 and 2018, in three regional cities, namely, Gondar, Hawassa and Mekelle, representing the emerging dairy belts of Ethiopia. The total number of herds involved was 128, comprising an average of 2303 cattle in each round. The Single Intradermal Comparative Cervical Tuberculin (SICCT) test was used to identify reactor status and data on herd-level risk factors were collected using a structured questionnaire. In the first survey, the apparent prevalence of bTB, as measured by the SICCT test, was 4.5% (95% CI 3.7-5.4%) at the individual animal-level and 24% (95% CI 17.5-32%) at the herd-level. There was no statistically significant change in the overall apparent prevalence or regional distribution at the second survey, consistent with the infection being endemic. The incidence rate was estimated at 3.6 (95% CI 2.8-4.5) and 6.6 (95% CI 3.0-12.6) cases/100 cattle (or herd)-years at the animal- and herd-levels, respectively. Risk factors significantly associated with the within-herd transmission of bTB were age group and within-herd apparent prevalence at the start of the observation period. We noted that farmers voluntarily took steps to remove reactor cattle from their herds as a consequence of the information shared after the first survey. Removal of reactors between surveys was associated with a reduced risk of transmission within these herds. However, with no regulatory barriers to the sale of reactor animals, such actions could potentially lead to further spread between herds. We therefore advocate the importance of setting up regulations and then establishing a systematic bTB surveillance programme to monitor the impact prior to implementing any control measures in Ethiopia.

Delayed-type hypersensitivity skin test against Neospora caninum in heifers with undetectable specific antibodies.

Delayed-type hypersensitivity (DTH) has been used in human and veterinary medicine as a skin testing for evaluating in vivo cell-mediated immune responses (CMIR). Whereas CMIR is a key process to control intracellular pathogens, its value at identifying cattle exposed to the abortigenic intracellular coccidian parasite Neospora caninum is unknown. In this work, we have evaluated a DTH skin testing in cattle exposed to N. caninum and still seronegative. Female calves were experimentally sensitized by subcutaneous (SC) inoculation with live tachyzoites of N. caninum (NC-Argentina LP1) in sterile phosphate-buffered saline (PBS) (group A; n: 8) whereas other calveswere mock-sensitized with PBS (group B; n: 6). Two DTH skin tests were performed by intradermal inoculation with a soluble lysate of N. caninum tachyzoites (NC-Argentina LP1) in the neck region at 60d and 960 d after sensitization. Skinfold thickness at the intradermal inoculation site was measured at 0, 24, 48 h post each DTH skin test and skin biopsies taken for microscopic evaluation. Specific N. caninum antibodies kinetics was evaluated all throughthe experiment. We found that whereas N. caninum specific antibodies remained below the ELISA cut-off, a distinctive skinfold thickness increase was detected in sensitized animals (group A) at the DTH skin test site, showing induration, swelling and inflammatory infiltration. Mock sensitized animals (group B) showed no skinfold thickness growth and lacked specific antibody response. Thus, N. caninum DTH skin testing could be a useful diagnostic tool for the detection of CMIR during N. caninum infection in non-humoral responders.

Post-mortem surveillance of bovine tuberculosis in Ireland: herd-level variation in the probability of herds disclosed with lesions at routine slaughter to have skin test reactors at follow-up test.

Post-mortem surveillance in Ireland discloses skin-test negative cattle with presumptive evidence of infection of Mycobacterium bovis (lesions at routine slaughter (LRS)), the causative agent of bovine tuberculosis (bTB). Laboratory confirmation of lesions has impacts on trade restrictions for herds, therefore if laboratory capacity was diminished, how herds are treated would require an informed risk policy. Here we report the proportion of herds with subsequent evidence of within-herd transmission, based on skin-test results. We assess how herd-size, herd-type, and bTB-history affect the probability of additional reactors at follow-up test using univariable and multivariable random-effects models. The study represents a rapid response to developing an evidential base for policy demands during an extraordinary event, the COVID-19 epidemic in Ireland. A dataset from 2005 to 2019 of breakdowns were collated. Overall, 20,116 breakdowns were initiated by LRS cases. During the index tests of these breakdowns, 3931 revealed ≥1 skin-test reactor animals (19.54%; ≥1 standard reactors: 3827; 19.02%). Increasing herd-size was associated with reactor disclosure on follow-up. For small herds (<33 animals), 11.74% of follow-up tests disclosed ≥1 reactor; 24.63% of follow-up tests from very large herds (>137) disclosed ≥1 reactors. Beef (13.87%) and "other" (13%) herd production types had lower proportion of index tests with reactors in comparison with dairy (28.27%) or suckler (20.48%) herds. Historic breakdown size during the previous 3-years was associated reactor disclosure risk on follow-up. Our results are useful for rapid tailored policy development aimed at identifying higher risk herds.

Preliminary Comparative Assessment of Brucellergene Skin Test for Diagnosis of Brucellosis in Dromedary Camels (Camelus dromedarius).

This study was conducted to evaluate the use of Brucellergene skin test (BST) for the diagnosis of Brucellosis in camels (Camelus dromedarius) in comparison with Rose Bengal test (RBT) and competitive enzyme-linked immunosorbent assay (c-ELISA). A total of 68 apparently healthy adult dromedary camels of either gender from three different geographical locations of Abu Dhabi Emirate, United Arab Emirates (UAE), were included in the study. The skin test was applied on two shaved areas at the middle of the neck: one for the test and the other area was injected with normal saline as a control. Reading was done 72 h postinjection. Results were subjected to Bayesian analysis to assess the test performances in camels. The model estimated the following sensitivity and specificity median values: BST: Se = 70.72%, Sp = 98.82%; RBT: Se = 93.27%, Sp = 97.79%; and c-ELISA: Se = 94.78%, Sp = 98.48%. As the BST investigated in this study proved to be a highly specific test, we propose using it as a confirmatory test in camels particularly when the serological tests give doubtful results on individual animals.

Co-reactivity between related and unrelated environmental allergens in equine allergen-specific IgE serology testing in the UK.

BACKGROUND: Identification of environmental allergens in horses with allergic disease facilitates allergen avoidance and targeted immunotherapy. HYPOTHESIS/OBJECTIVES: To evaluate allergenic co-reactivity between 44 environmental allergens. ANIMALS: Horses with suspected allergic disease (n = 344) whose sera were submitted for environmental allergen testing. METHODS AND MATERIALS: Allergen-specific IgE serological assays were performed using 44 allergens divided into six taxonomically related groups: house dust/storage mites, moulds, insects, grass, tree and weed pollens. Using pairwise comparisons, odds ratios (ORs) were calculated for each environmental pair to determine if there was increased or decreased likelihood of a positive result for one allergen, given a positive result to another. The OR significance was set (using Holm-Bonferroni correction) at P < 0.00006 for all horses (n = 344) and P < 0.00005 for horses with at least one positive reaction (n = 239). Using one-way ANOVA with Tukey's post hoc tests (significance at P < 0.05), differences in mean log e ORs between three groups, taxonomically related allergens with a statistically significant association (related-associated), related allergens lacking a significant association (related-nonassociated) and unrelated allergens were tested. RESULTS: Statistically significant associations were found between both related and unrelated allergen pairs, the former being more frequent. For all horses (n = 344) and horses with at least one positive reaction (n = 239), co-reactivity ranged from 100% (grasses) to 0% (moulds). The weeds group was exceptional in having more co-reactions with another group (grasses). CONCLUSIONS AND CLINICAL IMPORTANCE: Co-reactivity was shown within and between certain related allergen groups. Further studies are required to determine whether this is the result of antigenic cross-reactivity.

Agreement of serum allergen test results with unblocked and blocked IgE against cross-reactive carbohydrate determinants (CCD) and intradermal test results in atopic dogs.

BACKGROUND: Tests for allergen-specific immunoglobulin E (IgE) are used to select allergens for immunotherapy in atopic dogs. Antibodies against cross-reactive carbohydrate determinants (anti-CCD IgE) have been identified in serum samples of atopic dogs. Their presence in humans is a known cause of clinically irrelevant polysensitization to plant allergens. OBJECTIVES: To compare the results of an intradermal test (IDT) and a serum test for allergen-specific IgE, with and without blocking anti-CCD IgE, before testing in dogs. ANIMALS: Thirty-one privately owned dogs with atopic dermatitis. METHODS AND MATERIALS: Dogs were prospectively skin tested and their serum samples were analysed for anti-CCD IgE. An Fc-ε receptor-based serum test for allergen-specific IgE was performed with and without blocking anti-CCD IgE. RESULTS: In dogs with negative anti-CCD IgE samples, the agreement between the results of the serum test and the IDT was substantial (κ = 0.71). Dogs with positive anti-CCD IgE samples (38.7%) showed no agreement between serum and skin testing (κ = -0.35), blocking anti-CCD IgE in those samples resulted in a fair agreement (κ = 0.43). Anti-CCD IgE positive sera had multiple positive results for grass and weed allergens, and blocking decreased them markedly. CONCLUSION AND CLINICAL IMPORTANCE: Intradermal testing agreed best with serum testing in dogs with no detectable anti-CCD IgE. Sera containing anti-CCD IgE had no agreement with IDT. Test agreement was improved by blocking the anti-CCD IgE. Apparent serum test polysensitization to plant allergens was associated with anti-CCD IgE.

Genome-Wide Association Study of Cell-Mediated Response in Dogs Naturally Infected by Leishmania infantum.

A genome-wide association study (GWAS) could unravel the complexity of the cell-mediated immunity (CMI) to canine leishmaniasis (CanL). Therefore, we scanned 110,165 single-nucleotide polymorphisms (SNPs), aiming to identify chromosomal regions associated with the leishmanin skin test (LST), lymphocyte proliferation assay (LPA), and cytokine responses to further understand the role played by CMI in the outcome of natural Leishmania infantum infection in 189 dogs. Based on LST and LPA, four CMI profiles were identified (LST-/LPA-, LST+/LPA-, LST-/LPA+, and LST+/LPA+), which were not associated with subclinically infected or diseased dogs. LST+/LPA+ dogs showed increased interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) levels and mild parasitism in the lymph nodes, whereas LST-/LPA+ dogs, in spite of increased IFN-γ, also showed increased interleukin-10 (IL-10) and transforming growth factor ß (TGF-ß) levels and the highest parasite load in lymph nodes. Low T cell proliferation under low parasite load suggested that L. infantum was not able to induce effective CMI in the early stage of infection. Altogether, genetic markers explained 87%, 16%, 15%, 11%, 0%, and 0% of phenotypic variance in TNF-α, TGF-ß, LST, IL-10, IFN-γ, and LPA, respectively. GWAS showed that regions associated with TNF-α include the following genes: IL12RB1, JAK3, CCRL2, CCR2, CCR3, and CXCR6, involved in cytokine and chemokine signaling; regions associated with LST, including COMMD5 and SHARPIN, involved in regulation of NF-κB signaling; and regions associated with IL-10, including LTBP1 and RASGRP3, involved in T regulatory lymphocytes differentiation. These findings pinpoint chromosomic regions related to the cell-mediated response that potentially affect the clinical complexity and the parasite replication in canine L. infantum infection.

BAYESIAN ANALYSIS TO EVALUATE TESTS FOR THE DETECTION OF MYCOBACTERIUM BOVIS INFECTION IN FREE-RANGING WILD BISON (BISON BISON ATHABASCAE) IN THE ABSENCE OF A GOLD STANDARD.

We estimated the sensitivity and specificity of the caudal-fold skin test (CFT), the fluorescent polarization assay (FPA), and the rapid lateral-flow test (RT) for the detection of Mycobacterium bovis in free-ranging wild wood bison (Bison bison athabascae), in the absence of a gold standard, by using Bayesian analysis, and then used those estimates to forecast the performance of a pairwise combination of tests in parallel. In 1998-99, 212 wood bison from Wood Buffalo National Park (Canada) were tested for M. bovis infection using CFT and two serologic tests (FPA and RT). The sensitivity and specificity of each test were estimated using a three-test, one-population, Bayesian model allowing for conditional dependence between FPA and RT. The sensitivity and specificity of the combination of CFT and each serologic test in parallel were calculated assuming conditional independence. The test performance estimates were influenced by the prior values chosen. However, the rank of tests and combinations of tests based on those estimates remained constant. The CFT was the most sensitive test and the FPA was the least sensitive, whereas RT was the most specific test and CFT was the least specific. In conclusion, given the fact that gold standards for the detection of M. bovis are imperfect and difficult to obtain in the field, Bayesian analysis holds promise as a tool to rank tests and combinations of tests based on their performance. Combining a skin test with an animal-side serologic test, such as RT, increases sensitivity in the detection of M. bovis and is a good approach to enhance disease eradication or control in wild bison.

Assessment of immune response in periparturient dairy cows using ex vivo whole blood stimulation assay with lipopolysaccharides and carrageenan skin test.

The transition period is known to be the most critical phase in the life of high yielding dairy cow. Changes in the immune functions have been observed during the transition period which may account for the onset of clinical and subclinical (e.g. inflammatory response) problems at calving or at the beginning of lactation however this relationship has not yet been adequately investigated. Thus, to establish the potential of the periparturient dairy cow's immune system to respond to stimuli, two challenges [an ex vivo whole blood stimulation assay (WBA) with lipopolysaccharides and a carrageenan skin test (CST)] were performed in addition to characterizing the metabolic and inflammatory profile. The WBA was performed using 0, 0.01 and 5 µg LPS/mL on whole blood and CST was administered by subcutaneous injection of 0.7 mL solution containing 4.2mg of carrageenan to the shoulder region of the cows. These tests were performed on 10 Holstein-Friesian cows at -45 ± 2, -20 ± 2, -3, 3, 7, 28 ± 2 days from parturition (DFP). Cows were also monitored for health status, body condition score, milk yield. The results demonstrate a higher production of IL-1ß and IL-6 from leukocytes after LPS stimulation around calving (from -3 to 3 DFP) compared to -45 DFP (P < 0.05). Moreover, IL-6 (but not IL-1ß) was able to reach close to the maximum response at the lower stimulus intensity (0.01 µg LPS/mL), maintaining a higher response over a longer time in early lactation. The release of higher levels of IL-6 in the transition period, with low LPS dose, suggests its crucial role in the regulation of inflammatory response around calving. The response of cows to CST decreased a few days before calving (-3 DFP) compared with response at -45 and 28 DFP (P<0.05), and remained low in the first week of lactation. This result suggests the reduction of the functionality of some vascular factors, which decreases diapedesis. Overall, the WBA and CST tests confirm changes in immunocompetence around calving. These tests are able to better describe the changes of the innate immune response at a local and systemic level, mainly when combined with conventional metabolic and inflammatory indices.

Clinical and histological features of brucellin skin test responses in Brucella suis biovar 2 infected pigs.

Current serological tests for swine brucellosis detect antibodies to the Brucella O-polysaccharide (O/PS). However, when infections by bacteria carrying cross-reacting O/PS occur, these tests suffer from false positive serological reactions (FPSR), and the skin test with Brucella soluble protein extracts is the best diagnostic alternative to differentiate true Brucella suis infections from FPSR in pigs. Since this test has been seldom used in B. suis infected swine, the clinical and histological features involved have not been described properly. Here, we describe the clinical and histological events in B. suis biovar 2 infected pigs skin tested with a cytosoluble O/PS free protein extract from rough Brucella abortus Tn5::per mutant. A similar extract from rough Ochrobactrum intermedium was also used for comparative purposes. No relevant differences were evidenced between the homologous and heterologous allergens, and the main clinical feature was an elevated area of the skin showing different induration degrees. Moreover, an important vascular reaction with hyperemia and haemorrhage was produced in most infected sows 24-48 h after inoculation, thus facilitating the clinical interpretation of positive reactions. Histologically, combined immediate (type III) and delayed (type IV) hypersensitivity reactions were identified as the most relevant feature of the inflammatory responses produced.

Characterization of IgE-mediated cutaneous immediate and late-phase reactions in nonallergic horses.

OBJECTIVE: To characterize the response of skin of nonallergic horses following ID injection of polyclonal rabbit anti-canine IgE (anti-IgE) and rabbit IgG. ANIMALS: 6 healthy horses. PROCEDURES: Skin in the cervical area was injected ID with anti-IgE and IgG. Wheal measurements and skin biopsy specimens were obtained before and 20 minutes and 6, 24, and 48 hours after injection. Tissue sections were evaluated for inflammatory cells at 4 dermal depths. Immunohistochemical analysis for CD3, CD4, and CD8 was performed, and cell counts were evaluated. RESULTS: Anti-IgE wheals were significantly larger than IgG wheals at 20 minutes and 6 and 24 hours after injection. There were significantly more degranulated mast cells after anti-IgE injection than after IgG injection. There were significantly more eosinophils at 6, 24, and 48 hours and neutrophils at 6 hours after anti-IgE injection, compared with cell numbers at those same times after IgG injection. There were significantly more eosinophils in the deeper dermis of anti-IgE samples, compared with results for IgG samples. No significant differences between treatments were detected for CD3(+), CD4(+), or CD8(+) cells. CONCLUSIONS AND CLINICAL RELEVANCE: Injection of anti-IgE antibodies was associated with the development of gross and microscopic inflammation characterized by mast cell degranulation and accumulation of inflammatory cells, particularly eosinophils and neutrophils. This pattern appeared to be similar to that of horses with naturally developing allergic skin disease, although lymphocytes were not increased; thus, ID injection of anti-IgE in horses may be of use for evaluating allergic skin diseases of horses.

Role of soybean ß-conglycinin subunits as potential dietary allergens in piglets.

ß-Conglycinin, a major seed-storage protein in soybeans, is one of the primary antigenic proteins responsible for soybean-meal hypersensitivity in weaned piglets. The protein is a heterotrimer composed of subunits α, α' and ß. It is currently unknown which of the ß-conglycinin subunits are allergenic for piglets. The aim of this study was to identify potential allergenic subunits of ß-conglycinin for soybean sensitive piglets and to characterise these subunits by immunoglobulin (Ig) G and E immunoblotting, ELISA, 'skin prick' and whole blood histamine-release testing. The IgG and IgE binding capabilities of the purified α, α' and ß subunits of ß-conglycinin were determined by immunoblot analysis and ELISA with sera from ß-conglycinin sensitised piglets. Skin prick testing and whole blood histamine release testing were also performed to detect the activated effector cell response to specific allergens. Specific IgG and E antibodies were identified that recognised all three subunits of ß-conglycinin in the sera of ß-conglycinin sensitised piglets. All three subunits of ß-conglycinin elicited positive skin test and specific histamine release responses from the whole blood of ß-conglycinin sensitised piglets. These results suggest that all three ß-conglycinin subunits are potential allergens for piglets.

Effects of pentoxifylline on immediate and late-phase cutaneous reactions in response to anti-immunoglobulin E antibodies in clinically normal dogs.

OBJECTIVE: To characterize the effects of pentoxifylline on the gross and microscopic variables associated with immediate and late-phase inflammation following injection of IgE-specific antibodies in the skin of clinically normal dogs. ANIMALS: 6 healthy adult mixed-breed dogs. PROCEDURES: Intradermal injections (0.1 mL each) of PBS solution, histamine phosphate, and cross-linking rabbit-origin anti-canine IgE antibodies (3 injections/dog) were administered at 0 hours on day 0; wheal sizes were evaluated at 20 minutes, 6 hours, and 24 hours. Biopsy specimens of injected and noninjected skin were collected 24 hours after injection. On day 2, treatment with pentoxifylline (20 mg/kg, PO, q 8 h) was initiated and continued until day 30. For each dog, injection, measurement, and biopsy procedures were repeated on days 30 to 31 and on days 37 to 38 (ie, after discontinuation of pentoxifylline administration). RESULTS: Pentoxifylline administration was associated with a significant decrease in wheal size at 6 and 24 hours (but not at 20 minutes) after injection of anti-canine IgE. Repeated injections performed 1 week after drug discontinuation revealed partial recovery of the 6-hour cutaneous reaction and complete recovery of the 24-hour cutaneous reaction. Pentoxifylline administration was also associated with inhibition of mast cell degranulation and significant decreases in the total numbers of cutaneous inflammatory cells and eosinophils, compared with pretreatment findings. CONCLUSIONS AND CLINICAL RELEVANCE: In clinically normal dogs, pentoxifylline effectively impaired late-phase reactions but not immediate reactions at sites of intradermal injection of IgE-specific antibodies by inhibiting mast cell degranulation and recruitment of cutaneous inflammatory cells, especially eosinophils.

Produção e purificação parcial de PPD-maleína para diagnóstico do mormo em equídeos / Partially purified malleo-proteins production for glanders diagnosis in Equidae

Objetivou-se com este estudo produzir e purificar parcialmente a PPD-maleína a partir de amostras de Burkholderia mallei isoladas de equídeos no Brasil com potencial para uso no diagnóstico do mormo. As linhagens de B. mallei fenotipicamente caracterizadas e de virulência comprovada foram inoculadas em caldo Dorset-Henley para crescer e metabolizar. Em seguida, as proteínas foram separadas por precipitação com ácido tricloroacético e precipitadas com sulfato de amônia. As PPDs-maleínas foram concentradas em 1,0mg/mL e na avaliação realizada em cobaios foi eficaz no desenvolvimento da hipersensibilidade do tipo tardia e consequentemente na identificação de animais verdadeiro positivos e exclusão dos verdadeiro negativos, sendo uma possibilidade em potencial para utilização no diagnóstico do mormo.

The objective of this study was to produce and partially purify Malleo-protein from Burkholderia mallei samples isolates from Equidae in Brazil with potential for use in the diagnosis of glanders. The strain B. mallei phenotypically characterized and proven virulent was inoculated into broth Dorset-Henley to grow and metabolize. The proteins were separated by trichloroacetic acid precipitation and amonium sulfate precipitation. The PPD mallein was concentrated 1.0mg/mL and biologically tested in guinea pigs. It was effective in the development of delayed-type hypersensitivity and consequently to identify true-positive animals and to exclude of true negatives. There is the possibility for potential use in the glanders diagnosis in Equidae.

Performance of skin tests with allergens from B. melitensis B115 and rough B. abortus mutants for diagnosing swine brucellosis.

Swine brucellosis by Brucella suis biovar 2 is an emerging disease whose control is based on serological testing and culling. However, current serological tests detect antibodies to the O-polysaccharide (O/PS) moiety of Brucella smooth lipopolysaccharide (S-LPS), and thus lack specificity when infections by Yersinia enterocolitica O:9 and other gram-negative bacteria carrying cross-reacting O/PS occur. The skin test with the protein-rich brucellin extract obtained from rough B. melitensis B115 is assumed to be specific for discriminating these false positive serological reactions (FPSR). However, B115 strain, although unable to synthesize S-LPS, accumulates O/PS internally, which could cause diagnostic problems. Since the brucellin skin test has been seldom used in pigs and FPSR are common in these animals, we assessed its performance using cytosoluble protein extracts obtained from B. abortus rough mutants in manBcore or per genes (critical for O/PS biosynthesis) and B. melitensis B115. The diagnostic sensitivity and specificity were determined in B. suis biovar 2 culture positive and brucellosis free sows, and apparent prevalence in sows of unknown individual bacteriological and serological status belonging to B. suis biovar 2 naturally infected herds. Moreover, the specificity in discriminating brucellosis from FPSR was assessed in brucellosis free boars showing FPSR. The skin test with B. abortus ΔmanBcore and B. melitensis B115 allergens performed similarly, and the former one resulted in 100% specificity when testing animals showing FPSR in indirect ELISA, Rose Bengal and complement fixation serological tests. We conclude that O/PS-free genetically defined mutants represent an appropriate alternative to obtain Brucella protein extracts for diagnosing swine brucellosis.

Effects of butorphanol versus dexmedetomidine sedation on intradermal allergen and histamine responses in dogs with atopic dermatitis.

BACKGROUND: Sedation is commonly used during intradermal testing (IDT). Morphine and its derivatives have long been avoided because of their histamine-releasing effects. HYPOTHESIS/OBJECTIVES: Butorphanol, an opioid agonist/antagonist, will not adversely affect IDT in dogs. ANIMALS: Ten client-owned dogs diagnosed with atopic dermatitis. METHODS: Dogs were randomized to be sedated with butorphanol (0.4 mg/kg) or dexmedetomidine (5 µg/kg). Routine IDT along with intradermal injections of various dilutions of histamine were performed on the lateral thorax, followed 7 days later by the alternative sedative and IDT on the opposite side. The injection sites were subjectively scored and objectively measured by one investigator, blinded to the sedatives, and compared between groups. RESULTS: When the mean wheal diameters from the objective measurements of all antigens, including saline and histamine dilutions, were compared, butorphanol was associated with significantly smaller reactions than dexmedetomidine (P = 0.0001). There was a high level of agreement between sedatives when positive reactions subjectively scored as ≥3+ were compared (κ = 0.91). When mean wheal diameters of histamine at concentrations of 1:100,000, 1:400,000, 1:1,600,000 and 1:6,400,000 were compared, there were no significant differences between sedative types. Moreover, the percentage agreement when comparing subjective interpretation of all histamine dilutions between sedations was high (κ = 0.90). However, there was only 69% agreement beyond chance when objective and subjective interpretations of all antigens were compared between sedative groups. CONCLUSIONS: Although butorphanol resulted in significantly smaller wheal size in comparison to dexmedetomidine, it did not affect the overall subjective interpretation of the results of IDT.