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 ).     

Evaluation of three different histamine concentrations in intradermal testing of normal cats and attempted determination of 'irritant' threshold concentrations for 48 allergens

The purpose of this study was to determine the optimal histamine concentration and 'irritant' allergen threshold concentrations in intradermal testing (IDT) in normal cats. Thirty healthy cats were tested with three different histamine concentrations and four different concentrations of each allergen. The optimal histamine concentration was determined to be 1: 50,000 w/v (0.05 mg mL(-1)). Using this histamine concentration, the 'irritant' threshold concentration for most allergens was above the highest concentrations tested (4,000 PNU mL(-1) for 41 allergens and 700 PNU mL(-1) for human dander). The 'irritant' threshold concentration for flea antigen was determined to be 1:750 w/v. More than 10% of the tested cats showed positive reactions to Dermatophagoides farinae, Dermatophagoides pteronyssinus, housefly, mosquito and moth at every allergen concentration, which suggests that the 'irritant' threshold concentration for these allergens is below 1,000 PNU mL(-1), the lowest allergen concentration tested. Our results confirm previous studies in indicating that allergen and histamine concentrations used in feline IDT may need to be revised.     

Effects of propofol-induced sedation on intradermal test reactions in dogs with atopic dermatitis

We compared the effect of propofol and saline control on intradermal test reactions in dogs with atopic dermatitis undergoing outpatient intradermal testing (IDT). Nineteen dogs were used in this clinical study. Patients were randomly allocated to receive either intravenous (IV) propofol or IV 0.9% saline, and IDT was performed on the right or left (randomized) lateral thorax. One investigator, unaware of the treatments, interpreted all IDT results. Injection sites were analysed using a subjective and objective method. A value of P or= 1+ on all dogs, significantly more positive sites were apparent during propofol sedation than during saline administration. In addition, the greater number of individual dogs experiencing more positive reactions >or= 1+ during propofol sedation was significant. When subjectively analysing reactions >or= 2+, the greater number of positive reactions and the greater number of dogs with more positive reactions observed during propofol treatment was not significantly different from the saline control. When analysed objectively, the greater number of positive reactions observed during propofol sedation was not significant. A greater number of dogs had higher subjective scores and larger objective measurements during propofol sedation compared with saline administration. In summary, propofol sedation was associated with an overall greater number of positive IDT reactions compared with the saline control. Although not always significant, this difference should be considered when choosing propofol for skin testing dogs with atopic dermatitis.     

Results of intradermal testing for the investigation of atopic dermatitis and recurrent urticaria in 50 horses in the south of England

Atopic dermatitis (AD) and recurrent urticaria (RU) are common immune‐mediated conditions of horses and ponies associated with high morbidity. Effective pharmacological treatment options are limited but identification of the causal allergens allows avoidance strategies and immunotherapy regimens to be employed. Intradermal testing (IDT) is the most widely accepted means of identifying the relevant allergens but there are no published reports of this technique being used in the UK for the investigation of dermatological disease. This study presents the results of testing with a varied panel of allergens in 50 horses with dermatological disease living in the south of England. Intradermal testing was performed in horses presented to The Liphook Equine Hospital for further investigation of AD or RU between June 2002 and March 2009. Allergen selection was based upon availability, results of previous studies, pollen charts and the likelihood of allergens being prevalent in the stable or pasture environment in the south of England. Injection sites were evaluated at 1 h (immediate phase), 4 h (late phase) and 24 h (delayed phase) and skin responses compared to the response generated by the positive control (histamine) at 1 h. Total numbers of positive reactions and numbers of positive reactions to specific allergens were similar in horses with atopic dermatitis and those with urticaria (P = 0.39). There was a statistically significant difference in the number of reactions observed at different time points, with more positive reactions occurring at 1 h than 24 h (P<0.001), and at 4 h than 24 h (P<0.001). Reading the test at 24 h rarely provides additional information. Reaction patterns were similar to those of previous studies performed in other countries with large numbers of positive reactions reported to mites, dusts and insects. Positive reactions were also common to allergens not previously identified as irritants or common allergens in equids; nettle, daisy, dandelion, horse chestnut, cat, cattle, sheep and pigeon. These allergens may be important causes of allergic dermatitis in equids in the UK; however, further studies should be performed in both normal horses and horses with allergic dermatitis to investigate irritant thresholds and validate these findings. Intradermal testing may be shortened from the conventional 24 h to 4 h without significantly affecting the results of the test.     

The suitability of medetomidine sedation for intradermal skin testing in dogs

The objective of this study was to determine the suitability of medetomidine sedation for facilitating intradermal skin testing in dogs. Quality of sedation and immobilization, and effects of sedation on responses to intradermally injected histamine were evaluated. Ten clinically normal dogs were injected intradermally before and after medetomidine sedation (10 μg kg^?1 intravenously) with diminishing concentrations of histamine (100–10^?5μg mL^?1) and a negative control. Mean wheal responses at injection sites were compared before and during sedation, and no significant suppression of responses occurred during sedation. Medetomidine produced sedation that notably increased the ease of performing multiple intradermal injections in all dogs and sedative effects were rapidly reversed by the antagonist atipamezole. It was concluded that medetomidine may be an excellent sedative for facilitating intradermal skin testing in dogs provided further studies similarly reveal no inhibition of responses to intradermally injected allergens in atopic dogs.     

Comparison of intradermal testing and serum testing for allergen-specific IgE using monoclonal IgE antibodies in 84 atopic dogs

OBJECTIVE: To compare an ELISA measuring serum allergen-specific IgE with intradermal skin testing in canine atopic dermatitis. PROCEDURE: Eighty-four dogs with the clinical diagnosis of atopic dermatitis underwent intradermal skin testing and serum testing for allergen-specific IgE. Tests were performed in a blinded fashion. Positive reactions were compared and the sensitivity and specificity of the serum test (using intradermal skin test as the standard) were determined overall and for individual allergen groups (grass pollens, weed pollens, tree pollens, house dust mites and fleas). RESULTS: The sensitivity of the ELISA overall was 90.4%. Evaluating the individual allergen groups, the sensitivity for dust mite hypersensitivity was 95.1%, for fleas 85.4%, for tree pollens 84.3%, for grass pollens 95.1% and for weed pollens 96.4%. The specificity was 91.6% overall, for dust mites 96.3%, for fleas 92.7%, for tree pollens 95.2%, for grass pollens 94% and for weed pollens 80.7%. CONCLUSION: The evaluated ELISA seemed reliable for the diagnosis of atopy in practice and can be recommended as a screening test prior to intradermal skin testing or for use in dogs when immunotherapy is not a therapeutic option.     

Comparison between IMMUNODOT tests and the intradermal skin test in atopic dogs

This study evaluated a new perspective in the diagnosis of dermatitis in dogs with signs suggestive of allergic skin disease. The results obtained with CMG IMMUNODOT tests using the technique of allergen-specific strip tests, as employed for human allergy diagnosis, were compared with those obtained by the intradermal skin test (IDST). Forty-eight cases completed the diagnostic evaluation, which included IDST, flea-control program, exclusion of sarcoptes and, for some cases, a 1- to 2-month stabilization period on a restricted protein source diet and testing the serum in the presence of allergen-specific IgE and total IgE. The most common disorders included house and storage dust mites, allergic dermatitis and flea-allergic dermatitis together with atopy. This was confirmed serologically. In the case of positive IDST to pollens, Aspergillus spp. and cat epithelium, CMG IMMUNODOT strip tests were negative. A total of 25% of cases were considered to be primarily associated with food hypersensitivity, but only 4% were confirmed serologically. This study emphasizes the value of CMG IMMUNODOT tests as a support in the diagnosis of dog allergy.     

Diagnosis of flea allergy dermatitis: comparison of intradermal testing with flea allergens and a FcepsilonRI alpha-based IgE assay in response to flea control

The objective of this study was to evaluate the accuracy of in vivo and in vitro tests in the diagnosis of flea allergy dermatitis in comparison with history, clinical signs and response to flea control. Intradermal testing using four different sources of flea allergens and FcepsilonRIalpha-based immunoglobulin (Ig)E assays were performed in 15 flea-allergic dogs, 15 atopic dogs and 15 dogs infested with fleas but showing no clinical signs of skin disease. Sensitivity, specificity, negative predictive value, positive predictive value and accuracy were calculated for all five tests and results varied greatly. Sensitivity, specificity and overall accuracy were 27, 83 and 64%, respectively, for one extract (Isotec), 67, 90 and 82% for another extract (Greer), 93, 90 and 91% for flea saliva, 40, 90 and 73% for the recombinant Cte f 1 both produced by Heska Corp. and 87, 53 and 64% for a FcepsilonRIalpha-based IgE assay. These results indicate that intradermal testing with flea extracts is more accurate in the diagnosis of flea allergy dermatitis than in vitro tests. Moreover, pure flea saliva used as a reagent for intradermal testing provided the best results in terms of sensitivity, specificity and overall accuracy although the Greer extract, a whole body flea extract, also allowed a good correlation between intradermal testing results and clinical approach to flea allergy dermatitis diagnosis.     

Thermal and mechanical nociceptive threshold testing in pregnant sheep

ObjectiveAnalgesic regimes were compared in pregnant ewes after laparotomy by measuring thermal (TT) and mechanical (MT) nociceptive thresholds.Study designProspective randomised experimental study.AnimalsPregnant ewes at 121 days gestation underwent laparotomy as part of another research project.MethodsThermal and mechanical thresholds were measured before, and 2, 6, 24 and 48 hours after surgery. Thermal stimuli were delivered to the lateral aspect of the metatarsus via a skin-mounted probe, and mechanical stimuli to the contralateral site via a pneumatically driven 1.5 mm diameter pin. Each test was performed five times, alternating thermal and mechanical stimuli, with ten minutes between thermal stimuli. At the end of surgery ewes received either: 75 μg hour^?1 transdermal fentanyl patch (medial thigh) (group FP) (n = 8), or 3 μg kg^?1hour^?1 intra-peritoneal medetomidine via an osmotic pump (group IPM) (n = 8) inserted immediately prior to closure. Data were analysed using the Kruskal–Wallis RS Test (p < 0.05). Once a significant effect was identified, pairwise comparisons were performed using paired Wilcoxon RS tests. To compensate for multiple hypotheses testing, p < 0.005 was considered significant.ResultsPrior to surgery mean ± SD TT was 56.1 ± 5.0 °C (FP) and 55.6 ± 5.0 °C (IPM); MT was 5.3 ± 2.6 N (FP) and 8.0 ± 5.0 N (IPM). In FP there was no significant change in either TT or MT over time. In IPM there was no significant change in MT over time but TT increased at two hours to 59.2 ± 3.0 °C (p = 0.003). Skin temperature (ST) ranged from 33.0 to 34.7 °C and did not change over time. There were no significant differences between groups in TT, MT or ST.Conclusions and clinical relevanceAdministration of intra-peritoneal medetomidine (3 μg kg^?1hour^?1) by an osmotic pump increases the thermal nociceptive threshold in the immediate post operative period in pregnant sheep, suggesting that this agent may have a role in providing post-operative analgesia.     

Evaluation of intravenous fluorescein in intradermal allergy testing in psittacines

This study was designed to improve the clinical feasibility of intradermal skin testing of psittacine birds using intravenous fluorescein stain. Twenty-five healthy, anaesthetized Hispaniolan Amazon parrots (Amazona ventralis) were injected intravenously with 10 mg kg-1 fluorescein-sodium 1% followed by intradermal injections of 0.02 mL phosphate-buffered saline, histamine phosphate (1:100,000 w/v) and codeine phosphate (1:100,000 w/v) at the sternal apteria. Wheal diameters of reaction sites were measured grossly and under illumination with a Wood's lamp after 5 and 10 min. Fluorescence-enhanced injection sites were scored between 0 and 2, with 0 equivalent to normal skin and 2 equivalent to a plucked feather follicle. The presence of a fluorescent halo around intradermal injections was also recorded. Under Wood's light illumination at 10 min, histamine and saline were evaluated as positive and negative controls, respectively, based on a positive test having a halo and a score of 2. Sensitivity and specificity were each 76% for halo, 84 and 42% for score and 64 and 77% for combination of score and halo, respectively. Further, mean histamine reactions were significantly larger than codeine phosphate and saline (8.8 +/- 0.4 mm; 7.2 +/- 0.3 mm; 5.9 +/- 0.6 mm); however, this finding was not consistent in individual birds. Wheal size, halo presence and score were affected by site location independent from the injected compound. Intravenous fluorescein improved the readability of avian skin tests; however, the compounds tested raised inconsistent reactions in wheal size, score or halo presence. The compound-independent site effect raises concern on the validity of avian skin testing and warrants investigation of other techniques such as in vitro allergy testing. Based on our findings, intradermal allergy testing in psittacines with or without fluorescein is unreliable and cannot be recommended for practical clinical use.     

The use of compound 48/80 and codeine phosphate as positive controls for intradermal skin testing in dogs

The mast cell secretagogues compound 48/80 and codeine phosphate were evaluated as potential positive controls for intradermal skin testing in dogs. Wheal responses to both agents were compared with responses to histamine and saline in 11 normal dogs, and were strong and not significantly different from histamine responses in nine dogs ( P < 0.01), and significantly weaker than histamine in two dogs ( P < 0.05). Wheal responses to compound 48/80 (1 mg mL⁻¹) were evaluated in 82 suspected atopic dogs and were similar to histamine in 79 dogs and markedly weaker than histamine in three dogs. Of nine confirmed atopic dogs with weak responses to injected allergens, seven had strong responses to compound 48/80, and eight had strong responses to histamine. Compound 48/80 and codeine phosphate appear unreliable positive controls for skin testing in normal dogs. Compound 48/80 (1 mg mL⁻¹) may be a reliable positive control in atopic dogs but is a poor indicator of skin reactivity to allergens.     

Development of a pressure nociceptive threshold testing device for evaluation of analgesics in cats

A pressure analgesiometric device was developed for unrestrained cats. Eleven cats were studied. Stimulation was via three rounded pins within a bracelet on the forearm. The pins were advanced by manual bladder inflation. Bladder pressure was measured using a strain gauge pressure transducer. The threshold was recorded at the behavioural end point. Thresholds were measured at 5 and 15min intervals for 2-4h, after removal/replacement of the cuff, for 120min after SC butorphanol (0.4mg/kg), and with mild skin inflammation at the testing site. Data were analysed using ANOVA. Pressure thresholds in untreated cats were around 150mmHg. The minimum interval for testing was established as 15min. Data were reproducible over 4h and beyond 24h. Thresholds in 5 cats increased (P<0.05) above baseline for 45min after butorphanol with a maximum increase of 270+/-182mmHg at 10min. Thresholds decreased with inflammation. The method appears suitable for feline analgesia investigations.     

Determination of threshold concentrations of multiple allergenic extracts for equine intradermal testing using normal horses in three seasons

Forty-one normal horses were evaluated for reactivity to intradermally injected aqueous allergens to determine allergen threshold concentrations (TC), with potential relevance to equine intradermal testing (IDT). Horses were tested three times over 1 year to assess seasonal variation in reactivity, using three to five serial dilutions of 27 allergens each time. Injection sites were evaluated after 15 min, 1 h, 4 h and 24 h. The highest allergen concentration at which < 10% of horses demonstrated positive reactivity (subjective score of > or = 2, scale of 0 to 4) at 15 min was considered the TC. The TC was determined for nine pollens (2000 to > 6000 PNU mL(-1)), four moulds (4000 to > 6000 PNU mL(-1)), seven insects (ant, horse fly 125 PNU mL(-1); house fly, cockroach 250 PNU mL(-1); moth 60 PNU mL(-1); mosquito 1000 PNU mL(-1); Culicoides nebeculosis 1 : 5000 w v(-1)) and three of four storage mites (1 : 10,000 w v(-1)). The TC was not determined due to excessive reactivity at the lowest concentrations tested for dust mites (Dermatophagoides farinae [< 1 : 12,000 w v(-1)], D. pteronyssinus [< 1 : 30,000 w v(-1)]), and Acarus siro (< 1 : 10,000 w v(-1)). Minor variation in the TC for specific allergens occurred in different seasons. Progressive sensitization with repeat testing occurred for grain mill dust mix. Positive reactivity at 1 h and 4 h occurred in > 10% of horses for nine of 19 allergens (pollens, mosquito, storage mites) at their determined TC. Positive reactivity was rare at 24 h. This study in normal horses suggests that appropriate testing concentrations of allergens for equine IDT in atopic horses may be > or = 1000 PNU mL(-1) for pollens and moulds, 60 to 250 PNU mL(-1) for most insects and < 1 : 12,000 w v(-1) for dust mites; and that reactions at 1-4 h may be insignificant.     

Challenges of thermal nociceptive threshold testing in the donkey

ObjectiveTo evaluate a thermal nociceptive threshold (TNT) testing device in the donkey, and the influence of potential confounding factors on TNTs.AnimalsTwo groups (Group 1 and Group 2) of eight castrated male donkeys aged 4–9 years, weighing 105–170 kg.MethodsTNTs were measured by heating a thermal probe on skin until an end-point behaviour (threshold temperature) or a cut-out temperature (51 °C) was reached. The withers and the dorsal aspect of the distal limb were used as sites for TNT testing. The effects on TNT of different confounding factors: the limb tested; rate of heating; and ambient temperature were evaluated. Data were analyzed using general linear models, and Mann-Whitney tests, p < 0.05 was considered significant.ResultsEnd-point behaviours (skin twitch or donkey looking at test device) when the thermal probe heated the withers were observed in approximately half of tests. TNT was (mean ± SD) 46.8 ± 2.85 °C. Subsequently the limb was evaluated as the test site in Group 1 followed by Group 2 donkeys; end-point behaviour being a foot-lift. In Group 1, 72% of tests ended in an end-point behaviour but the response rate was lower in Group 2 (20%), although TNTs were similar [(47.6 ± 3.3) and (47.3 ± 3.0) °C respectively] for responding animals. Rate of heating, ambient temperature and laterality (right or left) did not affect thresholds, but mean TNT was significantly higher in the forelimb (48.5 ± 2.8 °C) than the hind limb (47.4 ± 2.8 °C) (p = 0.012).ConclusionsWhen a thermal probe cut-out temperature of 51 °C was used in TNT testing in the donkey a high proportion of tests did not produce an identifiable end point behaviour. Higher cut-out temperatures damaged the skin. Under these conditions, thermal nociceptive threshold testing appears not be an appropriate analgesiometry technique in the donkey.Clinical relevanceTNT testing under these conditions is not suitable form of analgesiometry for donkeys.     

Thermal and mechanical nociceptive threshold testing in horses: a review

OBJECTIVE: This review evaluates the thermal and mechanical nociceptive threshold testing techniques that have been used in horses and discusses them with reference to their applications, limitations and the factors which can influence both the testing procedure itself and the animal's responses. Methods to optimise the reliability and repeatability of the testing procedures are suggested and the potential clinical applications discussed. DATABASES USED: Web of Science and Medline. CONCLUSIONS: Thermal and mechanical nociceptive threshold testing techniques have valuable roles in both the identification of altered nociceptive function and the pre-clinical evaluation of analgesics in horses.