Determination of threshold concentrations of allergens and evaluation of two different histamine concentrations in canine intradermal testing

The purpose of this study was to determine the optimal histamine concentration and allergen threshold concentrations for canine intradermal testing. Thirty healthy dogs were tested using two different concentrations of histamine and four different concentrations of each allergen. The optimal histamine concentration was determined to be 1:10 000 w/v. The threshold concentration was at least 1750 PNU/mL for all tested grasses, weeds, trees, moulds and insects, except for fleas which was as least 1:500 w/v. For Dermatophagoides pteronyssinus, the optimal threshold concentration was 250 PNU/mL, whereas for Dermatophagoides farinae and Tyrophagus putrescentiae, it was 100 PNU/mL. Threshold concentration for all epidermals except human dander was at least 1250 PNU/mL. The optimal threshold concentration for human dander was 300 PNU/mL. Our results suggest that the currently used 1:100 000 w/v concentration of histamine and the 1000 PNU/mL concentration for most grasses, weeds, trees, moulds, epidermals and insects may not be appropriate for canine intradermal testing.     

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 ).     

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.     

Evaluation of cross-reactivity of allergens by use of intradermal testing in atopic dogs

OBJECTIVE: To examine cross-reactivity of aeroallergens in Colorado and surrounding states by evaluating concurrent positive reactions of related and nonrelated allergens of intradermal tests in dogs. SAMPLE POPULATION: Intradermal test results of 268 atopic dogs. PROCEDURE: A retrospective evaluation of skin test results for 268 dogs was performed. Pairs of closely related and nonrelated allergens were evaluated. Group 1 consisted of closely related allergens with demonstrated antibody cross-reactivity in humans. In group 2, allergens of the same plant group (ie, trees, grasses, or weeds) that were not closely related were paired. In group 3, allergen pairs were of different plant groups. Plant allergens were paired with dust mite allergens, animal dander, or mold spores in group 4. In the last group, allergens not derived from plants were paired. Data were evaluated twice by use of a different definition of a positive reaction. Significance of the difference between group means of log odds ratios was estimated by use of a boot-strap percentile confidence interval. RESULTS: Significant differences in the number of concurrent positive reactions were not found between related versus nonrelated grass, weed, or tree allergens. Significant differences in the number of concurrent positive reactions were found between plant allergens of different groups (ie, grasses, weeds, and trees) and plant allergens of the same groups, related or nonrelated, as well as between plant-derived and nonplant-derived allergens. Many dogs reacting to a specific allergen did not react to a closely related allergen at the same time. CONCLUSION: These results provide evidence against clinically relevant cross-reactivity and suggest that allergen-specific immunotherapy should be formulated on the basis of single allergen test results.     

Evaluation of the precision of intradermal injection of control substances for intradermal testing in clinically normal horses

OBJECTIVE: To evaluate the precision of intradermal testing (IDT) in horses. ANIMALS: 12 healthy adult horses. PROCEDURE: IDT was performed on the neck of each horse by use of 2 positive control substances (histamine and phytohemagglutinin [PHA]) and a negative control substance. An equal volume (0.1 mL) for each injection was prepared to yield a total of 20 syringes ([4 concentrations of each positive control substance plus 1 negative control substance] times 2 positive control substances times 2 duplicative tests) for each side of the neck. Both sides of the neck were used for IDT; therefore, 40 syringes were prepared for each horse. Hair was clipped on both sides of the neck, and ID injections were performed. Diameter of the skin wheals was recorded 0.5, 4, and 24 hours after ID injection. RESULTS: Intra- and interhorse skin reactions to ID injection of histamine and PHA resulted in wheals of uniform size at 0.5 and 4 hours, respectively. Significant intra- and interhorse variation was detected in wheals caused by PHA at 24 hours. CONCLUSIONS AND CLINICAL RELEVANCE: ID injection of histamine and PHA caused repeatable and precise results at 0.5 and 4 hours, respectively. Concentrations of 0.005 mg of histamine/mL and 0.1 mg of PHA/mL are recommended for use as positive control substances for IDT in horses. This information suggests that consistent wheal size is evident for ID injection of control substances, and variation in wheals in response to ID injection of test antigens results from a horse's immune response to specific antigens.     

A thermal threshold testing device for evaluation of analgesics in cats

A thermal analgesiometric device was developed for unrestrained cats. Heat was provided by an electrical element potted together with a temperature sensor in thermally conductive epoxy in a 5 gm probe. This was attached to an elasticated band round the cat's thorax with an inflated bladder maintaining constant pressure between probe and skin. A safety cut-off was set at 60 degrees C. End point was a skin flick, turning, or jumping. Threshold temperatures in untreated cats were around 40 degrees C and repeatable to 4 degrees C with 5, 10 or 15 minutes between tests. Threshold temperature was stable in tests at 15 minutes intervals without false positives or negatives. Tests repeated at weekly intervals were repeatable to within 4 degrees C. Treatment with the opioid analgesic pethidine increased the threshold temperatures 10.2 (6.7) degrees C 45 minutes after treatment. The device was well tolerated for at least 24 hours and the analgesic effect of an opioid was detected. The system appears suitable for use in investigations into analgesic pharmacology in cats.     

Plasma chloramphenicol concentrations in cats following intramuscular administration of three different chloramphenicol preparations

Five adult cats were given 30 mg chloramphenicol/kg by intramuscular injection on three separate occasions, using a different preparation on each occasion. The preparations were an aqueous suspension of chloramphenicol, a solution of chloramphenicol in methylpyrrolidone, and an aqueous solution of chloramphenicol sodium succinate. Plasma chloramphenicol concentrations were determined chemically at fixed intervals after dosing. Chloramphenicol sodium succinate solution gave higher and more rapidly attained plasma chloramphenicol concentrations than the other two preparations. The extent of chloramphenicol absorption up to 8 h after administration was greater with the succincate ester than with chloramphenicol dissolved in methylpyrrolidone. There was no difference in bioavailability between the aqueous suspension and the solution in methylpyrrolidone.     

Effects of otic betamethasone on intradermal testing in normal dogs

Otitis externa is common in atopic dogs and is frequently treated using potent glucocorticoids topically. These preparations can cause adrenal suppression and affect skin test reactivity. The purpose of this study was to determine if an otic product containing betamethasone could decrease skin reactivity in normal dogs. Sixteen laboratory beagles were used in a cross-over, blinded trial. Dogs were enrolled in two groups; one received placebo and the other a betamethasone-containing otic preparation (Otomax) twice daily for 2 weeks. After a 4-week wash-out period, treatments were switched. Dogs were intradermally tested on days 0 and 14 of each treatment period with histamine phosphate (1 : 100,000 and 1 : 200,000 w/v) and allergens common in the area. Adrenocorticotropic hormone (ACTH) stimulation tests were done before and after treatment to investigate adrenal suppression. After 2 weeks of otic betamethasone, Dermatophagoides farinae (P = 0.0034), Cynodon dactylon (P = 0.0459) and histamine 1 : 100,000 w/v (P = 0.0028) reactions were significantly reduced. Pre-treatment post-ACTH serum cortisol levels and those obtained after both treatments did not differ statistically (P = 0.6362). Betamethasone induced a slight but statistically significant elevation (P = 0.0002) of serum alkaline phosphatase. Despite the increase, values were within normal range. It is concluded that, although otic betamethasone did not suppress adrenal glands, it mildly suppressed intradermal reactions to 1 : 100,000 w/v histamine, D. farinae and C. dactylon.     

Determination of irritant threshold concentrations to weeds,trees and grasses through serial dilutions in intradermal testing on healthy clinically nonallergic dogs

Irritant threshold concentration (ITC) for intradermal testing (IDT) was determined in 31 healthy, clinically nonallergic dogs. Twenty‐three allergens were tested at five variable concentrations ranging from 1000 to 8000 PNU/mL. To distinguish irritant reactions from subclinical IgE‐mediated hypersensitivities, serum allergy testing was performed. ITCs were determined by evaluating the lowest concentration to which no dogs (0% cut‐off) and to which at least 10% of dogs (≥10% cut‐off) reacted. ITCs at the 0% cut‐off were: 1000 PNU/mL (Johnson grass), 2000 PNU/mL (Ash, Lamb’s Quarter and Bermuda), 3000 PNU/mL (Bahia, Rye, Pig Weed and Virginia Oak), 4000 PNU/mL (Marsh Elder and Maple), 5000 PNU/mL (Sorrel sheep) and 7000 PNU/mL (Cocklebur and Black Willow). ITC for Dog Fennel, Box Elder and Red Cedar was <1000 PNU/mL. ITCs at the ≥10% cut‐off were: 2500 PNU/mL (Johnson), 3000 PNU/mL (Box Elder), 5000 PNU/mL (Bahia), 6000 PNU/mL (Pigweed and Marsh Elder) and 8000 PNU/mL (Virginia Oak and Black Willow). For all other allergens, the ITC was >8000 PNU/mL and could not be determined. No significant agreement between positive values was found for the same allergen on IDT and serum allergy testing for each dog suggesting reactions caused by the determined ITCs are less likely subclinical IgE‐mediated reactions. These results suggest that ITCs may vary, also they may be very high for the allergens tested and that higher test concentrations may be used for IDT for the tested allergens without inducing an irritant reaction. Further studies are needed to evaluate the benefit of higher IDT concentrations in atopic dogs.     

Serial determination of thyroxine concentrations in hyperthyroid cats

Serum thyroxine (T4) concentrations of 10 hyperthyroid cats were measured at hourly intervals between 9 AM and 4 PM. In 5 cats, blood samples were obtained by jugular venipuncture; the remaining 5 cats had blood samples obtained from jugular catheters. Over the 7-hour period, a significant temporal (diurnal) variation was not identified in the serum T4 concentrations of the cats (P greater than 0.01). The lowest mean serum T4 concentration (9.1 micrograms/dl) was measured at 3 PM and was 14.2% less than the highest mean serum T4 concentration (10.6 micrograms/dl) measured at 9 AM. Though there were fluctuations in serum T4 concentrations during the 7-hour period, the differences were not systematic. The maximal variation in serum T4 concentrations over the 7-hour period averaged less than 21%. Despite the random variations during the 7-hour period, none of the measured serum T4 concentrations was in the normal range. Measurement of serum T4 concentration from randomly obtained blood samples was determined to be reliable for diagnosing feline hyperthyroidism.     

Comparison of three methods for cardiac output determination in cats

Cardiac output (CO) was measured in sodium pentobarbital-anesthetized cats over a wide range of blood flow rates. In 10 cats, CO was measured simultaneously, using Fick determination and thermodilution techniques. Echocardiography was used to estimate contractility of the heart by measuring percentage change in minor diameter and velocity of circumferential fiber shortening. These indices were compared with CO by the other techniques. Echocardiographic equations used for CO determination in man were evaluated for reliability in the cat. Thermodilution and Fick determination correlated best with low CO (r = 0.89) and less with intermediate (r = 0.69) and high (r = 0.75) CO. Percentage change in minor diameter and velocity of circumferential fiber shortening correlated with thermodilution measurements of the cardiac index (r = 0.71 and r = 0.84, respectively). The value of echocardiography for CO estimation was questionable, using existing equations. Fick determination of CO was more inconsistent and was more prone to technical error than was thermodilution.     

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.     

Erythrocyte protoporphyrin concentrations in clinically normal cats and cats with lead toxicity.

Erythrocyte protoporphyrin (EPP) and blood lead concentrations were determined in 91 clinically healthy cats living in the inner suburban area of Sydney, Australia. The mean EPP concentration was 223.4 +/- 186.1 micrograms litre-1 whole blood and the mean blood lead concentration 0.62 +/- 0.25 mumol litre-1. EPP concentrations were also monitored in three cats with confirmed lead toxicity--at the time of diagnosis and one week and one month after chelation therapy with calcium EDTA. EPP concentrations were elevated in two cats and within the normal range in the third cat at the time of diagnosis. EPP concentration were higher in two cats one week after treatment than at the time of diagnosis. One month after chelation therapy, EPP concentrations were normal in two cats but still substantially elevated in the third cat although its blood lead concentration had returned to normal and all clinical signs of lead toxicity had resolved. It was determined that the predominant form of protoporphyrin present in cats with lead toxicity was zinc protoporphyrin. The EPP assay may have limited value in the diagnosis of acute lead toxicity and in monitoring the success of chelation therapy in cats.     

Evaluation of skin test reactivity to environmental allergens in healthy cats and cats with atopic dermatitis

OBJECTIVE: To evaluate skin test reactivity to environmental allergens in healthy cats and in cats with atopic dermatitis (AD). ANIMAL: 10 healthy cats and 10 cats with AD. PROCEDURE: 10 allergens in serial dilutions were injected ID on the lateral aspect of the thorax of sedated cats. Histamine (0.01% solution) and buffer solutions were used as positive and negative controls, respectively. Immediately after the last injection, 10% fluorescein solution was administered IV. Skin test results were evaluated with ultraviolet light after 15 to 30 minutes and at 4 and 6 hours by 2 independent observers. In the control group, skin tests were repeated after 6 weeks. Skin test reactivity and the nature of the immunoglobulin involved were investigated by use of the Prausnitz-Küstner test with untreated and heat-treated cat sera. RESULTS: Intertest and interobserver agreement were high when measurement of the diameter of the fluorescent wheal was used to evaluate skin test responses, compared with assessment of its intensity. In both groups of cats, immediate skin test reactivity was observed as an IgE-mediated reaction, as an IgG-mediated reaction, and as a result of nonspecific mast cell degranulation. There was no correlation between allergen concentration and the type of reaction observed. CONCLUSIONS AND CLINICAL RELEVANCE: Skin test reactivity in cats should be evaluated after IV administration of 10% fluorescein solution by means of a Prausnitz-Küstner test to differentiate among IgE-mediated, IgG-mediated, and nonspecific reactions.