Evaluation of a commercially available enzyme-linked immunosorbent assay for the detection of allergen-specific IgE antibodies in dogs

Twenty-eight atopic dogs, 22 pruritic, non-atopic dogs and 10 healthy dogs were ELISA tested. For calculations of diagnostic specificity and sensitivity, positive ELISA test results in non-atopic dogs were considered false positive results. The absence of any positive results in the atopic dogs was considered false negative results. The atopic dogs were tested both with ELISA and an intradermal test, utilising allergen extracts from the same manufacturer, to determine the frequency of positive allergen reactions in the ELISA test compared with the intradermal test. The Prausnitz-Küstner test was performed to evaluate the significance of a positive ELISA test result. Based on cross-tabulations with clinically defined atopic dermatitis, the ELISA test showed a sensitivity of 53.6% and a specificity of 84.4%. The correlation between the ELISA and the intradermal test was poor. Positive Prausnitz-Küstner tests were not obtained using sera from dogs that were intradermal test negative for the tested allergens, even though sera had high levels of IgE as measured by the ELISA. These findings question the significance of a positive ELISA test result and indicate that the test is not measuring functional allergen-specific IgE.     

Evaluation of a commercially available ELISA kit for detection of antibodies to Anaplasma marginale and Anaplasma centrale in cattle in Australia and Zimbabwe

A newly available competitive inhibition ELISA kit for the serological diagnosis of anaplasmosis was evaluated in Australia and Zimbabwe. In Australia the performance of the test was compared with the card agglutination test (CAT).The assay was evaluated using negative sera collected from Anaplasma-free herds, positive sera from experimentally infected cattle and sera from Anaplasma marginale-endemic herds. The sensitivity and specificity of the ELISA in Australia were 100 % and 83,3 %, respectively, and the sensitivity and specificity of the CAT were both 100%. The agreement between the ELISA and CAT in the sera from endemic herds was 86,4 % (kappa = 0,718). The specificity of the ELISA in Zimbabwe was 100%. No meaningful estimate of sensitivity was possible in Zimbabwe because few known positive sera were available for testing, but all eight known positive sera that were available were clearly positive. We conclude that the ELISA is a useful alternative to the CAT for epidemiological studies.The ELISA kits have advantages over the CAT in that the ELISA is more robust and reagents are better standardized, but the kits are expensive.     

Evaluation of a commercially available enzyme-linked immunosorbent assay for detecting antibodies to Fasciola hepatica and Fasciola gigantica in cattle, sheep and buffaloes in Australia

A commercially available ELISA for detecting antibodies to liver fluke was evaluated for use in Australia. Milk and serum samples from cattle and sheep in which infection with Fasciola hepatica was confirmed by detection of eggs in faeces were used to estimate sensitivity. Similar samples collected from cattle and sheep outside the F. hepatica-endemic area were used to estimate specificity. The ELISA was also evaluated for detecting antibodies to F. hepatica in milk from sheep and antibodies to Fasciola gigantica in sera from cattle and buffaloes, but with small numbers of samples. In cattle, the sensitivity and specificity of the ELISA were 98.2% and 98.3% using serum and 97.7% and 99.3% using milk. In infected herds, 41.4% and 41.5% of animals were positive in the serum and milk ELISAs, respectively, whereas F. hepatica eggs were found in faecal samples from 26.5% of animals. In sheep, the sensitivity of the ELISA was 96.9% and the specificity was 99.4%. In infected flocks, 60.2% of animals were positive in the serum ELISA and F. hepatica eggs were found in faecal samples 52.2% of animals. There was perfect agreement in the ELISA between paired serum and milk samples collected from ewes. The assay detected antibodies in sera from cattle and buffaloes with natural and experimental F. gigantica infections. In the experimentally infected animals, antibodies were detected 2 weeks post-infection. We conclude that the ELISA will be a valuable tool for diagnosing F. hepatica infections in cattle and sheep. The assay may also be useful for diagnosing F. gigantica infections but further studies are required to establish sensitivity and specificity.     

Evaluation of commercially available assays for the measurement of equine insulin

Determining circulating equine insulin concentrations is becoming increasingly important in equine clinical practice and research. Most available assays are optimized for human medicine, but there is strong equine cross-reactivity because of the highly conserved nature of insulin. To identify an accurate and reliable assay for equine insulin, 6 commercial immunoassays were evaluated for precision, accuracy, and specificity. Only 1 assay initially reached the requisite standard: Mercodia Equine Insulin Enzyme-linked Immunosorbent assay (ELISA). Plasma matrix interferences were identified when the provided assay buffer was used with the Siemens Count-a-Coat Insulin radioimmunoassay (RIA) but not when charcoal-stripped equine plasma was used as the diluent. This modified RIA and the Mercodia Equine Insulin ELISA were evaluated further by directly examining accuracy by comparing their results for 18 equine plasma samples with values obtained using liquid chromatography and high-resolution/high-accuracy mass spectrometry (LC-MS). Compared with LC-MS measurements, the modified Siemens Insulin RIA rendered a moderate Lin's concordance coefficient (ρ_c) of 0.41, whereas the Mercodia Equine Insulin ELISA rendered a very poor ρ_c of 0.06. This suggests that the Siemens Insulin RIA is appropriate to use for routine evaluations when LC-MS is not available.     

Feline struvite crystalluria: a comparison of some commercially available dry cat diets in New Zealand

Thirteen adult cats were each fed consecutively five test diets: firstly a complete canned diet, and then four commercially available dry cat diets (New Formula Biscats, Cat Chow, Go-Cat and Whiskettes) for 7,13, 5, 4 and 4 weeks respectively. Urine samples were tested for pH and presence of struvite crystals. Body weight was recorded weekly. Urine pH was ranked from acid to alkaline as follows: Whiskettes (pH 5.8), canned diet (pH 5.9), Biscats (pH 6.1), Cat Chow (pH 6.45) and Go-Cat (pH 6.5). The pH of urine when cats were fed Whiskettes, Biscats and canned diet was not significantly different. Biscats produced a urine pH significantly higher than Whiskettes but, in each case, these three diets produced a significantly lower urine pH than Cat Chow and Go-Cat. Struvite crystals occurred spontaneously in 9% of urine samples from cats when fed Whiskettes and Biscats, 71% when fed Cat Chow and 38% when fed Go-Cat: spontaneous struvite was not seen in any cat fed CX9. Haematuria was seen in 2.9% (2/69) urine samples from cats fed Cat Chow. One tom cat had a blocked urethra after 24 days on Go-Cat. No abnormalities were observed in any cat when fed canned diet, Biscats or Whiskettes. It was concluded that of the four dry cat diets tested only Biscats and Whiskettes produce urine with an acceptably low rate of spontaneous struvite crystalluria.     

Evaluation of bacterial and protozoal contamination of commercially available raw meat diets for dogs

OBJECTIVE: To evaluate bacterial and protozoal contamination of commercially available raw meat diets for dogs. DESIGN: Prospective longitudinal study. SAMPLE POPULATION: 240 samples from 20 raw meat diets for dogs (containing beef, lamb, chicken, or turkey), 24 samples from 2 dry dog foods, and 24 samples from 2 canned dog foods. PROCEDURE: Each product was purchased commercially on 4 dates approximately 2 months apart. Three samples from each product at each sampling period were evaluated via bacterial culture for non-type-specific Escherichia coli (NTSEC), Salmonella enterica, and Campylobacter spp. Antimicrobial susceptibility testing was performed on selected isolates. Polymerase chain reaction assays were used to detect DNA from Cryptosporidium spp, Neospora spp, and Toxoplasma spp in samples obtained in the third and fourth sampling periods. RESULTS: One hundred fifty-three of 288 (53%) samples were contaminated with NTSEC. Both raw and prepared foods contained NTSEC during at least 1 culture period. Salmonella enterica was recovered from 17 (5.9%) samples, all of which were raw meat products. Campylobacter spp was not isolated from any samples. In 91 of 288 (31.6%) samples, there was no gram-negative bacterial growth before enrichment and in 48 of 288 (16.7%) samples, there was no aerobic bacterial growth before enrichment. Susceptibility phenotypes were variable. Cryptosporidium spp DNA was detected in 3 samples. CONCLUSIONS AND CLINICAL RELEVANCE: Bacterial contamination is common in commercially available raw meat diets, suggesting that there is a risk of foodborne illness in dogs fed these diets as well possible risk for humans associated with the dogs or their environments.     

Evaluation of a commercially available immunoassay for assessing adequacy of passive transfer in calves

OBJECTIVE: To evaluate diagnostic utility of a commercially available immunoassay for assessing adequacy of passive transfer of immunity in neonatal calves. DESIGN: Prospective study. ANIMALS: 123 calves. PROCEDURE: Blood and serum samples were obtained from the calves prior to 2 weeks of age. The immunoassay was performed, along with refractometry and an 18% sodium sulfite turbidity test. Serum IgG concentration was determined with a radial immunodiffusion assay. Sensitivity and specificity of the immunoassay, refractometry, and the sodium sulfite test were calculated by comparing results with results of the radial immunodiffusion assay. RESULTS: Sensitivity and specificity of the blood IgG immunoassay were 0.93 and 0.88, respectively, compared with 1.00 and 0.53 for the sodium sulfite test. For refractometry, sensitivity and specificity were 0.71 and 0.83, respectively, when a serum total solids concentration of 5.2 g/dl was used as the cutoff between positive and negative test results. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that the immunoassay performs well in detecting calves with inadequate passive transfer of immunity.     

The detection of bovine respiratory syncytial virus in formalin fixed bovine lung with commercially available monoclonal antibodies and avidin biotin complex immunohistochemistry.

Eight commercially available monoclonal antibodies directed against respiratory syncytial virus antigens were tested for ability to detect bovine respiratory syncytial virus (BRSV) antigen in formalin fixed, paraffin embedded bovine lung using avidin-biotin complex immunohistochemical staining. Monoclonal antibodies from clone 18B2 purchased from Biosoft, Paris, France and those from clone 8G12 purchased from the Department of Veterinary Sciences, University of Nebraska, Lincoln, Nebraska stained BRSV antigen in infected bovine lung with acceptable background staining of uninfected tissues. This method offers advantages over other techniques for BRSV diagnosis in that fresh tissue is not required and all reagents may be purchased commercially.     

Evaluation of a commercially available luteinizing hormone test for its ability to distinguish between ovariectomized and sexually intact bitches

OBJECTIVE: To determine the value of a commercially available test for measurement of luteinizing hormone (LH) concentrations for its use in distinguishing between ovariectomized and sexually intact bitches. DESIGN: Prospective longitudinal cohort study. ANIMALS: 101 sexually intact bitches and 199 ovariectomized bitches PROCEDURE: A commercial test kit was used to assay serum samples for LH concentration. A kappa test was used to assess the consistency of test interpretations. Sensitivity, specificity, predictive values, and consistency of test interpretation in the sample population and in 2 theoretical populations of bitches with differing frequencies of ovarian removal were determined. RESULTS: The test had excellent sensitivity (98%) but moderate specificity (78%) because of a high frequency of false-positive test results. This means that a single high serum LH concentration was not a reliable indicator of ovarian removal. However, a single low serum LH concentration was an excellent indicator that a bitch was sexually intact. The LH test results were positive within 10 days after ovarian removal and remained high in bitches from which ovaries had been removed > 5 years previously. Analysis of kappa values consistently revealed excellent agreement in test interpretation within and among evaluators and over a 6-hour time span. CONCLUSIONS AND CLINICAL RELEVANCE: A single low LH test result confirms the need for ovariectomy in bitches prior to adoption. The test is applicable to bitches that have been ovariectomized prior to puberty and probably applies over the lifespan of the animal.     

Variability in a commercially available enzyme-linked immunosorbent assay system. I. Assay variability.

Three experiments were conducted to characterize the variation in enzyme-linked immunosorbent assay (ELISA) kits for infectious bronchitis virus (IBV) and infectious bursal disease virus (IBDV). Expt. 1 was carried out to determine the variation in assay results when the same pools of low-, medium-, and high-titered serum were assayed. Significant variation occurred among separate lots and among test plates within the same lots for the IBV and IBDV assays. In most cases, variability between days and among technicians was not significant. Coefficients of variation were larger than is acceptable for immune-type assays. In the IBDV assay with high-titered serum, most of the wells in the plates reached maximum absorbance and were not capable of detecting titers above 1:8000-1:9000. Expt. 2 was conducted to determine the effects of varying the length of the ortho-phenylene-diamine (OPD) incubation time upon assay results. Either 7-, 12-, or 15-minute OPD incubation times were used. Incubation time significantly affected mean titer at all combinations of assay types and times, except determinations on the low-titered IBV samples. Expt. 3 was conducted to determine the effects of three different dilution methods on observed IBDV titer. The use of non-standard dilutions had significant effects on observed titer. In the medium- and high-titered samples, the use of two different dilution methods at 1:5000 rather than 1:500 resulted in titers that were three to four times those observed at the 1:500 dilution.     

Evaluation of a commercially available enzyme-linked immunosorbent assay for the diagnosis of canine sarcoptic mange

This study was designed to assess the accuracy of a commercial enzyme-linked immunosorbent assay for the diagnosis of canine scabies. Serum samples from 37 dogs were examined blind; 12 had sarcoptic mange confirmed by the identification of mites in skin scrapings, 12 were atopic (with positive intradermal reactions to one or more aeroallergens, including Dermatophagoides farinae), and 13 were healthy dogs with no history of skin disease. Optical density values of more than 0.16 were considered positive, 0.145 to 0.16 were considered questionable and less than 0.145 were considered negative. Ten of the 12 dogs with scabies were positive, all 12 atopic dogs were negative, and 11 of the 13 healthy dogs were negative and two were questionable.     

Evaluation of commercially available Escherichia coli J5 bacterin as protection against experimental challenge with Pasteurella multocida in rabbits.

OBJECTIVE: To evaluate the ability of commercially available Escherichia coli J5 bacterin to protect rabbits from experimental challenge with Pasteurella multocida. ANIMALS: 40 P multocida-free New Zealand White rabbits. PROCEDURES: Rabbits were assigned to 1 of 4 groups of 10 rabbits each. Three of the groups were inoculated SC with J5 bacterin at 8 weeks old. Inoculation was repeated 3 and 6 weeks later. The fourth group was not inoculated and served as controls. Groups 1, 2, and 3 were given 10(9), 10(8), and 10(7) colony forming units (CFU), respectively. Response was monitored by titer assessment, using an E coli J5 antigen capture ELISA. Five weeks after the last inoculation, all rabbits were challenged with P multocida and observed for an additional 5 weeks. Clinical, hematologic, serologic, culture, and necropsy data were collected. RESULTS: Inoculation of rabbits with 10(9) CFU of E coli J5 bacterin-induced titers that were significantly greater than titers of rabbits vaccinated with 10(8) or 10(7) CFU or those in controls. The incidence of acute bacteremia was lower in rabbits with high titers. At necropsy, prevalence of lesions typical of P multocida was not significantly different among groups. Prevalence of histologic lesions was also not significantly different among groups. CONCLUSIONS AND CLINICAL RELEVANCE: Although the bacterin induced considerable antibody response and possibly reduced the rate of bacteremia, antibodies were not protective against long-term colonization or infection of the frontal sinuses or tympanic bullae by the challenge strain of P multocida. This bacterin in its currently available form is unlikely to aid in reducing the prevalence of pasteurellosis in rabbits.     

Variability in a commercially available enzyme-linked immunosorbent assay system. II. Laboratory variability.

An experiment was conducted to determine the amount of variability that occurred in enzyme-linked immunosorbent assays when samples from common serum pools were assayed in five different labs on three consecutive days. Low- (approximately 1:2000), medium- (approximately 1:4000), and high-titered (approximately 1:8000) serum pools were distributed to five poultry industry laboratories that cooperated in the study. Results varied significantly among different laboratories and among different days in the same lab. Variation among days within the same laboratory and among laboratories were large. The greatest variability occurred among labs. Correlations between mean daily titer and laboratory ambient temperature were small and not significant. The amount of variability within and among different laboratories that were observed indicate that single determinations on individual serum samples are not likely to give a reliable estimate of antibody titer. The large variability within labs further indicates the need for standard reference pools of positive serum to be included in assays in order to substantiate assay results.     

Evaluation of a commercially available apparatus for measuring the acute phase protein serum amyloid A in horses

The aim of this study was to investigate the reliability of an immunoturbidometric assay for measuring the acute phase protein serum amyloid A (SAA) in horses in clinical practice. The assay was compared to a previously validated assay, and overlap performance was assessed by measuring the concentration of SAA in clinically healthy horses and horses with inflammatory and non-inflammatory diseases. In pools of serum with low and high SAA concentrations the assay's intra-assay coefficients of variation were 11.7 per cent and 4.6 per cent, and its interassay coefficients of variation were 9.1 per cent and 5.6 per cent, respectively. Slight inaccuracies were observed, but they were negligible in comparison with the range of the SAA response. The assay systematically underestimated the concentrations of SAA in comparison with the results of the validated assay. The assay detected the expected difference in SAA concentrations between the healthy and diseased horses.     

Taurine deficiency in Newfoundlands fed commercially available complete and balanced diets

OBJECTIVE: To determine taurine status in a large group of Newfoundlands related by environment, diet, or breeding to a dog with dilated cardiomyopathy and taurine deficiency. DESIGN: Prospective study. ANIMALS: 19 privately owned Newfoundlands between 5 months and 11.5 years old that had been fed commercial dry diets meeting established nutrient recommendations. PROCEDURE: Diet histories were obtained, and blood, plasma, and urine taurine concentrations and plasma methionine and cysteine concentrations were measured. In 8 dogs, taurine concentrations were measured before and after supplementation with methionine for 30 days. Ophthalmic examinations were performed in 16 dogs; echocardiography was performed in 6 dogs that were taurine deficient. RESULTS: Plasma taurine concentrations ranged from 3 to 228 nmol/mL. Twelve dogs had concentrations < 40 nmol/mL and were considered taurine deficient. For dogs with plasma concentrations < 40 nmol/mL, there was a significant linear correlation between plasma and blood taurine concentrations. For dogs with plasma concentrations > 40 nmol/mL, blood taurine concentrations did not vary substantially. Taurine-deficient dogs had been fed lamb meal and rice diets. Retinal degeneration, dilated cardiomyopathy, and cystinuria were not found in any dog examined for these conditions. The taurine deficiency was reversed by a change in diet or methionine supplementation. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicate a high prevalence of taurine deficiency among an environmentally and genetically related cohort of Newfoundlands fed apparently complete and balanced diets. Blood taurine concentrations indicative of taurine deficiency in Newfoundlands may be substantially less than concentrations indicative of a deficiency in cats.