Comparison of the complement-fixation and agar gel immunodiffusion tests for diagnosis of subclinical bovine paratuberculosis

The performance of the serum complement fixation (CF) test was compared with that of a serum agar gel immunodiffusion (AGID) test on 74 subclinically infected and 154 uninfected cattle in 6 commercial midwestern dairy herds with Mycobacterium paratuberculosis infection and on 30 cattle in a herd that was free of infection. Infection status of cattle within herds was established by performance of a series of 3 or more fecal cultures and of ileocecal lymph node cultures of culled cattle. In cattle with subclinical infection detected by culturing, the sensitivity estimates of the CF and AGID tests were 10.8% (3.6% SE) and 18.9% (4.5% SE), respectively. In the cattle classified as disease free, the specificity estimates of the CF and AGID tests were 97.4% (1.3% SE) and 99.4% (0.6% SE), respectively. Neither set of estimates was significantly different. Negative test results obtained with the use of either test in apparently normal cattle from suspect herds should be interpreted with caution because both tests suffer from low sensitivities in subclinically infected animals. However, the AGID test may be more useful in regulatory situations in which the CF test is currently used because the AGID test is easier to perform and to interpret.     

Comparison of agar gel immunodiffusion test, rapid slide agglutination test, microbiological culture and PCR for the diagnosis of canine brucellosis

The performance of the rapid slide agglutination test, with and without 2-mercaptoethanol (RSAT and 2ME-RSAT) and agar gel immunodiffusion test (AGID) was evaluated for the diagnosis of brucellosis in naturally infected dogs. The microbiological culture, PCR and clinical parameters were used as reference. A total of 167 dogs were clinically examined and tested by blood culture, culture of semen/vaginal swab and PCR in blood and semen/vaginal swab. According to the results observed the 167 dogs were divided into three groups: Brucella canis infected dogs (Group 1), B. canis non-infected dogs (Group 2) and dogs with suspected brucellosis (Group 3). The dogs were then tested by RSAT, 2ME-RSAT and AGID. Groups 1 and 2 were used to calculate the diagnostic sensitivity and specificity of the serological tests and the results observed in Group 3 were also discussed. The diagnostic sensitivity of RSAT, 2ME-RSAT and AGID was respectively 70.58%, 31.76%, and 52.94%. The diagnostic specificity of RSAT, 2ME-RSAT and AGID was respectively 83.34%, 100%, and 100%. In dogs with suspected brucellosis 15% were RSAT positive, none was 2ME-RSAT positive and 5% were AGID positive. Although the serological tests are the most commonly used methods for brucellosis diagnosis, a significant proportion of false-negative results were observed highlighting the importance of the direct methods of diagnosis, like blood culture and PCR to improve the diagnosis of canine brucellosis.     

Evaluation of the agar gel immunodiffusion test for diagnosis of subclinical paratuberculosis in cattle

Concurrent bacteriologic culture of feces and agar gel immunodiffusion (AGID) testing was performed on all cows and bred heifers over 14 months old in 10 dairy herds during a 32-month period to determine the effectiveness of the AGID test for the detection of subclinical paratuberculosis. Herds were sampled 5 times and, when possible, culled animals were tested again at slaughter. During 5 herd-wide samplings, Mycobacterium paratuberculosis was isolated from 139 fecal specimens obtained from 109 cattle. Results of the AGID test were simultaneously positive 40 of 139 times (28.8%). Thirty-six of the 109 cattle (33.0%) determined to be infected had a positive AGID test result at some point during the 5 herd-wide samplings. When results of tests performed at time of slaughter were included, 117 cattle were identified as infected by culture methods; 55 of these (47.0%) were AGID test-positive at some point during the study. The upper limit of the maximal false-positive rate for the AGID test was 2.1%. On the basis of colony counts from cultures, subclinically infected cows shedding higher numbers of M paratuberculosis in their feces were more likely to have positive AGID test results (P less than 0.0001). In known infected cattle, neither the culture nor AGID test results were consistently positive on repeated testing. Of 48 official calfhood paratuberculosis vaccinates tested as adults, 3 had positive AGID test results and in 1 of these, M paratuberculosis was also isolated from the feces, indicating that the rate of false-positive AGID test results in calfhood vaccinates is low.     

Detection of Aspergillus-specific antibodies by agar gel double immunodiffusion and IgG ELISA in feline upper respiratory tract aspergillosis

Feline upper respiratory tract aspergillosis (URTA) is an emerging infectious disease. The aims of this study were: (1) to assess the diagnostic value of detection of Aspergillus-specific antibodies using an agar gel double immunodiffusion (AGID) assay and an indirect immunoglobulin G (IgG) ELISA; and (2) to determine if an aspergillin derived from mycelia of Aspergillus fumigatus, Aspergillus niger and Aspergillus flavus can be used to detect serum antibodies against cryptic Aspergillus spp. in Aspergillus section Fumigati. Sera from cats with URTA (group 1: n = 21) and two control groups (group 2: cats with other upper respiratory tract diseases, n = 25; group 3: healthy cats and cats with non-respiratory, non-fungal illness, n = 84) were tested. Isolates from cats with URTA comprised A. fumigatus (n = 5), A. flavus (n = 1) and four cryptic species: Aspergillus felis (n = 12), Aspergillus thermomutatus (Neosartorya pseudofischeri, n = 1), Aspergillus lentulus (n = 1) and Aspergillus udagawae (n = 1).Brachycephalic purebred cats were significantly more likely to develop URTA than other breeds (P = 0.013). The sensitivity (Se) of the AGID was 43% and the specificity (Sp) was 100%. At a cut-off value of 6 ELISA units/mL, the Se of the IgG ELISA was 95.2% and the Sp was 92% and 92.9% for groups 2 and 3 cats, respectively. Aspergillus-specific antibodies against all four cryptic species were detected in one or both assays. Assay Se was not associated with species identity. Detection of Aspergillus-specific antibodies by IgG ELISA has high Se and Sp for diagnosis of feline URTA.     

Utility of fungal polymerase chain reaction on nasal swab samples in the diagnosis and monitoring of sinonasal aspergillosis in dogs

BackgroundIn dogs with sinonasal aspergillosis (SNA) the utility of PCR in the diagnosis and monitoring of the disease after treatment has not been assessed.ObjectivesTo evaluate the presence of fungal DNA using quantitative PCR targeting Aspergillus fumigatus (Aspfum) and Aspergillus spp. (PanAsp), and PCR targeting multiple fungal species (PanFun), in samples obtained from nasal cavities of dogs with SNA, other nasal diseases and healthy dogs.AnimalsSixty‐two dogs including 20 with SNA, 12 with cured SNA (of which 10 are from the SNA group), 20 dogs with Non‐SNA nasal disease, and 20 healthy dogs.MethodsProspective cross‐sectional study. Aspfum, PanAsp, and PanFun were performed on blindly collected nasal swabs obtained in anesthetized dogs.ResultsIn SNA dogs, Aspfum and PanAsp were positive in 13/20 and 14/20 dogs. In all dogs in the 3 other groups, A. fumigatus DNA was not detected using Aspfum. PanAsp was positive in 3 non‐SNA dogs: 1 with cured SNA and 2 with Non‐SNA nasal disease. A Ct cut‐off value of 33.3 for Aspfum demonstrated 65% sensitivity and 100% specificity. A Ct cut‐off value of 34.5 for PanAsp demonstrated 70% sensitivity and 96.2% specificity. PanFun was positive in 16/20, 12/12, 19/20, and 7/20 dogs in the SNA, cured SNA, Non‐SNA, and healthy groups, respectively.Conclusion and Clinical Importance Aspfum and PanAsp on blindly collected nasal swabs can be useful for the detection of SNA at diagnosis and at cure, especially when more invasive methods are not available.     

Comparison of commercial enzyme-linked immunosorbent assays and agar gel immunodiffusion tests for the serodiagnosis of equine infectious anemia

The purpose of this study was to estimate the performance characteristics (accuracy, detection limit, and precision) of commercially available enzyme-linked immunosorbent assay (ELISA) and agar gel immunodiffusion (AGID) kits in comparison with a reference AGID kit for the detection of equine infectious anemia (EIA) antibodies in horses for regulatory use in Canada. A total of 285 positive and 315 negative samples by the reference AGID were tested blindly on 2 other AGID and 4 ELISA kits. Commercially available AGID kits for the serodiagnosis of EIA were found equivalent. The 3 ELISAs directed against antibodies to the p26 core protein also performed relatively well in comparison with the reference AGID, with excellent relative accuracy and acceptable precision. The single ELISA directed against antibodies to the gp45 trans-membrane viral protein yielded a lower relative sensitivity. The performance characteristics of the ELISAs directed against antibodies to p26 are, therefore, adequate to support the implementation of ELISA for regulatory purposes in Canada.     

Comparison of agar gel immunodiffusion test, enzyme-linked immunosorbent assay and western blotting for the detection of BLV antibodies

An indirect enzyme-linked immunosorbent assay (ELISA) for the diagnosis of bovine leukaemia virus (BLV) infection was developed and compared with the agar gel immunodiffusion test (AGIDT). Western blotting (WB) was used as confirmatory test. ELISA and AGIDT had specificities that were comparable with that of WB, however, ELISA showed a higher sensitivity than AGIDT. The ELISA was useful for screening a large number of samples, whereas WB was important for detecting the antibody response against the individual BLV-proteins. Different types of positive serological reactions were discerned in WB, that correlated with reactions of sera in AGIDT and ELISA. The most important antigen in WB and ELISA was the BLV protein p24, whereas the BLV glycoproteins gp51 and gp30 were of special importance in AGIDT. The relevance of repeatedly testing the antibody response in BLV-infected herds for control and eradication programmes using assays with higher sensitivity than AGIDT was demonstrated.     

Evaluation of pseudorabies viral antigens in the agar gel immunodiffusion test.

Procedures designed to extract pseudorabies viral (PRV) antigens from PRV-infected tissue cultures were investigated to determine whether differences in extraction method had an effect upon the final concentrated antigenic product. All four of the preparations made from PRV-infected tissue culture cells (trypsin extract and disrupted cells) or entire PRV-infected cultures (polysorbate 80 extract and (NH4)2SO4 precipitate) contained relatively large amounts of the same antigen, whereas cell-free PRV-infected tissue culture fluids did not contain significant amounts of this antigen. Specific antibody directed against this antigen was present in all PRV antisera tested. Two other antigens were observed in some of the preparations, but PRV antisera varied in their ability to precipitate with these antigens. Therefore, the number of precipitation lines observed in agar gel immunodiffusion between PRV preparations and PRV-positive antisera depended both upon the extraction method used to obtain the antigen and upon the specificity of the selected antiserum.     

Comparative evaluation of the agar gel immunodiffusion test and two commercial ELISA kits for the serodiagnosis of equine infectious anemia

Selected sets of serum samples of horses were tested blindly in a comparative investigation for antibodies against Equine Infectious Anemia (EIA) virus. Three commercial kits were used, a well-established agar-gel immuno-diffusion kit which our laboratory has been using routinely for 14 years on one hand, a competitive ELISA kit (CELISA) and a non-competitive ELISA kit on the other hand. The American EIA Reference Laboratory in Ames cotested 56 serum samples with the same 3 products, with highest-level correlation, thereby ascertaining full dependability of our own results. Five EIA experts supplied us critically weak or doubtfully reacting serum samples of experimentally infected horses together with their own test results, by necessity limited to the then available AGID in most instances. A high degree of correlation was found between our and their AGID results. In our own laboratory good correlation was found between the AGID test and one lot of the CELISA product. Time of seroconversion was coincident in some experimentally infected horses, partly AGID, partly CELISA proved more sensitive. Another lot of the CELISA product deteriorated completely long before the warranted validity, an unpleasant finding experienced by many other laboratories alike. The non-competitive ELISA product showed unacceptable inter-lot differences, oscillation between positive and negative results on consecutive samples of one and the same horse, never reacted with the weak positive International Reference Serum, and one lot deteriorated well beyond its expiration date. We discuss our results with the background: high sensitivity versus false-positive horses and advocate to maintain at their present sensitivity levels the AGID and the CELISA tests and not to push them further, as would be technically possible.     

Whole blood and tissue fungal DNA quantification in the diagnosis of canine sino-nasal aspergillosis

Various combinations of tests are used to confirm the diagnosis of canine sino-nasal aspergillosis (SNA) because false-positive and false-negative results can occur with each test. Therefore, the aim of this study was to evaluate whether detection of fungal DNA in blood and nasal tissue samples was of value in the clinical diagnosis of this disease. Four groups were included in the study (dogs with SNA, lymphoplasmacytic rhinitis or nasal neoplasia, and control animals). Real-time PCR assays detecting DNA from all Penicillium and Aspergillus species (PenAsp assay) or species-specific DNA from A. fumigatus, A. terreus, A. flavus and A. niger were applied to whole blood and nasal tissue samples. Results obtained by PCR were compared between the groups. Sensitivity, specificity, positive and negative predictive values (PPV and NPV) for fungal DNA detection were compared with those for alternative diagnostic procedures including histopathology, serology and fungal culture. Significantly more fungal DNA was detected by the PenAsp assay in tissue biopsies from dogs with SNA than in the three other groups. Sensitivity, specificity, PPV and NPV for this method were 1.00, 0.06, 0.32 and 1.00. A. fumigatus DNA was detected in seven tissue biopsies from dogs with SNA and in one biopsy from a dog with a nasal tumour. Sensitivity, specificity, PPV and NPV for this diagnostic test were 0.50, 0.97, 0.87 and 0.82. No significant difference was found between the groups with respect to the amount of DNA detected in blood by the PenAsp assay. Sensitivity, specificity, PPV and NPV for this method were 0.71, 0.24, 0.31 and 0.64. A. fumigatus DNA was detected in the blood of three dogs with SNA and sixteen dogs without SNA. Sensitivity, specificity, PPV and NPV for this diagnostic tool were 0.21, 0.45, 0.15 and 0.54. Detection of A. fumigatus DNA in nasal tissue had the highest specificity, PPV and NPV but sensitivity of this method was low. Detection of fungal DNA in whole blood was of no value in the diagnosis of SNA.     

Bovine leukemia virus infection in Taiwan: evaluation of the enzyme-linked immunosorbent assay and agar gel immunodiffusion test.

I evaluated an enzyme-linked immunosorbent assay (ELISA) and agar gel immunodiffusion (AGID) test simultaneously for the detection of bovine leukemia virus (BLV) antibodies. Total 1,293 serum samples were tested for ELISA and AGID test and the results were compared. The results of ELISA and AGID agreed by 1,156 out of 1,293 (89.4%). All of AGID-positive 356 sera were positive by ELISA. However, of 451 ELISA-positive sera, 95 sera were either negative or equivocal by AGID test. Eleven animals which showed ELISA-positive but AGID-negative or equivocal became AGID-positive in a year. It may be inferred that ELISA detects infected cattle earlier and with greater sensitivity than AGID.     

Agar gel immunodiffusion test for diagnosis of clinical paratuberculosis in cattle

During a 19-month period, the agar gel immunodiffusion (AGID) test was used as an aid in differential diagnosis of paratuberculosis in 51 cattle with a history of chronic weight loss and/or chronic diarrhea. Thirty-three cattle were AGID test-positive for paratuberculosis. Twenty-eight cattle (87.5%) yielded Mycobacterium paratuberculosis from bacterial culture of feces. Four cattle were confirmed as having paratuberculosis on necropsy. One had a negative fecal culture but was lost to follow-up. Thus, 32 of the 33 AGID test-positive cattle (96.9%) were confirmed as paratuberculous by culture or necropsy. Of the 18 cattle that were AGID test-negative, 17 (94%) also were culture-negative after 12 to 20 weeks' incubation of the culture tubes. Direct fecal smears from 29 confirmed paratuberculous cattle were examined. Twenty-three (79.3%) had smears positive for M paratuberculosis. Fifteen culture-negative cattle were examined by direct smear; the results were negative for all.     

Standardization and validation of an agar gel immunodiffusion test for the diagnosis of equine infectious anemia using a recombinant p26 antigen

We developed and validated an agar gel immunodiffusion test (AGID) test for the diagnosis of equine infectious anemia (EIA) using as antigen the p26 protein of equine infectious anemia virus (EIAV) produced in the Escherichia coli expression system. The developed rp26-AGID test showed an excellent diagnostic relative sensitivity (100%) and specificity (100%) compared to a commercial AGID assay when 1855 field serum samples were analyzed. In addition, the rp26-AGID demonstrated to be a precise assay with excellent repeatability and reproducibility. In the analytical sensitivity trial, positive sera showed nearly the same endpoint dilutions for both compared tests. No positive-reactions were observed with 35 serum samples with antibodies related to other endemic agents and also with severely hemolysed samples, demonstrating that the rp26-AGID has an excellent analytical specificity. Complete concordance with blind previous results from five proficiency test panels confirmed the capability of the assay of accurate detection of EIAV antibodies. This is the first time that a recombinant AGID assay able to identify EIAV infections has been standardized and validated in Argentina according to international guidelines. Taking into account the results obtained, the p26-AGID could be adopted as an official test method for the diagnosis and control of EIA in this country.     

马传染性贫血琼扩抗原生产工艺的改进

马传染性贫血病毒(Equine infectious anemia virus，EIAV)是逆转录病毒科慢病毒属的成员之一，其基因组结构与人免疫缺陷病毒Ⅰ型(Human innunodeficiency virus type 1，HIV-Ⅰ)相似。马传染性贫血呈世界性分布，常引起慢性感染，以发热、贫血为特征，耐过马呈隐性感染并终身带毒。20世纪70年代初期，Coggins博士建立了检测感染马血清抗体的琼脂扩散试验(AGID)，成为检测EIA的标准方法。     

Comparison of selective culture and serologic agglutination of Treponema hyodysenteriae for diagnosis of swine dysentery

Samples of faeces and of serum were collected from pigs of various ages on 21 farms. Faecal samples were cultured on trypticase soy agar containing 5 per cent citrated bovine blood and 400 micrograms per ml spectinomycin, incubated at 42 degrees C in Gaspak jars under an atmosphere of 80 per cent hydrogen: 20 per cent carbon dioxide. Antibody titres to Treponema hyodysenteriae were determined by a microtitration agglutination method using merthiolate-inactivated whole cell antigen prepared from a beta-haemolytic isolate. Results indicated that mean titres in pigs from which beta-haemolytic T hyodysenteriae was isolated were significantly higher than in pigs which yielded isolates of weak beta-haemolytic T innocens or in culturally negative pigs (P less than 0.0225). Mean titres of herds where beta-haemolytic T hyodysenteriae was isolated were significantly higher (P less than 0.005) than the mean titres of either of the other two groups. However, mean titres of herds where no isolates were obtained were not significantly different from mean titres of herds where weak beta-haemolytic T innocens was isolated.     

Comparison of a new gold-immunochromatographic assay for the detection of antibodies against avian influenza virus with hemagglutination inhibition and agar gel immunodiffusion assays

A gold-immunochromatographic test-strip kit is used for the detection of IgG antibodies against the nucleocapsid protein of Avian Influenza Virus (AIV). Compared with the "gold standard", i.e. hemagglutination inhibition (HI) and agar gel immunodiffusion (AGID) assays, the gold-immunochromatographic test strip has many advantages, such as high specificity, high sensitivity, convenience, is rapid and has low cost. The gold-immunochromatographic test strip provides a unique tool for the on-site surveillance and diagnosis of Avian Influenza.     

An enzyme-linked immunosorbent assay for detection of antibodies to maedi-visna virus in sheep. II. Comparison to conventional agar gel immunodiffusion test.

A study was conducted to compare the indirect enzyme-linked immunosorbent-assay (i-ELISA) test using antigen prepared by a simple technique using sodium dodecyl sulfate (SDS) treatment to the conventional agar gel immunodiffusion test (AGID). Ten specific-pathogen-free (SPF) sheep were inoculated with maedi-visna virus (MVV) and serum antibody titers compared over a period of 14 weeks. All the sheep seroconverted by the i-ELISA compared to 90% by the AGID. The i-ELISA detected antibody at a mean of 2.6 weeks prior to the AGID. In both tests, fluctuations were observed in the serum antibody response of two sheep. The i-ELISA had a specificity of at least 98.8% and an increased relative sensitivity of 15.5% compared to the AGID, based on the analysis of sera from experimental sheep with MVV free status and sera from sheep from various sources. Of the sera from a seronegative flock which had been monitored with the AGID after a "test and remove" eradication program, 10.2% were positive by the i-ELISA. It was concluded that the AGID test may not be adequate to monitor samples for an eradication scheme.