Development of an enzyme-linked immunosorbant assay (ELISA) for the serodiagnosis of canine dermatophytosis caused by Microsporum canis

Abstract In dogs, dermatophytosis should be considered in any case of alopecic, papular or pustular lesion. The aim of this study was to develop an enzyme-linked immunosorbant assay (ELISA) as an aid in the diagnosis of canine dermatophytosis. The antigen used was a whole fungal extract obtained from an isolate of Microsporum canis cultured on a liquid medium from the parasitized hair of a cat with patches of alopecia. To assess the ELISA performances, sera from 18 dogs with dermatophytosis caused by M. canis (group A, n = 18), 20 dogs with skin diseases other than dermatophytosis and 22 healthy dogs (group B, n = 42) were tested. Four further animals were tested: three with dermatophytosis caused by M. gypseum and one by T. mentagrophytes. A significant difference (P < 0.01, Wilcoxon's test, w = 364) was found between IgG-specific levels of sera of recently M. canis-infected dogs (infection < 15 days) and controls (although three dogs had negative titres at this stage). A highly significant difference (P < 0.001, w = 462) was noted between controls and dogs with infection of longer duration (> 30 days). All dogs had positive titres at this stage. A highly significant correlation (P < 0.001, Spearman's test, rho = 0.86) between duration of infection and IgG concentration was noted. The test has good sensitivity (83.3%) and high specificity (95.2%) but some dogs retained positive titres after elimination of infection. The sensitivity is higher than that of direct microscopic hair examination and similar to that of fungal culture with DTM (dermatophyte test medium).     

Validation of an indirect enzyme-linked immunosorbent assay for the detection of antibody against Brucella abortus in cattle sera using an automated ELISA workstation

An automated indirect enzyme-linked immunosorbent assay (I-ELISA) for the serological diagnosis of bovine brucellosis was developed and validated in-house. A total of 4,803 cattle sera from South Africa (n = 3,643), Canada (n = 652), Germany (n = 240), France (n = 73) and the USA (n = 195) was used. The South African panel of sera represented 834 sera known to be positive by the Rose Bengal test (RBT), serum agglutination test (SAT) and complement fixation test (CFT), 2709 sera that were negative by CFT, and 100 sera from animals vaccinated with a standard dose of Brucella abortus strain 19. Overseas sera were obtained from reference non-vaccinated brucella-free cattle (n = 834), naturally infected (n = 72), experimentally infected (n = 71), and vaccinated animals (n = 83). Also 100 sera collected from cattle in Canada and known to be positive by competitive ELISA (C-ELISA) were used. The intermediate ranges ("borderline" range for the interpretation of test results) were derived from two-graph receiver operating characteristics analysis. The lowest values of the misclassification cost-term analysis obtained from testing overseas panels, covered lower I-ELISA cut-off PP values (0.02-3.0) than those from local panels (1.5-5.0). The relatively low cut-off PP values selected for I-ELISA were due to the fact that the positive control used represents a very strong standard compared to other reference positive sera. The greater overlap found between negative and positive cattle sera from South Africa than that between reference overseas panels was probably due to the different criteria used in classifying these panels as negative (sera from true non-diseased/non-infected animals) or positive (sera from true diseased/infected animals). The diagnostic sensitivity of the I-ELISA (at the optimum cut-off value) was 100% and of the CFT 83.3%. The diagnostic specificity of I-ELISA was 99.8% and of the CFT 100%. Estimate of Youden's index was higher for the I-ELISA (0.998) than that for the CFT (0.833). Analysis of distribution of PP values in sera from vaccinated and naturally infected cattle shows that in vaccinated animals all readings were below 31 PP where in infected ones these values represented 43%. Therefore, it appears that I-ELISA could be of use in identifying some naturally infected animals (with values > 31 PP), but more sera from reference vaccinated and infected animals need to be tested to further substantiate this statistically. Of 834 sera positive by RBT, SAT and CFT, 825 (98.9%) were positive in the I-ELISA. Compared to C-ELISA the relative diagnostic sensitivity of the I-ELISA was 94% and of the CFT 88% when testing 100 Canadian cattle sera. Of 258 South African cattle sera, of which 183 (70.9 %) were positive by the I-ELISA and 148 (57.4 %) by the CFT, 197 (76.4%) were positive by C-ELISA when re-tested in Canada. One has to stress, however, that Canadian C-ELISA has not been optimised locally. Thus, the C-ELISA was probably not used at the best diagnostic threshold for testing South African cattle sera. This study shows that the I-ELISA performed on an automated ELISA workstation provides a rapid, simple, highly sensitive and specific diagnostic system for large-scale detection of antibodies against B. abortus. Based on the diagnostic accuracy of this assay reported here, the authors suggest that it could replace not only the currently used confirmatory CFT test, but also the two in-use screening tests, namely the RBT and SAT.     

A fluorescence polarization assay for the detection of antibodies to Mycobacterium bovis in cattle sera

A fluorescence polarization assay (FPA) utilizing fluorescein-labelled MPB70 protein as the antigen was developed and evaluated for its ability to detect antibodies to Mycobacterium bovis in cattle sera. Three panels of sera were examined in this study. These included: (A) sera (n=28) obtained from cattle from which M. bovis was cultured; (B) sera (n=5666) from Canadian field cattle which were presumed to be free from M. bovis; (C) sera (n=10) from cattle infected with Mycobacterium paratuberculosis and known to contain antibodies to this organism. Receiver operating characteristic (ROC) curve analysis of the results of panels A and B yielded an area under the curve value of 0.975 (95% confidence interval=0.971-0.979), which indicated that this FPA is an accurate indicator of M. bovis infection. At the cut-off point recommended by the ROC curve analysis, the FPA sensitivity and specificity estimates were 92.9% (95% confidence interval=76.5-98.9%) and 98.3% (95% confidence interval=97.9-98.6%) respectively. The FPA results were compared to the results of the single intradermal (SID) test for the 28 infected cattle. Fifteen of these animals were scored positive with the SID test (sensitivity=53.6%). The FPA detected 15/15 (100%) of the SID test-positive animals and 11/13 (84.6%) of the SID test-negative animals. Two of the culture-positive cattle were not detected by either test. None of the sera that were obtained from the M. paratuberculosis-infected animals cross-reacted in this assay.     

Comparative evaluation of enzyme-linked immunosorbent assay based on crude and purified antigen in the diagnosis of canine visceral leishmaniasis in symptomatic and oligosymptomatic dogs

This study evaluated the performance of crude total antigen (CTA) and fucose-mannose ligand antigen (FML) in an enzyme-linked immunosorbent assay for diagnosis of canine visceral leishmaniasis (CVL). The assays used sera from known negative controls (n=30), clinically symptomatic (n=30) and oligosymptomatic (n=30) parasitologically proven infection (by microscopy). Aspirates of popliteal lymph node from infected canines were colleted to score parasitism and compared with the ELISA results. The study indicated that FML used in ELISA provided high sensitivity for detecting oligosymptomatic dogs (90%) and CTA showed greater sensitivity than FML for symptomatic canines (90%). In oligosymptomatic dogs, specificity was 100% for CTA-ELISA, but in symptomatic dogs, FML specificity was higher (96.7%) than CTA-ELISA (93.3%). A significant correlation was observed between the degree of parasitism and the results obtained in CTA-ELISA. Since no available antigen offers 100% specificity and sensitivity for CVL diagnosis, the choice of antigen used must depend on the aim of the investigation.     

Evaluation of an ELISA rapid device for the serological diagnosis of Leishmania infantum infection in dog as compared with immunofluorescence assay and Western blot

In this study we compared a commercial enzyme linked immunosorbent assay (ELISA) rapid test (Snap CLATK Canine Leishmania Antibody Test Kit, IDEXX-Snap) with indirect immunofluorescence assay (IFA) and Western blot (WB) for the detection of Leishmania infantum antibodies in dogs. In total sera from 234 dogs were collected: 59 positives and 51 doubtful sera (IFA 1:40-1:80) from an L. infantum endemic area and 124 negative sera from a non-endemic area were tested. To evaluate the Snap CLATK's performances on whole blood, blood in EDTA and sera from 37 dogs were tested in parallel with Snap CLATK. Snap CLATK sensitivity and specificity compared to IFA were 91.1% and 99.2%, while compared to WB were 93.4% and 98.3%, respectively. When IFA doubtful sera (titers of 1:40 or 1:80) were tested Snap CLATK, using WB as reference, sensitivity and specificity were 90.9% and 100%, respectively. Moreover, a complete concordance was observed when Snap CLATK rapid assay was carried out on whole blood or sera from 37 dogs. The Snap CLATK has demonstrated simplicity and performance and can be considered a quick and reliable alternative for the diagnosis of L. infantum infection in dogs.     

Evaluation of an automated colorimetric assay for the measurement of lipase activity in canine sera.

An automated colorimetric method for determining lipase activity in canine sera was evaluated for precision, linearity and correlation to existing assay methods. The colorimetric method was a commercial reagent that used a series of enzymatic reactions based on the hydrolysis of 1,2 diglyceride by pancreatic lipase. Within-run and between-run coefficients of variation were < 6.8% and < 8.3%, respectively. Linearity was determined to be at least 1366 U/L. Canine serum lipase concentrations attained using the colorimetric method were compared to both titrimetric and dry-film methods for measuring serum lipase activity, resulting in significant (P < or = 0.05) correlation coefficients of 0.92 and 0.77, respectively. Canine serum lipase concentrations measured using the colorimetric assay on 2 different automated analyzers had a significant (P < or = 0.05) correlation coefficient of 0.92. A laboratory reference range using serum samples from 56 healthy dogs (0-561 U/L) was established. There were no significant (P < or = 0.05) differences in mean serum lipase concentrations comparing male and female dogs or comparing young dogs (< or = 3 y) to mature (4-7 y) and older (> 7 y) dogs using this assay. It was concluded that the automated colorimetric assay was a reliable indicator of canine serum lipase activity and offered several advantages, including small sample volume and short analysis time.     

A fluorescent bead-based multiplex assay for the simultaneous detection of antibodies to B. burgdorferi outer surface proteins in canine serum

Lyme disease is a zoonotic, vector-borne disease affecting humans, dogs, horses and other species. It is caused by infection with spirochetes of the Borrelia burgdorferi sensu lato group which are transmitted to the mammalian host by infected ticks (Ixodes). Exposure to B. burgdorferi is commonly diagnosed by serological testing. The gold standard for the detection of antibodies to B. burgdorferi is a two-step procedure of an ELISA followed by confirmatory Western blotting (WB). Here, we developed and validated a new bead-based multiplex assay for the detection of antibodies to B. burgdorferi in canine serum which combined the testing by ELISA and WB in a single quantitative test. B. burgdorferi outer surface protein A (OspA), OspC and OspF were expressed in E. coli. The recombinant proteins were coupled to fluorescent beads providing the matrix of the assay. Two sets of canine sera were used for validation of the multiplex assay. First, sera from 79 dogs with known ELISA and WB results were used to establish the conditions of the assay. These samples were selected to provide similar numbers of pre-tested sera ranging from negative to high positive results and included sera from vaccinated and/or naturally infected dogs. A high correlation was observed for detection of antibodies to B. burgdorferi in the single and multiplex assays (n=79). Spearman's rank correlations were 0.93, 0.88 and 0.96 for OspA, OspC and OspF, respectively. Second, a total of 188 canine serum samples that were not tested previously were used for further multiplex assay validation. All samples were also blindly analyzed for antibodies to B. burgdorferi antigens by WB. The WB results provided a 'relative gold standard' for each antigen and were used to perform a receiver operating curve analysis. The areas under the curves were 0.93 for OspA, 0.82 for OspC, and 0.89 for OspF. Multiplex assay interpretation ranges for antibodies to all three B. burgdorferi antigens in canine serum were established by likelihood analysis. The diagnostic sensitivities of the individual OspA, OspC and OspF bead-based assays were 83%, 62% and 82%, respectively, and the diagnostic specificities were 90%, 89% and 86%, respectively. The new multiplex assay provides a sensitive and fully quantitative platform for the simultaneous evaluation of antibodies to B. burgdorferi OspA, OspC and OspF antigens and distinguishes between antibodies that originated from vaccination or natural exposure to B. burgdorferi.     

Validation of the fluorescence polarization assay as a serological test for the presumptive diagnosis of porcine brucellosis.

Sera from Canadian pigs (brucellosis free, n = 14037) and sera from pigs infected with Brucella suis (n = 401) were tested by the buffered antigen plate agglutination test, the complement fixation test, an indirect and a competitive enzyme immunoassay and a fluorescence polarization assay. The results were analysed and assay sensitivity and specificity estimates were calculated. The sensitivity and specificity of the tests were as follows: the buffered antigen plate agglutination test, 77.1 and 96.9%; the complement fixation test (considering anticomplementary sera as negative), 93.3 and 95.5%; the complement fixation test (considering anticomplementary sera as positive), 58.1 and 99.9%; the indirect enzyme immunoassay, 94.0 and 97.9%; the competitive enzyme immunoassay, 90.8 and 96.6%; and the fluorescence polarization assay, 93.5 and 97.2%; respectively. It was concluded that the fluorescence polarization assay was a valuable asset to the diagnosis of porcine brucellosis because of its accuracy, ease of performance and relative cost.     

Validation of an ELISA method for the serological diagnosis of canine brucellosis due to Brucella canis

In the present study, the validation of an enzyme-linked immunosorbent assay (ELISA) for serodiagnosis of canine brucellosis is described. Two different antigenic extracts, obtained by heat or ultrasonic homogenization of microbial antigens from a wild isolate of Brucella canis bacteria, were compared by ELISA and Western blot (WB). A total of 145 canine sera were used to define sensitivity, specificity and accuracy of the ELISA as follows: (1) sera from 34 animals with natural B. canis infection, confirmed by blood culture and PCR, as well as 51 sera samples from healthy dogs with negative results by the agar–gel immunodiffusion (AGID) test for canine brucellosis, were used as the control panel for B. canis infection; and (2) to scrutinize the possibility of cross reactions with other common dog infections in the same geographical area in Brazil, 60 sera samples from dogs harboring known infections by Leptospira sp., Ehrlichia canis, canine distemper virus (CDV), Neospora caninum, Babesia canis and Leishmania chagasi (10 in each group) were included in the study. The ELISA using heat soluble bacterial extract (HE-antigen) as antigen showed the best values of sensitivity (91.18%), specificity (100%) and accuracy (96.47%). In the WB analyses, the HE-antigen showed no cross-reactivity with sera from dogs with different infections, while the B. canis sonicate had various protein bands identified by those sera. The performance of the ELISA standardized with the heat soluble B. canis antigen indicates that this assay can be used as a reliable and practical method to confirm infection by this microorganism, as well as a tool for seroepidemiological studies.     

Antigen requirements and specificity of enzyme-linked immunosorbent assay for detection of canine IgG against canine distemper viral antigens

An enzyme-linked immunosorbent assay (ELISA) was developed for detection of specific immunoglobulin G (IgG) against canine distemper virus (CDV) antigens. Sucrose gradient separation of viral and cellular proteins was required to produce coating antigens for the ELISA. The specificity of the ELISA was demonstrated by blocking CDV-positive canine sera with CDV-specific antisera produced in goats and rabbits and adsorption of positive sera with CDV antigens. A comparison of the ELISA with the serum-neutralization technique for the detection of CDV antibodies was conducted. Anti-CDV IgG was detected in conventional dogs as early as 6 days after inoculation with a commercial vaccine to CDV. Paired sera from the immunized dogs were evaluated by both techniques and a statistically (P less than 0.01) significant agreement between the ELISA and the serum-neutralization technique was shown (r = 0.6121, n = 75).     

An enzyme-linked immunosorbent assay for antibodies to Hepatozoon canis

Canine hepatozoonosis is a tick-borne protozoal disease caused in the Old World and South America by Hepatozoon canis. An enzyme-linked immunosorbent assay (ELISA) using purified H. canis gamont antigen was applied for the detection of antibodies reactive with H. canis. Evaluation of the ELISA with sera from naturally infected parasitemic dogs indicated that it was sensitive (86%), specific (97%), and comparable to the indirect fluorescent antibody test (IFAT) for the detection of H. canis antibodies. A variable degree of serologic cross-reactivity was found between sera from H. americanum-infected dogs and the H. canis antigen. Dogs experimentally infected with H. canis seroconverted 1-4 weeks post-infection (PI). Antibody levels peaked at 7-9 weeks PI and gradually declined thereafter remaining above the cut-off value until the conclusion of the study 7 months PI. The ELISA will be valuable for serological evaluation of dogs suspected of exposure to H. canis and for epidemiological studies.     

Comparison of serological assays for the diagnosis of canine visceral leishmaniasis in animals presenting different clinical manifestations

Three serological methods, indirect fluorescent immunoassay (IFI), enzyme-linked immunosorbent assay (ELISA) and direct agglutination test (DAT) that are commonly employed in the diagnosis of canine visceral leishmaniasis (CVL), have been assessed. A total of 234 domestic dogs, drawn from an area in the municipality of Belo Horizonte, Minas Gerais, Brazil, endemic for visceral leishmaniasis, were submitted to clinical and parasitological examinations and serological assay. Sera collected from confirmed non-infected dogs (n=20), and from dogs with other parasitic diseases including Trypanosoma cruzi (n=7), Leishmania braziliensis (n=5), Toxoplasma gondii (n=5) and Ehrlichia canis (n=3), were also included in the study. IFI presented a lower sensitivity (72%) than ELISA (95%), although the specificities of these assays were low (52 and 64%, respectively) and both exhibited cross-reactivity with sera from dogs infected with T. cruzi, L. braziliensis and E. canis. In contrast, DAT exhibited a high sensitivity (93%) and a high specificity (95%) and cross-reacted with only one serum sample derived from an E. canis-infected dog. The reproducibilities of the ELISA and DAT assays were excellent, whilst that of IFI was considered to be acceptable. The results produced by ELISA and DAT were in complete agreement, those between ELISA and IFI were at an acceptable level of agreement, whilst the concurrence between the IFI and DAT results were either acceptable or poor depending on the clinical conditions of the group of dogs examined. Since there is no readily accessible method for the diagnosis of CVL that offers 100% specificity and sensitivity, the choice of technique employed must depend on the aim of the investigation.     

Indirect enzyme-linked immunosorbent assay for the detection of antibody against Rift Valley fever virus in domestic and wild ruminant sera

An indirect enzyme-linked immunosorbent assay (I-ELISA) for the detection of specific IgG immunoglobulins against Rift Valley fever virus (RVFV) was validated in-house. A total of 3055 sera from sheep (n = 1159), goats (n = 636), cattle (n = 203), African buffalo (n = 928), and other wild ruminants (n = 129), including eland, kudu, and black wildebeest, was used. Sera from domestic ruminants were collected in West (n = 10), South (n = 1654) and East Africa (n = 334), and sera from wild ruminants (n = 1064) were collected in South Africa. In addition, 136 sera from eight experimentally RVFV-infected sheep, taken during a period of 28 days post infection (dpi), were used to study the kinetics of RVFV antibody production. Field sera were tested by the serum neutralization (VN) test and experimental sera by VN and haemagglutination-inhibition (HI) test. Based on VN test results, negative sera were regarded as reference controls from RVFV-free, and positive sera were regarded as reference controls from RVFV-infected subpopulations of animals. ELISA data were expressed as the percentage positivity (PP) of an internal high positive control. The two-graph receiver operating characteristics approach was used for the selection and optimization of I-ELISA cut-offs including the misclassification costs term and Youden index (J). In addition, cut-off values were determined as the mean plus two-fold standard deviation of the result observed with the RVFV-free subpopulations. Established optimal cut-offs were different for each of the data sets analyzed, and ranged from 1.65 PP (buffalo) to 9.1 PP (goats). At the cut-off giving the highest estimate of combined measure of diagnostic accuracy (highest J value), the I-ELISA test parameters were determined as follows: (1) Diagnostic sensitivity (%): cattle--84.31, buffalo--94.44, sheep--98.91, goats--99.18. (2) Diagnostic specificity (%): cattle--99.34, buffalo--98.28, sheep--99.16, goats--99.23 and other game ruminants--99.26. In the group of RVFV-experimentally infected sheep, seroconversion In all individuals was detected by VN on 4-6 dpi, by HI on 5-7 dpi, and by I-ELISA on 6-7 dpi. All tests showed the same kinetic pattern of immunological response. Antibody levels were low for a very short period before increasing to high titres, after which it was easily detectable by all tests. Compared to traditional tests, the lower sensitivity of I-ELISA in the detection of the earliest stage of immunological response may be practically insignificant, particularily when this assay is used in population-based, disease-surveillance programmes. The high sensitivity and specificity of I-ELISA established in this study, especially for the statistically more representative subpopulations of animals tested, seem to support this prediction. Test parameters determined in this study should, however, be regarded as in-house diagnostic decision limits, for which further updating is recommended, particularly for specimens from other countries, and preferably by applying a standardized method for sampling of new subpopulations of animals to be targeted by the assay.     

Serodiagnosis of Babesia gibsoni infection in dogs by an improved enzyme-linked immunosorbent assay with recombinant truncated P50

The surface antigen P50 of Babesia gibsoni is an important candidate for the development of a diagnostic reagent for canine piroplasmosis. In order to establish an effective diagnostic method for practical use, the gene encoding truncated P50 (P50t) lacking a signal peptide and C-terminal hydrophobic regions were cloned and expressed in Escherichia coli as a fusion protein with glutathione S-transferase (GST). More than 90% portion of the GST-P50t was expressed as a soluble form, in contrast with GST-P50f (full-length), which was completely expressed as an insoluble form. This result indicates that removal of the hydrophobic signal peptide and C-terminus had dramatically improved its hydrophilicity. The purified GST-P50t was tested in an enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to B. gibsoni in dogs. The ELISA with GST-P50t clearly differentiated between B. gibsoni-infected dog sera and uninfected dog sera. In addition, the ELISA detected no cross-reactivity with sera from dogs experimentally infected with the closely related parasites, B. canis canis, B. canis vogeli, and B. canis rossi. Field serum samples collected from dogs in Japan and China were examined for the diagnosis of B. gibsoni infection by using the ELISA. 14.5% (9/62), 5.8% (7/120), and 5.4% (2/37) of tested samples were positive for dogs from Okinawa, Yamaguchi, and Osaka prefectures, Japan, respectively. On the other hand, 4.8% (2/41) of tested samples were positive for dogs from Nanjing, China. These results suggest that the GST-P50t could be a reliable reagent for practical use in ELISA for the serodiagnosis of canine piroplasmosis caused by B. gibsoni.     

Development and validation of an improved enzyme-linked immunosorbent assay for the detection of thyroglobulin autoantibodies in canine serum samples

An enzyme-linked immunosorbent assay to detect thyroglobulin autoantibodies (TGAB) in canine serum was developed and validated. The test result for each sample was derived from the optical density readings (OD) and expressed as an Ab-score(%) calculated from three in-house calibrators. The assay specifically detected TGAB as judged from lack of response in the assay after samples had been incubated with specific antigen. Intra- and interassay coefficients of variation ranged from 2.0–4.9% and 4.6–9.9%, respectively. The detection limit, an Ab-score of 5.6%, was close to the median Ab-score of 10% observed in healthy dogs (n = 132). The median Ab-score of dogs with primary hypothyroidism and lymphocytic thyroiditis (n = 11), skin diseases (n = 35), and non-thyroidal diseases (n = 63) was 340%, 12%, and 8%, respectively. The prevalence of TGAB in hypothyroid dogs with lymphocytic thyroiditis (sensitivity) was 91% (95% confidence limits: 59%–99%). In dogs with dermatological diseases without lymphocytic thyroiditis the prevalence of TGAB was 3% corresponding to a specificity of 97% (95% confidence limit: 85%–100%). In dogs with non-thyroidal diseases and healthy dogs the prevalence of TGAB was 5% and 6%, respectively. The diagnostic accuracy of serum TGAB was evaluated by subjecting the data from 11 dogs with lymphocytic thyroiditis and 35 control dogs without lymphocytic thyroiditis to receiver-operating characteristic curve analysis. The area under the receiver-operating characteristic curve (W = 0.966; 95% confidence limit 87%–100%) was significantly higher than that of a worthless test (0.5) (P < 0.0001), thereby indicating that serum TGAB measurements distinguished between dogs with and without lymphocytic thyroiditis.     

Prospective clinical evaluation of an ELISA B-type natriuretic peptide assay in the diagnosis of congestive heart failure in dogs presenting with cough or dyspnea

BACKGROUND: B-type natriuretic peptide (BNP) is increased in dogs with congestive heart failure (CHF). HYPOTHESIS: The purpose of this study was to evaluate the clinical utility of a novel canine-specific enzyme-linked immunosorbent assay of BNP for the diagnosis of CHF in dogs presenting with either cough or dyspnea. ANIMALS: Three hundred and thirty dogs from 2 large university teaching hospitals. METHODS: We prospectively measured plasma BNP concentrations in 3 groups of dogs: (1) normal adult dogs (n = 75), (2) dogs with asymptomatic heart disease (n = 76), and (3) dogs with cough or dyspnea (n = 179). The final diagnosis of dogs with cough or dyspnea and the severity of CHF (International Small Animal Cardiac Health Council Heart Failure Classification [ISACHC]) were determined by medical record review by a study cardiologist who was blinded to the results of the BNP assay. RESULTS: Dogs with CHF had a higher median BNP concentration (24.6 pg/mL) than dogs with noncardiac causes of cough or dyspnea (2.6 pg/mL) (P < .0001). The area under the curve was 0.91 for the receiver operating curve analysis of the diagnostic accuracy of the BNP measurement to differentiate CHF from other causes of cough or dyspnea. The median BNP concentrations in dogs were 3.0 pg/mL with ISACHC I, 17.8 pg/mL with ISACHC II, and 30.5 pg/mL with ISACHC III. (P < .0001) CONCLUSION AND CLINICAL IMPORTANCE: Measurement of BNP is useful in establishing or in excluding the diagnosis of CHF in dogs with cough or dyspnea. B-type natriuretic peptide concentrations rose significantly as a function of severity of CHF.     

Antibodies to spotted fever-group rickettsiae in dogs and prevalence of infected ticks in southern Connecticut

Blood samples and ticks were obtained from dogs to assess canine exposure to spotted fever-group (SFG) rickettsiae during 1978-1980 in southern Connecticut. Of the 1,576 dog sera screened by microimmunofluorescence. 174 (11.0%) contained specific antibodies at titers greater than or equal to 1:64 against Rickettsia montana (n = 34), R rickettsii (n = 31), R rhipicephali (n = 19), or the unclassified 369-C rickettsia (n = 90). End points greater than or equal to 1:8,192 to R rickettsii and to R rhipicephali were recorded for 6 and 3 sera, respectively. Seropositivity rates from southwestern and southeastern Connecticut were similar (about 11%), with positive sera obtained from each region in nearly all months of the investigation. Rates were between 10% for dogs 2 to 7 years old and 14% for those greater than or equal to 8 years. Eight of 629 Dermacentor variabilis, 1 of 18 Ixodes dammini, and 2 of 3 Amblyomma americanum were positive by direct immunofluorescence for SFG rickettsiae. Thirteen D variabilis contained unidentified, long, bacillus-like organisms that differed from the short, ovoid (coccal) forms typical fo the spotted-fever agent, R rickettsii. With the exposure to infected ticks and production of type-specific antibodies against at least 4 SFG antigens, dogs may serve as suitable enzootic or epizootic indicators of rickettsial activity.     

Detection of autoantibodies against thyroid peroxidase in serum samples of hypothyroid dogs

OBJECTIVE: To establish a sensitive test for the detection of autoantibodies against thyroid peroxidase (TPO) in canine serum samples. SAMPLE POPULATION: 365 serum samples from dogs with hypothyroidism as determined on the basis of serum concentrations of total and free triiodothyronine (T3), total and free thyroxine (T4), and thyroid-stimulating hormone, of which 195 (53%) had positive results for at least 1 of 3 thyroid autoantibodies (against thyroglobulin [Tg], T4, or T3) and serum samples from 28 healthy dogs (control samples). PROCEDURE: TPO was purified from canine thyroid glands by extraction with detergents, ultracentrifugation, and precipitation with ammonium sulfate. Screening for anti-TPO autoantibodies in canine sera was performed by use of an immunoblot assay. Thyroid extract containing TPO was separated electrophoretically, blotted, and probed with canine sera. Alkaline phosphatase-conjugated rabbit anti-dog IgG was used for detection of bound antibodies. RESULTS: TPO bands were observed at 110, 100, and 40 kd. Anti-TPO autoantibodies against the 40-kd fragment were detected in 33 (17%) sera of dogs with positive results for anti-Tg, anti-T4, or anti-T3 autoantibodies but not in sera of hypothyroid dogs without these autoantibodies or in sera of healthy dogs. CONCLUSIONS AND CLINICAL RELEVANCE: The immunoblot assay was a sensitive and specific method for the detection of autoantibodies because it also provided information about the antigen. Anti-TPO autoantibodies were clearly detected in a fraction of hypothyroid dogs. The value of anti-TPO autoantibodies for use in early diagnosis of animals with thyroid gland diseases should be evaluated in additional studies.     

Serological survey of Neospora caninum in dogs in the Czech Republic and a long-term study of dynamics of antibodies

The main aim of the present study was to establish the prevalence of antibodies against Neospora caninum dogs from the Czech Republic and to examine the dynamics of antibody titers during a long-term period. For this purpose, sera of 858 dogs were examined for the presence of anti-N. caninum antibodies using an indirect immunofluorescent antibody test (IFAT). Four groups of dogs of various origins were included in the survey: the first group (A, n=470) comprised dogs purchased by the Czech Army from the civilian sector throughout the Czech Republic, with 22 (4.7%) N. caninum-positive dogs, second group (B, n=115) represented police dogs with no seropositive animal, third group (C, n=195) were pet dog sera collected for veterinary clinic with 5 (2.6%) anti-N. caninum sera and the fourth group (D, n=78) of canine shelter dogs with the seroprevalence of 19.2%. The differences in seroprevalence were significant (P< or =0.01) between groups B and A, and between D and A. None of the serologically positive animals had clinical signs of neurological disorders. Coprological examination did not reveal any dog shedding N. caninum oocysts. The seropositivity rates for N. caninum were analyzed in relation to other data, such as age, breed and gender. Increased prevalence rates of anti-N. caninum antibodies were found in the older age strata of the dog population sample tested in the present study. We found significantly higher (P=0.02) prevalence in 3-3.5-year-old dogs (11.1% of 36), as compared to 1-1.5-years-old dogs (2% of 98). A longitudinal study of antibody dynamics was carried out in 19 initially seropositive dogs over a period of 4 years. The second and third examinations revealed that antibody titers decreased in majority of positive dogs (10, 52.6%), of which in seven cases (36.8%) the titers fell to levels that are currently considered as being seronegative (titer <1:50), or even became undetectable (titer <1:25).     

Evaluation of an enzyme-linked immunosorbent assay (ELISA) for serodiagnosis of infectious bovine rhinotracheitis infection, with results of a preliminary survey

An indirect enzyme linked immunosorbent assay (ELISA) procedure was evaluated against the serum neutralisation test (SNT) for the detection of antibodies to infectious bovine rhinotracheitis virus (bovine herpesvirus type l), using 2028 sera from 166 dairy and 172 beef cattle herds. The results showed the ELISA to give high levels of agreement with the SNT in classifying positive and negative sera (98% and 97% respectively). Such disagreements as did occur involved weakly reactive sera with SNT titres of % or less. A number of sera (n=123) with trace neutralising activity of doubtful diagnostic significance were found to give marginal reactivity with ELISA. ELISA absorbance values were found to be highly correlated with SNT titres (r=0.909) on an overall basis, though agreements were lower with individual sera. The ELISA procedure was quicker, cheaper, and detected more reactors than the SNT. It also allowed results to be obtained with a number of sera which were unsuitable for testing by SNT because of their cytotoxic nature. Analysis of ELISA results showed reactors to be present in 57% of tested sera, representing 81% of cattle herds. Reactor rates for sera and herds in the South Island, (37% and 58%), were significantly lower than for those in the North Island (64% and 88%). Antibody prevalence was also found to be significantly lower in districts having a low annual rainfall (<850 mm), and to be lower in beef cattle than in dairy cattle. A surprising exception to the latter occurred in low rainfall districts, where dairy cattle showed significantly lower reactor rates than local beef animals.