Evaluation of diagnostic tests for the detection of classical swine fever in the field without a gold standard.

Knowledge of the sensitivity of diagnostic tests for infectious diseases under field conditions can be used to design a surveillance program that increases the effectiveness of the control policy. In this study, the sensitivity of tests for the detection of classical swine fever (CSF) virus (CSFV) under field conditions was estimated without knowledge of the true disease status of the animals tested. During the CSF epidemic of 1997-1998 in The Netherlands, tonsil samples from pigs of CSF suspect farms were collected for laboratory diagnosis of CSE These specimens were tested in a fluorescence antibody test (FAT1) for the presence of CSFV antigen. When at least 1 specimen in a particular sample series from a farm was positive, this farm was declared CSFV infected. Specimens of that series, either FAT1 negative (98) or FAT1 positive (127), were subsequently tested again (FAT2). After that, a suspension was made of the remaining tissue, and this suspension was evaluated with a virus isolation test. In total, 225 tonsil specimens were examined. A statistical model was formulated, and the sensitivity of the 3 tests and the prevalence of positive specimens in the sample were estimated by the method of maximum likelihood. The sensitivity of the FAT1, the test that was used for confirmation of CSFV infection in a pig herd, was approximately 78% (95% confidence interval [CI] = 62-92%). The effectiveness of the selection process of animals on the farm by the veterinarian was estimated to be 77% (64-87%). The sensitivity of the combination of FAT1 and FAT2 (60%) indicates that at least 5 animals should be selected on a CSF-suspect farm to gain a detection probability for CSFV of 99%.     

Comparison of fluorescent antibody tests for tuberculosis and paratuberculosis with antigens coupled to insoluble spheres or taken up by macrophages

Sera of 106 cattle from farms with histories of Mycobacterium johnei infection and sera from 15 human tuberculous patients as well as a number of control sera were examined by means of two different fluorescent antibody tests (FAT) for the occurrence of antibodies against M. johnei and M. tuberculosis respectively. The antigens used were PPD johnin and PPD tuberculin. In the macrophage uptake FAT (MU/FAT) mouse macrophages after phagocytosis of the tuberculins served as the matrix; in the tests performed using the defined antigen substrate spheres (DASS) system, Sepharose beads activated by CNBr were used for the matrix. A good correlation was found between the results of the DASS/FAT and those of the MU/FAT, which is known to be a sensitive and specific test in the diagnosis of Johne's disease in cattle. It is suggested that the FAT, with utilization of the DASS system, might have good prospects for routine examination for antibodies against species of Mycobacterium.     

Fluorescent Antibody Test for the Rapid Diagnosis of Infectious Hematopoietic Necrosis

Abstract

A fluorescent antibody test (FAT) was developed for the rapid detection of infectious hematopoietic necrosis virus (IHNV). Both polyclonal and monoclonal antisera prepared against IHNV were evaluated. Test variables investigated included type of fixative, dilution rate of antibody reagents, staining time, and type of fluorescent conjugate that would be optimal for detection of IHNV. Specificity tests of the FAT indicated no cross-reactivity of the two antisera with other viruses or with cell lines of salmonid and nonsalmonid origin. All strains of IHNV tested, which included different electropherotypes, those isolated from selected salmonids at different life stages, and those from different geographic regions, reacted with both antisera. The FAT has been used for the detection of IHNV in blood smears and organ imprints from clinically infected juveniles, and in IHNV-infected cells in ovarian fluid from adult carriers. With this FAT, IHNV was detected after 48 h in cell lines inoculated with infected fish tissue. The test was equal in sensitivity to the plaque assay method and required less time to obtain a definitive diagnosis.     

Relative efficiency of techniques for detecting avian reticuloendotheliosis virus as a vaccine contaminant

Three techniques were compared for sensitivity in detecting reticuloendotheliosis virus (REV) in a deliberately contaminated Marek's disease vaccine. The most sensitive and rapid test was the indirect fluorescent antibody test (FAT). The indirect immunoperoxidase test, although simple to perform and only marginally less sensitive than the FAT, was difficult to interpret at low levels of REV. Using immunoelectron microscopy, virions were seen only after three subcultures and then not to the same level as that detected by the FAT or immunoperoxidase test. Serum raised against the HPRS-1 strain of REV detected other strains of this virus in the FAT.     

Detection of rinderpest antibodies

Rinderpest antibodies were detected by employing the fluorescent antibody test (FAT) and the immunoperoxidase test (IPT) and the results were compared with the counterimmuno electrophoresis test (CIE). FAT was found to be the most sensitive in detecting post-vaccinal antibodies followed by IPT and CIE tests.     

In situ hybridization for the detection of transmissible gastroenteritis virus in pigs and comparison with other methods.

Archived formalin-fixed, paraffin-embedded tissues from 25 pigs naturally infected with transmissible gastroenteritis virus (TGEV) were examined by in situ hybridization for TGEV nucleic acid using a nonradioactive digoxigenin-labeled cDNA probe that targeted the nucleocapsid sequence of TGEV strains. The results of in situ hybridization for the detection of TGEV were compared with virus isolation (VI), a fluorescent antibody test (FAT), and transmission electron microscopy (TEM). VI, FAT, and TEM were tested over a course of time before the in situ hybridization was performed. Positive hybridization signals were detected in duodenal, jejunal, and ileal enterocytes from 21 pigs. Hybridization signals were confined to the cytoplasm. Intestinal specimens from 25 piglets were evaluated by 4 tests. Twenty-one of 25 were positive by in situ hybridization. Of these 21 samples, 5 (24%) were positive for TGEV by all 4 tests, 15 (71%) were positive by FAT, 14 (67%) were positive by VI, and 6 (29%) were positive by TEM. In situ hybridization for the detection of TGEV in formalin-fixed, paraffin-embedded tissues provides a rapid means of confirmation of a histopathological diagnosis of TGEV without virus isolation, or when only formalin-fixed intestinal specimens were available.     

Diagnosis and prevalence of leptospira infection in aborted and stillborn horses

A study was conducted to evaluate a recently available fluorescent antibody test (FAT) conjugate for the detection of leptospires in tissues of aborted and stillborn horses, to determine the leptospira antibody titers and compare serologic test results with FAT results, and to determine the prevalence of leptospira-induced abortions and stillbirths in the equine population of central Kentucky. From July 1, 1988 through June 30, 1989, 15 (2.5%) of 594 submissions (fetuses, stillborn foals, and/or placentas) were diagnosed as leptospirosis by the FAT (14 of 15 tested) and/or microscopic agglutination test (12 of 14 tested). Of the 12 serologically positive fetal fluids, 10 had high tigers against Leptospira interrogans serovar pomona and 2 against serovar grippotyphosa.     

Vibriosis: Demonstration of Vibrio fetus and Vibrio bubulus Organisms in Preputial Fluid by Immunofluorescence and Cultural Techniques

Fluorescent conjugates were prepared from the sera of calves immunized with four Vibrio fetus strains and one Vibrio bubulus strain. The fluorescent antibody technique (FAT) was then used to detect vibrio organisms in preputial fluid collected from 67 bulls belonging to a Canadian artificial insemination (AI) unit. The V. fetus conjugates reacted with both V. fetus var venerealis and V. fetus var intestinalis. V. fetus was found in 20 animals (29.9%), 13 of which also harboured V. bubulus. In two cases, the FAT failed to detect V. fetus which was isolated by concurrent bacteriological examinations.

It was concluded that the FAT can be a rapid method of detecting some carrier bulls but more reliable results are obtained when a combination of FAT and bacteriological methods is employed. It was found that a single sample giving negative results is inconclusive and additional tests are required before making a final diagnosis. The FAT can also be used to differentiate V. fetus isolates from V. bubulus.

     

A capture-enzyme immunoassay for rapid diagnosis of transmissible gastroenteritis virus.

Two enzyme immunoassays (EIA) were developed for the detection of swine transmissible gastroenteritis virus (TGEV) antigens. The 2 EIAs used the same detecting system, a monoclonal antibody conjugated to horseradish peroxidase, but used different capture systems including a monoclonal antibody (m-EIA) or a polyclonal antibody (p-EIA). The EIAs were compared with the fluorescent antibody test (FAT) and electron microscopy (EM) for the detection of TGEV in intestinal samples of experimentally inoculated gnotobiotic piglets and of conventional diarrheic pigs submitted for diagnosis. In the gnotobiotic piglets experimentally inoculated with TGEV, 81.8% (9/11) were positive for TGEV by p-EIA, and 72.7% (8/11) were positive by m-EIA. In comparison, 81.8% (9/11) were positive by FAT and 27.2% (3/11) were positive by EM. Three noninfected controls were negative by all tests. In the diagnostic samples, 86.0% (43/50) were positive by p-EIA, 68.2% (30/44) were positive by m-EIA, 28.6% (14/49) were positive by IFA, and 38.0% (19/50) were positive by EM. The m-EIA had a higher agreement with FAT and EM than did p-EIA.     

Detection of rabies viral antigens in non-autolysed and autolysed tissues by using an immunoperoxidase technique

The objectives of this study were to determine the potential of an immunoperoxidase technique involving the avidin-biotin complex (ABC) stain for the diagnosis of rabies in fresh tissues and compare it with other standard methods, including the fluorescent antibody test (FAT), haematoxylin and eosin and Seller's stain, and to investigate its capacity to detect rabies antigen in autolysed tissues. Samples of non-autolysed brain from 81 domestic and wild animals suspected of having rabies were examined. Rabies antigen was detected by FAT in 41 of these samples and Negri bodies were detected in 40 (97.6 per cent) of them by the immunoperoxidase technique, in 25 by haematoxylin and eosin and in 22 by Seller's stain. The sensitivity of the immunoperoxidase technique decreased as the tissues were left to autolyse; after two days it was 91.2 per cent, after four days 70.6 per cent, and after seven days 11.8 per cent.     

SEROLOGICAL METHODS IN THE DIAGNOSIS OF BLUETONGUE

The viral antibodies in the serum of cattle, sheep and deer are not detectable in the ordinary direct CF test. We have shown that the so-called "non complement-fixing" viral antibodies in the serum of these animal species can be demonstrated by the MDCF test. The MDCF test, as well as the micro AGP test, are group reactive, with all BT isolates studied. However, it is possible with these tests to differentiate antigenically BT virus from EHD virus. The FAT was also useful in differentiating BT virus grown in TC cells from the EHD virus. In the PRN test, all the BT virus isolates studied cross reacted and although quantitative differences were frequently observed, no obvious antigenic classification was possible with the antiserums used in this work. Reactions between the BT viral isolates and the EHD virus were all within the limits of what is presently considered to be non-specific inhibition.     

A dot enzyme linked immunosorbent assay (dot ELISA): Comparison with standard fluorescent antibody test (FAT) for the diagnosis of rabies in animals

A modified enzyme linked immunosorbent assay (Dot ELISA) is described for visual detection of rabies antigen in animals. The test materials were dotted onto the nitrocellulose paper and allowed to react with rabies antiserum. The bound antigen—anti-body were reacted with a peroxidase conjugated antirabbit immunoglobulin. Positive reactions were easily visualized as brown dots after enzyme degradation of the substrate. A total of 400 specimens from various geographical locations were tested with the dot ELISA technique, and also with the fluorescent antibody test (FAT), which was used as a reference method. The concordance between the two tests was 95.25%. The dot ELISA may have potential applications as a rapid, simple and economical field test in the diagnosis of rabies.     

Demonstration of Rabies Antigen in Salivary Glands of Rabies Suspected Animals

Submaxillary salivary glands from 129 rabies suspected animals were studied by the following methods: a) microscopic examination of frozen sections stained by the Fluorescent antibody technique (FAT), and b) mouse infectivity test (MIT). Flourescent antibody staining of frozen sections from the salivary glands of rabid animals proved to be a satisfactory method for demonstrating rabies antigen, when compared with the mouse infectivity test.     

Direct and maternal genetic relationships of lamb live weight and carcass traits in Swedish sheep breeds

Possibilities to include carcass traits recorded at commercial slaughterhouses in the genetic evaluation of sheep in Sweden were investigated by estimating direct and maternal genetic parameters for 4‐month weight (4MW), carcass weight (CW), carcass fatness grade (FAT), and carcass fleshiness (FLESH) using multiple‐trait animal models. Data included two sets of breeds, the so‐called white breeds (Swedish landrace breeds, Texel, Dorset, Oxford Down, Suffolk, East Friesian Milk Sheep, and Swedish crossbred) and the Gotland breed. There were 30 625 observations on 4MW and 5062 observations on carcass traits for the white breeds. For the Gotland breed the numbers were 43 642 and 7893, respectively. The results showed that it is feasible to use field‐recorded carcass traits in the genetic evaluation. To consider the effects of selection and to utilize all information in an optimal way multiple trait animal models should be used. Direct and maternal heritabilities for 4MW and CW varied between 0.04 and 0.18 and heritabilities for FAT and FLESH between 0.21 and 0.29. Direct and maternal genetic correlations between 4MW and CW were high (0.61–0.97). Genetic correlations were higher between the weights and FLESH (0.11–0.62) than between the weights and FAT (?0.23 to 0.40). Genetic correlations between FAT and FLESH were moderate (0.38–0.45). Heritabilities for CW were higher if 4MW was included in the analyses and the effect of selection on 4MW was stronger for CW than for FAT or FLESH. The importance of maternal effects on carcass traits was discussed.     

Electrophysiologic Characteristics and Topographic Distribution of Focal Atrial Tachycardias in Dogs

Background: Focal atrial tachycardia (FAT) is a common supraventricular tachycardia in dogs. Objective: To evaluate electrophysiologic characteristics and topographic distribution of FAT. Animals: Sixteen dogs with symptomatic FAT. Methods: Retrospective case series. Electrophysiological studies were performed to test the inducibility of documented and no documented arrhythmias. Once induced for each dog, FAT was analyzed for electrogenic mechanism, endocardial electrogram, and location. Results: Nineteen FATs could be studied in 16 dogs, 12 were automatic, 4 nonautomatic, and 3 incessant. Two dogs had >1 focus. Mean atrial cycle length (CL) was 238.2 ± 69.2 (SD) milliseconds, mean ventricular CL of 292.7 ± 72.5 (SD) milliseconds, with atrioventricular block in 6 cases. Mean presystolic atrial activity recorded at the ectopic focus was –39.9 ± 17.7 (SD) milliseconds. Atrial potentials were fragmented in 11 dogs and were low amplitude in 6 dogs. Sixty‐three percent of ectopic foci were distributed within the right atrium (5 crista terminalis, 3 triangle of Koch, 2 tricuspid valve annulus, 1 interatrial septum, and 1 right auricle) and 37% in the pulmonary veins (PVs) (4 right superior PV, 2 left superior PV, and 1 right inferior PV). Persistent atrial fibrillation (AF) and paroxysmal AF were triggered by FATs in 7 dogs (2 with multiple ectopic foci and 4 with at least one PV focus). Conclusion and Clinical Relevance: According to our findings, dogs have a predominance of right‐sided FAT. The majority of FATs are automatic and can trigger AF, particularly in the case of PV location.