Application of a modified indirect fluorescent antibody test to the detection of antibodies to bovine respiratory syncytial virus in Ontario cattle.

A modified indirect fluorescent antibody test for the detection of serum antibodies to bovine respiratory syncytial virus was developed. The test made use of Terasaki plastic microtiter plates in which bovine respiratory syncytial virus (Saskatchewan strain) infected Georgia bovine kidney cells were grown and fixed in situ by a modified acetone fixation procedure. Evans blue dye was used as a counterstain to reduce nonspecific fluorescence. In a study of 986 field sera from a geographically broad cross-section of mature Ontario cattle, 95% of the samples were found to be positive at or above a 1:2 dilution. No seronegative regions, counties or herds were identified. When representative samples covering a range of indirect fluorescent antibody titers were further examined by a microtiter virus neutralization assay, a significant agreement was found between the two tests. Up to a fourfold decrease in titer was observed when antigen coated plates were stored at -70 degrees C for four months. The modified indirect fluorescent antibody test for bovine respiratory syncytial virus antibody detection proved to be a rapid, practical procedure for use in the diagnostic laboratory. This study confirms that bovine respiratory syncytial virus is widespread in the Ontario cattle population and that most mature cattle can be assumed to have been exposed to this virus.     

Serologic diagnosis of infectious cyclic thrombocytopenia in dogs using an indirect fluorescent antibody test

An indirect fluorescent antibody test was used for detection of serum antibodies to the platelet-specific rickettsial organism that is the causative agent of infectious cyclic thrombocytopenia (ICT) in dogs. The test converted from negative to positive in 7 of 7 experimentally inoculated dogs. One of 2 attempts to recover the rickettsial agent of ICT from naturally occurring seropositive dogs, by blood inoculation of experimental dogs, was successful. Seemingly, the test did not detect antibodies to Ehrlichia canis, nor did a similar test, using E canis antigen slides, detect antibodies to the rickettsial agent of ICT. The rickettsial agent of ICT has been classified tentatively as E platys. When applied to sera from a group of healthy random-source dogs, the test revealed a relatively low (5%) occurrence of positive reactions. A higher occurrence of positive reactions (35%) was noticed in sera from a group of thrombocytopenic dogs from the University of Florida. A majority of these positive sera were also positive for antibodies to E canis. The highest occurrence of positive reactions was found (greater than 50%) in E canis-positive sera from dogs at the University of Florida, as well as from dogs from 9 other states.     

The indirect fluorescent antibody technique as a method for detecting antibodies in aborted fetuses.

In this investigation the indirect fluorescent antibody technique was used to titrate antibodies in bovine sera to parainfluenza 3, infectious bovine rhinotracheitis virus and bovine viral diarrhea virus. These results were compared to those determined on the same samples by hemagglutination inhibition for parainfluenza 3 virus and serum neutralization for bovine virus diarrhea and infectious bovine rhinotracheitis virus. The results of the serological methods agreed closely. The indirect fluorescent antibody technique is a rapid and sensitive method for detecting antibodies and the procedure lends itself to use in diagnostic laboratories. In addition to the above viruses the presence or absence of antibodies to bovine coronavirus and bovine adenovirus 3 were determined by the indirect fluorescent antibody technique in thoracic fluids from 100 aborted fetuses and 50 nonaborted fetuses. Results on these samples were not compared to hemagglutination inhibition or serum neutralization as the condition of fluid samples from aborted fetuses renders interpretation of such tests unreliable. Antibodies to one or more viruses were detected in 30 of the 100 aborted fetuses and in seven of the 50 nonaborted fetuses. Antibodies to more than one agent were detected in eleven of the 100 aborted and in one of the 50 nonaborted fetuses. Reasons for this occurrence and application of the test in determination of causes of abortion are discussed.     

Modified indirect fluorescent antibody test for the serodiagnosis of Anaplasma marginale infections in cattle

A modified indirect fluorescence antibody technique was used for the serodiagnosis of Anaplasma marginale infections in cattle. Nonspecific antibodies adherent to infected erythrocytes were removed, using acidic glycine buffer. Evans blue was used as a counterstain.     

Prevalence of antibodies to porcine adenovirus in swine by indirect fluorescent antibody test

An indirect fluorescent antibody test was developed for routine identification of a porcine adenovirus and its specific antibody. Two specific-pathogen-free young pigs were inoculated with the viral antigen prepared in continuous porcine kidney cell cultures, and their sera were used as an antibody reagent to standardize the test. Sera of adult pigs with respiratory problems, obtained from pig farms in Quebec, were tested for antibodies to this virus; 83 of 540 sera tested (15.2%) were found to be positive.     

Results from an indirect fluorescent antibody test using three different strains of Ehrlichia canis

An indirect fluorescent antibody (IFA) test is usually performed to detect antibodies in dogs naturally infected by Ehrlichia canis. In this work, results obtained using three different E. canis strains as antigen (a commercial antigen, the E. canis Oklahoma strain and the E. canis Madrid strain) were compared. One hundred and forty-nine serum samples obtained from dogs living in the centre of Spain were analysed. When qualitative results were evaluated, identical results were detected in 87.2% of samples for the three antigens tested. When comparing antibody titre results, differences between the Madrid strain and the commercial antigen, and between the Madrid and Oklahoma strains were statistically significant (P < 0.0001). No differences were found when comparing the Oklahoma strain with the commercial antigen (P = 0.562). Subtle intra-laboratory variations shown in this study suggest a higher sensitivity of the IFA test when an autochthonous strain is used as antigen.     

Development of an indirect fluorescent antibody test, using microfluorometry as a diagnostic test for bovine anaplasmosis

The indirect fluorescent antibody test for the diagnosis of Anaplasma marginale infection in cattle was modified for use with microfluorometry. The test was standardized by use of a fluorometer that measures intensity of fluorescence. Standardization included A marginale-infected blood smears on microscope slides as antigen, serum from an inoculated calf as a positive control containing specific antibody, and an affinity-purified fluorescein-conjugated anti-bovine immunoglobulin as 2nd antibody. The modified test and microfluorometry allowed for titration of sera from A marginale (Florida isolate)-inoculated cattle with a degree of accuracy exceeding visual determinations. In addition, the fluorometric test was more sensitive than the complement fixation or card agglutination tests in identifying cattle that had previous Anaplasma infections.     

An indirect fluorescent antibody test for antibodies to transmissible gastroenteritis of swine.

The indirect fluorescent antibody test was modified to provide a rapid technique for the detection, screening and titration of antibodies to transmissible gastroenteritis of pigs. Large numbers of slides containing transmissible gastroenteritis antigen were prepared by planting mixtures of infected and uninfected swine testicular cells onto multiwelled teflon-coated slides. After overnight incubation, about one-half of the cells in each well were infected which provided contrast to aid in detecting specific fluorescence in the presence of varying degrees of background staining. Following fixation, antigen slides were stored at -20 degrees C until used. The indirect fluorescent antibody test was compared to the virus neutralization test in both the screening and titration of swine sera containing transmissible gastroenteritis antibodies. The test was found to be sensitive and reliable and to offer certain advantages over the virus neutralization test.     

Comparison of a commercial ELISA with an indirect fluorescent antibody test to detect antibodies to Lawsonia intracellularis in experimentally challenged pigs

Objective The ability of a new commercial ELISA to detect pigs with subclinical proliferative enteropathy (PE) was compared with the traditional indirect fluorescent antibody test (IFAT). Methods Serum samples were selected from pigs with known Lawsonia intracellularis infection status and clinical signs of PE, but the sample population consisted predominantly of pigs subclinically affected by PE. Results Significant association and agreement were shown between the ELISA and IFAT assays. ELISA results correlated well with the duration of L. intracellularis shedding as detected by polymerase chain reaction. Conclusion ELISA can be successfully used to monitor L. intracellularis infection in pigs.     

Development and persistence of Cowdria ruminantium specific antibodies following experimental infection of cattle, as detected by the indirect fluorescent antibody test.

Different breeds of cattle were experimentally infected with Palm River, a Zimbabwean isolate, or Ball-3, a South African isolate of Cowdria ruminantium, derived from tissue culture or tick or blood stabilates. C. ruminantium specific antibody responses were detected by an indirect fluorescent antibody test (IFAT) using C. ruminantium-infected bovine aortic endothelial (BAE) cell cultures as antigen. The first detection of antibodies to C. ruminantium generally coincided with the peak of the febrile reaction and the antibodies remained detectable for a period of 8-30 weeks in the Palm River infected group and 18-30 weeks in the Ball-3 infected group. Peak reciprocal antibody titres in both groups ranged from 64 to 2048 between 3 and 6 weeks post-infection. No apparent serological differences were observed among the various C. ruminantium isolates when tested in homologous and heterologous IFATs. Post-infection sera to Anaplasma marginale, Theileria parva parva, Babesia bigemina and Rickettsia conorii did not exhibit reactivity with the C. ruminantium antigen. These results indicate the possible use of C. ruminantium-infected cultures as antigen in IFATs to detect similar C. ruminantium-specific antibody responses in the field in clinically sick, recovered and carrier animals.     

Comparison of blood smear and indirect fluorescent antibody techniques in detection of haemoparasite infections in trade cattle in Nigeria

The incidence of blood parasites in trade cattle was surveyed with emphasis on tick-borne parasites, using blood smears and immunofluorescent antibody (IFA) techniques. With the blood smear method, about 9 and 8.9% of cattle examined were found positive for Babesia bigemina and Anaplasma marginale, respectively. Percentage infections with other parasites were 3.33, 1.92, 0.75, 0.75 and 0.58, respectively, for Babesia bovis, Trypanosoma brucei, Anaplasma centrale, Eperythrozoon and Theileria species as well as Trypanosoma congolense. The incidence of A. marginale infection was at its peak during the rainy season while B. bigemina was most prevalent during the dry season. There were mixed infections of Anaplasma and Babesia (1.42%); Babesia and trypanosomes (1.00%); Babesia and Eperythrozoon (0.75%) and Babesia and Theileria (0.75%). Using the indirect fluorescent antibody test, 93, 55 and 68% of cattle sera examined were found to be positive for B. bigemina, B. bovis and A. marginale, respectively. Forty-nine percent of the positive sera of B. bigemina had highest titres. The importance of using serological means for determining the endemic levels of tick-borne diseases in cattle in Nigeria is discussed.     

Immunofluorescence of spirochetes with serum from swine recovered from swine dysentery using an indirect fluorescent antibody test.

Using an indirect fluorescent antibody test, immunofluorescence of large spirochetes was observed with serum from swine that had recovered from swine dysentery. The spirochetes were obtained from scrapings of the colonic mucosa on the first day of diarrhea which was the time when the spirochete population was observed to be the highest. Of 29 exposed nonmedicated swine which developed and recovered from a diarrhea characteristic of swine dysentery 27 had antispirochete serum titers which ranged from 1:2 to 1:16. None of the 50 nonexposes swine developed a titer. Of 19 swine with a serum titer and reexposed with infective swine dysentery inoculum, 18 did not develop a diarrhea and were presumed to be immune. Considering these findings it is possible that this test could be used to detect antispirochete antibody in unknown swine serum.     

Development of an indirect ELISA-NcSRS2 for detection of Neospora caninum antibodies in cattle

Neosporosis is of alarming economic concern in the cattle industry. The effectiveness of diagnostic tests for detecting specific antibodies against Neospora caninum is hampered by potential cross-reaction with other coccidia. Use of a single specific antigen might improve test specificity. An indirect enzyme-linked immunosorbent assay (ELISA) was developed using the truncated protein NcSRS2 expressed in Escherichia coli. The ELISA results were compared with those of the indirect fluorescence antibody test (IFAT). Receiver Operating Characteristic (ROC) and Tests in the Absence of a Gold Standard (TAGS) analysis revealed an assay having 96% specificity and 95% sensitivity when applied to 145 positive and 352 negative sera from two distinct cattle populations. Using OD ≤ 0.095 as the cut-off point, the assay's negative and positive predictive values ranged from 98.8% to 50.8% and from 58.8% to 99.1%, respectively, depending on neosporosis prevalence in a given area. The novel ELISA-NcSRS2 format described in the present report constitutes a specific and sensitive method for detecting N. caninum in cattle.