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Topic:Immunofluorescence Assay
Immunofluorescence assay (IFA) is a laboratory technique used to detect and visualize specific antigens or antibodies in equine tissue samples or bodily fluids. This method employs fluorescent-labeled antibodies to bind target molecules, allowing for the observation of fluorescence under a microscope. In horses, IFA is employed in various research and diagnostic applications, including the study of infectious diseases, immune responses, and cellular localization of proteins. The technique provides valuable insights into the distribution and expression of specific proteins within equine cells and tissues. This page aggregates peer-reviewed research studies and scholarly articles that explore the methodology, applications, and advancements of immunofluorescence assay in equine research.
Characterisation of Chlamydia psittaci isolated from a horse. This paper describes the isolation and characterisation of a strain of Chlamydia psittaci obtained from a nasal swab taken from a horse with serous nasal discharge. Initial isolation was achieved in cycloheximide-treated McCoy cell monolayers. Chlamydial inclusions stained by immunofluorescence either with a rabbit antiserum raised against C. psittaci or with a monoclonal antibody directed against the genus-specific lipopolysaccharide antigen were single and compact. They did not stain with iodine or with a monoclonal antibody reactive against Chlamydia trachomatis. The agent was re-isolated i...
Rapid diagnosis of equine influenza. During an epizootic of equine influenza in Norway caused by influenza A/equine (H3N8) virus the efficacy of rapid virus diagnosis by the indirect immunofluorescence technique was evaluated. The antiserum used in the test was a polyclonal influenza A virus antiserum with reactivity directed mainly against the common nucleoprotein and matrix protein. This antiserum possessed sufficient reactivity for the detection of virus-infected exfoliated nasopharyngeal cells. Nasopharyngeal smear samples from 92 horses were examined and a positive diagnosis was obtained for 57 (62 per cent). Paired serum sa...
Cryopreservation of equine mononuclear cells for immunological studies. A rapid and simple technique for the cryopreservation and recovery of equine mononuclear cells was developed. Buffy-coat leukocytes were frozen in autologous plasma containing 10% DMSO and mononuclear cells were recovered by gradient sedimentation using a standard Ficoll-Hypaque purification procedure. The total numbers of mononuclear cells recovered from cryopreserved samples were 94%-82% of those recovered from fresh blood samples. The functional capabilities of the mononuclear cells from cryopreserved buffy coat preparations were compared with those of mononuclear cells from fresh samples b...
Transcript analysis of the equine herpesvirus 1 glycoprotein B gene homologue and its expression by a recombinant vaccinia virus. Transcript mapping of the equine herpesvirus 1 (EHV-1) glycoprotein B (gB) gene homologue by Northern blot, S1 nuclease and primer extension analyses indicated that two overlapping transcripts of 3.4 and 4.6 kb originated from the same strand and were transcribed from left to right between coordinates 0.40 and 0.43 of the EHV-1 genome. The 3.4 kb transcript encoded EHV-1 gB and the 5' RNA terminus was located approximately 30 bases downstream from a probable TATA element. The coding region of the gB gene homologue was reconstructed from two subclones using oligonucleotide mutagenesis and inser...
Laboratory diagnosis of African horse sickness: comparison of serological techniques and evaluation of storage methods of samples for virus isolation. Five serological methods of diagnosing African horse sickness were evaluated, using a battery of serum samples from experimental horses vaccinated and challenged with each serotype of African horse sickness virus (AHSV1 through AHSV9): agar gel immunodiffusion (AGID), indirect fluorescent antibody (IFA), complement fixation (CF), virus neutralization (VN), and enzyme-linked immunosorbent assay (ELISA). The 5 tests were also compared using a panel of field samples, convalescent equine sera with antibodies to domestic equine viral diseases, and sera from horses awaiting export. The ELISA describ...
Comparison of heparan sulfate proteoglycans from equine and human glomerular basement membranes. 1. Proteoglycans extracted from human and equine glomerular basement membranes (GBM) were purified by ion-exchange chromatography and gel filtration. 2. The glycoconjugates had an apparent molecular mass of 200-400 kDa and consisted of 75% protein and 25% glycosaminoglycan. Glycosidase and HNO2 treatment and the amino sugar and sulfate composition of both proteoglycan preparations identified heparan sulfate (HS) as the predominant saccharide chain. 3. Hydrolysis with trifluoromethanesulfonic acid yielded comparable core proteins with molecular masses of ca 160 and 120 kDa. 4. The HS chains had...
Monthly prevalence (in 1986) of antibody titers against equine monocytic ehrlichiosis in apparently healthy horses in Illinois. The seroprevalence and seasonal trend of antibody titers against equine monocytic ehrlichiosis (Potomac horse fever) were determined in apparently healthy horses in selected areas of Illinois in 1986. Sera from 1,367 horses (6 months to 29 years old) were evaluated for the presence of antibodies against Ehrlichia risticii with indirect immunofluorescence. The majority (88%) of the horses were Thoroughbred or Standardbred racehorses. The number of horses with antibodies against E risticii was 229/1,367 (16.75%). The titers in these horses ranged from 1:10 to 1:640. As the year progressed, the n...
Leptospira abortion in horses. Leptospira infection was diagnosed as the cause of 4 late-term equine abortions/stillbirths and 1 neonatal death in Louisiana. The most consistent gross and microscopic lesions were icterus and interstitial nephritis, respectively. Diagnoses were based on visualization of compatible spirochetes in Warthin-Starry-stained sections of kidney, liver, and placenta. Confirmation by immunofluorescence was made in 2 cases.
Antibody response to Ehrlichia risticii and antibody reactivity to the component antigens in horses with induced Potomac horse fever. The antibody response and the antibody reactivity to component antigens of Ehrlichia risticii were studied in horses with induced Potomac horse fever. These horses had no detectable antibodies to E. risticii in their preinoculation (PrI) sera by indirect fluorescent-antibody assay and enzyme-linked immunosorbent assay (ELISA). All the horses exhibited typical disease features following experimental infection and responded with specific antibodies, as measured by ELISA and indirect fluorescent-antibody assay. A primary antibody response was detected in 70% of the horses, while a secondary-type ...
Light and electron microscopy of keratinization in the laminar epidermis of the equine hoof with reference to laminitis. The laminar epidermis (epidermis parietis) of hooves from 14 clinically normal horses, 6 months to 15 years old, was examined by light and electron microscopy and immunofluorescence to measure the contributions of this region to the formation of the hoof wall. By their progressive keratinization to form primary epidermal laminae, the secondary epidermal laminae ultimately contributed about 20% of the thickness of the hoof wall (as revealed in the white line [zona alba]). The keratinized, primary epidermal laminae were developed to a height of 4 mm during their proximodistal-course, much of thi...
Experimental trichinellosis in horses: biological and parasitological evaluation. Three groups of three horses each were, respectively, infected with 5000, 20,000 and 50,000 larvae of Trichinella spiralis. The strain used was isolated from a human biopsy during horsemeat-related outbreaks of trichinellosis in France. Transient muscular disorders were only observed in two of the horses infected with 50,000 larvae but none of the horses had fever. A significant increase in blood eosinophils was noticed in 5 horses. Serum LDH, aldolase and CPK peaked at the fifth week post-infection. Specific IgG assayed by indirect immunofluorescence and ELISA, appeared 2-5 weeks post-infecti...
Surface antigens on equine sarcoid cells and normal dermal fibroblasts as assessed by xenogeneic antisera. To characterise the expression of surface antigens on equine sarcoid cells compared to normal equine fibroblasts, immune sera were produced in rabbits against transformed cells of a virus-containing sarcoid cell line (Mc-1) and normal dermal fibroblasts, respectively. The specificities of the sera were analysed by antibody-dependent cellular cytotoxicity against 51Cr-labelled target cells using human lymphocytes as effector cells. Anti-Mc-1 antiserum induced strong cytotoxicity against transformed cells of two sarcoid cell lines (Mc-1 and Bay Mc-1), whereas the cytotoxicity against transformed...
[Contagious equine metritis in The Netherlands]. Contagious Equine Metritis (CEM) was detected in the Netherlands for the first time in 1987. A total number of five mares (Dutch saddle-horse) were infected in three separate outbreaks. The origin of the infection could not be determined in any of the cases. As the isolates of the causal organism, Taylorella equigenitalis, showed auto-agglutination, diagnosis was difficult. Therefore, an indirect immune fluorescence test as used to diagnose the second isolate. Five strains were isolated, which all were resistant to streptomycin. The prevalence of CEM since 1981 is summarised. The importance of...
Changes in quality of stallion spermatozoa during cryopreservation: Plasma membrane integrity and motion characteristics. Better procedures for freezing and thawing equine sperm are needed since variable fertility is obtained when cryopreserved sperm are used. To evaluate current methods of freezing equine sperm, we examined spermatozoal quality by means of two new techniques. These measured the integrity of plasma-acrosomal membranes by immunofluorescent analyses of binding of an antibody specific to the acrosome and evaluated eight parameters of spermatozoal motion using a fully automated computerized system. Five ejaculates from each of eight stallions were processed for freezing in egg yolk-lactose extender w...
Contraception in mares heteroimmunized with pig zonae pellucidae. Ten fertile feral mares and 6 domestic horses (4 fertile mares, 1 infertile mare, 1 gelding) were immunized with heat-solubilized pig zonae pellucidae by 4 injections equivalent to 2000 or 5000 zonae each at 2-4-week intervals and a booster injection of 20,000 zonae 6-10 months after the last of the initial inoculations. The immune response was reflected by high antibody levels as measured by an enzyme-linked immunosorbent assay (ELISA) using immobilized pig zona antigen. In-vivo inhibition of fertility occurred in 12 (86%) of the 14 fertile mares studied and persisted for a minimum of 7 month...
Urogenital paraneurons in several mammals. Amines and/or peptide-producing cells, deserving to be called paraneurons, were demonstrated in the urethro-prostatic complex of the man, rabbit, buffalo and sheep and in the uterine horns of the pig, horse and mouse, by means of histochemical, immunohistochemical, immunofluorescent and double labeling immunofluorescent techniques. In particular, the urethro-prostatic complex of the sheep contains cells producing serotonin, chromogranin A and somatostatin. Often the amine and the "marker"-protein were colocalized in the same cells. Chromogranin A- and somatostatin-containing cells were found i...
Identification of Highlands J virus from a Florida horse. A virus, strain 64A-1519, isolated from the brain of a horse dying of encephalitis in Florida in 1964, was identified as western equine encephalomyelitis (WEE) virus. Recently, we used polyclonal and monoclonal immune reagents to identify this isolate by comparing it to 2 strains of WEE virus and to Highlands J (HJ) virus in hemagglutination-inhibition, immunofluorescent antibody, and plaque-reduction neutralization tests. These tests demonstrate that strain 64A-1519 is a strain of HJ virus distinct from WEE virus.
Clinical, histopathological, and immunological responses of ponies to Ehrlichia sennetsu and subsequent Ehrlichia risticii challenge. Ehrlichia risticii has a close antigenic relationship to E. sennetsu. Sera of ponies experimentally infected with E. risticii, the etiologic agent of Potomac horse fever, consistently reacted with E. sennetsu, a human pathogen, in indirect fluorescent-antibody (IFA) testing, while human E. sennetsu convalescent serum reacted with E. risticii by IFA testing and immunoferritin labeling of cells infected in vitro. Two ponies injected intravenously with live E. sennetsu did no develop clinical illness. Subsequent injection with live E. sennetsu did not develop clinical illness. Subsequent injectio...
Use of a monoclonal antibody to evaluate integrity of the plasma membrane of stallion sperm. Transmission electron microscopy was used to confirm that a monoclonal antibody (F79.3E2; class IgG1 kappa) was specifically localized to an antigen in the acrosomal ground substance of stallion sperm. This antibody was used to develop and validate an indirect immunofluorescent procedure to evaluate integrity of the plasma-acrosomal membranes of stallion sperm. The concept was that primary monoclonal antibody would be "shielded" from its acrosomal antigen by an intact plasma membrane. Conversely, sperm with damaged plasma-acrosomal membranes would exhibit green acrosomal fluorescence when view...
Use of a monoclonal antibody in the diagnosis of infection by Dermatophilus congolensis. A monoclonal antibody (McAb) to Dermatophilus congolensis was produced from murine hybridoma cultures and purified by affinity chromatography. Species specificity was demonstrated using indirect immunofluorescent staining; the McAb was shown to react with 10 D congolensis isolates but not with 10 Nocardia species isolates, a Rhodococcus and a Streptomyces species isolate. The McAb was used to demonstrate D congolensis in clinical material from confirmed bovine and ovine cases and presumptive equine cases of dermatophilosis by indirect immunofluorescent staining.
An investigation into alternative methods for the serodiagnosis of dourine. The complement fixation test (CFT), indirect fluorescent antibody test (IFAT), card agglutination test for trypanosomiasis (CATT) and enzyme-linked immunosorbent assay (ELISA) were compared in their application to the serological diagnosis of Trypanosoma equiperdum infection in 43 horses. The CFT remains a reliable test for dourine, especially in countries where other members of the subgenus Trypanozoon do not occur. The IFAT is a good 'back-up' test, but, requiring skilled operators it has the disadvantage of making it labour intensive, and interpretation of results subjective. This makes it ...
Borreliosis in equids in northeastern United States. During 1982 and 1985, blood samples from 705 equids were examined for antibodies to Borrelia burgdorferi. By indirect immunofluorescence staining, IgM and total immunoglobulin (IgM and IgG) antibodies were detected in 37 (5.3%) and 90 (12.8%) serum specimens, respectively. The geometric mean titer for IgM antibody (140.4) was highest during July, whereas total immunoglobulin ranged from 94.1 in October to 338 in May. Eighty-six equids with total immunoglobulin to B burgdorferi lived in areas of Connecticut where the primary tick vector, Ixodes dammini, was present. Of the 86 equids, 9 from Lym...
Borrelia burgdorferi infection in Wisconsin horses and cows. Blood samples from Wisconsin horses and cows suspected of having clinical disease due to Borrelia burgdorferi infection were submitted by veterinary practitioners. All serum, milk, colostrum, and synovial samples were tested for B. burgdorferi antibodies by immunofluorescence. Whole blood, milk, colostrum, and synovial fluid samples were cultured for B. burgdorferi. Records were kept on the clinical signs of antibody-positive animals, date of sample, and location of the animal by county. Of the samples tested for antibodies 282/430 cow sera, 118/190 horse sera, 5/10 cow synovial fluids, 3/6 ho...
Serologic response of Babesia equi-infected horses as measured by complement-fixation and indirect fluorescent antibody tests. Both the complement-fixation test (CFT) and the indirect fluorescent antibody test (IFAT) were conducted on weekly serum samples from nine Arab geldings for 28 days before and 256 days after their exposure to Babesia equi of European origin. On an average the IFAT became positive 8 days before the CFT and showed higher relative serum titer increases. Both test procedures successfully detected infection and neither showed an appreciable drop in titer during this time frame, with the exception of the CFT, which showed a transient drop immediately following treatment with imidocarb. A test conduc...
Evaluation of the presence of a specific histocompatibility protein on equine embryonic cells. An indirect immunofluorescence assay was used to detect the presence of H-Y antigen on equine blastocysts. A total of 33 blastocyst stage horse embryos were collected 6 to 7 days post-ovulation by trans-cervical flush and were immediately evaluated for the presence of H-Y antigen. Additionally, 17 embryos, were collected and cultured for 72 h to the expanded blastocyst stage and similarly evaluated. Embryos were placed in medium containing monoclonal antibodies to H-Y antigen followed by incubation in medium containing 1/10 (v/v) fluorescein isothiocyanate conjugated goat anti-mouse IgM Fc spe...
A monoclonal antibody identifying a T-cell marker in the horse. A cell surface molecule of equine T lymphocytes was identified and characterized using a mouse monoclonal antibody, HT23A. The molecule was detected on all T cells but not on other cells in peripheral blood, with the possible exception of a small subpopulation (about 5%) of B cells, as assessed by indirect immunofluorescence and flow cytometry. HT23A labelled T cell areas of horse lymph nodes and spleen when used in an indirect immunoperoxidase assay on frozen sections. Macrophages and neutrophils were not labelled by the antibody nor were frozen sections of horse liver, kidney, or brain. HT23...
Defective thymocyte maturation in horses with severe combined immunodeficiency. Six monoclonal antibodies, designated EqT2, EqT3, EqT6, EqT7, EqT12, and EqT13, which identify T lymphocyte antigens present at different stages of T cell maturation were used to examine T lymphocyte development in foals with severe combined immunodeficiency (SCID). Flow microfluorimetry demonstrated the presence of EqT12+ and EqT13+ prothymocytes and a few phenotypically mature EqT2+ and EqT3+ thymocytes within the thymic remnants of SCID foals. However, very few EqT6+ and EqT7+ resident cortical thymocytes were detected. The near absence of EqT6+ and EqT7+ cortical thymocytes was confirmed b...
Eumycotic mycetoma: review and report of a cutaneous lesion caused by Pseudallescheria boydii in a horse. A cutaneous mass (1.5 cm in diameter) was removed from the head of a horse and was diagnosed histologically as eumycotic mycetoma. Immunofluorescence, performed on formalin-fixed, paraffin-embedded tissue, identified Pseudallescheria boydii as the etiologic agent. Findings from earlier reports of eumycotic mycetoma were compared with those of this horse.
