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.
Antibodies to surface antigens of pigmented cells in animals with vitiligo. All of 24 animals (dogs, cats, and horses) with vitiligo were found to have antibodies to pigmented cells that could be detected by specific immunoprecipitation of radioiodinated, detergent-soluble surface macromolecules, and by indirect immunofluorescence on viable cells. These antibodies were not detected in 17 normal animals of the same species. The antibodies were directed to an 85-kDa surface antigen selectively expressed by pigmented cells that was not present on nonpigmented control cells. These observations suggest that vitiligo in animals is an autoimmune disease mediated to pigmented...
Serodiagnosis of experimental and natural Babesia equi and B. caballi infections. The sensitivity and specificity of the complement fixation (CF) test for the diagnosis of Babesia infections in equines was assessed, using the indirect fluorescent antibody (IFA) test as a reference. Antibodies were first detected between 11 and 20 days post infection (dpi) in the CF test and between 7 and 14 dpi in the IFA test in ponies infected experimentally with B. equi (USDA strain). The CF test became negative in four of five ponies 63-174 dpi although B. equi was demonstrated microscopically in two of these four ponies up to 364 and 455 dpi. The IFA test remained positive up to 476 dp...
Species-specific serodiagnosis of equine piroplasma infections by means of complement fixation test (CFT), immunofluorescence (IIF), and enzyme-linked immunosorbent assay (ELISA). The increasing horse trade requires a reliable immunodiagnosis of equine piroplasma infections due to import restrictions imposed by various countries, including the United States of America. It was the aim of our investigations to establish the suitability of serological tests for the detection of parasite carriers and, eventually, to differentiate between Babesia caballi and B. equi infections. The investigations were carried out on 11 ponies with experimentally-induced B. caballi and/or B. equi infection. The infections were confirmed by the demonstration of parasites in blood smears 2-13 d...
Immunodiagnosis of autoimmune skin disease in the dog, cat and horse. Skin biopsies from 47 dogs, 6 cats and 5 horses with suspected autoimmune skin disease were submitted for immunofluorescence from 1978 to 1985. These cases were predominantly Western Australian in origin, although a number were also referred from Queensland and Victoria. In 5 dogs, 2 cats and 2 horses immunoglobulin binding to intercellular cement substance and/or basement membrane was demonstrated by direct immunofluorescence. Antinuclear antibody was also demonstrated in several of these cases. Immunofluorescence was used in combination with histopathological examination to confirm the clini...
Endothelial cell infection and thrombosis in paralysis caused by equid herpesvirus-1: equine stroke. Eight mares were infected with equid herpesvirus-1 subtype 1 isolated from a case of equine paresis. In two mares killed at 4 d.p.i. immunofluorescence showed endothelial cell infection together with thrombosis in the rete arteriosus of the nasal mucosa and also in the spinal cord of one of these mares. Circulating platelet counts in the other six mares fell as early as 2 d.p.i. and remained depressed for seven days. Circulating immune complexes started to appear at 2 d.p.i., reached maximum levels at 10 d.p.i., but were undetectable at 28 d.p.i. Three of the six remaining mares developed vary...
Characteristics of cells derived from the girdle region of the pre-implantation blastocyst of the donkey. The establishment of a monolayer culture of cells derived from the girdle region of a 34-day-old donkey conceptus is described. These cells have had over 100 repeated passages in culture. Low levels of pregnant mares' serum gonadotrophin (PMSG, eCG) could be detected in the cells by indirect immunofluorescence using some monoclonal anti-eCG antibodies, but the cells did not secrete eCG as measured by radioimmunoassay or inhibition of haemagglutination. There was marked nuclear polymorphism with binucleate and occasional multinucleate cells. The cells were strongly reactive with wheatgerm agglu...
Effects of horse and fetal calf serum on the expression of tumor-associated antigen and tumorigenicity of L5178Y leukemia/lymphoma cells. A tumor antigen (TA) associated with murine leukemia-lymphoma L5178Y cells has been identified by the enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence (IIF) techniques. The antigen was present in both non-solubilized and 0.5% NP-40 solubilized membrane extracts. Rabbit anti-L5178Y lymphoma serum (RALS), extensively absorbed with normal mouse tissues, identified TA in extracts of L5178Y lymphoma and L5178Y leukemia cells grown in horse serum (L5178Y/HS), but not in extracts of L5178Y cells grown in fetal calf serum (L5178Y/FCS). Similarly, absorbed rabbit anti-L5178Y/HS...
Antibodies to Borrelia burgdorferi in New England horses: serologic survey. Twelve of 50 randomly selected horses from areas endemic for Borrelia burgdorferi had indirect fluorescent antibody titers of 1:8 to 1:2,048 against B burgdorferi. One of 50 horses from nonendemic areas had a titer of 1:8. This difference in the number of horses seropositive for B burgdorferi (P less than 0.002) and our finding that seropositive horses did not have agglutinating antibodies against potentially cross-reacting Leptospira spp indicated that horses in endemic areas were exposed to B burgdorferi and that the spirochete induced an antibody response in the horses.
Experimental demonstration of an antigenic relationship between Leptospira and equine cornea. Horses inoculated with either equine cornea or killed Leptospira interrogans serovars pomona, tarassovi, icterohaemorrhagiae, wolffi and hardjo, developed corneal opacity and produced antibodies which made it possible to demonstrate partial antigenic identity between equine cornea and four of those serovars employed. These antibodies were isolated by means of immunoadsorptions, purified by ion-exchange chromatography (DEAE-Sephadex A-50) and run by immuno-electrophoresis in agar gel. Both antibodies, anti-equine cornea and anti-leptospira, showed that they corresponded to the IgGb subclass. Th...
Healing of surgically created defects in the equine superficial digital flexor tendon: collagen-type transformation and tissue morphologic reorganization. Full-thickness defects were surgically created in the superficial digital flexor tendons of the front limbs of 20 horses. Tissues formed within the defect were evaluated histologically, and the collagen composition of the tissue was determined by immunofluorescence. Transformation occurred from loose fibrillar areas of types I and III collagen and pericellular types IV and V collagen to dense bundles of type I collagen fibers. Loose fibrillar areas of types I and III collagen were present after 24 weeks. Histologically, in horses killed after 2 weeks, the tissue within the defect was a randoml...
Healing of surgically created defects in the equine superficial digital flexor tendon: effects of pulsing electromagnetic field therapy on collagen-type transformation and tissue morphologic reorganization. The effect of pulsing electromagnetic field (PEMF) therapy on the healing of surgically created defects in equine superficial digital flexor tendons was evaluated. Defects were created in both front superficial digital flexor tendons of 20 horses. The defect in 1 limb was exposed to a PEMF for 2 hours daily. The other limb served as a control. Histologic and immunofluorescent evaluations were done in horse killed at postsurgical weeks 2, 4, 8, 12, and 24. Therapy with the PEMF significantly (P less than 0.05) delayed the maturation of the tissue formed within the defect at postsurgical weeks 8...
Recent vesicular stomatitis virus infection detected by immunoglobulin M antibody capture enzyme-linked immunosorbent assay. We developed an enzyme-linked immunosorbent assay (ELISA) that was capable of detecting immunoglobulin M (IgM) antibody to vesicular stomatitis virus (VSV) in the sera of experimentally and naturally infected cattle and horses. The detection of IgM in the sera of these animals permitted an estimate of the recency of infection by VSV serotype New Jersey. A VSV serotype New Jersey epizootic strain isolated from a horse and passed once in an Aedes albopictus cell line was used to infect a horse and a calf. Sera from these animals were used to standardize the ELISA. This assay was used to test ser...
Causative ehrlichial organisms in Potomac horse fever. An ehrlichia was consistently isolated from the peripheral blood leukocyte fraction of ponies that had been experimentally infected with Potomac horse fever by whole blood transfusion from naturally infected horses. The organism was propagated in a human histiocyte cell line for 3 to 5 weeks and then inoculated intravenously or intradermally into healthy adult ponies. Clinical signs of Potomac horse fever, which varied in the degree of severity, occurred 9 to 14 days post-inoculation in all of the ponies. One pony died 20 days post-inoculation. The ehrlichial organism was reisolated in the hum...
Experimental reproduction of Potomac horse fever in horses with a newly isolated Ehrlichia organism. Potomac horse fever, a recently recognized disease of equines, characterized by high fever, leukopenia, and a profuse diarrhea, was studied for its etiology. An Ehrlichia organism was isolated in equine macrophage-fibroblast cell cultures and mouse macrophage cell cultures from the mononuclear cells of blood of infected horses. The agent was continuously propagated in mouse macrophage cell cultures. The organism multiplied in the cytoplasm of mouse macrophage cells and was identified by Giemsa staining, acridine orange staining, and by indirect immunofluorescence with convalescent sera from in...
Microtubular mass defect of spermatozoa in the stallion. A microtubular mass (MM) defect was found in the spermatozoa of 7 Standardbred stallions; 3 stallions were sons of the same sire. Two of these 3 stallions and 2 other stallions (for a total of 4 out of the 7 stallions) were considered subfertile when the defect was first observed. Fertility improved with time, either during the first breeding season or when a given stallion was used less frequently; however, the MM defect persisted, consisting of tortuous arrays of small abnormal microtubules visible only by transmission electron microscopy. The MM probably contained the protein tubulin as ind...
[Babesia infections in horses, cattle and dogs in southern Germany]. Babesia infections serologically diagnosed in horses, cattle and dogs in Southern Germany during the last few years are described. 321 sera of horses were examined for specific antibodies to Babesia by means of CFT and IIF in 1984; 18 sera reacted to Babesia equi and 4 to Babesia caballi antigen. In a cattle breeding area in the Western Allgäu 13% of 1616 cattle reacted positive to Babesia divergens antigen using IIF and ELISA; during the grazing season 1982 new latent infections were observed in 25 of 266 calves and heifers. Cases of introduced canine babesiosis are more frequent; 10 of 34 s...
A new surface marker on equine peripheral blood lymphocytes. I. Subpopulations of lymphocytes with receptors for Helix pomatia A hemagglutinin (HP). Untreated and neuraminidase-treated equine peripheral blood lymphocytes were analysed for binding of the A hemagglutinin of the snail Helix pomatia (HP). For optimal staining by direct immunofluorescence, the concentration of neuraminidase had to be increased as compared to that needed for other species. Moreover, higher concentrations of HP were required for optimal staining of equine lymphocytes as compared to lymphocytes from other species. Even so, the maximal number of equine lymphocytes exhibiting positive staining was only about 20%. No, or very few, HP-positive lymphocytes were seen wh...
A new surface marker on equine peripheral blood lymphocytes. II. Characterization and separation of purified blood lymphocytes with receptors for Helix pomatia A hemagglutinin (HP). In a preceding report we have shown that the lectin Helix pomatia A hemagglutinin (HP) binds to two subpopulations of neuraminidase-treated equine peripheral blood lymphocytes (PBL), constituting about 20% and 75% of PBL, respectively. The aim of the present study was to further characterize these HP+ cells in regard to other surface markers such as receptors for guinea pig erythrocytes (GPR+ cells), membrane-bound immunoglobulins (sIg+ cells), receptors for activated complement (C3R+ cells) and receptors for IgG (Fc alpha R+ cells). This was done by double marker analysis and by lymphocyte fr...
Serologic evidence of Legionella infection in horses. The indirect fluorescent antibody test was used to examine 109 samples of equine sera randomly selected from serum pools. Results were compared with titers obtained by the microagglutination (MA) test. A high correlation (r = 0.89) was found between titers measured by the 2 tests. Blood samples were obtained serially from a total of 156 horses at a research farm and the sera were tested against Legionella pneumophila serogroups 1 through 4 using the MA test; 29 horses (19%) seroconverted to at least 1 serogroup of L pneumophila. The indirect fluorescent antibody test substantiated the results ...
Pitfalls in immunofluorescence testing in dermatology. III. Pemphigus-like antibodies in the horse and direct immunofluorescence testing in equine dermatophilosis. Indirect immunofluorescence testing for pemphigus-like antibodies was performed on 79 horses: 28 horses with various nonpemphigus dermatologic diseases, 21 horses with various nondermatologic diseases, and 30 normal horses. Pemphigus-like antibodies were detected in 6 horses: 3 normal horses with titers of 1:40, 2 horses with dermatophilosis at titers of 1:10 and 1:80, and 1 horse with lymphosarcoma at a titer of 1:320. It was concluded that equine pemphigus-like antibodies are a potential source of misinterpretation and misdiagnosis in indirect immunofluorescence testing. Direct immunofluores...
Equine adenovirus 1 isolated from cauda equina neuritis. Equine adenovirus 1 was recovered after four to six passages from two out of three cases of cauda equina neuritis (CEN) using kidney monolayers. Similar treatment of lumbo-sacral spinal cord from six normal horses did not yield adenovirus. All three cases of CEN had antibodies to the neuritogenic myelin protein P2 while immunofluorescence demonstrated that autologous IgG bound to the myelin of affected nerves. Adenovirus was not detected in neural tissue by immunofluorescence.
Xenogeneic monoclonal antibodies to cell surface antigens of equine lymphocytes. Monoclonal antibodies to equine lymphocyte antigens were produced, using normal peripheral blood lymphocytes as the immunogen and standard hybridoma techniques. Antibody producing hybridomas were detected by a solid-phase enzyme-linked immunosorbent assay. Antibodies produced by 6 cloned hybrids were characterized further by microlymphocytotoxicity, indirect immunofluorescence, and agglutination assays on peripheral blood lymphocytes, platelets, and erythrocytes. Reaction patterns on leukocytes indicated that these antibodies may recognize at least 3 different cell-surface antigens: (1) an ant...
[Method of isolating and controlling fluorescent Fab fragments of antibodies against horse serum proteins]. For the first time the lyophilized fluorescent Fab-fragments of rabbit antibodies to horse serum protein, suitable for detecting different antigens and antibodies to these antigens, have been obtained by the specially developed method. The criteria to be used in the control of the antispecific fluorescent fragments of antibodies have been described and the methods of their control before and after lyophilization have been developed. The use of the antispecific fluorescent Fab-fragments of antibodies has been shown to considerably accelerate and simplify the indirect immunofluorescent assay.
Coestablishment of persistent infection and oncogenic transformation of hamster embryo cells by equine cytomegalovirus. Semipermissive, primary hamster embryo (HE) cells were morphologically transformed in vitro by infection with UV-irradiated equine cytomegalovirus (equine herpesvirus type 2; ECMV). Cell lines (designated EC-1-3) were established independently from foci and were shown to exhibit growth and biological properties typically associated with transformed cells: altered morphology, loss of contact inhibition, increased saturation density, decreased generation time, immortality in culture, normal growth in low concentrations of serum, colony formation in soft agar, and resistance to ECMV superinfectio...
The distribution of types I and III collagen and fibronectin in the healing equine tendon. During tissue response to injury the glycoproteins fibronectin and Type III collagen are synthesized in increased amounts. We have studied the distribution of these molecules in the healing tendon at various times after injury by comparison with that of the major constituent of normal tendon, Type I collagen. Immunofluorescent localization demonstrated the presence of fibronectin throughout the tendon within one week after injury. Staining was found in the matrix, both around capillaries and around fibroblast-like cells. Fibronectin was still apparent in the healing tendon at one month after i...
Granules of blood eosinophils are stained directly by anti-immunoglobulin fluorescein isothiocyanate conjugates. Direct staining of the granules of blood eosinophils by anti-immunoglobulin fluorescein isothiocyanate (FITC) conjugates was observed when feline blood smears were tested for presence of feline leukemia virus (FeLV) antigen by immunofluorescent antibody. When blood smears of other species including swine, horses, cattle, dogs, sheep, birds, and human beings were examined, direct staining of eosinophils by FITC conjugates was also detected. This FITC staining was restricted to eosinophils and was not observed in neutrophils, lymphocytes, and platelets. Direct FITC staining of eosinophils does n...
Leptospiral infection in aborted equine foetuses. During an investigation of equine abortion, leptospiral infection was demonstrated in nine out of 22 foetuses examined by immunofluorescence and culture. Strains belonging to four serogroups (Australis, Pomona, Hebdomadis and Icterohaemorrhagiae) were isolated. The age of leptospira infected foetuses ranged from six months to term.
Analysis of serum and lymphocyte surface IgM of healthy and immunodeficient horses with monoclonal antibodies. Nine monoclonal antibodies which reacted with equine immunoglobulin (Ig)M and not other equine Ig and serum proteins were prepared. Cells producing antibodies (C 1.9) which precipitated with IgM and bound to staphylococcal protein A were triple-cloned (C 1.9/3.2) and the antibodies further characterized. Monoclonal antibody C 1.9/3.2 reacted with an IgM determinant present on serum IgM from horses of several breeds. Studies with 125I-labeled IgM revealed the presence of this determinant on all IgM molecules. The monoclonal antibody enabled quantitation of IgM in presuckling foal and adult hors...