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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.
Epidemiology of equine Cryptosporidium and Giardia infections. Prevalence and infection patterns of Cryptosporidium and Giardia infections in horses were studied by a direct immunofluorescence staining method. Faecal examinations of 222 horses of different age groups revealed Cryptosporidium infection rates of 15-31% in 66 foals surveyed in central Ohio, southern Ohio and central Kentucky, USA. Only 1 of 39 weanlings, 0 of 46 yearlings, and 0 of 71 mares were positive. Giardia infection was found in all age groups, although the infection rates for foals were higher (17-35%). Chronological study of infection in 35 foals showed that foals started to excrete...
Granulomatous enteritis and cutaneous arteritis in a horse. A 5-year-old Quarter Horse mare was examined because of weight loss of 2 months' duration. The horse was anemic and in poor body condition, and had several areas of cutaneous ulceration, including areas on the muzzle and distal portion of the limbs. Histologic examination of a rectal mucosa biopsy specimen revealed a lymphoplasmacytic infiltrate in the lamina propria mucosa. Results of a glucose absorption test were suggestive of malabsorption. Histologic examination of a biopsy specimen from the cutaneous lesions revealed pronounced arteritis, and weak, patchy immunofluorescent staining for i...
Serologic evidence of canine and equine ehrlichiosis in northeastern United States. In a retrospective study, indirect fluorescent-antibody staining methods were used to detect immunoglobulins to Ehrlichia canis and Ehrlichia risticii in canine and equine sera that had originally been analyzed for antibodies to Borrelia burgdorferi. Analyses of 60 dog serum specimens collected in Connecticut and New York State during 1986 revealed antibodies to E. canis in 7 (11.7%) specimens; titration endpoints ranged from 1:40 to 1:320. Three of these dogs had anemia. Of the 187 equine serum specimens obtained in Connecticut during 1985 and analyzed by indirect fluorescent-antibody stainin...
Characterization of Ehrlichia risticii binding, internalization, and proliferation in host cells by flow cytometry. The binding, internalization, and proliferation of Ehrlichia risticii in P388D1 cells and equine polymorphonuclear (PMN) leukocytes were studied by immunofluorescent staining and flow cytometric analysis. The binding of ehrlichiae to P388D1 cells at 4 degrees C was dose dependent, and the antigens of bound organisms were susceptible to pronase treatment. Additionally, the binding of ehrlichiae to P388D1 cells was diminished when either P388D1 cells or ehrlichiae were treated with 1% paraformaldehyde for 30 min or 0.25% trypsin for 15 min. These results indicate that the ehrlichial ligand and h...
Characterization of the myristylated polypeptide encoded by the UL1 gene that is conserved in the genome of defective interfering particles of equine herpesvirus 1. Equine herpesvirus 1 (EHV-1, Kentucky A strain) preparations enriched for defective interfering particles (DIPs) can readily establish persistent infection. The UL1 gene, which is conserved in the genome of DIPs that mediate persistent infection, maps between nucleotides 1418 and 2192 (258 amino acids) from the L (long) terminus. UL1 has no homology with any known gene encoded by herpes simplex virus type 1 but has limited homology to open reading frame 2 of varicella-zoster virus and the "circ" gene of bovine herpesvirus type 1. Previous work showed that the EHV-1 UL1 gene belongs to the earl...
Production and characterization of a monoclonal antibody recognizing a cytoplasmic antigen of equine mononuclear phagocytes. An IgG1 mouse monoclonal antibody, designated 1.646, is described which recognizes a cytoplasmic antigen of equine mononuclear phagocytes. Indirect fluorescent antibody staining of peripheral blood leukocytes reveals a granular cytoplasmic staining, predominantly in adherent blood mononuclear cells. Indirect fluorescent antibody staining is positive for alveolar and peritoneal macrophages. In some horses, a few neutrophils are also stained. In equine tissue samples stained by immunohistochemistry, the distribution of positive cells is consistent with the distribution of tissue macrophages. The...
Phenotypic analysis of peripheral blood and bronchoalveolar lavage fluid lymphocytes in control and chronic obstructive pulmonary disease affected horses, before and after ‘natural (hay and straw) challenges’. Phenotypic analysis of lymphocytes in peripheral blood (PB) and bronchoalveolar lavage fluid (BALF) of control and chronic obstructive pulmonary disease (COPD) affected horses, both before and after 'natural (hay and straw) challenge', were performed using immunofluorescent labelling with monoclonal antibodies and flow cytometry. BALF lymphocytes were shown to be predominantly EqCD5+ cells, approximately half of which were also EqCD8+, with a smaller proportion of B cells. In comparison with PB, BALF contained higher proportions of EqCD5+ cells and EqCD8+ cells and a lower proportion of B cell...
Detection of antibodies against Borna disease virus in sera and cerebrospinal fluid of horses in the USA. Sera from 295 horses in the USA were examined by an indirect immunofluorescence assay and Western blot assays to determine the prevalence of Borna disease virus infection. Eight (2.7 per cent) of the samples were positive in both assays, and 18 (6.1 per cent) were positive only in the Western blot assay. The indirect fluorescence titres ranged from 1:20 to 1:80 of antibodies recognising the virus-specific antigen from Borna disease virus-infected cells. The purified virus-specific proteins isolated from infected rat brains were recognised by positive equine serum samples after immunostaining b...
Analysis of multiple mRNAs from pathogenic equine infectious anemia virus (EIAV) in an acutely infected horse reveals a novel protein, Ttm, derived from the carboxy terminus of the EIAV transmembrane protein. Transcription of pathogenic equine infectious anemia virus (EIAV) in an acutely infected horse was examined by using the polymerase chain reaction and nucleotide sequencing. Four spliced transcripts were identified in liver tissue, in contrast to the multiplicity of alternatively spliced messages reported for in vitro-propagated human immunodeficiency virus, simian immunodeficiency virus, and, to a lesser extent, EIAV. Nucleotide sequence analysis demonstrated that three of these mRNAs encode known viral proteins: the envelope precursor, the product of the S2 open reading frame, and the regula...
Equine protozoal myeloencephalitis: antigen analysis of cultured Sarcocystis neurona merozoites. Antigens of cultured Sarcocystis neurona merozoites were examined using immunoblot analysis. Blotted proteins were probed with S. cruzi, S. muris, and S. neurona antisera produced in rabbits, S. fayeri (pre- and post-infection) and S. neurona (pre- and post-inoculation) sera produced in horses, immune sera from 7 histologically confirmed cases of equine protozoal myeloencephalitis (EPM), and pre-suckle serum from a newborn foal. Eight proteins, 70, 24, 23.5, 22.5, 13, 11, 10.5, and 10 Kd, were detected only by S. neurona antiserum and/or immune serum from EPM-affected horses. Equine sera were ...
Characterization of African horsesickness virus serotype 4-induced polypeptides in Vero cells and their reactivity in Western immunoblotting. The structural and non-structural proteins induced by African horsesickness virus serotype 4 (AHSV-4) in infected Vero cells were analysed by SDS-PAGE. Twenty-two virus-induced polypeptides were detected in infected cells by comparison with the polypeptides of mock-infected cells, of which four major (VP2, VP3, VP5 and VP7) and three minor (VP1, VP4 and VP6) structural proteins and four non-structural proteins (P58, P48, P21 and P20) were shown to be virus-coded, as deduced from electrophoretic and antigenic studies of purified virions and infected cells. The proteins that elicit the major ant...
[The use of ELISA and indirect immunofluorescence technics for the rapid detection of eastern equine encephalomyelitis]. We present the results attained in the identification of Eastern equine encephalomyelitis virus isolations in Vero and XL-2 cell systems, using a double-antibody ELISA technique and the indirect immunofluorescence method. The results attained through these two techniques coincided by 100% with identification through neutralization. With the former, the virus was detected within 6-8 hours after inoculation. Better results were attained with XL-2 cells.
A type-specific conformational epitope on the nucleocapsid of equid herpesvirus-1 and its use in diagnosis. A type-specific monoclonal antibody was produced by immunizing mice with purified equid herpesvirus-1 (EHV-1). The EHV-1 specific mAb reacted with all the EHV-1 strains tested so far by indirect ELISA, immunofluorescence, and immunoperoxidase tests. No reactions were detected with the EHV-4, EHV-2, or EHV-3 strains tested. The indirect immunofluorescence and immunoperoxidase tests showed that the nuclei of infected cells were predominantly stained by this mAb. Triton treatment of the virus and immunogold labeling experiments indicated that the nucleocapsid of EHV-1 was the target antigen of th...
Diagnostic methods for African horsesickness virus using monoclonal antibodies to structural and non-structural proteins. A panel of 32 hybridoma cell lines secreting monoclonal antibodies (MAbs) reactive with African horsesickness virus serotype 4 (AHSV-4) has been developed. Four of the MAbs recognized the major core antigen VP7, twenty recognized the outer capsid protein VP2 and eight reacted with the non-structural protein NS1. With the VP7-specific MAbs a rapid and sensitive double antibody sandwich immunoassay has been developed to detect viral antigen in infected Vero cells and in spleen tissue from AHSV-infected horses. The sensitivity of the assay is 10 ng viral antigen per 100 microliters. The NS1-speci...
Demonstration of the humoral immune response of horses to Babesia caballi by western blotting. Babesia caballi-infected or normal equine erythrocytes were solubilized in sodium dodecyl sulfate (SDS) buffer and analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Antigens were allowed to react with sera from horses experimentally or field-infected with B. caballi and with sera from non-infected horses. Major babesial antigens recognized by immune sera had apparent mol. wts of 141, 112, 70, 50, 48, 34, and 30 kDa. The polypeptides at 50 and 48 kDa were recognized earliest and throughout infection, but also weakly by 3/100 equine sera tested negative and 1/33...
Immunocytochemistry of paraneurons in the female urethra of the horse, cattle, sheep, and pig. The aim of this study is to describe the presence of neuroendocrine (NE) cells (paraneurons), producing biogenic amines and/or peptidergic hormones, in the female urethra of cattle, sheep, pigs, and horses, by means of histochemical and double labeling immunofluorescent techniques. 5-Hydroxy-tryptamine-, chromogranin A-, cholecystokinin- and somatostatin-containing NE cells are present in the urethral epithelium of all the species studied, with the unique exception of the lack of somatostatin cells in the horse. Paraneurons containing 5-hydroxytryptamine colocalized with chromogranin A or chol...
Equine glomerulonephritis and renal failure associated with complexes of group-C streptococcal antigen and IgG antibody. A 12-year-old thoroughbred gelding died from diffuse global glomerulonephritis, 3 months after a lower respiratory infection from which Streptococcus zooepidemicus was isolated. Immunopathological studies (immunofluorescence, immunodiffusion, immunoperoxidase testing and immunoblotting) indicated the presence of an immune reactant renal disease associated with IgG antibody and streptococcal antigens.
[Causes of prenatal foal loss in Switzerland]. In Switzerland during the foaling season 1988 and 1989 the cause of abortion in 60 foals was investigated. Special attention was paid to infections with equine herpesvirus 1 (EHV 1). Diagnosis were based on post-mortem, histopathological, bacteriological and immunofluorescence investigation. The results confirm data from other countries, that EHV 1 is the most prevalent viral (20%) cause of abortion, followed by various bacterial agents (12%). Other causes were umbilical torsion, twin pregnancy and malformations. In 18% of the cases the investigation of fetuses did not give any results as to t...
Equine monocytic ehrlichiosis (Potomac horse fever): a review. In the late 1970s, a new infectious disease in horses, involving acute enteritis, was recognised in the Potomac River area of Maryland, U.S.A. The causative agent was identified subsequently as a new species of rickettsial organism, later named Ehrlichia risticii. Since then, the disease has been reported in many other states, and in enzootic areas vaccination is common. Signs associated with the clinical disease included depression, fever, anorexia, decreased or absent intestinal sounds, profuse watery diarrhoea and laminitis. However, considerable variation in clinical manifestations has bee...
Comparative growth of different rotavirus strains in differentiated cells (MA104, HepG2, and CaCo-2). The production of viral antigen after infection of MA104, HepG2 (derived from human liver), and CaCo-2 (derived from human colon) cells with various cultivatable human and animal rotavirus strains was compared using immunofluorescence tests. All rotavirus strains examined expressed antigen in CaCo-2 cells and MA104 cells, but only some virus strains, namely, SA11-Cl3 (simian), RRV (simian), CU-1 (canine), and Ty1 (turkey), produced antigen in numbers of infected HepG2 cells comparable to infections in MA104 and CaCo-2 cells. Fl-14 (equine), OSU (porcine), NCDV (bovine), and Ch2 (chicken) strai...
Three monoclonal antibodies identifying antigens on all equine T lymphocytes, and two mutually exclusive T-lymphocyte subsets. The aim of this study was to produce monoclonal antibodies (mAb) recognizing equine lymphocyte surface antigens. Fusions were conducted using BALB/c mice hyperimmunized with equine thymocytes. Hybridoma supernatants were screened by flow cytometry and positive hybridomas were cloned twice by limiting dilution. These mAb were then characterized for tissue distribution by immunohistology and flow cytometry, and by precipitation and analysis of the lymphocyte antigens which they recognized. Three mAb (CVS5, CVS4 and CVS8) are described which recognize only T lymphocytes in peripheral blood. Two-c...
The role of endothelial cell infection in the endometrium, placenta and foetus of equid herpesvirus 1 (EHV-1) abortions. One of three mares in the last trimester of pregnancy became paraplegic 7 days after experimental infection with EHV-1 and was killed 10 days after infection (d.p.i.). The other two mares aborted foetuses at 12 and 14 d.p.i. In the first mare, virus was detected by immunofluorescence (IIF) and immunoperoxidase (IP) staining in endothelial cells of the endometrium, placenta and umbilical vein, but not in any other foetal tissues. In the experimentally aborted foetuses, and in two other independent field cases of abortions, endothelial cell infection was also detected in the foetuses, both in ma...
[The differentiation of viruses in the Venezuelan equine encephalomyelitis complex by using monoclonal antibodies and lanthanide immunofluorescence analysis]. Potentialities of differentiation between Venezuelan equine encephalomyelitis (VEE) complex viruses by time-resolved fluoroimmunoassay and enzyme immunoassay were studied. For this, 4 test systems were used based on different combinations of native and labeled polyclonal antibodies to VEE virus, strain Trinidad, and monoclonal (MCA) antibody MAK 14-7 to protein EL of this virus. The maximal sensitivity and specificity was achieved in the test system formed from native MCA MAK 14-7 for sensitization of the solid phase and labeled polyclonal immunoglobulins for demonstration of the test results....
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 agglutin...
[Symptomatology and diagnosis of Borna encephalitis of horses. A case analysis of the last 13 years]. In a retrospective study of 38 horses with Borna encephalitis which were clinically and histopathologically examined in the "I. Medizinische Tierklinik" in Munich between 1977 and 1990, the epidemiology, the clinical symptomatic and the diagnostic procedures available are presented. Indirect immunofluorescence showed antibodies in the serum of 12 out of 29 cases (41%) and in the cerebrospinal fluid (CSF) of 17 out of 28 cases (61%). The evaluation of 23 cases in which indirect immunofluorescence of serum and CSF, and also the post mortem virological and histopathological examination of the bra...
[A horse seropositive for Ehrlichia risticii]. A four year old Dutch warmblooded mare was born and raised in the province of North-Brabant, the Netherlands. On May 16, 1989, she showed signs of colic, anorexia, depression, ileus, severe dehydration and leukopenia. When the mare collapsed, euthanasia was carried out. Acute colitis and cytoplasmic inclusion bodies in macrophages were observed at autopsy. When an indirect immunofluorescence assay was performed, the Ehrlichia risticii titre of the serum was found to be 1:640.
[Serologic studies of the occurrence of Borrelia burgdorferi in domestic animals in Berlin (West)]. The prevalence of B. burgdorferi, the causative agent of Lyme Borreliosis in humans, was determined in domestic animals living in Berlin. 189 dogs, 29 cats, 224 horses and 194 cows were investigated. Using the indirect immunofluorescence test (IFT) 5.8% of the dogs and 24.5% of the cows investigated showed a positive reaction at titres of 1:128 or higher. Horses and cats gave negative results. ELISA was more sensitive than IFT. 10.1% of the dogs, 16.1% of the horses and 66% of the local cows showed positive reaction. Domestic animals seem to be in contact with B. burgdorferi and can be a reser...
Acrosome reaction of stallion spermatozoa evaluated with monoclonal antibody and zona-free hamster eggs. The acrosome of the stallion spermatozoon was visualized by indirect immunofluorescence with monoclonal antibody (18.6) which recognized an integral acrosomal membrane component. Localization was confirmed by electron microscopy using peroxidase labelled antibody. In fresh semen samples (n = 19), 73.9 +/- 9.1% of the spermatozoa from five fertile stallions displayed a uniform bright fluorescence over their acrosome region. In two semen samples from an infertile stallion only 28% and 35% of spermatozoa showed the same pattern of fluorescence. Spermatozoa from fertile stallions incubated for up ...
Modulation of an adhesion-related surface antigen on equine neutrophils by bacterial lipopolysaccharide and antiinflammatory drugs. The essential role of the CD11/CD18 family of leukocyte adhesion molecules (LeuCams) in neutrophil-substrate adhesion is well documented. We have found that a monoclonal antibody designated 60.3 (MoAb 60.3) that recognizes the common beta-subunit (CD18) on human neutrophils (PMN) also recognizes a surface antigen on equine PMN. Antigen expression as assessed by immunofluorescence flow cytometry was enhanced by zymosan-activated serum (ZAS) or phorbol 12-myristate 13-acetate (PMA) stimulation. Pretreatment of equine PMN with MoAb 60.3 inhibited ZAS-stimulated aggregation, indicating that the mo...
Methods for detection of immune-mediated neutropenia in horses, using antineutrophil serum of rabbit origin. Equine neutrophil antibody was raised in rabbits inoculated with equine neutrophils isolated to purity greater than 99.0%, using Percoll density-gradient sedimentation. Neutrophil antibody was detected by use of agar gel diffusion, leukoagglutination, indirect immunofluorescence, staphylococcal protein A and streptococcal protein G binding, and phagocytic inhibition techniques. Precipitin lines and leukoagglutination were seen in antiserum dilutions of 1:4 and 1:64, respectively. The specific nature of leukoagglutination was characterized by the formation of rosette-like clumps of neutrophils....
