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.
Hernández-Jáuregui , González-Angulo A.Electron microscopy and immunofluorescence techniques were used to study the various epithelial cells in the endometrial cups of ten pregnant mares, and in the uterine epithelium of one non-pregnant mare. Evidence was obtained which suggested that epithelial gland cells within the endometrial cups, as distinct from the hypertrophied decidual-like cells in the cup, may synthesize PMSG. The suggestion does not agree with the findings of other workers in the fetal origin (chorionic girdle cells) of this gonadotrophin.
Erickson GA, Maré CJ.Goat Venezuelan equine encephalomyelitis (VEE) antiserum and normal serum were conjugated and evaluated for staining sensitivity and specificity. Cross-staining with either eastern or western equine encephalomyelitis virus-infected cells did not occur. The baby hamster kidney (BHK-21) cell line when combined with highly specific VEE conjugate detected 100 medium suckling mouse intracerebral lethal doses (suckling mouse LD-50/IC) of the 1B subtype of VEE virus per milliliter of equine tissue suspension. Conjugated goat antiserum was assayed for sensitivity for detection of VEE virus-infected eq...
Joncas JH, Gilker JC, Chagnon A.The relative value of heterophil agglutinins (HA) and of specific EBV antibodies in the diagnosis of infectious mononucleosis (IM) was assessed in 108 cases of the disease and in 280 controls. Among the 108 cases 93 were HA-positive by sheep cells in at least one of their sera, while 15 were HA-negative by the same test. Among the 280 controls false-positive HA tests were not encountered except in eight cases with the horse cell microtitre tests. With one of the two slide tests at least two false-positive tests and 12 false-negative tests were also found but these sera had low titres in microt...
Watson RE, Larson KA.Indirect immunofluorescence and lymphocyte cytotoxicity experiments demonstrated the presence of a tumor-specific antigen(s) on the surface of cells from an equine sarcoid cell line (Mc1). Autologous serum (taken from the horse from which the Mc1 cells were derived) and sera from three other sarcoid-bearing horses revealed a similar membrane immunofluorescence when reacted with Mc1 cells, indicating the existence of cross-reacting antibodies. Results of serum colony inhibition experiments indicate that these antibodies are not cytotoxic. Incubation of Mc1 cells with autologous lymphocytes resu...
Banks KL, Henson JB.A defect in lymphocyte function could be responsible for persistent infection by the equine infectious anemia virus. The number of lymphocytes bearing surface immunoglobulin, as detected by immunofluorescence, and lymphocyte response to mitogens were the same in uninfected and equine infectious anemia-infected animals. A defect in T or B lymphocyte numbers or ability to respond to stimuli was not detected in this chronic virus disease.
Larghi OP, González E, Held JR.The corneal test (CT) for rabies diagnosis was evaluated in samples from 313 subjects of different species. Some of the subjects were inoculated experimentally and others were naturally infected. When the CT was compared with immunofluorescence staining and mouse inoculation tests on brains of the same subjects, a sensitivity of 41.7% and a specificity of 100% were found. The authors conclude that a positive CT result would confirm the diagnosis of rabies, but a negative one would not exclude the possibility of disease.
Smith IM, Girard A, Corner AH, Mitchell D.Using two known positive equine viral rhinopneumonitis (EVR) sera, conjugates were prepared with fluorescein isothiocyanate and tested for specificity using EVR infected tissue culture cells. The conjugate was then applied to selected tissues from 32 aborted fetuses and foals submitted during a natural outbreak of EVR. Antigen was detected in various tissues by immunofluorescence in 20 cases (62.5%). In 24 cases bovine fetal kidney cell monolayers were inoculated with a pool of lung and liver and EVR virus was isolated from 15 (62.5%). Histological examination of various tissues from 29 cases ...
Ushimi C, Henson JB, Gorham JR.Primary horse leukocyte cultures were inoculated with 2 or 10 50% tissue culture infective doses (TCID(50)) of equine infectious anemia (EIA) virus per cell, and the titer of cell-associated and fluid-phase virus was determined from 1 to 72 hr postinoculation (PI). Cover slips were collected from 4 to 72 hr PI and stained for EIA viral antigen by the indirect immunofluorescent (FA) technique. Viral replication was detected after a latent period of approximately 18 to 24 hr and reached peak titers of approximately 10(4.5) to 10(6) TCID(50)/0.5 ml from 48 to 72 hr PI. The fluid phase contained 1...
Tessler J.The fluorescent antibody reaction was studied in tissues of ponies infected with African horsesickness virus (AHSV). Lung, spleen, lymph node, liver, skeletal muscle, intestine, stomach, nerve ganglion and kidney were sectioned and stained by the direct fluorescent antibody technique (FA). Fluorescence was demonstrated only in the spleen and could be inhibited by using unconjugated antiserum.
Green JH, Bolin RC, Carver RK, Gross H, Pigott N, Harrell WK.The serological response in burros and horses to the viable LVS strain of Pasteurella tularensis was studied. High-titered agglutinating antisera and fluorescent-antibody conjugates were obtained in both groups of animals. Maximum titers were obtained in horses 14 to 21 days after the start of vaccination and in burros 21 to 28 days after the start of vaccination. The use of Woodhour's adjuvants or booster inoculations did not result in increased titers.
Bochsler PN, Slauson DO, Neilsen NR.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...
Erickson GA, Maré CJ.Goat Venezuelan equine encephalomyelitis (VEE) antiserum and normal serum were conjugated and evaluated for staining sensitivity and specificity. Cross-staining with either eastern or western equine encephalomyelitis virus-infected cells did not occur. The baby hamster kidney (BHK-21) cell line when combined with highly specific VEE conjugate detected 100 medium suckling mouse intracerebral lethal doses (suckling mouse LD-50/IC) of the 1B subtype of VEE virus per milliliter of equine tissue suspension. Conjugated goat antiserum was assayed for sensitivity for detection of VEE virus-infected eq...
Broström H, Hellström U, Hammarström S, Obel N, Perlmann P.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...
Gaĭdamovich SIa, Pomelova VG, Lavrova NA, Mel'nikova EE, Sokolova MV, Kharitonenkov IG, Zlobin VN.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....
Broström H, Hellström U, Ziverts I, Obel N, Perlmann P.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...
Lopez BS, Hurley DJ, Giancola S, Giguère S, Felippe MJB, Hart KA.The impact of culture conditions on equine monocyte-derived dendritic cells (MoDC) generation has not been fully characterized. We hypothesized that 1) MoDC could be cultured in a commercially available serum-free medium (AIM-V); and 2) that differential culture conditions would influence MoDC viability, yield and phenotype. MoDC generated from adult horses were cultured under variable conditions in a series of experiments. Viability was assessed using trypan blue and propidium iodide staining. Yield was determined by manual hemocytometer counting. Phenotype was assessed by flow cytometric ana...
Rabinovsky ED, Yang TJ.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...
Kawanishi N, Kinoshita Y, Kambayashi Y, Bannai H, Tsujimura K, Yamanaka T, Cullinane A, Nemoto M.Equine influenza virus (EIV) infection is one of the most important respiratory diseases in the equine industry around the world. Rapid diagnosis, facilitated by point-of-care testing, is essential to implement movement restrictions and control disease outbreaks. This study evaluated a microfluidic immunofluorescence assay kit, which detects influenza virus and SARS-CoV-2 antigens in human specimens with a 12 min turnaround time, for its potential use in detecting EIV. The microfluidic immunofluorescence assay kit succeeded in detecting 11 EIV strains. Using the real-time reverse transcription...
Yi M, Asgenbaatar N, Wang X, Ulaangerel T, Shen Y, Wen X, Du M, Dong X, Dugarjav M, Bou G.DNA methyltransferases (DNMTs) are important epigenetic modification during spermatogenesis. To further evaluate the pattern of DNMTs in horse testes during development, we investigated the expression and localization of DNMT1, DNMT3a and DNMT3b at different time points. The qRT-PCR results showed that DNMT1 expression was maintained in testes tissue from 6-month-old (0.5y) to 2-year-old (2y) of age and decreased after 3-year-old (3y) (P < 0.01). The expression levels of DNMT3a and DNMT3b peaked in testes tissue at 3y (P < 0.01). At 4-year-old (4y), the expression of DNMT3a and DNMT3b was ...
Guenther MC, Borowicz PP, Hirchert MR, Semanchik PL, Simons JL, Fridley J, White-Springer SH, Hammer CJ.The neonatal Fc receptor (FcRn) is the receptor responsible for bidirectional transport of immunoglobulin G (IgG) across cells, maintenance of IgG levels in serum, and assisting with antigen presentation. Unfortunately, little is known about FcRn in horses. Therefore, the objective of this study was to provide fundamental information regarding the location of FcRn in equine tissues. Tissues were collected from six horses of mixed breed, age, and sex immediately following euthanasia. Sampling locations included the respiratory tract, gastrointestinal tract (GIT), other visceral organs, cornea, ...
Baatar T, Song D, Weng Y, Wang G, Jin L, Guo R, Li B, Dugarjaviin M.The epigenetic regulation of gene expression through the covalent modification of histones is crucial for developing germline cells. To study the regulatory role of alternative splicing (AS) of euchromatic histone lysine methyltransferase 2 (EHMT2/G9A) in spermatogenesis in Mongolian horses, this study first examines the localization of the EHMT2 gene in testicular support cells and then predicts the higher-order structures of sequences with and without AS. Two types of lentiviral vectors for overexpression were subsequently constructed for the EHMT2 gene, one with AS and one without, to infec...
Granella MCS, Mendes RP, da Silva Casa M, Ribeiro GSN, Sangioni LA, Vogel FSF, Braünig P, Ferian PE, Salbego FZ, Schwarz DGG, Fonteque JH.Protozoan Neospora spp. is known to negatively affect horses reared in rural and urban environments, being studied for causing abortion, neonatal mortality and central nervous system disorders in horses. This study aimed to verify the occurrence of anti-Neospora spp. antibodies in horses bred in rural and urban areas from southern Brazil and the risk factors associated with infection. The study consisted of 400 horses, which 241 were bred in rural areas and 159 in urban areas. The detection of serum anti-Neospora spp. antibodies was determined using Immunofluorescence Antibody Test (IFAT). The...
Heilen LB, Roßgardt J, Dern-Wieloch J, Vogelsberg J, Staszyk C.Horses' hypsodont (high-crowned) teeth face permanent dental wear. This is compensated for by a continuous eruption, which requires a high adaptability of odontoblasts; otherwise, the dental pulp would be exposed. Here, we report on how equine odontoblasts respond to the challenge of maintaining a high production rate of dentin. We analyzed CD90, a marker of odontoblastic differentiation, and nestin, a marker of mature odontoblasts, in equine pulpal tissue via immunofluorescence. For comparison, we examined the hypselodont (ever-growing) incisors and brachydont (short-crowned) molars of rats. ...
Durham AE.This study aimed to further define and quantify possible cross-reactive peptides when measuring plasma adrenocorticotropic hormone (ACTH) concentration in equids. Equine plasma samples were spiked with known concentrations of exogenous manufactured peptides comprising human ACTH, ACTH (corticotropin-like intermediate lobe peptide, CLIP) and ACTH (corticotropin inhibiting peptide, CIP). All samples were assayed in duplicate using Siemens Immulite 2000xpi chemiluminescent assay (CLA) and Tosoh AIA-900 immunoflurorescent assay (IFA). As expected, ACTH was measured by both assays although higher v...
Valderrama-Martinez C, Packham A, Smith W, Mendoza-Flores JE, Zheng S, Chigerwe M, Plancarte M, Aleman M.Long-term freezing storage can alter the stability of proteins, thereby compromising accurate determination of indirect fluorescent antibody test (IFAT) titers that support the diagnosis of equine protozoal myeloencephalitis. Objective: Assess the effect of long-term storage at -80°C on IFAT against S. neurona and N. hughesi in equine serum and cerebrospinal fluid (CSF). Methods: Paired serum and CSF (n = 46), and serum only (n = 25) samples. Methods: Prospective study of samples stored 6-12, 13-18, and 19-24 months. Comparing antibody titers across time points, McNemar and Wilcoxon...
Lugo T, Myers S, Nguyen TA.In vitro models have revolutionized our understanding of biological pathways and mechanisms, offering a viable alternative to direct patient testing. However, there is a significant lack of models for different animals, particularly equine models. This study presents a novel primary cell culture extracted from a 3-year-old horse diagnosed with multisystemic eosinophilic epitheliotropic disease. Tissue samples were collected from lymph nodes at various locations. Growth curves of extracted primary cells were analyzed and the optimal conditions were assessed. Biomarkers, such as CD31, ZO-1, CD79...
Hobbs KJ, Ludwig EK, Martin EM, Bayless R, Bauck AG, Freeman DE, Gonzalez LM.To determine if blood and peritoneal fluid levels of syndecan-1, heparan sulfate, and hyaluronan are elevated in horses with small intestinal disease compared to healthy counterparts. Unassigned: 61 horses were categorized into control (n = 20), inflammatory (11), and strangulating (30) groups. Paired peritoneal fluid and blood samples were obtained from each group from December 2016 through December 2024 and analyzed via equine-validated ELISAs for syndecan-1, heparan sulfate, and hyaluronan concentrations. Patient survival to discharge was recorded for all groups. Immunofluorescence staining...
Partusch L, Rutland CS, Martens A, Du Cheyne C, De Spiegelaere W, Michler JK.Exuberant granulation tissue (EGT) is a second intention wound healing disorder. It commonly occurs in the distal limb of horses. EGT causes significant increase in the duration and cost of treatment, potentially leading to the decision not to pursue treatment and euthanize the patient. The underlying pathomechanisms of this fibroproliferative disorder remain unclear, particularly in terms of collagen composition and the association between myofibroblasts and blood vessels. This study investigated the collagen composition in naturally occurring EGT following trimming in 19 horses (EGT group). ...
de Oliveira UV, Waap H, Gomes J, da Silva AN, Lacerda LC, da Silva Oliveira Costa T, de Sousa Lima T, Schares G, Pinheiro AM, Gondim LFP, Munhoz AD.The study aimed to assess the prevalence of horses reactive to Sarcocystis neurona, Neospora caninum, and Besnoitia besnoiti in horses from the state of Rio Grande do Sul, Brazil. Sera reactivity was tested by the Immunofluorescent Antibody Test (IFAT) in 354 equids sampled at slaughter, using a cut-off of 1:80 for S. neurona and 1:50 for N. caninum and B. besnoiti. Detection of N. caninum was attempted by PCR and bioassay in gerbils (Meriones unguiculatus) using brain samples collected from seropositive horses. Sera from 158 horses (44.63 %) (158/354; CI: 39.54-49.84 %) reacted to S. neuron...
Meeremans M, Devriendt B, Bairiot S, Van Poucke M, Peelman L, Demeyere K, Meyer E, Van Vlierberghe S, De Schauwer C.Macrophages play key roles in tissue homeostasis and regeneration-associated inflammation. Unlike humans, a reliable protocol to obtain and polarise equine monocyte-derived macrophages is lacking. In this study the polarisation of equine macrophages, derived from CD172a peripheral blood monocytes is described. After differentiation, IFN-γ/LPS or IL-4 were used to induce pro- and anti-inflammatory phenotypes, respectively. Evaluation criteria included morphology, mRNA (RT-qPCR) and protein expression (flow cytometry, immunofluorescence), nitric oxide and arginase production, cytokine secretion...
Schwarz S, Kummer S, Klang A, Walter I, Nell B, Brandt S.Squamous cell carcinoma (SCC) is the most common malignant tumor disease in horses. It predominantly affects the ocular, oronasal, and anogenital region. Equine SCC is difficult to treat, also because important aspects of SCC development and metastasis are still unclear. We previously provided evidence that equine SCC cells can adopt a stem cell-like phenotype as a hallmark of malignant progression. Here, we investigated whether equine SCCs harbor endothelial-like tumor cells that form an alternative network of pseudo-vessels better known as vasculogenic mimicry (VM). Following histopathologic...
Pettersson J, Levanov L, Tervo S, Hautala K, Aaltonen K, Utriainen M, Kareinen L, Gadd T, Sironen T, Vapalahti O, Kinnunen PM.Parapoxviruses (PPV) cause skin and mucous membrane signs to several animal species and humans worldwide. Equine parapoxvirus (EqPPV) was first detected in a sick horse in Finland in 2013. It is potentially zoonotic, and a similar virus has been detected in humans in the USA. In winter 2021–2022, EqPPV caused a large-scale pastern dermatitis epidemic in racehorses all over Finland. Field reports suggest that similar epidemics of unverified cause have also occurred in 2015 and 2019. The aim of this study was to develop a serological test and study the immune response, seroprevalence, and hist...
