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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.
Nonsteroidal anti-inflammatory drug administration alters crypt distribution of the peroxisomal marker acyl-coenzyme A oxidase 1 in the equine right dorsal colon. To characterize and compare the distribution of peroxisomal markers in the right dorsal colon (RDC) and left ventral colon (LVC) of healthy horses and horses treated with NSAIDs. Unassigned: Archived colonic tissues were evaluated from 10 horses (n = 5/group) administered phenylbutazone (4.4 mg/kg, PO, twice daily for 10 days) or placebo prior to euthanasia. Samples from the RDC and LVC were analyzed using immunofluorescence to assess peroxisomal biogenesis factor 14 (PEX14) and acyl-coenzyme A oxidase 1 (ACOX1) distribution. Marker abundance was quantified within colonic crypts and compared b...
Recombinant EHV-1 Vector Expressing Immunodominant Hemagglutinin Protein of Equine Influenza Virus H3N8 (Sub-Lineage Florida Clade 2). Equine herpesvirus type 1 (EHV-1) and equine influenza virus (EIV) are major respiratory pathogens in horses, causing significant economic losses in domesticated horses. Bacterial Artificial Chromosome (BAC) technology can be used to precisely manipulate the EHV-1 genome for the development of live-attenuated vector vaccines. Earlier, our group developed a live-attenuated EHV-1 vaccine by deleting virulence-associated genes using this technology and the mutant EHV-1 has been exploited for expressing foreign gene in the current study. Specifically, in this study, a mutant EHV-1 virus expressing...
Immunohistological characterization of equine synovial tissue in metacarpophalangeal joints of different ages and osteoarthritis status. Synovial macrophages' role in equine osteoarthritis (OA) is not well understood, despite horses being a model for human degenerative joint disorders. Equine synovial macrophages' characterization is limited and age-related changes are not described. This study aimed to evaluate surface marker distribution and expression from potential synovial tissue macrophages in foals, adult non-affected, and OA-affected horses. Unassigned: Surface markers (CD14, CD16, CD206) were quantitatively evaluated on dorsal villous synovial biopsies from the metacarpophalangeal joint of foals (F), non-affected (cont...
Comparison of in vitro degradation of immunoreactive adrenocorticotropic hormone and insulin in equine blood measured by two immunoassay methods. This study compared the in vitro stability of equine plasma adrenocorticotrophic hormone (ACTH) and serum insulin measured by chemiluminescent assay (CLA; Siemens Immulite 2000XPi) and immunofluorescent assay (IFA; Tosoh AIA-900) after storage at 4°C or room temperature (approximately 21°C) for clinically relevant periods. Fresh equine EDTA blood for ACTH and coagulated blood for insulin were aliquoted and analysed immediately (T0) and after storage. ACTH was assessed at 24, 48 and 72 hours; insulin was assessed at 24 hours, 72 hours and 7 days. Percentage recovery relative to T0 was analyse...
In vitro cultivation of Trypanosoma equiperdum: Comparison of in vitro and in vivo antigens for an ethical and sustainable serological diagnosis of Dourine. The protozoan Trypanosoma equiperdum is the causative agent of Dourine, a contagious sexually transmitted disease of equines that can manifest itself in chronic or acute form. Dourine has serious veterinary and economic implications, as there are no vaccines available for prevention and the management of the disease requires the culling of infected animals, together with rigorous sanitary control measures. This study compares the traditional production of T. equiperdum antigen grown in vivo, with an in vitro cultivation methodology developed to eliminate the use of animals. The in vitro and in...
Downregulation of a choline transporter gene in equine colitis indicates a role for choline supplementation to support the large intestinal epithelial barrier. Acute colitis is an inflammatory condition of the equine colon associated with high morbidity and mortality. In these horses, epithelial barrier function is critical but the mechanisms underlying large intestinal epithelial damage and recovery are incompletely understood. Objective: Our objectives were to (1) identify intestinal colitis-associated transcriptional pathways, and (2) determine the effect of in vitro choline supplementation on the epithelial barrier. Methods: We performed RNA-sequencing on rectal biopsies from healthy and colitis horses (n=3/group). Downregulated SLC5A7, a gene en...
Seroprevalence of Sarcocystis neurona and Neospora hughesi in healthy horses from the province of Alberta, Canada. Literature on the seroprevalence of antibodies to the causative agents of equine protozoal myeloencephalitis (EPM) in Canada is limited. Objective: To determine the prevalence of serum antibodies against Sarcocystis neurona and Neospora hughesi in apparently healthy horses without signs of neurologic disease which have always resided in Alberta, Canada, using commercially available immunodiagnostic tests. Methods: Samples from 116 healthy horses were obtained from a biobank containing horse serum collected across Alberta in 2021 and 2022. Samples were selected based on owner survey responses, ...
Antibody responses to equine parapoxvirus reveal a re-emerging pattern. 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...
Odontoblasts in Equine Hypsodont Teeth-How They Cope with Permanent Occlusal Wear. 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. ...
Detection of vasculogenic mimicry in equine ocular, oronasal, and genital squamous cell carcinoma. 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...
Equine monocyte-derived macrophages revisited: isolation and comprehensive characterization of pro- versus anti-inflammatory polarisation. 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...
Construction and Immunogenicity Evaluation of a Recombinant Fowlpox Virus Expressing VP2 Gene of African Horse Sickness Virus Serotype 1. African horse sickness (AHS) is a lethal vector-borne disease caused by African horse sickness virus (AHSV) and represents a major threat to equine health and the horse industry. In 2020, outbreaks of AHS caused by AHSV serotype 1 (AHSV-1) were reported in Thailand, increasing the risk of AHS introduction into China. Given the safety issues associated with currently available live attenuated AHS vaccines, the development of safer and more effective vaccination strategies is urgently needed. In this study, we constructed a recombinant fowlpox virus (rFPV) expressing the AHSV-1 VP2 protein as a ...
Seroprevalence of Sarcocystis spp., Neospora spp. and Besnoitia spp. in horses (Equus caballus) intended for slaughter in the state of Rio Grande do Sul, Brazil. 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...
Collagen composition in equine exuberant granulation tissue reflects tissue immaturity. 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). ...
Endothelial glycocalyx damage is associated with small intestinal disease in horses. 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...
Isolation and Characterization of Equine Lymph Node Endothelial Cells. 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...
Effect of Long-Term Freezing on Indirect Fluorescent Antibody Titers for the Diagnosis of Equine Protozoal Myeloencephalitis. 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...
Cannabinoid Receptors in the Horse Lateral Nucleus of the Amygdala: A Potential Target for Ameliorating Pain Perception, Stress and Anxiety in Horses. The amygdala is composed of several nuclei, including the lateral nucleus which is the main receiving area for the input from cortical and subcortical brain regions. It mediates fear, anxiety, stress, and pain across species. Evidence suggests that the endocannabinoid system may be a promising target for modulating these processes. Cannabinoid and cannabinoid-related receptors have been identified in the amygdala of rodents, carnivores, and humans, but not in horses. This study aimed to investigate the gene expression of cannabinoid receptors 1 (CB1R) and 2 (CB2R), transient receptor potential...
Investigation of peptide cross reactivity in equine plasma using two adrenocorticotropic hormone immunoassays. 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...
Development of a two-layer 3D equine endometrial tissue model using genipin-crosslinked collagen scaffolds and 3D printing. Advances in endometrial tissue engineering have enabled the combination of modified scaffolding materials with modern cell culture technologies. Genipin and three-dimensional (3D) printing have advanced cell-tissue engineering by enabling the precise layering of cell-containing matrices while ensuring low cytotoxicity. This study aimed to advance equine endometrial tissue engineering by designing customized collagen scaffolds using 3D printing technology, while optimizing the genipin concentration to avoid toxicity. Genipin was tested at concentrations of 4, 2, 1, 0.5, 0.25, 0.125, and 0 mM on...
Seroprevalence of Neospora spp. in horses reared in rural and urban areas in southern Brazil. 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...
Effect of Alternative Splicing Euchromatic Histone Lysine Methyltransferase 2 (EHMT2/G9A) on Spermatogenesis in Mongolian Horses. 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...
Effect of refrigeration, room temperature, and processing time on serum immunofluorescent antibody titers for Sarcocystis neurona. Evaluating antibody titers for Sarcocystis neurona for the diagnosis of equine protozoal myeloencephalitis from serum samples is a common practice. However, ensuring timely and proper refrigeration is not always possible. Objective: To evaluate immunofluorescent antibody (IFA) titers for S. neurona from serum samples stored at room temperature and 4°C. Methods: Twenty-two serum samples. Methods: Prospective longitudinal study. Two serum aliquots of 1 mL each were stored at room temperature (20-23.3°C) and 4°C. The unrefrigerated aliquot was immediately tested for IFA titers. Both aliquots...
Occurrence and risk factors of equine piroplasmosis in Portugal: A five-year retrospective study. Equine piroplasmosis (EP) is a tick-borne disease of equids caused by Theileria equi, Theileria haneyi, and Babesia caballi. EP is endemic in most tropical and subtropical regions worldwide, and there is a likelihood that it is also endemic in Portugal. This retrospective study aimed to determine the seroprevalence, prevalence, and potential risk factors of EP in our country over the past five years. A total of 3063 diagnostic test records were analysed. Results from the competitive enzyme-linked immunosorbent assay (cELISA) revealed a seroprevalence of 32.7 % and 15.7 % for T. equi and B....
Identification and Characterization of Linear Epitopes of Monoclonal Antibodies Against African Horse Sickness Virus Serotype 1 VP2 Protein. African horse sickness (AHS) is an acute, fatal, contagious disease of animals of the family Equidae and is caused by infection with the African horse sickness virus (AHSV). Based on the outer capsid protein VP2, AHSV is classified into nine serotypes (AHSV-1 to -9) with little or no serological cross-reactivity between them. In 2020, AHS outbreaks caused by AHSV-1 were reported in Thailand and Malaysia, marking the first occurrences of AHS in Southeast Asia. However, little is known about the antigenic profile of AHSV-1 VP2. In this study, a recombinant VP2 protein was expressed in and used ...
Investigations into the role of platelet-activating factor in the peri-conception period of the mare. In many mammals, the lipid platelet-activating factor (PAF) has important functions in female reproduction and fertility. This study shows that PAF is present in the reproductive tissues of mares and is involved in processes related to ovulation and early pregnancy. Platelet-activating factor (PAF) has been implicated in a number of reproductive processes ranging from ovulation to embryo motility but has not been widely explored in the mare. To identify the presence and examine the role of PAF in the equine periconception processes, targeted mass spectrometry coupled with chromatographic separ...
Identifying the immunoglobulin G transporter in equine tissues: A look at the neonatal Fc receptor. 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, ...
A fresh look at the SarcoFluor antibody test for the detection of specific antibodies to Sarcocystis neurona for the diagnosis of equine protozoal myeloencephalitis. Equine protozoal myeloencephalitis (EPM) is a challenging disease to diagnose in horses with neurological signs. To optimize contemporary diagnostic testing, including the use of serum:CSF antibody ratios, the SarcoFluor antibody test for Sarcocystis neurona requires revalidation. The SarcoFluor, a previously validated immunofluorescent antibody test (IFAT) for the detection of antibodies specific to S. neurona in serum and cerebrospinal fluid (CSF) of naturally infected horses was analyzed using recent data and considering a serum:CSF antibody ratio threshold. Utilization of serum and CSF pho...
Development and application of a colloidal-gold immunochromatographic strip for detecting Getah virus antibodies. Getah virus (GETV) is a re-emerging mosquito-borne alphavirus that is highly pathogenic, mainly to pigs and horses. There are no vaccines or treatments available for GETV in swine in China. Therefore, the development of a simple, rapid, specific, and sensitive serological assay for GETV antibodies is essential for the prevention and control of GETV. Current antibody monitoring methods are time-consuming, expensive, and dependent on specialized instrumentation, and these features are not conducive to rapid detection in clinical samples. To address these problem, we developed immunochromatograph...
Deregulation of Metalloproteinase Expression in Gray Horse Melanoma Ex Vivo and In Vitro. The ability of human melanoma cells to switch from an epithelial to a mesenchymal phenotype contributes to the metastatic potential of disease. Metalloproteinases (MPs) are crucially involved in this process by promoting the detachment of tumor cells from the primary lesion and their migration to the vasculature. In gray horse melanoma, epithelial-mesenchymal transition (EMT) is poorly understood, prompting us to address MP expression in lesions versus intact skin by transcriptome analyses and the immunofluorescence staining (IF) of gray horse tumor tissue and primary melanoma cells. RNAseq re...
