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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.
Seroprevalence of Neospora spp. in asymptomatic horses in Italy. One hundred and fifty horses from Italy were tested for serum antibodies to Neospora caninum by indirect fluorescence antibody test (IFAT). Antibodies were present in 42 horses (28%). The positive titres were: 1:50 (16/150-10.5%), 1:100 (26/150-17%), 1:200 (8/150-5.3%) and 1:800 (1/150-0.6%). No neurological anomalies were observed in the seropositive horses. This is the first serologic survey for Neospora spp. antibodies performed on horses born and raised in Italy.
Prevalence of antibodies to spotted fever group rickettsiae in humans and domestic animals in a Brazilian spotted fever-endemic area in the state of São Paulo, Brazil: serologic evidence for infection by Rickettsia rickettsii and another spotted fever group Rickettsia. In serum samples obtained from all the healthy humans, horses, dogs, and donkeys present on three farms in the Pedreira Municipality, an endemic area for Brazilian spotted fever, an indirect immunofluorescence assay (IFA) detected antibodies against Rickettsia rickettsii in 17 (77.3%) horses, 5 (31.3%) dogs (titers ranging from 64 to 4,048), and none of 4 donkeys or 50 humans. Five canine and eight equine sera with high antibody titers to R. rickettsii were also tested by IFA against R. bellii, R. akari, and R. africae antigens. Sera from two horses and two dogs that showed similar high antibo...
Evaluation and comparison of an indirect fluorescent antibody test for detection of antibodies to Sarcocystis neurona, using serum and cerebrospinal fluid of naturally and experimentally infected, and vaccinated horses. The objectives of this study were to evaluate the accuracy of the indirect fluorescent antibody test (IFAT) using serum and cerebrospinal fluid (CSF) of horses naturally and experimentally infected with Sarcocystis neurona, to assess the correlation between serum and CSF titers, and to determine the effect of S. neurona vaccination on the diagnosis of infection. Using receiver-operating characteristic analysis, the areas under the curve for the IFAT were 0.97 (serum) and 0.99 (CSF). Sensitivity and specificity were 83.3 and 96.9% (serum, cutoff 80) and 100 and 99% (CSF, cutoff 5), respectively...
Equine Cryptosporidium parvum infections in western Poland. A total of 564 fecal specimens from 318 horses used for recreational riding, child hippotherapy, and racing at ten commercial and government-run stables in western Poland were tested for Cryptosporidium parvum oocysts by microscopic examination of Ziehl-Neelsen stained smears, enzyme immunoassay, and combined direct immunofluorescent antibody and fluorescent in situ hybridization. Also, seven stool specimens from five personnel who had repeated contact with these horses were tested for C. parvum oocysts. Eleven horses that shed C. parvum oocysts were found in five of ten stables (50%). The pre...
Equine laminitis: cleavage of laminin 5 associated with basement membrane dysadhesion. The key lesion of laminitis is separation at the hoof lamellar dermal-epidermal interface. For this to happen the structural and adhesion proteins of the basement membrane zone must be altered. Which proteins and how damage to them leads to the lamellar separation of laminitis is unknown. Objective: To investigate lamellar hemidesmosome and cytoskeleton damage and basement membrane dysadhesion using light microscopy (LM) and immunofluorescence microscopy (IFM). Methods: Cryostat sections of lamellar tissues from 2 control and 6 Standardbred horses with oligofructose induced laminitis were stud...
The first isolation of equine arteritis virus in Argentina. This paper describes the first isolation of equine arteritis virus (EAV) in Argentina. The virus was isolated from the semen of an imported seropositive stallion held in isolation at a breeding farm in Tandil in the Buenos Aires Province. In addition, viral nucleic acid was detected in seminal plasma using the reverse-transcription polymerase chain reaction. The isolated virus was propagated in cell cultures and confirmed as EAV by indirect immunofluorescence and virus neutralisation, using a serum specific for the reference Bucyrus strain of EAV. As far as the authors are aware, this is the f...
Immunohistochemical identification and fiber type specific localization of protein kinase C isoforms in equine skeletal muscle. To investigate whether protein kinase C (PKC) isoforms are expressed in equine skeletal muscle and determine their distribution in various types of fibers by use of immunofluorescence microscopy. Methods: 5 healthy adult Dutch Warmblood horses. Methods: In each horse, 2 biopsy specimens were obtained from the vastus lateralis muscle. Cryosections of equine muscle were stained with PKC isoform (alpha, beta1, beta2, delta, epsilon, or zeta)-specific polyclonal antibodies and examined by use of a fluorescence microscope. Homogenized muscle samples were evaluated via western blot analysis. Results...
Peripheral blood neutrophil function and lymphocyte subpopulations in cycling mares. The purpose of this study was to evaluate different parameters of the immune status in the mare, during the follicular and the luteal phases of the oestrous cycle, in two consecutive years. Functional competence of peripheral blood neutrophils, such as chemotaxis, phagocytosis and oxidative burst was assessed under physiological cyclic conditions (Exp. I). In the second year of this study (Exp. II), besides peripheral blood neutrophil phagocytosis and oxidative burst analysis, circulating lymphocyte subsets were also characterized. The reproductive status in a total of 17 adult cycling mares w...
Long-term study of Cryptosporidium prevalence on a lowland farm in the United Kingdom. A longitudinal sample survey testing for Cryptosporidium in livestock and small wild mammals conducted over 6 years (1992-1997) on a lowland farm in Warwickshire, England, has shown the parasite to be endemic and persistently present in all mammalian categories. Faecal samples were taken throughout the year and oocysts concentrated by a formal ether sedimentation method for detection by immunofluorescence staining using a commercially available genus specific monoclonal antibody. Cryptosporidium parvum was identified by morphology and measurement of modified Ziehl-Neelsen stained oocysts. C. m...
Development of competitive ELISA for serodiagnosis on African horsesickness virus using baculovirus expressed VP7 and monoclonal antibody. VP7, the sero-group common antigen, of African horsesickness virus (AHSV-4) was expressed in insect cells by recombinant baculovirus. To develop a specific diagnostic method, monoclonal antibody (Mab) against VP7 was prepared and investigated as diagnostic reagent with the baculovirus expressed VP7. However, the Mab against VP7 of AHSV cross-reacted with Chuzan virus by the indirect immunofluorescence assay (IFA), confirming the presence of conserved domain of VP7 among Orbiviruses. This study describes two types of ELISA; Mab linked indirect (I-ELISA) and competitive-ELISA (C-ELISA) using bac...
[The occurrence of animal sarcocystiosis in Algeria]. Investigations to occurrence of Sarcocystiosis of domestic animals in Algeria were carried out by different methods--Immunofluorescence (IFAT), macroscopic and microscopic examinations of muscle, coproscopia. Positive results could be found in intermediate hosts--362 of 537 cattle, 188 of 292 sheep, 13 of 57 horses and in the final hosts dogs and cats. Muscle-probes from cattle contained S. cruzi (58.7 respectively 61.0%), S. hirsuta (48.9 respectively 35.0%), from sheep S. tenella (61.0 respectively 59.6%, S. arieticanis (38.8 respectively 40.3%), from horses S. bertrami (24.5%). IFAT-data of...
Monoclonal anti-equine IgE antibodies with specificity for different epitopes on the immunoglobulin heavy chain of native IgE. In this study we describe the generation of monoclonal antibodies (mAbs), which recognize different epitopes of the equine IgE constant heavy chain. Equi-murine recombinant IgE (rIgE), composed of the murine V(H)186.2 heavy chain variable region, linked to the equine IgE constant heavy chain and expressed together with the murine lambda(1) chain in J558L cells was used to immunize BALB/C mice. A total of 17 different mAbs were obtained, which recognized the rIgE heavy chain constant region. None of the mAbs reacted with monoclonal equine isotypes IgM, IgG1 (IgGa), IgG3 (IgG(T)), IgG4 (IgGb) or...
Immunolocalization of aromatase in stallion Leydig cells and seminiferous tubules. High levels of plasma estrogens constitute an endocrine peculiarity of the adult stallion. This is mostly due to testicular cytochrome p450 aromatase, the only irreversible enzyme responsible for the bioconversion of androgens into estrogens. To identify more precisely the testicular aromatase synthesis sites in the stallion, testes from nine horses (2-5 years) were obtained during winter or spring. Paraplast-embedded sections were processed using rabbit anti-equine aromatase, followed by biotinylated goat anti-rabbit antibodies, and amplified with a streptavidin-peroxidase complex. Immunoreac...
Comparison of a serum indirect fluorescent antibody test with two Western blot tests for the diagnosis of equine protozoal myeloencephalitis. A serum indirect fluorescent antibody test (IFAT) was compared with a Western blot (WB) and a modified Western blot (mWB) for diagnosis of equine protozoal myeloencephalitis (EPM). Using receiver-operating characteristic (ROC) analysis, the area under the curve of the IFAT was greater than the areaunder the curves of the WB and the mWB (P = 0.025 and P = 0.044, respectively). There was no statistically significant difference between the areas under the curves of the WBs (P > 0.05). On the basis of an arbitrarily chosen cut-off titer for a positive test result of 1:80 for the IFAT and interp...
Qualitative evaluation of selective tests for detection of Neospora hughesi antibodies in serum and cerebrospinal fluid of experimentally infected horses. Neospora hughesi is a newly recognized protozoan pathogen in horses that causes a myeloencephalitis similar to Sarcocystis neurona. There are no validated serologic tests using the gold standard sera that are currently available to detect specific N. hughesi antibodies and, thus, no tests available to detect antemortem exposure or estimate seroprevalence in the horse. The objectives of the present study were to establish a bank of gold standard equine sera through experimental infections with N. hughesi and to assess several serologic tests for the detection of related protozoan antibodies. Se...
Immunohistochemical localization of chromogranin a in the acinar cells of equine salivary glands contrasts with rodent glands. We investigated the existence of chromogranin A (CgA) in salivary glands of the horse by Western blotting and enzyme immunoassay (EIA) using an antiserum against a peptide sequence of equine CgA. We also compared its cellular distribution between the horse and rat salivary glands with a tyramide signal amplification immunofluorescence technique. Western blotting gave three significant immunoreactive bands (74, 56 and 48 kDa) in adrenal medulla and three major salivary glands of horses. Immunoreactivities for CgA measured by EIA in horses were 154.05 +/- 41.46, 20.32 +/- 5.59 and 4.43 +/- 2.23 ...
Animal models for skin blistering conditions: absence of laminin 5 causes hereditary junctional mechanobullous disease in the Belgian horse. Recent achievements in the genetic correction of keratinocytes isolated from patients with junctional epidermolysis bullosa have paved the way to a gene therapy approach for the disease. Because gene therapy protocols require preclinical validation in animals, we have characterized spontaneous animal models of junctional epidermolysis bullosa. In this study we have elucidated the genetic basis of the hereditary junctional mechanobullous disease in the Belgian horse, a condition characterized by blistering of the skin and mouth epithelia, and exungulation (loss of the hoof). Immunofluorescence ...
The use of chosen serological diagnostic methods in Lyme disease in horses. Part I. Indirect immunofluorescence and enzyme-linked immunosorbent assay (ELISA). The investigations aimed to establish the reliability of the chosen serological tests designed for the diagnosis of Lyme borreliosis in horses. The investigations were carried out in five Horse Breeding Centres (OHK). Statistical analysis methods were used to determine sample size for particular centres: Krasne (Kr)--49, Łack (Ł)--21, Walewice (W)--111, BogusŁawice (B)--17, Kozienice (K)--61. The experimental material comprised the chosen horses from which blood samples were collected in order to obtain sera. The test used for indirect immunofluorescence assay (IFA No 75941, Bio-Mérieux) i...
Equid herpesvirus 1 is neurotropic in mice, but latency from which infectious virus can be reactivated does not occur. Equid herpesvirus 1 (EHV-1) is the most common cause of virus-induced abortion in horses. After primary infection the virus becomes latent predominantly in the respiratory tract lymph nodes and the genome can also be detected in the peripheral nervous system. The role of mouse as a feasible model for the establishment of latency and reactivation of EHV-1 was investigated. Intracerebral and intranasal infections of 3- and 17-day-old mice were made and virus replication was confirmed by virus isolation and detected by indirect immunofluorescence (IIF) in brain. For reactivation studies, the mice...
Seroprevalence of antibodies against Neospora caninum in diagnostic equine serum samples and their possible association with fetal loss. A case-control study of the association between the presence of serum antibodies against Neospora spp. and fetal loss was performed on serum samples submitted to a veterinary diagnostic laboratory in northwestern United States. Control sera were randomly selected from those submitted from healthy horses for routine equine infectious anemia testing required for regulatory health certification. Case sera were randomly selected from those submitted from aborting mares for diagnostic workup. Based on a 1:50 or greater titer on the indirect fluorescent antibody test, 8% of the 160 control sera and ...
Increased susceptibility of peripheral blood mononuclear cells to equine herpes virus type 1 infection upon mitogen stimulation: a role of the cell cycle and of cell-to-cell transmission of the virus. Equine herpesvirus-1 (EHV-1) is an important pathogen of horses, causing abortion and nervous system disorders, even in vaccinated animals. During the cell-associated viremia, EHV-1 is carried by peripheral blood mononuclear cells (PBMC), mainly lymphocytes. In vitro, monocytes are the most important fraction of PBMC in which EHV-1 replicates, however, mitogen stimulation prior to EHV-1 infection increases the percentage of infected lymphocytes. The role of the cell cycle in viral replication and the role of cluster formation in cell-to-cell transmission of the virus were examined in mitogen-s...
Characterization of monoclonal antibodies developed against Sarcocystis neurona. Equine protozoal myeloencephalitis (EPM), caused by a protozoal parasite infection of the central nervous system, is the most commonly diagnosed neurologic disease of horses in North America. In specific regions of the United States approximately 50% of the horse population is seropositive to Sarcocystis neurona. However, not all seropositive horses develop clinical signs. Detailed clinical examination, along with cerebrospinal fluid antibody evaluation are often used to diagnose EPM. Postmortem evaluation of the brain stem and spinal cord for histopathologic lesions compatible with nonsuppura...
Specific antibody levels and antigenic recognition of Wistar rats inoculated with distinct isolates of Trypanosoma evansi. "Mal de Cadeiras", an enzootic disease caused by Trypanosoma evansi, is one of the most important trypanosomiases in the Brazilian Pantanal region. The disease affects mainly horses, which are widely used in extensive cattle production, an activity of greatest economical significance for the region. The parasite also infects sylvan (coatis and capybaras) and domestic (dogs) animals, respectively considered wild and domestic reservoirs of T. evansi. For a better understanding of the interaction of T. evansi with its rodent host, we evaluated the differences in the specific antibody level patter...
Posttranslational processing of PH-20 during epididymal sperm maturation in the horse. It is generally accepted that spermatozoa become functionally mature during epididymal transit. The objective of this study was to determine whether the cellular location of equine PH-20 is modified during epididymal transit and, if so, the mechanism for such modification. Sperm were isolated from caput and cauda epididymal regions from stallions undergoing castration (n = 7) and used as whole sperm cell or subjected to nitrogen cavitation for isolation of plasma membrane proteins. Both caput and cauda sperm and sperm protein extracts were subjected to N-deglycosylation, O-deglycosylation, or ...
Construction of chimeric arteriviruses reveals that the ectodomain of the major glycoprotein is not the main determinant of equine arteritis virus tropism in cell culture. The recent development of arterivirus full-length cDNA clones makes possible the construction of chimeric arteriviruses for fundamental and applied studies. Using an equine arteritis virus (EAV) infectious cDNA clone, we have engineered chimeras in which the ectodomains of the two major envelope proteins, the glycoprotein GP(5) and the membrane protein M, were replaced by sequences from envelope proteins of related and unrelated RNA viruses. Using immunofluorescence microscopy, we monitored the transport of the hybrid GP(5) and M proteins to the Golgi complex, which depends on their heterodime...
Detection of equine herpesvirus type 2 (EHV-2) in horses with keratoconjunctivitis. The prevalence of EHV-2 in 27 horses with keratoconjunctivitis and 21 clinically healthy horses of different ages and stocks were analyzed. We demonstrated that EHV-2 was present in 12 keratoconjunctivitis cases as shown by nested PCR on ocular swabs. This is statistically more often than in the control group, where only two ocular swabs were EHV-2 positive. Cocultivation was successful on peripheral blood leukocytes of healthy and diseased horses but not on swabs. We isolated ten EHV-2 strains from diseased and nine from control horses, whereas 16 isolates showed different restriction enzyme ...
Immunohistochemical demonstration of chromogranin A in endocrine organs of the rat and horse by use of region-specific antibodies. Chromogranin A (CgA) is an acidic glycoprotein that is co-stored with hormones or neurotransmitters in granular components of endocrine cells and neurons, and released together with them in response to adequate stimulation. In addition to acting as a packaging protein, CgA functions as a precursor molecule that yields several bioactive peptides by proteolytic cleavage. The purpose of this study is to elucidate how different the processing of CgA is among endocrine tissues by immunostaining using multiple region-specific antisera, and to evaluate the availability of region-specific antisera. Wh...
Candidate vaccine against botulinum neurotoxin serotype A derived from a Venezuelan equine encephalitis virus vector system. A candidate vaccine against botulinum neurotoxin serotype A (BoNT/A) was developed by using a Venezuelan equine encephalitis (VEE) virus replicon vector. This vaccine vector is composed of a self-replicating RNA containing all of the VEE nonstructural genes and cis-acting elements and also a heterologous immunogen gene placed downstream of the subgenomic 26S promoter in place of the viral structural genes. In this study, the nontoxic 50-kDa carboxy-terminal fragment (H(C)) of the BoNT/A heavy chain was cloned into the replicon vector (H(C)-replicon). Cotransfection of BHK cells in vitro with t...
Validation of a competitive enzyme-linked immunosorbent assay for diagnosing Babesia equi infections of Moroccan origin and its use in determining the seroprevalence of B. equi in Morocco. A highly specific and sensitive competitive enzyme-linked immunosorbent assay for detection of specific antibody to Babesia equi in serum from equids was validated for use in Morocco. The assay is based on the specific inhibition of binding of a monoclonal antibody to a conserved epitope within a recombinant parasite peptide by serum from infected animals. The assay was compared to an established indirect immunofluorescence assay, with a concordance of 91%. The assay was used to determine seroprevalence for B. equi infections in donkeys and horses throughout Morocco. A total of 578 sera (163 h...
