Topic:Polymerase Chain Reaction
Polymerase Chain Reaction (PCR) is a molecular biology technique used to amplify specific DNA sequences, allowing for detailed genetic analysis in horses. This method enables the detection and quantification of genetic material, facilitating research in areas such as genetic disorders, infectious diseases, and population genetics in equine species. PCR applications in horses include identifying pathogens, verifying parentage, and studying genetic variations. The technique's sensitivity and specificity make it a valuable tool in equine veterinary diagnostics and research. This page compiles peer-reviewed research studies and scholarly articles that explore the applications, methodologies, and advancements of PCR in equine science.
Evidence of p-glycoprotein sequence diversity in cyathostomins. P-glycoproteins (Pgps) are adenosine triphosphate-binding transporter proteins thought to be associated with multi-drug resistance in mammals and protozoans and have been suggested to be involved in the mechanism of ivermectin (IVM) resistance in Haemonchus contortus. Until now, resistance to IVM has not been reported in cyathostomins in horses in spite of its widespread and frequent use. Reasons for this might be differences in the molecular mechanism of the development of resistance. Based on this hypothesis, the present study was carried out to find homologues of Pgp in cyathostomins. A 416...
Molecular characterization of equine P-selectin (CD62P) and its regulation in ovarian follicles during the ovulatory process. Ovulation is accompanied by a marked infiltration of leukocytes into thecal layers after the gonadotropin surge. P-selectin is known to play a critical role in the initial steps of leukocyte recruitment from the bloodstream during inflammation. Thus, the objective was to investigate the potential regulation of P-selectin by gonadotropins in equine preovulatory follicles. The full-length equine P-selectin cDNA was cloned by a combination of reverse transcription-polymerase chain reaction (RT-PCR) and 5'- and 3'-rapid amplification of cDNA ends. Results showed that equine P-selectin cDNA encodes...
Infection of immunodeficient horses with Sarcocystis neurona does not result in neurologic disease. Equine protozoal myeloencephalitis is a progressive neurologic disease of horses most commonly caused by infection with the apicomplexan parasite Sarcocystis neurona. Factors affecting neuroinvasion and neurovirulence have not been determined. We investigated the pathogenesis of infection with S. neurona in horses with severe combined immune deficiency (SCID). Two immunocompetent (IC) Arabian horses and two Arabian horses with SCID were infected orally with 5 x 10(5) sporocysts of S. neurona. Four IC horses and one SCID horse were infected intravenously (i.v.) with 5 x 10(8) merozoites of the ...
African horsesickness virus serotyping and identification of multiple co-infecting serotypes with a single genome segment 2 RT-PCR amplification and reverse line blot hybridization. Since protection against African horsesickness (AHS) is serotype-specific, rapid serotyping of AHSV is crucial to identify the correct vaccine serotype for efficient control of the spread of AHS outbreaks, especially when they occur in non-endemic regions. This paper describes the first one-day serotyping procedure that requires only a single RT-PCR and hybridization and which can identify multiple serotypes in mixed infections in one assay. The same region of genome segment 2 of all nine AHSV serotypes is amplified in a single RT-PCR. A universal primer set, designed to amplify the 5'-termina...
Enzootiology of Trypanosoma evansi in Pantanal, Brazil. In order to better understand the enzootiology of trypanosomiasis caused by Trypanosoma evansi in the Brazilian Pantanal we examined domestic and wild mammals by microhematocrit centrifuge technique (MHCT), immunofluorescence antibody test (IFAT) and polymerase chain reaction (PCR). T. evansi infection was detected in all species sampled with exception of the sheep and the feral pig. High parasitemias were observed in capybaras (5/24), coatis (18/115), horses (31/321) and dogs (3/112). Among these species, only the capybaras did not develop anemia. Low parasitemias, only detected by PCR, were ...
2000 Year-old ancient equids: an ancient-DNA lesson from pompeii remains. Ancient DNA extracted from 2000 year-old equine bones was examined in order to amplify mitochondrial and nuclear DNA fragments. A specific equine satellite-type sequence representing 3.7%-11% of the entire equine genome, proved to be a suitable target to address the question of the presence of aDNA in ancient bones. The PCR strategy designed to investigate this specific target also allowed us to calculate the molecular weight of amplifiable DNA fragments. Sequencing of a 370 bp DNA fragment of mitochondrial control region allowed the comparison of ancient DNA sequences with those of modern hor...
Leukoencephalitis associated with selective viral replication in the brain of a pony with experimental chronic equine infectious anemia virus infection. Neurologic disease occurs sporadically in horses infected with the equine infectious anemia virus (EIAV). This report describes a case of clinically severe neurologic disease in a pony experimentally infected with EIAV. This pony did not have fever or anemia, which are the characteristic clinical signs of disease. The histopathologic changes were characterized as lymphohistiocytic periventricular leukoencephalitis. Polymerase chain reaction and in situ hybridization data showed that the brain lesions were directly associated with viral replication and that high-level viral replication occurred...
Use of the meridian test for the detection of equine herpesvirus type 1 infection in horses with decreased performance. To evaluate use of the acupuncture meridian test for detection of recent or recently reactivated equine herpesvirus type 1 (EHV-1) infection in horses with decreased performance. Methods: Case-control study. Methods: 40 horses. Methods: Physical and neurologic examinations were performed, and acupuncture points on the bladder meridian were tested for sensitivity reactions in case and control horses. Polymerase chain reaction assays were performed to determine whether EHV-1 or equine herpesvirus type 4 (EHV-4) DNA could be detected in peripheral blood mononuclear cells. Complement fixation (CF)...
[Feline leishmaniasis: what’s the epidemiological role of the cat?]. Feline leishmaniasis (FL) is a quite uncommon feature. Clinical disease has been described in cats since nineties begin. More than 40 reports in world literature have been referred, but the clinical cases have been only recently well defined. Most of the reports focus on infected cats living in endemic areas, even if, more recently FL due to Leishmania infantum was found in Sao Paulo State, in Brazil where autochthonous human or canine leishmaniasis cases have never reported. In Europe clinical cases of FL have been described from Portugal, France, Spain and Italy from 1996 to 2002. When a typ...
Detection of bacterial DNA in synovial fluid from horses with infectious synovitis. Standard culturing techniques are often unrewarding in confirming diagnosis of synovial infection in the equine patient. Several human studies report the use of sensitive polymerase chain reaction (PCR) techniques for the detection of bacterial involvement in acute synovitis. However, successful extraction of bacterial DNA directly from clinical samples from horses without prior culture has not been reported yet. The goal of this study was to develop a sensitive and reliable method for molecular detection and identification of bacterial species in synovial fluid from horses with infectious syn...
Specific identification of Habronema microstoma and Habronema muscae (Spirurida, Habronematidae) by PCR using markers in ribosomal DNA. Gastric or cutaneous habronemosis caused by Habronema microstoma Creplin, 1849 and Habronema muscae Carter, 1865 is a parasitic disease of equids transmitted by muscid flies. There is a paucity of information on the epidemiology of this disease, which is mainly due to limitations with diagnosis in the live animal and with the identification of the parasites in the intermediate hosts. To overcome such limitations, a molecular approach, based on the use of genetic markers in the second internal transcribed spacer (ITS-2) of ribosomal DNA, was established for the two species of Habronema. Charact...
Detection of Anaplasma phagocytophilum in animals by real-time polymerase chain reaction. The aim of this study was to detect Anaplasma phagocytophilum in wild and domesticated animals and to identify the phylogenetic relationships of different strains of this bacterium. We adapted six published conventional methods targeting 16S fragments for real-time polymerase chain reaction. Initial screening of samples from 419 animals found 37 Anaplasma positives, later confirmed with several different primers and a TaqMan probe. We also performed DNA quantification and melting curve analysis. The nucleic acid of Anaplasma sp. was detected in a higher percentage of cases in members of the de...
Molecular detection of Culicoides spp. and Culicoides imicola, the principal vector of bluetongue (BT) and African horse sickness (AHS) in Africa and Europe. Bluetongue (BT) and African Horse Sickness (AHS) are infectious arthropod-borne viral diseases affecting ruminants and horses, respectively. Culicoides imicola Kieffer, 1913, a biting midge, is the principal vector of these livestock diseases in Africa and Europe. Recently bluetongue disease has re-emerged in the Mediterranean Basin and has had a devastating effect on the sheep industry in Italy and on the islands of Sicily, Sardinia, Corsica and the Balearics, but fortunately, has not penetrated onto mainland France and Spain. To survey for the presence of C. imicola, an extensive light-trap ...
Two cases of Neorickettsia (Ehrlichia) risticii infection in horses from Nova Scotia. Two horses from Nova Scotia were diagnosed with Potomac horse fever (PHF). Polymerase chain reaction analysis was performed on formalin-fixed colon tissue or whole blood to show the presence of Neorickettsia risticii DNA, the causative agent of PHF. These are the first reported cases of PHF in the Maritime Provinces. Un diagnostic d’ehrlichiose monocytaire équine (EME) a été posé sur 2 chevaux de Nouvelle-Écosse. Une analyse d’amplification en chaîne par polymérase a été effectuée sur du tissu de côlon fixé au formol ou sur du sang complet afin de démontrer la présence dâ€...
Use of a real-time polymerase chain reaction-based fluorogenic 5′ nuclease assay to evaluate insect vectors of Corynebacterium pseudotuberculosis infections in horses. To develop and use a sensitive molecular assay for detecting the phospholipase D (PLD) exotoxin gene of Corynebacterium pseudotuberculosis in an attempt to identify insect vectors that may be important in transmission of clinical disease in horses. Methods: 2,621 flies of various species. Methods: A real-time polymerase chain reaction (PCR)-based fluorogenic 5' nuclease (TaqMan) system (ie, TaqMan PCR assay) was developed for the detection of the PLD gene in insects. Flies were collected monthly (May to November 2002) from 5 farms in northern California where C. pseudotuberculosis infection in...
Detection of EHV-1 and EHV-4 DNA in unweaned Thoroughbred foals from vaccinated mares on a large stud farm. A silent cycle of equine herpesvirus 1 infection has been described following epidemiological studies in unvaccinated mares and foals. In 1997, an inactivated whole virus EHV-1 and EHV-4 vaccine was released commercially in Australia and used on many stud farms. However, it was not known what effect vaccination might have on the cycle of infection of EHV-1. Objective: To investigate whether EHV-1 and EHV-4 could be detected in young foals from vaccinated mares. Methods: Nasal and blood samples were tested by PCR and ELISA after collection from 237 unvaccinated, unweaned foals and vaccinated an...
Clinical West Nile virus infection in 2 horses in western Canada. Two horses had a history of ataxia and weakness or recumbency. One recovered and was diagnosed with West Nile virus (WNV) infection by serologic testing. The other was euthanized; it had meningoencephalomyelitis, WNV was detected by polymerase chain reaction. West Nile virus infection is an emerging disease. Year 2002 is the first year in which cases have been seen in Saskatchewan. Deux chevaux présentaient une histoire d’ataxie et de faiblesse ou de décubitus. Un cheval s’est rétabli et un diagnostic d’infection au virus du Nil occidental (VNO) a été posé par épreuve sérologiqu...
Identification and differentiation of avirulent and virulent Rhodococcus equi using selective media and colony blotting DNA hybridization to determine their concentrations in the environment. Selective agar media have been used for many years to facilitate the isolation of Rhodococcus equi from environmental and clinical samples. However, characterisation of R. equi still requires the use of immunochemical or polymerase chain reaction (PCR) analysis to differentiate between virulent and avirulent isolates. Here, we describe a novel method to detect and differentiate between R. equi isolates using colony blotting and DNA hybridization. Radiolabelled PCR product derived from the R. equi rrnA gene and specific hybridization conditions enabled differentiation of colonies of R. equi fro...
Occurrence of gastrointestinal parasites in horses in metropolitan Perth, Western Australia. To assess the occurrence of gastrointestinal parasites in horses in Perth. To apply polymerase chain reaction (PCR) for the identification of some species of encysted larval cyathostomes. Methods: Between February and September of 2000, the gastrointestinal tracts of 29 horses submitted to a local knackery and Murdoch University Veterinary hospital in Perth were examined post mortem for the presence of gastrointestinal parasites. Methods: The gastrointestinal tract was divided into six sections, which were screened for the presence of parasites such as Gasterophilus sp, Anoplocephala sp and Pa...
Polymerase chain reaction tests for the identification of Ross River, Kunjin and Murray Valley encephalitis virus infections in horses. To develop and validate specific, sensitive and rapid diagnostic tests using RT-PCR for the detection of Ross River virus (RRV), Kunjin virus (KV) and Murray Valley encephalitis virus (MVEV) infections in horses. Methods: Primer sets based on nucleotide sequence encoding the envelope glycoprotein E2 of RRV and on the nonstructural protein 5 (NS5) of KV and MVEV were designed and used in single round PCRs to test for the respective viruses in infected cell cultures and, in the case of RRV, in samples of horse blood and synovial fluid. Results: The primer pairs designed for each of the three vir...
Rapid determination of vapA/vapB genotype in Rhodococcus equi using a differential polymerase chain reaction method. Rhodococcus equi is a facultative pathogen of foals. Infection causes an often fatal pulmonary pneumonia. The organism has also been isolated from pigs, cattle, humans and the environment. Equine virulence has a high positive correlation with the expression of a 17.4 kD polypeptide of unknown function, VapA, the product of the plasmid-encoded vapA gene. More recently an isogene of vapA, referred to as vapB and encoding an 18.2 kDa polypeptide, has been identified among pig and human isolates. The two genes share > 80% sequence identity, yet their host strains apparently exhibit different patho...
Detection and nucleotide sequencing of a DNA-packaging protein gene of equine gammaherpesviruses. In previous studies, novel putative viral pathogens designated that asinine herpesvirus 4 (AsHV4) and asinine herpesvirus 5 (AsHV5) were associated with fatal interstitial pneumonia in donkeys (Equus asinus). Nucleotide sequence analysis of a portion of the DNA polymerase gene identified these putative pathogens as herpesviruses and possibly as members of the Gammaherpesvirinae subfamily. Although similar to equine herpesvirus 2 (EHV2) and equine herpesvirus 5 (EHV5), sequence diversity was observed among the detected viruses. In this study, novel sequence is reported for a DNA-packaging prote...
Detection of equine herpesvirus 3 in equine skin lesions by polymerase chain reaction. During a recent breeding season, ulcerative, pustular skin lesions were observed on the external genitalia of 2 mares and 1 stallion within a small herd. Based on the location and description of the skin lesions plus the clinical history, equine coital exanthema, caused by equine herpesvirus 3 (EHV3), was the primary differential diagnosis. Scrapings of skin lesions from the perineum of 2 mares were submitted for diagnostic evaluation. Virus isolation was attempted by inoculation of several cell lines of equine origin, but no cytopathic agent was detected. The skin scrapings were processed for...
Diagnosis of West Nile virus infection in horses. The North American West Nile virus (WNV) epizootic, which began in 1999, has caused significant morbidity and mortality in horses. Because experimental infection has failed to consistently produce encephalitis in inoculated horses, investigation of naturally occurring cases was used to optimize strategies for diagnosis of this disease. Although WNV RNA could be detected by reverse transcriptase-polymerase chain reaction (RT-PCR) performed on whole blood collected from both clinically affected horses and unaffected herdmates, the diagnostic sensitivity of this approach was low compared with IgM...
Comparison of sensitivities of virus isolation, antigen detection, and nucleic acid amplification for detection of equine influenza virus. Four seronegative foals aged 6 to 7 months were exposed to an aerosol of influenza strain A/Equi/2/Kildare/89 at 10(6) 50% egg infective doses (EID(50))/ml. Nasopharyngeal swabs were collected for 10 consecutive days after challenge. Virus isolation was performed in embryonated eggs, and the EID(50) was determined for all positive samples. The 50% tissue culture infective dose was determined using Madin-Darby canine kidney (MDCK) cells. Samples were also tested by an in vitro enzyme immunoassay test, Directigen Flu A, and by reverse transcription-PCR (RT-PCR) using nested primers from the nucl...
Expression of bone morphogenetic protein-6 and -2 and a bone morphogenetic protein antagonist in horses with naturally acquired osteochondrosis. To determine the mRNA expression of bone morphogenetic protein (BMP)-6 and -2 and a BMP antagonist (Noggin) in horses with osteochondrosis. Methods: Samples of articular cartilage from affected stifle or shoulder joints of 10 immature horses with naturally acquired osteochondrosis and corresponding joints of 9 clinically normal horses of similar age; additionally, samples of distal femoral growth plate cartilage and distal femoral articular cartilage were obtained from a normal equine fetus. Methods: Cartilage specimens were snap-frozen in liquid nitrogen, and total RNA was isolated. Adjacent ...
Detection of Salmonella organisms and assessment of a protocol for removal of contamination in horse stalls at a veterinary teaching hospital. To assess methods of detecting environmental contamination with Salmonella organisms and evaluate a cleaning and disinfection protocol for horse stalls in a veterinary teaching hospital. Methods: Original study. Methods: 37 horses with diarrhea likely to be caused by Salmonella infection and their stall environments. Methods: Fecal samples were collected from horses daily during hospitalization; samples were obtained from stall sites after cleaning and application of disinfectants. Fecal and environmental samples were cultured for Salmonella spp and tested via polymerase chain reaction (PCR) a...
Generation and characterization of an EICP0 null mutant of equine herpesvirus 1. The EICP0 gene (gene 63) of equine herpesvirus 1 (EHV-1) encodes an early regulatory protein that is a promiscuous trans-activator of all classes of viral genes. Bacterial artificial chromosome (BAC) technology and RecE/T cloning were employed to delete the EICP0 gene from EHV-1 strain KyA. Polymerase chain reaction, Southern blot analysis, and DNA sequencing confirmed the deletion of the EICP0 gene and its replacement with a kanamycin resistance gene in mutant KyA. Transfection of rabbit kidney cells with the EICP0 mutant genome produced infectious virus, indicating that the EICP0 gene is not...
Recent advances in molecular epidemiology and detection of Taylorella equigenitalis associated with contagious equine metritis (CEM). In the present review article, recent molecular advances relating to studies with Taylorella equigenitalis, as well as the recently described second species of the genus Taylorella, namely Taylorella asinigenitalis, have been described. Molecular genotyping of T. equigenitalis strains by pulsed-field gel electrophoresis (PFGE) after digestion with the suitable restriction enzyme(s) enabled the effective discrimination of strains, thus allowing the examination of the scientific mechanism(s) for its occurrence and transmission of contagious equine metritis (CEM). Alternatively, polymerase chain ...
Equine viral arteritis in a newborn foal: parallel detection of the virus by immunohistochemistry, polymerase chain reaction and virus isolation. A 4-days-old foal died after a short course of respiratory syndrome and fever. Large areas of the alveoli, bronchioles and bronchi were partly or completely filled by hyaline membranes. Pronounced oedema and mild interstitial pneumonia were present and, in the small muscular arteries, fibrinoid necrosis and vasculitis or perivasculitis could be seen. Vasculitis was found in several other organs, and it was most severe in the thymus. The virus was detected in the lung, kidney and spleen using virus isolation and in the lung and spleen using polymerase chain reaction. The virus was also detected...