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.
Dimsoski P.To describe the development and performance of the new horse genotyping kit. Methods: Highly discriminatory 17-Plex horse genotyping kit was designed by adding the fifth dye to the StockMarks kit for genotyping horses and taking advantage of the new instrument platforms. This was accomplished by using a new set of five fluorescent dyes developed by Applied Biosystems (DS-31), with four of the dyes used to label the forward amplification primers (6-FAM, VIC, NED, and PET) in each primer set. Results: The new equine kit contained five extra loci (ASB17, LEX3, HMS1, CA425, and ASB23) in addition ...
Rampersad J, Cesar E, Campbell MD, Samlal M, Ammons D.We report on a study that evaluated the usefulness of PCR for the routine detection of Babesia equi in horses. The blood from a total of 105 horses comprising both sick and apparently healthy animals were examined for the presence of B. equi using both Wright-Giemsa-stained blood smears and PCR. Microscopic analysis of Giemsa-stained blood smears revealed 10/105 animals positive for Babesia, compared to 16/105 for the primary PCR and 36/105 for the nested PCR. Three of the 10 samples positive by Wright-Giemsa-stain were negative by PCR for B. equi. However, evidence is presented that these sam...
Lannergård J, Frykberg L, Guss B.Streptococcus equi subspecies equi is an important horse pathogenic bacterium causing a serious disease called strangles. Using bioinformatics we identified a gene denoted cne (gene encoding collagen-binding protein from S. equi) coding for a novel potential virulence factor of this species called protein CNE. The protein is composed of 657 amino acids and has the typical features found in cell surface-anchored proteins in Gram-positive bacteria. CNE displays amino acid sequence similarities to the previously well-studied collagen-binding protein CNA from Staphylococcus aureus, a proven virule...
Clark RJ, Valderrama XP, Furlan MA, Chedrese PJ.We report the equine (Equs equs) and elk (Cervus elaphus) pituitary pre-prolactin (PRL) cDNA cloning, and their nucleotide and deduced amino acid sequences. Pre-PRL cDNA was obtained by RNA ligation mediated-rapid amplification of cDNA ends (RLM-RACE) and polymerase chain reaction (PCR). The elk pre-PRL cDNA exhibits two polymorphisms at positions 96 and 672, which are silent since they encode for the same amino acids, proline and isoleucine, respectively. We found no polymorphisms in the equine pre-PRL cDNA. The deduced amino acid sequence of the equine pre-PRL is 99% identical to the previou...
Westcott DG, King DP, Drew TW, Nowotny N, Kindermann J, Hannant D, Belák S, Paton DJ.Routine detection of equine arteritis virus (EAV) can be achieved by virus isolation (VI) in cell culture, or by the amplification of viral genome by molecular methods. To simplify molecular diagnosis, a number of different Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) and RT-nested PCR (RT-nPCR) assays were compared, and a one-tube method was developed and optimised utilizing a fluorogenic probe (TaqMan). An artificial RNA template (Mimic) and associated probe were also constructed to provide in-tube validation of the RT-PCR system. To assess the utility of the RT-PCR TaqMan assay,...
Schulman FY, Krafft AE, Janczewski T, Reupert R, Jackson K, Garner MM.Five camelid mucocutaneous fibropapillomas with histologic features similar to equine sarcoids were diagnosed. They were characterized by a dermal fibroblastic proliferation and overlying, often ulcerated hyperplastic epidermis with thin rete pegs extending down into the dermis. Two of the tumors came from llamas and three from alpacas. Four of the animals were 6-year-old females. The fifth was a 6-year-old castrated male. The fibropapillomas were located on the nose, lip, and cheeks. One of the llama tumors waxed and waned before surgery and recurred and spread after surgery. None of the othe...
Teifke JP, Kidney BA, Löhr CV, Yager JA.We examined 12 formalin-fixed paraffin-embedded feline skin tumours which had the histopathological features of fibropapillomas for the presence of papillomavirus (PV) DNA using touchdown polymerase chain reaction (PCR), DNA sequencing and nonradioactive in situ hybridization. Nine of the tumours contained a 102-bp PCR product demonstrated using consensus PV primers that amplify a portion of the L1 gene. The nucleotide sequences are closely related, but not identical to that of ovine PV type 2, rabbit oral PV and reindeer PV. The deduced amino acid sequences had strong homologies with the majo...
Sato F, Hasegawa T, Katayama Y, Ishida N.Complementary DNA (cDNA) encoding equine dopamine beta-hydroxylase (DBH) was amplified with a combination of reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) method, and their nucleotide sequences (Accession No. AB029430: the DDBJ nucleotide sequence database) was determined. A total of 3842 bp cDNA sequence was consisted with 5 bp of 5' flanking untranslated sequence, 1833 bp of open reading frame encoding 610 amino acids, and 2004 bp of 3' flanking untranslated sequence. The deduced amino acid sequence of equine DBH was very similar to the ...
Patterson-Kane JC, Caplazi P, Rurangirwa F, Tramontin RR, Wolfsdorf K.Encephalitozoon cuniculi is a microsporidial parasite, which has rarely been reported to cause placentitis in animals. A late-term aborted fetus and placenta from a Quarterhorse were presented to the Livestock Disease Diagnostic Center, University of Kentucky, for diagnostic examination. There was a necrotizing placentitis, with distension of many chorionic epithelial cells by intracytoplasmic vacuoles containing 1-2-microm-diameter, elongated, gram-positive organisms. The organisms were identified as E. cuniculi by electron microscopy and by polymerase chain reaction using primers to microspo...
Klonisch T, Steger K, Kehlen A, Allen WR, Froehlich C, Kauffold J, Bergmann M, Hombach-Klonisch S.We employed molecular and immunological techniques to investigate the expression of INSL3, a member of the insulin-like superfamily, in prepubertal testis, postpubertal testes exhibiting normal and disturbed spermatogenesis, and cryptorchid testes of male horses. In addition, the partial cDNA coding sequences of the equine homologue of the human relaxin/INSL3-receptor Lgr8 were determined. Nonradioactive in-situ hybridization with a cRNA probe for equine Insl3 and immunohistochemistry with a specific rabbit INSL3 antiserum localized Insl3 transcripts and immunoreactive INSL3 ligand to Leydig c...
Daly P, Doyle S.Equine herpesvirus-1 (EHV-1) infection is of significant animal welfare and economic importance. Yet, no standardised molecular techniques are available for diagnosis or confirmation of viral infection. The purpose of this study was to develop a standardised and quantitative assay system for the reliable detection of EHV-1 infection which was capable of eliminating the likelihood of false negative results. A region within the EHV-1 glycoprotein B gene was amplified by polymerase chain reaction (PCR), cloned and subjected to site-directed mutagenesis to generate a control plasmid, amplifiable b...
Anzai T, Wada R, Okuda T, Aoki T.The effectiveness of the polymerase chain reaction (PCR) as a field application test for the eradication of contagious equine metritis (CEM) was evaluated. Seven-thousands five-hundred and thirty-four genital swabs were collected from 4,026 Thoroughbred broodmares and stallions in Japan to test "high risk" horses as well as for general surveillance testing from 1998 to 2001. Bacterial isolation as well as PCR testing of original specimens and cultured specimens was performed for detection of Taylorella equigenitalis from genital swabs. As a result, T. equigenitalis was detected in 12 mares and...
Pusterla N, Chae JS, Kimsey RB, Berger Pusterla J, DeRock E, Dumler JS, Madigan JE.Most human granulocytic ehrlichiosis (HGE) studies carried out in horses use needle inoculation of infected leucocytes or cell cultures. This route of inoculation does not accurately reflect natural infection of the tick-borne agent. To investigate whether tick transmission influences the course of granulocytic ehrlichiosis in the horse model, experimental transmission through infected laboratory-reared Ixodes scapularis ticks was attempted into two healthy horses. One additional horse served as negative control and was exposed to uninfected ticks. Eleven days after exposure to nymphal or adul...
Semevolos SA, Brower-Toland BD, Bent SJ, Nixon AJ.Early changes in parathyroid hormone-related peptide (PTH-rP) and Indian hedgehog (Ihh) expression were examined in equine articular osteochondrosis (OC) as a model of a naturally acquired dyschondroplasia. Cartilage was harvested from OC-affected femoropatellar or scapulohumeral joints from immature horses and normal control horses of similar age. PTH-rP expression levels were assessed by semi-quantitative PCR, in situ hybridization, and immunohistochemistry. Ihh protein expression levels were assessed by immunohistochemistry. Elevated PTH-rP protein and mRNA expression were identified in the...
Tallmadge RL, Lear TL, Johnson AK, Guérin G, Millon LV, Carpenter SL, Antczak DF.A clone containing beta(2)-microglobulin (beta(2)-m), the light chain of the major histocompatibility complex class I cell surface molecule, was isolated from an equine bacterial artificial chromosome library. This clone was used as a template for polymerase chain reaction (PCR) and unidirectional sequencing to elucidate the genomic sequence and intron/exon boundaries. We obtained 7,000 bases of sequence, extending from 1,100 nucleotides (nt) upstream of the coding region start through 1,698 nt downstream of the stop codon. The sequence contained regulatory elements in the region upstream of t...
Taouji S, Collobert C, Gicquel B, Sailleau C, Brisseau N, Moussu C, Breuil MF, Pronost S, Borchers K, Zientara S.Equine herpesviruses type 1 and 4 (EHV-1 and EHV-4) are ubiquitous in the equine population. One of their main properties is their ability to establish life-long latent infections in their hosts even in those with natural or vaccine-induced immunity. However, effect of vaccination status on prevalence and tissue tropism was not established. In this study, EHV-1 and EHV-4 were detected by polymerase chain reaction and by classical virus isolation from neural, epithelial and lymphoid tissues collected from unvaccinated (33) or vaccinated (23) horses. The percentage of EHV-1- and EHV-4-positive h...
Koehler K, Stechele M, Hetzel U, Domingo M, Schönian G, Zahner H, Burkhardt E.This report describes a case of cutaneous leishmaniosis in a horse in southern Germany. Diagnosis is based on histopathology, immunohistochemistry and electron microscopy. The protozoan was identified as Leishmania infantum via PCR and restriction fragment length polymorphism. The horse did not show specific Leishmania antibodies. The lesions healed completely within 6 months without any specific treatment. Since neither the infected horse nor its dam had ever left their rural area, autochthonous infection in Germany cannot be excluded. Factors possibly influencing the epidemiological situatio...
Battsetseg B, Lucero S, Xuan X, Claveria F, Byambaa B, Battur B, Boldbaatar D, Batsukh Z, Khaliunaa T, Battsetseg G, Igarashi I, Nagasawa H....Babesia equi (EMA-1) and Babesia caballi (BC48) gene fragments were amplified by polymerase chain reaction (PCR), in blood samples, and partially fed-females and egg and larval progenies of Dermacentor nuttalli, collected from horses in Altanbulag, Tuv Province, Mongolia. While Babesia parasite DNA was detected in some horse blood samples during the first PCR, all positive cases in partially fed-female ticks, eggs and larvae were confirmed by nested PCR. Present study reinforces earlier similar findings in unfed D. nuttalli ticks collected from an open space vegetation in Bayanonjuul, Tuv Prov...
Anzai T, Timoney JE, Kuwamoto Y, Wada R, Oikawa M, Higuchi T.To develop polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis for molecular typing of strains of Streptococcus zooepidemicus and to use the new typing method to analyze a collection of isolates from the respiratory tract of Thoroughbreds. Methods: 10 strains of S zooepidemicus, 65 isolates from the respiratory tract of 9 yearlings following long distance transportation, and 89 isolates from tracheal aspirates of 20 foals with pneumonia. Methods: Phenotypic variations in the SzP protein were detected by western immunoblot analysis. Using PCR-RFLP analysis, ge...
Kurowski PB, Traub-Dargatz JL, Morley PS, Gentry-Weeks CR.To use real-time polymerase chain reaction (PCR) technology to develop a highly sensitive and specific diagnostic assay for the detection of Salmonella spp in fecal specimens. Methods: 299 fecal specimens from cattle, horses, and dogs. Methods: Enrichment of fecal specimens was followed by genomic DNA extraction by use of commercially available isolation kits. Real-time PCR assay was performed to target a Salmonella spp-specific DNA segment. Results of real-time PCR assay were compared with bacterial culture results to determine relative sensitivity and specificity. Results: Use of the spaQ pr...
Makrai L, Takai S, Tamura M, Tsukamoto A, Sekimoto R, Sasaki Y, Kakuda T, Tsubaki S, Varga J, Fodor L, Solymosi N, Major A.Rhodococcus equi isolates (204) obtained from foals (lung abscesses, lymph nodes, nasal discharge, rectal swabs) bred in 15 studs located throughout Hungary, isolates from soil samples, lymph nodes of pigs and from lesions of human patients were examined to determine genotypic diversity of virulence-associated plasmids. Isolates were tested for the presence of 15-17 kDa virulence-associated protein antigen (VapA) and 20k Da (VapB) genes by polymerase chain reaction (PCR). Plasmid DNAs were isolated and analysed by digestion with restriction endonucleases for estimation of size and comparison o...
Kim HY, Mott J, Zhi N, Tajima T, Rikihisa Y.Human granulocytic ehrlichiosis (HGE), a tick-borne zoonosis, is caused by an obligatory intragranulocytic bacterium, the HGE agent, a strain of Anaplasma phagocytophila. The equine model of HGE is considered valuable in understanding pathogenic and immune mechanisms of HGE. In the present study, cytokine mRNA expression by peripheral blood leukocytes (PBLs) in horses was examined during the course of infection by intravenous inoculation of A. phagocytophila or by allowing feeding by infected ticks. The p44 genes encoding the major outer membrane protein P44s of A. phagocytophila were detected...
Cook RF, Cook SJ, Li FL, Montelaro RC, Issel CJ.A single-tube reverse transcriptase-polymerase chain reaction (RT-PCR) using a fluorogenic real-time PCR detection method is described for the quantitation of equine infectious anemia virus (EIAV) RNA in the plasma of equids. To compensate for variations inherent in sample preparation a multiplex real-time RT-PCR system was developed that permitted the simultaneous calculation of the nucleic acid recovery rate along with the copy number of viral RNA molecules. Detection of EIAV RNA was linear from 10(9) to 10(1) molecules with intra- and inter-assay variability of less than 1% at 10(8), 10(6),...
Kleiboeker SB, Schommer SK, Johnson PJ, Ehlers B, Turnquist SE, Boucher M, Kreeger JM.Over a period of 6 years, antemortem and postmortem examinations were performed on a number of donkeys suffering from respiratory disease. For many cases, initial diagnostic efforts failed to identify an etiology consistent with the pathologic findings. However, retrospective examination of these cases using consensus primer polymerase chain reaction, designed to recognize herpesviruses from all 3 subfamilies of the Herpesviridae, amplified a fragment of the highly conserved herpesvirus DNA polymerase gene from a number of these animals. Two novel herpesviruses, herein designated asinine herpe...
Brooks SA, Terry RB, Bailey E.An MspI polymorphism was identified in intron 13 of the equine homologue of proto-oncogene c-kit (KIT) by comparing DNA sequences from horses with solid coat colour and horses homozygous for the tobiano spotting (To) gene. The allele associated with solid coat colour was designated KM0, while the allele associated with the tobiano pattern created an additional MspI restriction site and was designated KM1. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) studies using DNA from hair follicles demonstrated that all 129 of 129 tobiano patterned horses possessed the KM1...
Davidson AJ, Edwards GB, Proudman CJ, Cripps PJ, Matthews JB.The aim of this study was to investigate cytokine expression patterns in the large intestinal mucosa of horses, particularly in diseases associated with inflammation. Many cases of equine colitis remain without a definitive diagnosis and survival rates are poor. In humans, colitis is associated with increased expression of pro-inflammatory cytokines. To examine if similar responses occur in horses, we investigated il -2, il -4, il -5, il -10, tnfalpha, ifngamma and tgfbeta messenger rna expression in large intestinal mucosa. Samples were obtained from animals with large intestinal disease (n=1...
Iqbal J, Edington N.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...
Nordengrahn A, Merza M, Ros C, Lindholmc A, Palfl V, Hannant D, Belák S.Equineherpesvirustypes 2 and 5 (EHV-2andEHV-5)have a rather unclearpathogenicity and distribution within the equid population. In order to gain more information on the prevalence of these two viruses, type-specific PCR assays were developed to detect viral DNA in nasal specimens and in peripheral blood leukocytes (PBLs) of adult horses and foals from various regions of Europe, i.e. Sweden, Hungary and the United Kingdom. In adult horses, the prevalence of EHV-2 in PBLs was up to 68% in Sweden and 71% in the United Kingdom. EHV-2 DNA was detected in the PBLs from all the foals tested in all cou...
Spergser J, Aurich C, Aurich JE, Rosengarten R.Mycoplasma equigenitalium and M. subdolum have been implicated in genital disorders and infertility of horses. The reported cytopathic effects of M. equigenitalium observed in vitro underscore its potential pathogenic role in reproductive dysfunction in mares. This study was initiated to determine the prevalence of mycoplasmas in the genital tract of stallions in relationship to age, clinical signs, geographic location and semen quality. For this purpose the mycoplasma flora of the genital tract of 116 stallions of the Noric breed was determined by isolation and colony immunoblotting and by po...
Moulay S, Zientara S, Sailleau C, Cruciere C.In order to develop a non-radioactive dot-blot hybridization assay, for the detection of African-horse sickness virus (AHSV), genome segment 7 from 9 serotypes was amplified by RT-PCR. The resulting PCR products were denatured, immobilized on nylon membranes and then hybridized to a non-radioactive digoxigenin-labelled probe. This probe (265 bp in length) was generated by nested-PCR using genome segment 7 of AHSV, serotype 4 as a template. The dot-blot was visualized by chemiluminescence. Positives were obtained from the PCR products amplified from all 9 AHSV serotypes, but not from any other ...
Thieulent CJ, Carossino M, Balasuriya UBR, Graves K, Bailey E, Eberth J, Canisso IF, Andrews FM, Keowen ML, Go YY.Equine arteritis virus (EAV) is the causative agent of equine viral arteritis (EVA), a respiratory, systemic, and reproductive disease of equids. Following natural infection, up to 70% of the infected stallions can remain persistently infected over 1 year (long-term persistent infection [LTPI]) and shed EAV in their semen. Thus, the LTP-infected stallions play a pivotal role in maintaining and perpetuating EAV in the equine population. Previous studies identified equine C-X-C motif chemokine ligand 16 (CXCL16) as a critical host cell factor determining LTPI in the stallion's reproductive trac...
Mizukoshi N, Sakamoto K, Iwata A, Ueda S, Kamada M, Fukusho A.The reverse transcription followed by the polymerase chain reaction (RT-PCR) technique was applied to the detection of African horsesickness virus (AHSV) using primers specific for attenuated AHSV serotype 4 segment 5 (NS1 gene). Total RNA which contains both messenger RNA and genomic dsRNA was extracted by the acid guanidinium-phenol-chloroform method from the AHSV infected Vero cells and was used as templates to optimize the RT-PCR. A pair of primer (NP2-NP32) amplified the product of the expected size from all serotypes of attenuated AHSV when four pairs of primers were tested. Using this p...
Moyaert H, Decostere A, Pasmans F, Baele M, Ceelen L, Smits K, Ducatelle R, Haesebrouck F.A novel urease-negative Helicobacter species has been isolated from faecal samples of clinically healthy horses, but no information is available about the main sites of colonisation in the equine gastrointestinal tract nor is the pathogenic potential of this microorganism known. An experimental infection in horses was therefore carried out. Methods: Four horses were infected with H. equorum strain CCUG 52199T and subjected to euthanasia at 10 (n = 2) and 30 days (n = 2) post inoculation. A fifth animal was inoculated with phosphate buffered saline and used as control. Gastrointestinal samples ...
Tozaki T, Ohnuma A, Kikuchi M, Ishige T, Kakoi H, Hirota KI, Takahashi Y, Nagata SI.Gene doping, which is prohibited in horseracing and equestrian sports, can be performed by introducing exogenous genes, known as transgenes, into the bodies of postnatal animals. To detect exogenous genes, a method utilizing quantitative polymerase chain reaction (qPCR) with a hydrolysis probe was developed to test whole blood and plasma samples, thereby protecting the fairness of competition and the rights of stakeholders in horseracing and equestrian sports. Therefore, we aimed to develop sample storage methods suitable for A and B samples in gene doping tests using blood. For sample A, suff...
The goal of this work was the development of suitable (real-time) RT-PCR techniques for fast and sensitive diagnosis of EAV and for molecular-epidemiological characterisation of viral strains, as an alternative to virus isolation. To this purpose two conventional RT-PCR methods and one real-time RT-PCR were adapted to detect the broadest possible spectrum of viral strains. Several dilutions with Bucyrus strain showed a 100-fold higher sensitivity of real-time RT-PCR and heminested RT-PCR compared to simple RT-PCR. Making use of 11 cell culture supernatants of different EAV isolates and 7 semen...
Teifke JP.From 932 equine skin lesions 421 were diagnosed as sarcoids (about 45%). The most common locations were the ventral body regions, head, neck and sites of thin skin. Most often the fibroblastic type, less frequently the mixed type and most infrequent the verrucous type of sarcoid were diagnosed. Detection of BPV-DNA was performed by polymerase chain reaction (PCR) using an oligonucleotide primer pair located in the E5-open reading frame. DNA of BPV 1 and BPV 2 could be differentiated by digestion with restriction endonucleases. In 97 out of 108 sarcoids BPV-DNA was detected by PCR. Most samples...
Spyrou V, Papanastassopoulou M, Psychas V, Billinis Ch, Koumbati M, Vlemmas J, Koptopoulos G.There appears to be a lack of information concerning responses of mules to natural infection or experimental inoculation with equine infectious anemia virus (EIAV). In the present study EIAV was isolated from mules, for the first time, and its pathogenicity in naturally infected and experimentally inoculated animals was investigated. Two naturally infected (A and B) and three EIAV free mules (C, D and E) were used for this purpose. Mule A developed clinical signs, whereas mule B remained asymptomatic until the end of the study. Mules C and D were each inoculated with 10ml of blood from mule A ...
Semevolos SA, Nixon AJ, Strassheim ML.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 ...
Serpa PB, Garbade P, Natalini CC, Pires AR, Tisotti TM.OBJECTIVE To develop a high-resolution melting (HRM) assay to detect the g.66493737C>T polymorphism in the myostatin gene (MSTN) and determine the frequency of 3 previously defined g.66493737 genotypes (T/T, T/C, and C/C) in warmblood horses. SAMPLES Blood samples from 23 horses. PROCEDURES From each blood sample, DNA was extracted and analyzed by standard PCR methods and an HRM assay to determine the MSTN genotype. Three protocols (standard protocol, protocol in which a high-salt solution was added to the reaction mixture before the first melting cycle, and protocol in which an unlabeled p...
Tonekaboni FR, Narenjisani R, Staji H, Ahmadi-Hamedani M.This investigation aimed to compare the cell-free fetal DNA (cffDNA) plasma present in three trimesters of pregnancy in Torkaman pregnant mare. Peripheral blood samples of 32 pregnant mares in three trimesters of pregnancy were collected in tubes containing ethylenediaminetetraacetic acid at three time points. Circulating cffDNA was extracted from 3 mL of maternal plasma. Using outer and inner primers, a conventional polymerase chain reaction was performed for the sex-determining region Y (SRY) gene present in the Y chromosome. Of the total 32 Torkaman pregnant mares, 24 were carrying male fe...
Apprich V, Licka T, Freiler S, Gabriel C.Impaired keratinocyte differentiation has recently been suggested as a key event in equine hoof canker development. Koilocytotic appearance of keratinocytes, one of the most characteristic morphological alterations in hoof canker tissue, is also a common marker for papillomavirus (PV) infection, and bovine PV-1 and/or -2 (BPV-1/2) has previously been detected in equine canker patients. Therefore, the present study aimed to correlate the frequency and severity of koilocytotic keratinocytes with BPV detection in hoof canker samples. Hoof tissue of 5/18 canker-affected horses and 2/6 control hors...
Stasiak K, Rola J, Zmudzinski JF.A highly sensitive and specific real-time PCR assay was used for detection and quantitation of equine herpesvirus type 1 (EHV-1) in the different internal organs of aborted fetuses. Tissue samples from 23 aborted fetuses submitted to the Department of Virology of the National Veterinary Research Institute in Pulawy between 2012 and 2013 were used for testing. Total DNA was extracted using a phenol-chloroform-isoamyl alcohol standard protocol. A real-time PCR with forward and reverse primers encompassing a highly conserved region encoding viral glycoprotein B was adapted for diagnosis of EHV-1 ...
Song KD, Cho HW, Lee HK, Cho BW.The objective of this study was to determine the molecular characteristics of the horse vascular endothelial growth factor alpha gene (VEGFα) by constructing a phylogenetic tree, and to investigate gene expression profiles in tissues and blood leukocytes after exercise for development of suitable biomarkers. Using published amino acid sequences of other vertebrate species (human, chimpanzee, mouse, rat, cow, pig, chicken and dog), we constructed a phylogenetic tree which showed that equine VEGFα belonged to the same clade of the pig VEGFα. Analysis for synonymous (Ks) and non-synonymous sub...
Peippo J, Huhtinen M, Kotilainen T.A rapid and reliable method for sex determination of preimplantation-stage equine embryos has not been available. The aim of the present study was to find an enzyme which would distinguish sexes in the horse by finding a polymorphic restriction site between the ZFY and ZFX homologues amplified by the polymerase chain reaction (PCR). Altogether, 38 different restriction enzymes were tested using female and male DNA extracted from blood. The primers used for amplification were selected from conserved sequences between human ZFY and ZFX genes and mouse Zfy-1 and Zfy-2 genes. Nine enzymes cut the ...
Ren WX, Zhang XX, Long CY, Zhao Q, Cheng T, Ma JG, Xu P, Hou G, Ni HB.Horse meat and milk are an important source of nutrition for many Chinese. Previous studies have shown a fairly high prevalence of infection in horses. However, there is no data concerning the molecular characterization of in horses in China. The present study tested 231 cervical lymph node samples of horses from northern China (97 from Jilin, 54 from Liaoning, and 80 from Xinjiang) for the presence of the B1 gene by seminested PCR. The B1-positive samples were genotyped at nine nuclear loci, that is, SAG1, alternative SAG2, SAG3, BTUB, c22-8, GRA6, c29-2, PK1, and an apicoplast locus, usin...
Thompson D, Thirumalapura NR, Tewari D.To determine the prevalence of Anaplasma phagocytophilum and Borrelia burgdorferi infections in Pennsylvania horses. 271 horses. A survey was conducted with PCR and serology to evaluate anaplasmosis and Lyme disease infections in horses from Pennsylvania that were suspected for tick-borne infection. A phagocytophilum was detected in 19/271 (7.0%) Pennsylvania horses tested by the duplex PCR. B burgdorferi was not detected in any horse blood tested by PCR. Overall, 120/271 (44.3%) horses tested positive for presence of A phagocytophilum antibodies by at least the IDEXX SNAP 4Dx Plus lateral flo...
Pusterla N, Naranatt P, Swadia H, Winfield L, Hartwig A, Barnum S, Mendonsa E.The introduction of microfluidic card technology has opened the field for rapid point-of-care (POC) molecular assays, including fecal and environmental Salmonella spp. testing. The purpose of this study was to evaluate a novel POC PCR assay for the detection of Salmonella spp. in feces and environmental samples. A total of 143 fecal samples and 132 environmental samples were collected for POC PCR Salmonella spp. testing as well as qPCR testing. Each sample was inoculated into selenite broth and incubated for 18 to 24 hours. For the POC PCR assay, 14 μl of selenite broth were mixed with 126 μ...
Cappelli A, Capone A, Valzano M, Bozic J, Preziuso S, Mensah P, Varotto Boccazzi I, Rinaldi L, Favia G, Ricci I.Tick-borne diseases are an increasing problem for the community. Ticks harbor a complex microbial population acquired while feeding on a variety of animals. Profiling the bacterial population by 16S rDNA amplification and denaturing gradient gel electrophoresis enables detection of the broad spectrum of bacteria that settles in the ticks. This study identified known and unknown tick-infecting bacteria in samples from Italy. Seven adult ticks from different hosts and origins were analyzed: two ticks from dogs (Lombardia), two ticks from bovines (Lazio), and three ticks from humans (Marche). ...
Löfgren M, Ekman S, Ekholm J, Engström M, Fjordbakk CT, Svala E, Holm Forsström K, Lindahl A, Skiöldebrand E.Autologous conditioned serum (ACS) is used to treat osteoarthritis in horses, although its effects are not fully investigated. Objective: To investigate the effects of equine serum and conditioned serum on chondrocytes stimulated with interleukin (IL)-1β and cartilage explants with mild osteoarthritis. Methods: In vitro experimental study. Methods: The effect of three different serum preparations (unincubated control [PS], serum incubated 24 h [PS24h], and serum incubated 24 h in ACS containers [PCS]) pooled from lame horses were tested in two in vitro models. IL-1β and IL-1 receptor antagon...
Magdesian KG, Barnum S, Pusterla N. and cause significant morbidity and mortality in foals. Antemortem diagnosis of infection has been complicated by a paucity of tests available for toxin detection. Fecal PCR panels have assays for a variety of toxin gene sequences as well as for several other foal gastrointestinal pathogens. We evaluated results of a comprehensive fecal diarrhea PCR panel in 28 foals that had been presented to a referral hospital because of diarrhea. Sixteen (57%) foals were positive for and/or toxin gene sequences on fecal PCR, including 3 foals positive for NetF toxin. These foals were younger ( = ...
Prutton JSW, Barnum S, Pusterla N.Equine coronavirus (ECoV) is considered an emerging enteric virus with reported morbidity rates ranging from 10 to 83% and fatality rates ranging from 7 to 27% in adult horses; a vaccine for ECoV is currently not available. This study investigated the safety, humoral response and viral shedding in horses inoculated with a commercially available modified-live bovine coronavirus (BCoV) vaccine. Twelve healthy adult horses were vaccinated twice, 3 weeks apart, either orally, intranasally or intrarectally. Two healthy unvaccinated horses served as sentinel controls. Following each vaccine adminis...
Räsänen LA, Karvonen U, Pösö AR.In situ hybridization was used to localize xanthine dehydrogenase (XDH) mRNA in horse skeletal muscle. Capillary endothelial cells were found to express XDH, but muscle cells did not give any signal. The digoxigenin-labelled probe was produced by PCR with primers based on the cDNA sequence of mouse XDH and horse lung cDNAs. A 4.3 kb mRNA was detected in a Northern blot.
Timms M, Ganio K, Forbes G, Bailey S, Steel R.CJC-1295 is a 30 amino acid peptide-based drug that stimulates the release of growth hormone (GH) from the pituitary gland. It is unique among performance-enhancing peptides due to the presence of a reactive maleimidopropionic acid group that covalently links the peptide to free thiols on the surface of plasma proteins. Once conjugated, CJC-1295 remains active in the bloodstream for significantly longer than non-conjugated peptide-based drugs that are rapidly excreted. Conjugation of CJC-1295 to plasma proteins prevents its detection by top-down mass-spectrometry-based peptide screening protoc...
Dărăbuş G, Badea C, Oprescu I, Morariu S, Mederle N, Ilie M, Suici T, Imre M.Resistance of cyathostomins to benzimidazole (BZ) anthelmintics is widespread in horses in many parts of the world. This study compared three methods for the determination of benzimidazole resistance of Cyathostominae in 18 horses from a stud farm in Romania. The horses were treated with Fenbendazole. The resistance test was performed by FECRT, ERP and PCR. On Day 0, larvae of species belonging to the Cyathostominae subfamily, types A, B, C, D and Gyalocephalus, as well as Strongylus vulgaris species of the Strongylinae subfamily, were identified. At 42 days post treatment with fenbendazole on...
Selvaggi M, D'Alessandro AG, Dario C.The aims of this study were to assess the genetic variability in the exon 1 of the κ-casein gene in four Italian horse populations (Italian Saddle horse, Italian Trotter, Italian Heavy Draught horse, and Murgese horse) and in a sample of Martina Franca donkey by estimating genotype, allele and haplotype frequencies, as well as several population genetic indices. Genotyping of the selected polymorphisms was performed using the PCR-RFLP technique with two restriction enzymes: PstI and BseYI aimed to discover the presence of c.-66A>G and c.-36C>A polymorphism, respectively. Both these loci...
Miserez R, Frey J, Krawinkler M, Nicolet J.A polymerase chain reaction (PCR) for identification of Taylorella equigenitalis was developed. The oligonucleotide primers are based on the DNA sequence of the rrs gene of T. equigenitalis, encoding for the 16S ribosomal RNA. Analysis of 21 strains of T. equigenitalis from England, USA and Switzerland showed an amplification product of 410 bp with identical Sau3A restriction profile. The sensitivity of the PCR-Assay was estimated to detect 50 to 500 bacteria of T. equigenitalis in a mixture with frequently found contaminants. Further analysis of culture from 60 genital swabs, taken in the cou...
Xu J, Lasry JB, Svaren J, Wagner B, Darien BJ.P-selectin glycoprotein ligand-1 (PSGL-1, CD162) is a dimeric, mucin-like, transmembrane glycoprotein constitutively expressed on leukocytes. A high baseline level of P-selectin expression in circulating equine platelets suggests a primed state toward inflammation and thrombosis via P-selectin/PSGL-1 adhesion. To investigate the potential role of equine P-selectin in these events, we first identified the cDNA sequence of equine PSGL-1 (ePSGL-1) using degenerate PCR and RACE-PCR and then compared the predicted sequence with that of human PSGL-1 (hPSGL-1). ePSGL-1 protein subunit is predicted to...
Hu Z, Zhu C, Chang H, Guo W, Liu D, Xiang W, Wang X.The objective of this study was to develop a novel EvaGreen (EG) based real-time PCR technique for the simultaneous detection of Equine herpesvirus 1 (EHV-1) and Equine herpesvirus 4 (EHV-4) genomes from equine nasal swabs. Viral genomes were identified based on their specific melting temperatures (T m), which are 88.0 and 84.4 °C for EHV-1 and EHV-4, respectively. The detection limitation of this method was 50 copies/μl or 0.15 pg/μl for EHV-1 and 5 copies/μl or 2.5 fg/μl for EHV-4. This assay was 50-1,000 times more sensitive than the SYBR Green (SG)-based assay using the same primer...
Del Piero F, Wilkins PA, Timoney PJ, Kadushin J, Vogelbacker H, Lee JW, Berkowitz SJ, La Perle KM.A case of fatal nonneurological equine herpesvirus 1 (EHV-1) infection in a yearling filly is described. Gross lesions included extensive pulmonary edema, prominent laryngeal lymphoid follicles, and congestion and edema of the dorsal third ventricle choroid plexus. Histologically, there was vasculitis, hemorrhage, and edema in the lungs and dorsal third ventricle choroid plexus as well as mild intestinal crypt necrosis with occasional intranuclear inclusion bodies. The perivascular and vascular inflammatory infiltrates were comprised mainly of T lymphocytes and macrophages. EHV-1 antigen was i...
