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.
Bell KS, Philp JC, Christofi N, Aw DW.Two regions in the gene coding for 16S rRNA in Rhodococcus equi were selected as species-specific primer sequences for the polymerase chain reaction (PCR). PCR using these primers was tested against 10 strains of R. equi (including the type strain) and gave positive results for all but was negative for all other tested species of Rhodococcus; representatives of the most closely related genera and a number of other bacterial species. This method could therefore be used to identify this species which can infect the lungs or other organs of horses, pigs, humans and other animals.
Marsh AE, Barr BC, Madigan J, Lakritz J, Conrad PA.To identify Sarcocystis neurona-specific DNA sequences in the nuclear small subunit ribosomal RNA (nss-rRNA) gene that could be used to distinguish S neurona from other closely related protozoal parasites, and to evaluate a polymerase chain reaction (PCR) test, using broad based primers and a unique species-specific probe on CSF for detection of S neurona in equids. Methods: Sequencing of the nuclear small subunit ribosomal RNA gene from a new S neurona isolate (UCD 1) was performed. The sequence was compared with that of other closely related Sarcocystidae parasites. From this sequence, conse...
Cohen ND, Martin LJ, Simpson RB, Wallis DE, Neibergs HL.To compare the sensitivity of polymerase chain reaction (PCR) with microbiological culture for detecting salmonellae in equine fecal samples and equine environmental swab specimens. Methods: Samples and specimens were tested by PCR and microbiological culture. Methods: A fecal sample from each of 152 horses admitted consecutively to the clinic for evaluation by the outpatient service, 282 fecal samples from 110 hospitalized horses that had been submitted to the clinical microbiology laboratory, and 313 environmental swab specimens were examined. Methods: Each sample and specimen in the study w...
Barlough JE, Madigan JE, DeRock E, Bigornia L.A nested polymerase chain reaction for detecting Ehrlichia equi in horses and ticks (Ixodes pacificus) was developed. A major second-round PCR product of 928 bp could be readily visualized in ethidium bromide-stained agarose minigels. An internal probe was used to verify the identity of the amplified product by non-radioactive (digoxigenin-based) Southern blotting; additional confirmation was provided by DNA sequence analysis. A dilution study testing the sensitivity of the PCR indicated that DNA derived from 3 infected neutrophils was sufficient to generate a PCR signal. The specificity of t...
Langemeier JL, Cook SJ, Cook RF, Rushlow KE, Montelaro RC, Issel CJ.Control of equine infectious anemia (EIA) is currently based on detection of anti-EIA virus (EIAV) antibodies. However, serologic diagnostic methods may give false-negative results in infected horses that fail to respond adequately or are in the early stages of infection. We developed a reverse transcriptase nested PCR (RT-nPCR) assay for the detection of viral gag gene sequences in plasma from EIAV-infected horses. The ability of RT-nPCR to detect field strains of EIAV was investigated by assaying plasma samples from 71 horses stabled on EIA quarantine ranches. Positive PCR signals were detec...
Crabill MR, Cohen ND, Martin LJ, Simpson RB, Burney N.Equine synovial fluid aliquots were inoculated with Salmonella enteritidis, Escherichia coli, Actinobacillus equuli, Staphylococcus aureus, and Streptococcus zooepidemicus to obtain approximate concentrations of 1000, 100, 10, and 1 colony forming U/mL. Synovial fluid aliquots were also inoculated with an unquantitated inoculum of Bacteroides fragilis and Clostridium perfringens. Inoculated synovial fluid was incubated in trypticase-soy broth or Columbia broth for approximately 12 hours. Then aliquots were removed for DNA extraction and polymerase chain reaction (PCR) analysis for detection of...
Lebelt J, Hagenau K.Borna disease (BD) is a naturally occurring enzootic encephalomyelitis of horses and sheep. The aetiological agent, Borna disease virus (BDV) is an unclassified, neurotropic, negative stranded RNA virus. The study aimed at providing further information on BD of naturally infected animals. Samples obtained from 20 animals (18 horses, 1 donkey, 1 sheep) were investigated by a series of virological and molecular biological tests. The highly sensitive reverse transcription-polymerase chain reaction (RT-PCR) method was used to analyze the tissue distribution of BDV-specific RNA. BDV-specific RNA wa...
Munderloh UG, Madigan JE, Dumler JS, Goodman JL, Hayes SF, Barlough JE, Nelson CM, Kurtti TJ.The equine granulocytic ehrlichiosis agent, Ehrlichia equi, is closely related or identical to the human granulocytic ehrlichiosis (HGE) agent. Both are suspected of being transmitted by ticks. We have successfully isolated E. equi in a cell line, IDE8, derived from a putative vector, the tick Ixodes scapularis. Peripheral blood leukocytes from an experimentally infected horse were inoculated onto IDE8 monolayers. Cultures were incubated in a candle jar at 34 degrees C in tick cell culture medium with NaHCO3 and an organic buffer [3-(N-morpholino)-propanesulfonic acid] (MOPS). Within 2 weeks, ...
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...
Fraser DG, Bailey E.Single-strand conformational polymorphism (SSCP) gel electrophoresis and DNA sequencing were used to characterize the second exon of the horse DRB homologue as well as to identify eight new DRB alleles. The SSCP gels presented a complex pattern, with phenotypes exhibiting between 4 and 13 bands. The DRB SSCP patterns were studied for two families (6 to 13 bands per pattern). For both families, the patterns showed simple Mendelian inheritance. The polymerase chain reaction products from two individuals possessing homozygous major histocompatibility complex (MHC) alleles by descent were cloned a...
Browning GF, Begg AP.Variant types of VP4 and VP7 gene segments of faecal rotaviruses from diarrhoeic foals were identified by restriction endonuclease digestion of reverse transcription/polymerase chain reaction (RT/PCR) products. The variants observed were correlated with serotypes by determination of the sequence of representative RT/PCR products (entire coding sequence for VP7 and the VP8 region of VP4) and comparison to published sequences of equine G and P serotype genes. Both G and P serotypes could be predicted for 95/116 (82%) strains, P serotype only for a further 8 (7%) strains and G serotype only for 1...
Granstrom DE.This article reviews recent advances in laboratory diagnosis of equine parasitic diseases. Laboratory diagnosis of most equine parasitic diseases continues to rely on standard methods. Only laboratory diagnostic tests for EPM, cryptosporidiosis, and giardiasis were included. The criteria for testing and interpretation of results for each new diagnostic method were explained. Western blot and PCR testing for EPM and immunofluorescent staining with monoclonal antibodies for cryptosporidiosis and giardiasis were reviewed.
Fenger CK, Granstrom DE, Langemeier JL, Stamper S, Donahue JM, Patterson JS, Gajadhar AA, Marteniuk JV, Xiaomin Z, Dubey JP.Sarcocystis neurona is an apicomplexan that causes equine protozoal myeloencephalitis (EPM) in North and South America. Horses appear to be an aberrant host, because the merozoites continually divide in the central nervous system, without encysting. The natural host species has not previously been identified. The small subunit ribosomal RNA (SSURNA) gene of S. neurona was compared to those of Sarcocystis muris, Sarcocystis cruzi, Toxoplasma gondii, and Cryptosporidium parvum to identify a unique region suitable for a species-specific amplification primer. The S. neurona SSURNA primer was used ...
Dame JB, MacKay RJ, Yowell CA, Cutler TJ, Marsh A, Greiner EC.Equine protozoal myeloencephalitis (EPM) is a neurologic disease of horses caused by Sarcocystis neurona. The horse is a dead-end host for S. neurona and the definitive and intermediate hosts have not previously been identified. We hypothesized that S. neurona is actually Sarcocystis falcatula, a parasite that cycles in nature between Virginia opossums (Didelphis virginiana) and any of a variety of avian intermediate hosts. We extracted DNA from S. falcatula sarcocysts in the muscle of a brown-headed cowbird (Molothrus ater) and from schizonts in a fixed specimen of lung from a Moluccan cockat...
Arriaga C, Yépez-Mulia L, Viveros N, Adame LA, Zarlenga DS, Lichtenfels JR, Benitez E, Ortega-Pierres MG.Human trichinellosis outbreaks related to horsemeat consumption have been reported in France and Italy in recent years. In order to determine if Trichinella is present in horses slaughtered at an abattoir in the State of Mexico, diaphragm muscle tissue samples (22-37 g) from 80 horses were examined by artificial digestion. Four of these samples had larvae that were characterized as Trichinella sp. by morphological criteria and as Trichinella spiralis by the polymerase chain reaction.
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 ...
Nakamura Y, Kishi M, Nakaya T, Asahi S, Tanaka H, Sentsui H, Ikeda K, Ikuta K.Borna disease (BD) is a progressive poliomeningoencephalomyelitis which occurs naturally in horses and sheep. Here, peripheral blood mononuclear cells (PBMC) derived from 57 healthy horses in Japan were examined by a nested reverse transcription-polymerase chain reaction to determine the prevalence of BD virus (BDV) infection. Seventeen (29.8%) of the samples were positive by this examination and the specificity of the amplified product was confirmed by hybridization with authentic oligomer probes. About 60% of the BDV RNA-positive individuals also showed seropositivity by Western blotting. Th...
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 ...
Dudhia J, Platt D.Investigation of the structure of the equine articular cartilage link protein (LP) from individuals ranging in age from 1 to 15 years identified 3 distinct isoforms having molecular weights of 46,000, 43,000 and 41,000. The relative amounts of each of the 3 isoforms altered with age. The largest form did not change with age; however, amounts of the Mr 43,000 and 41,000 forms increased with increasing age. The results suggested that an accumulation, in the extracellular matrix of cartilage, of these 2 smaller products may have arisen from proteolytic cleavage. The complete amino acid sequence o...
Madewell BR, Tang YJ, Jang S, Madigan JE, Hirsh DC, Gumerlock PH, Silva J.Intestinal colonization with toxigenic strains of Clostridium difficile was documented in 9 of 10 horses with acute onset diarrhea in a veterinary medical teaching hospital, whereas a similar isolate was detected in only 1 of 23 other horses without diarrhea in the hospital. One horse with diarrhea was infected simultaneously with both C. difficile and Salmonella krefeld. Clostridium difficile was detected by fecal culture on selective medium, confirmed with a latex particle agglutination test, and identified as toxigenic by polymerase chain reaction amplification of toxin A and toxin B gene s...
Marklund S, Chaudhary R, Marklund L, Sandberg K, Andersson L.The hypervariable D-loop region of mitochondrial DNA (mtDNA) was amplified with the polymerase chain reaction using total horse DNA samples. Analysis of single strand conformation polymorphism (SSCP) of denatured amplification products was carried out by native polyacrylamide (8%) gel electrophoresis followed by silver staining. As many as 15 distinct SSCP variants were revealed when screening a total of 78 maternally unrelated horses representing five different breeds. All breeds showed a high degree of polymorphism and the estimated probability (PImt) that two maternally unrelated individual...
Epe C, Bienioschek S, Rehbein S, Schnieder T.Genomic DNA isolated from the four Dictyocaulus species D. viviparus, D. eckerti, D. filaria and D. arnfieldi was compared by random amplified polymorphic DNA polymerase chain reaction (RAPD)-PCR to get additional information whether lungworms from fallow deer belong to a separate species (D. eckerti) or have to be regarded as an isolate of D. viviparus in wild ruminants. The resulting banding patterns of the electrophoresed PCR products were compared to assess the degree of genetic differences between the different lungworms. For the two D. viviparus isolates a similarity coefficient of 93.4%...
Zientara S, Sailleau C, Moulay S, Wade-Evans A, Cruciere C.The development of a coupled reverse transcriptase-polymerase chain reaction assay (RT-PCR) is described for the detection of African horse sickness virus (AHSV) double-stranded RNA. Genome segments 7 and 10 were chosen as target templates for primers selected for use in the RT-PCR. Using these AHSV-specific primers all 9 serotypes were detectable. The sensitivity and specificity of the RT-PCR results were compared to those obtained by competition ELISA.
Wen B, Rikihisa Y, Fuerst PA, Chaichanasiriwithaya W.Ehrlichia risticii is the causative agent of Potomac horse fever. Variations among the major antigens of different local E. risticii strains have been detected previously. To further assess genetic variability in this species or species complex, the sequences of the 16S rRNA genes of several isolates obtained from sick horses diagnosed as having Potomac horse fever were determined. The sequences of six isolates obtained from Ohio and three isolates obtained from Kentucky were amplified by PCR. Three groups of sequences were identified. The sequences of five of the Ohio isolates were identical ...
Cohen ND, Wallis DE, Neibergs HL, Hargis BM.Salmonella was identified in feces from horses, using the polymerase chain reaction (PCR) and genus-specific oligonucleotide primers. Feces from healthy horses were determined to be culture negative and PCR negative for Salmonella. Fecal samples were inoculated with known numbers of colony-forming units (CFU) of S. enteritidis. The fecal samples were enriched overnight in tetrathionate broth, and then DNA was extracted and amplified by PCR using genus-specific primers. Sensitivity of the assay extended to 10 degrees CFU Salmonella enteritidis/g feces; sensitivity of microbiologic culture with ...
Campbell AJ, Gasser RB, Chilton NB.In the current study, molecular techniques were evaluated for the species identification of individual strongyle eggs. Adult worms of Strongylus edentatus, S. equinus and S. vulgaris were collected at necropsy from horses from Australia and the U.S.A. Genomic DNA was isolated and a ribosomal transcribed spacer (ITS-2) amplified and sequenced using polymerase chain reaction (PCR) techniques. The length of the ITS-2 sequence of S. edentatus, S. equinus and S. vulgaris ranged between 217 and 235 nucleotides. Extensive sequence analysis demonstrated a low degree (0-0.9%) of intraspecific variation...
McCann SH, Mumford JA, Binns MM.A search for variable restriction sites has been carried out for equine herpesvirus-1 (EHV-1) in an attempt to develop markers which can be used to group epidemiologically related viruses into groups, and to learn more about the dynamics of EHV-1 disease. Crude viral DNA extracts of EHV-1, prepared by Hirt extraction, were digested with AluI, HaeIII, or RsaI, and Southern blotted following electrophoresis. DNA fingerprints, produced by probing the Southern blots with the EHV-1 EcoR1-I fragment, separated 56 isolates into 16 groups. The variable sites within the EcoR1-I fragment were mapped app...
Johansson KE, Pettersson B, Uhlén M, Gunnarsson A, Malmqvist M, Olsson E.Seven Swedish isolates of Ehrlichia species from the blood of four dogs and three horses with clinical granulocytic ehrlichiosis, were identified by direct solid phase DNA sequencing of polymerase chain reaction (PCR) products from the 16S rRNA gene. The amplified DNA fragments were produced with primers complementary to the universal regions, U1, U2, U5 and U8 of the 16S rRNA molecule. Identical sequences were obtained from all seven isolates. This nucleotide sequence was similar to the sequences deposited in GenBank for Ehrlichia phagocytophila and E equi. The sequence of the Swedish ehrlich...
Grünig G, Antczak DF.The distribution of four cytokines was analyzed in the endometrium and trophoblast of the horse between Days 30 and 55 of gestation. Endometrial tissues, invasive trophoblast (chorionic girdle), and noninvasive trophoblast (chorion and allantochorion) were examined separately. Cytokine expression was determined by amplification of specific mRNA via the reverse transcriptase polymerase chain reaction (RT-PCR). Messenger RNA for interleukin 2 (IL-2), interleukin 4 (IL-4), and interferon gamma (IFN gamma) was detected in endometrial tissues, unstimulated peripheral blood lymphocytes, and control ...
Navarro P, Barbis DP, Antczak D, Butler JE.The cDNA from a transcript encoding the complete heavy chain of the equine immunoglobulin IgE has been cloned and sequenced. A fragment of the equine epsilon gene was amplified from cDNA using PCR and this fragment was then used to probe a horse cDNA library prepared from peripheral blood lymphocytes. A recombinant clone containing the cDNA encoding the complete horse epsilon chain and its associated V-D-J and leader, was subsequently isolated and sequenced. Comparison of the deduced amino acid sequence of equine IgE with the C epsilon heavy chains of other species indicates it to be most simi...
Niwa H, Anzai T, Hobo S.Contagious equine metritis (CEM) is a highly contagious bacterial venereal disease of horses caused by Taylorella equigenitalis. CEM-PCR is a semi-nested PCR method for detecting this bacterium. Although this technique is regarded as a sensitive diagnostic method for CEM, there are risks of it generating false positive and false negative results. In this study, we constructed a recombinant plasmid (CEM-POS) as reaction control to assure adequate PCR reaction and prevent false positive results caused by contamination of the reaction control in routine CEM-PCR examinations. CEM-POS was construct...
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...
Dong J, Bao H, Mang L.Rhinoestrus sp. (Diptera: Oestridae) is an economically important parasite that can cause severe nasal myiasis in equids and can also affect humans. The ultrastructure of all Rhinoestrus sp. larval instars from Mongolian horse was examined by light and scanning electron microscopy to characterize the features of Rhinoestrus. The structure of the anterior region, posterior region, and the spines of the third segment was analyzed for 10 specimens in each larval stage. Additionally, 34 third-instar (L3) larvae of Rhinoestrus sp. from Mongolian horse were subjected to molecular characterization by...
Bailey E, Lear TL.We compared pools of DNA from 10 Thoroughbred horses and 10 Arabian horses for the presence of randomly amplified polymorphic DNA (RAPD) markers which might be useful in distinguishing between the breeds. Using 212 decamer oligonucleotides and our polymerase chain reaction (PCR) conditions, 173 of the primers produced scoreable bands. The number of bands ranged from 0 to 9 with an average of 3.6. In family studies using 11 arbitrarily selected primers, five of the 11 primers produced polymorphic bands which exhibited Mendelian inheritance as dominant markers. When comparing the pooled DNA from...
Lambertini C, Bombardi C, Zannoni A, Bernardini C, Dondi F, Morini M, Rinnovati R, Spadari A, Romagnoli N.Proteinase activated receptor 4 (PAR) in the gastrointestinal tract is involved in the regulation of inflammation and pain pathways. The aim of the present study was to evaluate the distribution and expression of PAR in the jejunum of healthy horses and in the pathologic tracts from horses undergoing surgery for herniation of the small intestine through the epiploic foramen. Eight healthy horses (Group H) and eight horses with epiploic hernia (Group EH) were included; the jejunum samples were collected at the slaughter or intraoperatively after enterectomy, respectively. To evaluate PAR expres...
Onen EA.The aim of this study was to evaluate formalin-inactivated autovaccination to treat cutaneous papillomatosis and to perform molecular typing of the papillomavirus in four horses (two foals, one 3-year-old filly and a 5-year-old stallion). Methods: Histopathological slides of lesions were prepared and stained with haematoxylin and eosin (H&E) to establish a diagnosis that was based on observation koilocytosis, which is a pathognomonic cytopathic change that is associated with papillomatosis, using light microscopy. Polymerase chain reaction (PCR) and DNA sequencing were performed using the ...
Allano M, Grimes C, Boivin R, Smith G, Dumaresq J, Leclere M.A gelding from eastern Canada was presented for cough and exercise intolerance 14 months after it had travelled on Vancouver Island. Cryptococcus gattii pneumonia was diagnosed based on cytology, antigen titers, and polymerase chain reaction (PCR). The horse was treated with fluconazole for 10 months. Delayed C. gattii infection can occur after travel in an endemic area. Pneumonie à Cryptococcus gattii chez un cheval adulte ayant voyagé dans une région endémique. Un cheval hongre de l’est canadien fut présenté pour de la toux et de l’intolérance à l’exercice 14 mois après avoir ...
Oaks JL, Long MT, Baszler TV.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...
da Silva TRO, Gonçalves PNC, Marcus VB, Mucellini CI, Dos Santos IR, Kommers G, Driemeier D, Flores EF, Cargnelutti JF, Flores MM.For approximately one decade, a novel papillomavirus termed Equus caballus papillomavirus-2 (EcPV-2) has been associated with equine penile/preputial papillomas and squamous cell carcinomas (SCCs). It is currently believed that the virus has a carcinogenic activity, being able to induce such neoplastic lesions. After being first described, EcPV-2 has been detected in many countries from North America, Europe, and Asia; however, to date, it has not been reported in Brazil. The aim of this research was to investigate the presence of EcPV-2 in penile/preputial papillomas and SCCs of Brazilian hor...
Gysens L, Martens A, Haspeslagh M.Bovine papillomavirus (BPV) types 1 and 2 are causally associated with equine sarcoid, the most common mesenchymal neoplasm of horses, but the viral load (VL) differs between lesions. Sensitive and accurate BPV detection and quantification is essential for clinicians to confirm clinical suspicion, as well as in research settings for stratifying these skin lesions. Due to the limitations of histopathology in sarcoid diagnosis, PCR screening of superficial swabs constitutes the principal sampling method for BPV detection. This study aimed to investigate the ability of superficial swabs and fine-...
Mawhinney I, Bollard A.Detection of Taylorella equigenitalis (CEMO) in the horse uses genital swabs. These swabs traditionally have been put in Amies charcoal transport medium for detection by culture but are also used for PCR. We determined the suitability of swabs without transport medium (Dry swabs) for CEMO PCR compared to swabs in Amies charcoal transport medium. The experiment was a factorial design using swab type and dilution of organism in culture suspensions, done in two parts. Simulated genital swabs were prepared in the laboratory by dipping in pairs into culture suspensions containing T. equigenitalis w...
Breuil MF, Duquesne F, Sévin C, Laugier C, Petry S.Contagious equine metritis is a horse disease that causes endometrial inflammation due to Taylorella equigenitalis. Since Taylorella asinigenitalis was characterized, genital swab culture has proved to be an insufficient method for distinguishing between the two Taylorella species. Here, we developed an indirect immunofluorescence (IIF) test using polyclonal antibodies. Specificity, sensitivity, and detection limit were assessed using isolated bacteria (55 T. equigenitalis strains, 46 T. asinigenitalis strains and 18 other bacterial species), experimental and genital swabs in comparison to bac...
Oakey J, Hawkesford T, Smith C, Hewitson G, Tolosa X, Wright L, Moody N, Rodwell B, Corney B, Waltisbuhl D.Describe the in-house validation of a previously reported influenza virus type A 5'Taq nuclease assay for detecting equine influenza virus A RNA in nasal swab material. Methods: The validation compares the 5'Taq nuclease assay with a gel-based reverse transcription nested polymerase chain reaction (PCR) previously reported by the Irish Equine Centre for detection of H3N8 and H7N7 equine influenza viruses. This test was chosen because it targets a different region of the viral genome to the real-time test, so it is not merely a repeat of the same test in a different format. Moreover, nested PCR...
Spiegel IB, White SD, Foley JE, Drazenovich NL, Ihrke PJ, Affolter VK.Nine horses from ages 5 to 21 years were diagnosed with cutaneous equine sarcoidosis (ES) over an 18-year period. In addition to skin, the lungs were frequently involved, with other organ systems affected less commonly. A predisposition for thoroughbreds and geldings was noted. Cutaneous lesions and signs included crusts, scales, alopecia and pruritus. These were found at various sites, particularly the legs/thighs/elbows, thorax, neck, face and ventral abdomen. Three horses were euthanized shortly after hospitalization; others survived as long as 12 years. Histopathologic stains, immunohistoc...
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 ...
Kuhar U, Završnik J, Toplak I, Malovrh T.In 2009, a surprisingly high number of animals seropositive for equine infectious anaemia virus (EIAV; 26 horses from 13 farms) were detected in Slovenia. Objective: To develop a polymerase chain reaction (PCR) assay for the detection of the proviral nucleic acid, to phylogenetically characterise the Slovenian EIAV strains and to investigate whether transmission in utero occurred. Methods: Cross-sectional clinical study. Methods: In total, 26 horses (including 2 foals and 4 pregnant mares) and 4 fetuses were examined in this study. A PCR assay using the EIAV F1 and EIAV R1 primers was designed...
Kniazev SP, Reissmann M, Wagner HJ, Kuraĭ MV, Samovolov NV.Results of the first in Russia survey of the gene pool of the breeding nucleus of the Russian population of thoroughbred horses by means of PCR analysis of the E (Extension) locus MC1R gene mutations are presented. The data on the structure of breeding populations from the leading stud farms Voskhod and Oros with regard to color phenotypes as well as genotype and allele frequencies are presented. The population structure parameters are discussed with respect to possible specific features of microevolution processes.
Dimsoski P.To show that application of the polymerase chain reaction (PCR) method modified for amplification of a low-copy number DNA samples, ie, the isolation of PCR products (IPCRp), would represent improvement in obtaining genotypes from a fecal DNA compared with previously used genotyping methods. Methods: The DNA from the horse fecal matter was extracted by modified Qiagen DNA Stool Mini Kit protocol. Following the extraction, the DNA genotypes from fecal samples were obtained by the most powerful PCR amplification method, the IPCRp. The IPCRp-based multiplex kit amplified biotin-labeled strands we...
Lim QL, Tan YL, Ng WL, Yong CSY, Ismail A, Rovie-Ryan JJ, Rosli N, Annavi G.A molecular sexing method by polymerase chain reaction (PCR) amplification of a portion of the sex-determining region Y (SRY) and the zinc finger (ZF) gene, as well as six equine Y-chromosome-specific microsatellite markers, were tested in the Malayan tapir (Tapirus indicus). While the microsatellite markers did not yield any male-specific amplicons for sex-typing, the SRY/ZF marker system produced reliable molecular sexing results by accurately sex-typing 31 reference Malayan tapirs, using whole blood, dried blood spot (DBS), or tissue samples as materials for DNA extraction. The marker syste...
Martins AV, Coelho AL, Corrêa LL, Ribeiro MS, Lobão LF, Palmer JPS, Moura LC, Molento MB, Barbosa ADS.The frequency of gastrointestinal parasites with an emphasis on Strongylus vulgaris was investigated among the Brazilian Pony breed kept on farms in the municipality of Teresópolis, state of Rio de Janeiro. Fecal samples were collected in three stud farms: A (n= 22 animals), B (n= 3), and C (n= 2). Fecal samples were subjected to the quantitative Mini-FLOTAC technique, using three different solutions, and to qualitative techniques. The parasite prevalence was found to be 81.4%. Eggs from strongylids were identified in 74% of the ponies. Eggs of Parascaris spp. were detected in 22.7% of the an...
Csellner H, Walker C, Love DN, Whalley JM.An equine herpesvirus 1 (EHV-1) mutant was constructed by inserting a lacZ expression cassette into the intergenic region upstream of gene 62 (glycoprotein L; gL) and downstream of gene 63 (a homologue of the herpes simplex virus transcriptional activator ICP0). The recombinant lacZ62/63-EHV-1 had similar growth kinetics in cell culture to those of the parental wild type (wt) virus, with indistinguishable cytopathic effects and plaque morphology. Reverse transcriptase PCR confirmed that the lacZ insertion did not interfere with transcription of gL and immunoblot analysis indicated there was no...
Stumpf G, Fietz D, Ezer J, Litzke LF, Bergmann M.Surgically removed testicular tissue in cryptorchid stallions is sometimes difficult to identify because of morphological and histological malformation. Therefore, a sure method to characterise the removed tissue is required. A 2-year-old Haflinger stallion was castrated after diagnosis of cryptorchidism to remove the left intra-abdomnial testis. Intra-operative exploration of the abdominal cavity revealed a firm, dysmorphic structure, which could not be identified as testis based on macroscopic anatomy. The removed tissue was Bouin-fixed and paraffin-embedded for histological examination. We ...
Pomelova VG, Gaĭdamovich SIa, Demenev VA, Kadoshnikov IuP.A three-step concentration of Venezuelan equine encephalomyelitis (VEE) virus from tissue culture fluid was carried out in a two-phase system of polyethyleneglycol (PEG)--sodium dextran sulphate (SDS). The concentration method was based on the dependence of virus distribution coefficient upon NaCl content in the system which allowed alternating transfer of the virus from one phase of the system into the other. The infectious activity of the virus increased approximately 100-fold after the first step, 190-fold after the second, and 300-fold after the third step. The process of concentration was...
Xiang W, Ma J, Wang XF, Zhao YJ, Zhou JH.In this article, we report the analysis of genetic polymorphisms of horse MHC-I molecules by SSCP and HMA, which are methods based on the technique of polyacrylamide gel electrophoresis (PAGE). Our results showed that SSCP was not a suitable method for the analysis of genetic polymorphisms of horse MHC-I molecules due to the failure in generating satisfied separation of DNA fragments, even if experimental conditions were optimized. However, the HMA method produced clearly separated DNA fragments of horse MHC-I molecules, after the experimental conditions, such as the running temperature and th...
Kato H, Youn HY, Ohashi T, Watari T, Goitsuka R, Tsujimoto H, Hasegawa A.Using lipopolysaccharide (LPS)-stimulated equine peripheral blood mononuclear cell (PBMC) cDNA as a template, we performed polymerase chain reaction (PCR) amplification with equine interleukin-1 beta (IL-1 beta) specific primers. Electrophoresis of the PCR product on agarose gel revealed an additional smaller fragment that hybridized with an equine IL-1 beta cDNA probe. Sequencing of this fragment demonstrated that it was shorter than normal equine IL-1 beta cDNA by 162 nucleotides, which corresponded to exon 5 of the human and murine IL-1 beta genes. The deletion of 162 nucleotides did not re...
Gupta AK, Kaur D, Rattan B, Yadav MP.Three abortigenic Indian isolates of equine herpesvirus-1 (EHV-1) (Tohana, Hisar and Bikaner), along with two exotic abortigenic isolates (AB4 and V592) and another EHV-1 isolate (Jind) obtained from a case of perinatal foal mortality, were studied for variability. For this purpose, PCR and restriction endonuclease (RE) digestion techniques were used simultaneously as a DNA fingerprinting system. Nine different regions of EHV-1 virus were amplified by PCR using primer pairs specific for the regions and the products obtained from these regions were subsequently subjected to various restriction ...
Velineni S, Timoney JF, Artiushin SC, Donahue JM, Steinman M.Foals of mares infected with Leptospira interrogans serovar Pomona type kennewicki (Lk) may be aborted/stillborn or delivered as healthy foals. Is fetal survival explained in part by the immune response of the fetus to Leptospira antigens? Objective: To describe an outbreak of Leptospira abortion in which infected mares delivered dead/sick or normal foals and determine specificities of antibody in a collection of 54 fetuses from similar outbreaks. Methods: Outbreak investigation in combination with a case-control study of a larger set of samples from aborted fetuses. Methods: Serology and poly...
O'Keefe JS, Julian A, Moriarty K, Murray A, Wilks CR.A detection system incorporating the polymerase chain reaction was compared with the use of histopathology and virus isolation to determine the presence of equid herpesvirus type 1 or equid herpesvirus type 4 in equine tissues submitted to a diagnostic laboratory. When the polymerase chain reaction was performed, these tissues had been stored for up to 3 years. Thirty-eight tissues representing 14 cases had been stored embedded in paraffin wax. Analysis of these tissues using the PCR gave predictive values of 1.0 and 0.91 for a positive and negative result respectively, and sensitivity and spe...
Sarma PN, Tang YJ, Prindiville TP, Osborne PD, Jang S, Silva J, Cohen SH.In order to determine genetic relatedness of Bacteroides fragilis isolates from different clinical sources, arbitrarily primed polymerase chain reaction (PCR) (AP-PCR) was used to compare 17 strains isolated from patients with inflammatory bowel disease (IBD) and 20 strains isolated from foals with diarrhea. Three reference ATCC strains were also analyzed. Eighteen unique types were identified with a 22-mer arbitrary primer (ERIC-2) among the 20 patient isolates. Types 1 (enterotoxigenic) and 9 (nonenterotoxigenic), were each found in the stools of two patients. All other isolates showed a dis...
Nadruz V, Beard LA, Delph-Miller KM, Larson RL, Bai J, Chengappa MM.Prevention of spread of Streptococcus equi subspecies equi (S. equi) after an outbreak is best accomplished by endoscopic lavage of the guttural pouch, with samples tested by culture and real time, quantitative polymerase chain reaction (qPCR). Disinfection of endoscopes must eliminate bacteria and DNA to avoid false diagnosis of carrier horses of S. equi. Objective: Compare failure rates of disinfection of endoscopes contaminated with S. equi using 2 disinfectants (accelerated hydrogen peroxide [AHP] or ortho-phthalaldehyde [OPA]). The null hypothesis was that there would be no difference bet...
