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.
Henning K, Sachse K, Sting R.The isolation and identification of a chlamydial agent from an equine fetus is reported. The fetus was aborted by a mare with respiratory disease and fever in the 9th month of pregnancy. The serum of the mare was investigated by the compliment fixation test. Specific antibodies were detected for chlamydial antigen in a titer of > 1:40 and for equine herpes virus 1 antigen in a titer of 1:32. Pathological lesions were not found in the organs of the fetus. Chlamydiae were detected in the placenta by ELISA and subsequently isolated by cell culture. Using PCR technique the agent was identified ...
Cañon J, Checa ML, Carleos C, Vega-Pla JL, Vallejo M, Dunner S.Partition of the genetic variability, genetic structure and relationships among seven Spanish Celtic horse breeds were studied using PCR amplification of 13 microsatellites on 481 random individuals. In addition, 60 thoroughbred horses were included. The average observed heterozygosity and the mean number of alleles were higher for the Atlantic horse breeds than for the Balearic Islands breeds. Only eight percentage of the total genetic variability could be attributed to differences among breeds (mean FST approximately 0.08; P < 0.01). Atlantic breeds clearly form a separate cluster from th...
Abdulmawjood A, Lämmler CH.The 16S rRNA gene of 39 S. equi subsp. zooepidemicus strains and two S. equi subsp. equi strains was amplified by polymerase chain reaction and subsequently digested with the restriction enzyme Hinc II. A restriction profile with two fragments with sizes of 1250 bp and 200 bp could be observed for both S. equi subsp. equi strains and for 30 of the 39 S. equi subsp. zooepidemicus strains indicating a sequence variation within the V2 region of the 16S rRNA gene of the remaining nine S. equi subsp. zooepidemicus isolates. A segment of the 16S rRNA gene including the hypervariable V2 region of 11 ...
Anzai T, Eguchi M, Sekizaki T, Kamada M, Yamamoto K, Okuda T.In order to establish a rapid diagnostic method for contagious equine metritis (CEM), we developed and evaluated a polymerase chain reaction (PCR) test. Species-specific PCR primer sets were derived from the DNA sequence of a cloned DNA fragment of Taylorella equigenitalis that did not hybridize with the genome of a taxomonically related species, Oligella urethralis. Single step PCR with primer set P1-N2 and two-step semi-nested PCR with primer sets P1-N2 and P2-N2 detected as low as 100 and 10 CFU of the bacteria, respectively. Single-step PCR detected T. equigenitalis from genital swabs of e...
Chang YF, Novosol V, McDonough SP, Chang CF, Jacobson RH, Divers T, Quimby FW, Shin S, Lein DH.Seven specific-pathogen-free (SPF) ponies, 1-5 years old, were exposed to Borrelia burgdorferi-infected adult ticks while being treated with dexamethasone over 5 consecutive days. One SPF pony (pony No. 178) was first exposed to laboratory-reared nymphs without B. burgdorferi infection and 3 weeks later was exposed to B. burgdorferi-infected adult ticks with concurrent dexamethasone treatment for 5 consecutive days. Four uninfected ponies treated with dexamethasone, exposed to laboratory-reared ticks without B. burgdorferi infection served as uninfected controls. Clinical signs, bacteriologic ...
Clausen PH, Gebreselassie G, Abditcho S, Mehlitz D, Staak C.A field study of horses was conducted in the province of Bale, Ethiopian highlands. A rapid questionnaire analysis indicated that dourine, known as "Dirressa", is a major health problem of equines in this area. A total of 121 horses suspected of dourine were examined by use of clinical, parasitological, serological and DNA based techniques. Incoordination of hindlegs (76%), swelling of external genitalia (48.8%) and emaciation (39.7%) were the most common clinical signs observed. Using the haematocrit centrifugation technique (HCT), no trypanosomes were detected in blood, genital washes or tis...
Chang YF, McDonough SP, Chang CF, Shin KS, Yen W, Divers T.A pony was vaccinated with recombinant OspA vaccine (rOspA) and then exposed 3 months later to Borrelia burgdorferi-infected ticks (Ixodes scapularis) collected in Westchester County, N.Y. At 2 weeks after tick exposure, the pony developed a high fever (105 degrees F). Buffy coat smears showed that 20% of neutrophils contained ehrlichial inclusion bodies (morulae). Flunixin Meglumine (1 g daily) was given for 2 days, and the body temperature returned to normal. PCR for ehrlichial DNA was performed on blood samples for 10 consecutive days beginning when the pony was first febrile. This pony was...
Leutenegger CM, von Rechenberg B, Huder JB, Zlinsky K, Mislin C, Akens MK, Auer J, Lutz H.Specific amplification and quantitation of nucleic acid sequences by the polymerase chain reaction (PCR) has been extensively used for the detection of viral infection and gene expression. Although successful amplification of DNA and RNA sequences extracted from paraffin embedded tissue have been described, there are presently no reports available regarding RNA analysis from bone and calcified tissues embedded in hydrophobic acrylic resin. Here we describe a general method for quantitation of specific mRNA sequences extracted from undecalcified bone sections, fixed in paraformaldehyde, and emb...
Pusterla N, Pusterla JB, Braun U, Lutz H.Four cows and four horses were infected experimentally with Ehrlichia phagocytophila, the cause of tickborne fever in ruminants, and with human granulocytic ehrlichia-like agent, a recently discovered species that infects people, horses and dogs in the USA and Europe. They were infected in either order, 30 days apart, to investigate serological cross-reactivity within the Ephagocytophila genogroup. The course of infection was assessed by routine clinical, haematological, serological and polymerase chain reaction (PCR) examinations. Two of the cows infected with Ephagocytophila and two of the h...
Akiba M, Sameshima T, Anzai T, Wada R, Nakazawa M.Most Salmonella choleraesuis subsp. choleraesuis serovar Abortusequi strains of equine origin harbor a 95kb plasmid, pSA95. Results of PCR and Southern blot analysis suggest that pSA95 contains spv genes. A pSA95-cured strain of S. Abortusequi was 48 times less virulent to mice than its parental strain. Virulence was restored by reintroduction of pSA95. These results provide clear evidence that pSA95 confers virulence on S. Abortusequi in mice. This is the first report describing a virulence plasmid of S. Abortusequi.
Pfeffer M, Burck G, Meyer H.Five case reports on cowpox virus infections in cats, humans, and for the first time in a horse are presented. It becomes obvious that in most cases the diagnosis cowpox is suspected rather late, although fast and reliable diagnostic tools such as pathohistological examination and polymerase chain reaction are available. The threat of a zoonotic transmission mainly through cats is gaining importance. Although wild rodents have been claimed to be the reservoir and source for cowpox viruses in cats, very little is known about the epidemiology of cowpox virus. Based on the different genome organi...
Brees DJ, Sondhoff AH, Kluge JP, Andreasen CB, Brown CM.A 7-month-old foal was admitted to the hospital with a history of lethargy, weight loss, mild diarrhea, and anorexia. A diagnosis of proliferative enteritis caused by Lawsonia intracellularis-like organisms was made after necropsy and histologic examination of the small intestine. Although infection with L intracellularis-like organisms is a rare cause of enteritis in foals, it should be considered in the differential diagnosis, especially if the foal was housed in the proximity of pigs or pig feces. Antemortem diagnosis remains challenging because isolation of the organism in fecal material r...
Ehlers B, Borchers K, Grund C, Frölich K, Ludwig H, Buhk HJ.A consensus primer PCR approach was used to (i) investigate the presence of herpesviruses in wild and zoo equids (zebra, wild ass, tapir) and to (ii) study the genetic relationship of the herpesvirus of pigeons (columbid herpesvirus 1) to other herpesvirus species. The PCR assay, based on degenerate primers targeting highly conserved regions of the DNA polymerase gene of herpesviruses, was modified by using a mixture of degenerate and deoxyinosine-substituted primers. The applicability of the modification was validated by amplification of published DNA polymerase genes of 16 herpesvirus specie...
Hung GC, Gasser RB, Beveridge I, Chilton NB.The first and second internal transcribed spacer sequences of 28 morphologically-defined species of horse strongyle were characterized, and specific oligonucleotide primers were designed for some species based on the nucleotide differences. Utilizing these primers, a PCR approach was developed for the specific amplification of ribosomal DNA of Strongylus vulgaris, Cyathostomum catinatum, Cylicocyclus nassatus, Cylicostephanus longibursatus or Cylicostephanus goldi. The method allowed the species-specific amplification of parasite DNA derived from faecal samples and/or copro-cultures, demonstra...
Bashiruddin JB, Cammà C, Rebêlo E.Babesia equi and Babesia caballi are tick-borne haemoparasites that may cause babesiosis of Equidae. In southern Europe B. equi is enzootic and infections may occur asymptomatically and more frequently than those due to B. caballi. Complement fixation test (CFT) is the official serological test for the diagnosis of equine babesiosis, but it has low sensitivity during early and latent stages of the disease. With the aim of developing more sensitive and rapid direct diagnostic alternatives, PCR systems that amplified DNA targets of 664 or 659 bp regions of the 16S rRNA genes were designed and de...
Oxburgh L, Hagström A.In this paper we describe the development of a nested RT-PCR assay for the rapid diagnosis and characterisation of influenza virus directly from clinical specimens. Viral RNA is extracted from nasal swabs by the guanidine thiocyanate extraction method, and subsequently reverse transcribed. The complementary DNA is then used as template in a nested PCR reaction. Primers designed for use in this assay are specific for three templates; (1) the nucleoprotein (NP) gene, (2) the haemagglutinin gene of the H7N7 equine influenza virus (A1), and (3) the haemagglutinin gene of the H3N8 equine influenza ...
Ramina A, Dalla Valle L, De Mas S, Tisato E, Zuin A, Renier M, Cuteri V, Valente C, Cancellotti FM.The reverse transcriptase polymerase chain reaction (RT-PCR) assay was used to detect Equine Arteritis Virus (EAV) in the semen of 88 horses and 2 donkeys, with neutralising antibodies against EAV, on the basis of the amplification of a 279 bp long fragment located in the viral polymerase gene. The RT-PCR assay revealed the virus at 4 TCID50/ml in cell culture and showed a greater sensitivity (54.4%) than cell culture isolation (33.3%). Moreover, the two samples of donkey semen were found positive. The cDNAs obtained from 14 samples of horse and 2 of donkey semen were sequenced. Comparing the ...
Borchers K, Frölich K, Ludwig H.In blood samples of seven captive equid species from four German zoos EHV-1 specific antibodies were detected in 76% and EHV-4 specific antibodies in 73% of the 55 animals, whereas 93% were tested positive for EHV-2 and EHV-5, respectively. In only one blood sample from a Przewalski's wild horse EHV-4 DNA was amplified by PCR. From seven Przewalski's wild horses EHV-2, and from another one EHV-5 was isolated by cocultivation. The identity of the virus isolates was verified by PCR and restriction enzyme digestion.
Starick E.A reverse transcription-polymerase chain reaction (RT-PCR) assay using four different primer pairs for the detection of equine arteritis virus (EAV) RNA in semen and tissue samples was evaluated. A fragment encoding the leader sequence of the EAV genome was most successfully amplified. The specificity and sensitivity of RT-PCR was assessed by virus isolation in cell culture, restriction analysis, dot blot hybridisation and nested PCR. To this end, 23 semen samples from seropositive stallions and 11 tissue samples from 4 aborted foals were tested. Compared to the virus isolation test in cell cu...
Ishida N, Katayama Y, Sato F, Hasegawa T, Mukoyama H.The entire cDNA sequences were determined by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) techniques for equine copper/zinc superoxide dismutase (Cu/Zn-SOD) and manganese superoxide dismutase (Mn-SOD) through the use of total RNA extracted from the testis of an adult Thoroughbred. The results revealed a protein coding region for equine Cu/Zn-SOD with bases totaling 465 bp, accompanied by an estimated 154 residues of amino acids. As for equine Mn-SOD, its coding region contained a total of 669 bp and an estimated 222 residues of amino acid...
Magnarelli LA, Van Andel AE, Ijdo JW, Heimer R, Fikrig E.To diagnose granulocytic ehrlichiosis in horses, compare results of indirect fluorescent antibody (IFA) staining procedures with those of immunoblot analysis, and compare serologic test findings with polymerase chain reaction (PCR) results. Methods: 69 horses with high rectal temperatures (> or = 39 C) and lethargy, anorexia, or limb edema. Methods: 43 convalescent serum samples obtained from 38 horses 2 to 18 weeks after onset of illness were analyzed by use of immunoblot procedures and IFA staining methods, using the NCH-1 or BDS ehrlichial strains. Blood samples from 69 acutely ill horse...
Raudsepp T, Chowdhary BP.A pilot study comparing horse and donkey karyotypes on a molecular basis was initiated using the chromosomal microdissection approach. All equine meta- and submetacentric chromosomes, viz. ECA1 to ECA13 and the X and Y chromosomes, were microdissected. The DNA was PCR amplified, non-radioactively labelled and used as probes on equine metaphase chromosomes to confirm their origin. Once tested, the paints were used as probes on donkey metaphase chromosomes to detect homologous chromosomal segments between the two species. The results not only detected conservation of whole chromosome and/or arm ...
Tanhauser SM, Yowell CA, Cutler TJ, Greiner EC, MacKay RJ, Dame JB.Studies designed to investigate the causative agent of equine protozoal myeloencephalitis and its life cycle have been hampered by the marked similarity of Sarcocystis neurona to other Sarcocystis spp. present in the same definitive host. Random-amplified polymorphic DNA techniques were used to amplify DNA from isolates of S. neurona and Sarcocystis falcatula. DNA sequence analysis of polymerase chain reaction (PCR) products was then used to design PCR primers to amplify specific Sarcocystis spp. DNA products. The ribosomal RNA internal transcribed spacer was also amplified and compared betwee...
Hammond RA, Hannon R, Frean SP, Armstrong SJ, Flower RJ, Bryant CE.To determine the amount of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) enzymes induced in vitro in equine alveolar macrophages in response to lipopolysaccharide (LPS). Sample Population-Alveolar macrophages obtained from 12 horses. Methods: Alveolar macrophages were collected by bronchoalveolar lavage from 12 horses and incubated for 6 hours with LPS (0.001 to 10 microg/ml) or vehicle. Total RNA was extracted and purified. After first-strand cDNA synthesis, mRNA induction was measured, using a polymerase chain reaction (PCR) technique for COX-2, iNOS, and glyceraldehyde...
Wijesundera WS, Chandrasekharan NV, Karunanayake EH.A sensitive PCR assay for the detection of Setaria digitata has been developed. Two oligonucleotide primers (17 nt) were designed from a previously cloned and characterized tandemly arranged repetitive sequence of Setaria digitata. Using these primers, it was possible to amplify small quantities (100 fg) of S. digitata genomic DNA. A simple procedure, using proteinase K and non-ionic detergent NP 40, was followed to process the host blood samples and mosquitoes harbouring L3 larvae. The sensitivity of the polymerase chain reaction based assay surpasses the microscopic detection and the previou...
Caetano AR, Pomp D, Murray JD, Bowling AT.Polymerase chain reaction primers designed from horse cDNA sequences and from consensus sequences highly conserved in mammalian species were used to amplify markers for synteny mapping 18 equine type I genes. These markers were used to screen a horse-mouse somatic cell hybrid panel (UCDavis SCH). Fourteen primer sets amplified horse-specific fragments, while restriction enzyme digests of PCR products were used to distinguish the fragments amplified from horse and mouse with four primer sets. Synteny assignments were made based on correlation values between each marker tested and other markers ...
Swiderski CE, Klei TR, Horohov DW.Quantification of cytokine mRNA using reverse transcription coupled with the polymerase chain reaction (RT-PCR) has become a corner stone of the study of cytokine regulation. Quantitative competitive RT-PCR (QCRT-PCR) is commonly accepted as a reliable method for quantifying differences in mRNA levels but is both labor- and reagent-intensive. A noncompetitive polymerase chain reaction method that utilizes cytokine-specific, plasmid-derived, standard curves was developed for the quantification of equine cytokine mRNA. The assay can be performed on minute samples of cellular material, utilizes s...
Giguère S, Prescott JF.A reverse transcription-competitive polymerase chain reaction (RT-cPCR) method was developed to quantitate equine interleukin (IL)-1alpha, IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12 p35, IL-12 p40, interferon-gamma (INF-gamma), tumor necrosis factor-alpha (TNF-alpha), and beta-actin mRNA expression. Using primers based on equine-specific sequences, these cytokines could be detected in concanavalin A-stimulated peripheral blood mononuclear cells. The specificity of the amplified product was confirmed by sequencing. For each cytokine, the assay was made quantitative by generating competitor ...
Herholz C, Miserez R, Nicolet J, Frey J, Popoff M, Gibert M, Gerber H, Straub R.The incidence of a new, yet unassigned toxin type of Clostridium perfringens containing the genes for the alpha-toxin and the recently described beta2-toxin in horses with intestinal disorders is reported. The study included 18 horses suffering from typical typhlocolitis, 7 horses with atypical typhlocolitis, 16 horses with other intestinal disorders, and 58 horses without intestinal disease. In total, 20 samples of ingesta of the small and large intestines, five biopsy specimens of the intestinal wall, and 74 fecal samples were analyzed bacteriologically. C. perfringens isolates were typed fo...
Miragliotta V, Lefebvre-Lavoie J, Lussier JG, Theoret CL.Horses suffer from a debilitating impediment in repairing wounds located on the lower limb that leads to the development of a fibroproliferative disorder (exuberant granulation tissue). This condition is a source of wastage since it often forces retirement from competition. Treatments that resolve or prevent this condition are still lacking, maybe due to deficient knowledge of the underlying molecular mechanisms. Fibroblast-to-myofibroblast conversion is an essential step allowing contraction during wound repair and is accompanied by an increase in OB-cadherin expression. Objective: To clone e...
Starick E.A reverse transcription-polymerase chain reaction (RT-PCR) assay using four different primer pairs for the detection of equine arteritis virus (EAV) RNA in semen and tissue samples was evaluated. A fragment encoding the leader sequence of the EAV genome was most successfully amplified. The specificity and sensitivity of RT-PCR was assessed by virus isolation in cell culture, restriction analysis, dot blot hybridisation and nested PCR. To this end, 23 semen samples from seropositive stallions and 11 tissue samples from 4 aborted foals were tested. Compared to the virus isolation test in cell cu...
Guèrand M, Mahla R, Lagneaux D, Amigues Y, Palmer E, Bézard J.Paternity analysis was performed on the DNA of 21 equine embryos collected nonsurgically 10 days after ovulation from known mares, but involving 3 possible sires. After extraction, the DNA of each embryo was typed by radioactive PCR amplification using 10 characterised microsatellites; HMS 1, 2, 5, 6, 7 and 8 (Guérin et al. 1994) and HTG 3, 4, 6 and 10 (Marklund et al. 1994). The 21 dams and 3 sires were genotyped using DNA extracted from blood and amplified by PCR. After electrophoresis and autoradiography of the PCR products of the embryo and parents, the alleles of the embryo were compared...
Huhtinen M, Peippo J, Bredbacka P.Embryo biopsy has been used to detect inherited disorders and to improve the phenotype by analyzing of linkages between marker loci and the desired characteristics. Unfortunately, early procedures required the removal of a large portion (one-half) of the embryo for analysis, and the transfer of bisected equine embryos has not been particularly successful. Recent discovery of the polymerase chain reaction (PCR) has made possible the detection of specific DNA sequences from only a few cells. We investigated whether the removal of a small biopsy would allow for successful PCR and normal embryonic...
Peano A, Arnoldi S, Čmoková A, Hubka V.This article reports the first verified cases of infection by Trichophyton bullosum in Africa since the description of the fungus, isolated in 1933 from the coat of horses in Tunisia and Mali. We found the fungus in cutaneous samples obtained from donkeys suffering from severe dermatitis with areas of alopecia and scaling in the surroundings of Cairo (Egypt). Fungal elements (arthroconidia and hyphae) were seen at the microscopy of material collected by skin scraping and digested in NaOH. Fungal colonies grown on various culture media were identified through PCR and sequencing of the ITS rDNA ...
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.
Zeiss C, Neaderland M, Yang FC, Terwilliger G, Compton S.To assess the diagnostic utility of fungal polymerase chain reaction (PCR) in forty-three horses with naturally acquired corneal ulcers presenting to a private practice. Methods: Routine evaluation of cytologic, histologic, and microbiologic samples was performed. Two PCR approaches were compared - generic and specific fungal nested PCR followed by sequencing and quantitative PCR (qPCR). PCRs were applied to pure control fungal cultures, corneal tissue from ulcerated eyes and in a subset of 9 horses, to swabs from contralateral normal eyes. Results: The expected fungus was identified by nested...
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.
de la Fuente J, Massung RF, Wong SJ, Chu FK, Lutz H, Meli M, von Loewenich FD, Grzeszczuk A, Torina A, Caracappa S, Mangold AJ, Naranjo V, Stuen S....The causative agent of human granulocytic ehrlichiosis was recently reclassified as Anaplasma phagocytophilum, unifying previously described bacteria that cause disease in humans, horses, dogs, and ruminants. For the characterization of genetic heterogeneity in this species, the homologue of Anaplasma marginale major surface protein 4 gene (msp4) was identified, and the coding region was PCR amplified and sequenced from a variety of sources, including 50 samples from the United States, Germany, Poland, Norway, Italy, and Switzerland and 4 samples of A. phagocytophilum-like organisms obtained f...
Anzai T, Eguchi M, Sekizaki T, Kamada M, Yamamoto K, Okuda T.In order to establish a rapid diagnostic method for contagious equine metritis (CEM), we developed and evaluated a polymerase chain reaction (PCR) test. Species-specific PCR primer sets were derived from the DNA sequence of a cloned DNA fragment of Taylorella equigenitalis that did not hybridize with the genome of a taxomonically related species, Oligella urethralis. Single step PCR with primer set P1-N2 and two-step semi-nested PCR with primer sets P1-N2 and P2-N2 detected as low as 100 and 10 CFU of the bacteria, respectively. Single-step PCR detected T. equigenitalis from genital swabs of e...
Bakheit MA, Torra D, Palomino LA, Thekisoe OM, Mbati PA, Ongerth J, Karanis P.Three LAMP (loop-mediated isothermal DNA amplification) assays were applied to detect Cryptosporidium species DNA in a total number of 270 fecal samples originating from cattle, sheep and horses in South Africa. DNA was extracted from 0.5 g of fecal material. Results of LAMP detection were compared to those obtained by nested PCR targeting the Cryptosporidium 18 small subunit rRNA (18S) gene. All samples were negative by nested PCR, while up to one-third of samples were positive by LAMP assays. The SAM-1 LAMP assay, shown to detect C. parvum, C. hominis and C. meleagridis, amplified Cryptospor...
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...
M'ghirbi Y, Yaïch H, Ghorbel A, Bouattour A.Anaplasma phagocytophilum , the causative agent of granulocytic anaplasmosis, affects several species of wild and domesticated mammals, including horses. We used direct and indirect methods to compare and evaluate exposure to A. phagocytophilum in horses in northern Tunisia. Methods: Serum from 60 horses was tested by IFA for antibodies to A. phagocytophilum , and whole blood was tested for A. phagocytophilum 16S rRNA gene using a nested-PCR. To examine the risk of A. phagocytophilum transmission, 154 ticks that had been collected from horses were examined for the presence of A. phagocytophilu...
Pusterla N, Mapes S, Johnson C, Slovis N, Page A, Gebhart C.The purpose of the current study was to compare the molecular detection rate of Lawsonia intracellularis between feces and rectal swabs collected from 42 foals with suspected equine proliferative enteropathy (EPE). Fecal samples and rectal swabs were processed for DNA purification by using an automated extraction system. The purified DNA was then analyzed by real-time polymerase chain reaction (PCR) for the presence of the aspartate ammonia lyase (aspA) gene of L. intracellularis. Absolute quantitation was calculated by using a standard curve for L. intracellularis and expressed as copy number...
