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...
Weese JS, Saab M, Moore A, Cai H, McClure JT.To compare PCR and culture results for the detection of subspecies . Respiratory tract samples (N = 158) from horses being tested for Bacterial culture was carried out on samples from which was detected by quantitative real-time PCR. was isolated from 12 (7.6%) samples: 4/9 (44%) samples when the PCR cycle threshold (C) was ≤ 30, 7/30 (23%) when the C was 30.1 to 35, and 1/119 (0.8%) when the C was 35.1 to 40. The highest C sample from a sample that yielded a positive culture was 36.9. The optimal Youden's J value was at a C of 34.2, the same value as determined by number needed to misdi...
Boldbaatar B, Bazartseren T, Koba R, Murakami H, Oguma K, Murakami K, Sentsui H.In the current study, primers described previously and modified versions of these primers were evaluated for amplification of full-length gag genes from different equine infectious anemia virus (EIAV) strains from several countries, including the USA, Germany and Japan. Each strain was inoculated into a primary horse leukocyte culture, and the full-length gag gene was amplified by reverse transcription polymerase chain reaction. Each amplified gag gene was cloned into a plasmid vector for sequencing, and the detectable copy numbers of target DNA were determined. Use of a mixture of two forward...
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...
Jaramillo-Morales C, Gomez DE, Renaud D, Arroyo LG.The objectives of this study were to determine the prevalence of S. equi in a horse population in Colombia, to determine the risk factors associated with its detection in the guttural pouches and to report the antimicrobial susceptibility of the isolates. Fifteen farms and 137 horses >6 months of age were enrolled. Sampling was randomly, stratified and proportional to the population size of each farm. The guttural pouch (GP) was swabbed via endoscopic guidance and culture was performed. DNA extraction and conventional PCR were performed in colonies compatibles with S. equi, the PCR products we...
Saleem S, Ijaz M, Farooqi SH, Ghaffar A, Ali A, Iqbal K, Mehmood K, Zhang H.Equine granulocytic anaplasmosis (EGA) is an important tick borne disease of equines that is caused by Anaplasma phagocytophilum (A. phagocytophilum). The etiological agent has veterinary as well as public health importance because of its zoonotic nature. A. phagocytophilum causes an acute illness in equines with loss of appetite, lethargy, hemorrhages and lameness. Clinically, EGA is diagnosed upon examination of morulae within neutrophils especially granulocytes in the blood. The best diagnostic tool for the detection of EGA is Polymerase chain reaction (PCR). Previous studies suggested that...
East LM, Savage CJ, Traub-Dargatz JL, Dickinson CE, Ellis RP.To identify clinical signs, physical examination findings, results of diagnostic tests, treatments administered, and clinical outcome of neonatal foals with enterocolitis associated with Clostridium perfringens infection. Methods: Retrospective study. Methods: 54 neonatal foals. Results: Most foals had acute onset of obtunded mentation, colic, or diarrhea and developed leukopenia, neutropenia, an abnormally high number of band neutrophils, toxic WBC, and hypoproteinemia within 24 hours after admission, despite high serum IgG concentrations (> 800 mg/dl). Abdominocentesis and abdominal radiogra...
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 ...
Thean S, Elliott B, Riley TV.During a 24 month period from 2007 to 2009, 174 faecal specimens from horses in Australia (predominantly from Western Australia) were tested for Clostridium difficile. C. difficile was isolated from 14 (23 %) of 62 diarrhoeal animals (including 10 foals) and from none of 112 healthy adult horses. These isolates were toxin profiled by PCR for toxin A, toxin B and binary toxin, and ribotyped. Ten of the equine isolates were A(+)B(+)CDT(-). Other toxin profiles detected were A(-)B(-)CDT(+) (one isolate), A(+)B(+)CDT(+) (two isolates) and A(-)B(-)CDT(-) (three isolates). There were six different...
Bodaan CJ, Wise LM, Wakelin KA, Stuart GS, Real NC, Mercer AA, Riley CB, Theoret C.Healing is delayed in limb wounds relative to body wounds of horses, partly because of sustained inflammation and inefficient angiogenesis. In laboratory animals, proteins derived from orf virus modulate these processes and enhance healing. We aimed to compare immune cell trafficking and the inflammatory, vascular, and epidermal responses in body and limb wounds of horses and then to investigate the impact of orf virus interleukin-10 and vascular endothelial growth factor-E on these processes. Standardized excisional wounds were created on the body and forelimb of horses and their progression ...
Sacco SC, Ortega J, Navarro MA, Fresneda KC, Anderson M, Woods LW, Moore J, Uzal FA.Gas gangrene occurs in several animal species and is caused by one or more clostridial species. In horses, the disease is most often caused by Clostridium perfringens type A. Although Clostridium sordellii has been associated with gas gangrene in ruminants and humans, cases of the disease associated with this microorganism have not been described in horses, to our knowledge. We report herein 8 cases of gas gangrene caused by C. sordellii in horses. These cases were characterized by myonecrosis and cellulitis, associated with systemic changes suggestive of toxic shock. The diagnosis was confirm...
Andrade DGA, Basso RM, Castiglioni MCR, Silva JP, Machado VMV, Laufer-Amorim R, Borges AS, Oliveira-Filho JP.Four causative mutations (D1, D2, D3*, and D4) of chondrodysplastic dwarfism have been described in the equine () gene. Homozygotes for one of these mutations and heterozygotes for any combination of these mutations exhibit the disproportionate dwarfism phenotype. However, no case description of homozygotes for D4 (D4/D4) has been reported in the literature, to our knowledge. We report 2 Miniature horses with the genotype D4/D4 in the gene. Clinically, the 2 dwarfs had a domed head that was large compared to the rest of the body, mandibular prognathism, and short and bowed limbs, mainly in t...
Mawhinney I, Davis N, Carson T, Torrens N, Wales A.The study examined and compared the sensitivity of culture and a quantitative PCR assay for screening equine semen for the presence of Taylorella equigenitalis (CEMO). Chilled semen samples, both raw and treated with extender, from two stallions were spiked with the organism at seven or 23 days postejaculation and prepared in serial dilutions. Culture of the 7-day raw semen readily detected CEMO at all dilutions, but extended semen yielded counts that were two log cycles lower at equivalent dilutions, with the organism being nearly undetectable at the maximal dilutions. By contrast, PCR sensit...
Dynon K, Black WD, Ficorilli N, Hartley CA, Studdert MJ.To examine the association of viruses with acute febrile respiratory disease in horses. Design Nasal swab and serum samples were collected from 20 horses with acute febrile upper respiratory disease that was clinically assessed to have a viral origin. Methods: Each of the samples was inoculated onto equine fetal kidney, RK13 and Vero cell cultures, and viral nucleic acid was extracted for polymerase chain reaction (PCR) or reverse transcription PCR. PCR primers were designed to amplify nucleic acid from viruses known to cause or be associated with acute febrile respiratory disease in horses in...
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...
Drolet BS, Reeves WK, Bennett KE, Pauszek SJ, Bertram MR, Rodriguez LL.In 2006, vesicular stomatitis New Jersey virus (VSNJV) caused outbreaks in Wyoming (WY) horses and cattle after overwintering in 2004 and 2005. Within two weeks of the outbreak onset, 12,203 biting flies and 194 grasshoppers were collected near three equine-positive premises in Natrona County, WY. Insects were identified to the species level and tested by RT-qPCR for VSNJV polymerase (L) and phosphoprotein (P) gene RNA. Collected dipterans known to be competent for VSV transmission included Simulium black flies and Culicoides biting midges. VSNJV L and P RNA was detected in two pools of female...
Cohen ND, Flores-Ahlschewde P, Gonzales GM, Kahn SK, da Silveira BP, Bray JM, King EE, Blair CC, Bordin AI.Diagnostic accuracy of real-time, quantitative PCR (qPCR) assays to quantify virulent Rhodococcus equi using rectal swab samples has not been systematically evaluated. Objective: To evaluate the accuracy of qPCR of rectal swab samples to differentiate foals with pneumonia from healthy foals of similar age from the same environment. Methods: One hundred privately owned foals born in 2021 from 2 farms in New York. Methods: An incident case-control study design was used. Rectal swabs were collected from all foals diagnosed with R. equi pneumonia at 2 horse-breeding farms (n = 47). Eligible pneu...
Swerczek TW.Reports of 148 cases of Tyzzer's disease in foals in central Kentucky were analyzed to identify features of the disease and factors associated with it. The records indicate that Tyzzer's disease is a rapidly progressive, highly fatal hepatitis caused by Clostridium piliforme. Common clinical findings are lethargy, fever, anorexia, and icterus. Seizures, coma, and death may rapidly ensue. Laboratory findings are leukopenia, metabolic acidosis, hypoglycemia, and increased activity of hepatic enzymes. Diagnosis is primarily based on clinical signs and postmortem findings but a polymerase chain re...
Brandt S, Schoster A, Tober R, Kainzbauer C, Burgstaller JP, Haralambus R, Steinborn R, Hinterhofer C, Stanek C.Equine hoof canker is a chronic proliferative pododermatitis of as yet unknown aetiology. Like equine sarcoid disease, canker is a therapy-resistant disorder characterised by hyperkeratosis, acanthosis and a marked tendency to recur. Objective: There is an association of sarcoid-inducing bovine papillomaviruses of types 1 and 2 (BPV-1, BPV-2) with hoof canker disease. Methods: Using PCR-based techniques, we assessed canker tissue, intact skin and/or peripheral blood mononuclear cells (PBMCs) of 25 canker-affected horses for the presence of sarcoid-associated BPV-1 and -2. Results: Conventional...
Vázquez-Morón S, Avellón A, Echevarría JE.The Lyssavirus genus includes seven species or genotypes named 1-7. Rabies genotypes correlate with geographical distribution and specific hosts. Co-circulation of different lyssaviruses, imported cases, and the presence of unknown viruses, such as Aravan, Khujand, Irkut and West Caucasian Bat Virus, make it necessary to use generic methods able to detect all lyssaviruses. Primer sequences were chosen from conserved regions in all genotypes in order to optimise a generic RT-PCR. Serial dilutions of 12 RNA extracts from all seven Lyssavirus genotypes were examined to compare the sensitivity of ...
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.
Hensel M, Meason-Smith C, Plumlee QD, Myers AN, Coleman MC, Lawhon S, Rodrigues Hoffmann A, Rech RR.Pulmonary mycosis secondary to enterocolitis is an uncommon diagnosis in equine medicine, but is thought to result from mucosal compromise and translocation of enteric fungi. The aetiological agent associated with translocation is often identified based on fungal culture or hyphal features in histological sections. In order to understand better the aetiological agents involved, six horses diagnosed with Salmonella enteritis and concurrent pulmonary mycosis were identified retrospectively through a database search of veterinary teaching hospital records. Samples from these cases were subjected ...
Riihimäki M, Aspán A, Ljung H, Pringle J.The aim of the study was to use culture, qPCR and seM sequencing to map Streptococcus equi subspec. equi (S.equi) isolates in long term carrier animals. A strangles outbreak affecting 41 Icelandic horses was followed to determine strangles free status using nasal and/or guttural pouch lavages collected serially on eleven separate occasions over 13 months. Ten persistent carriers, of which eight had repeated culture positive samples for S. equi, were selected for the study. Of 115 samples collected, 61 were S. equi positive on qPCR; from which 32 were also culture positive. Amplification of par...
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...
