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Topic:Molecular biology
Molecular biology in horses involves the study of molecular processes and genetic mechanisms that underpin equine physiology and health. This field encompasses the analysis of DNA, RNA, proteins, and other biomolecules to understand gene expression, genetic variation, and cellular functions in horses. Techniques such as genomic sequencing, gene expression profiling, and molecular diagnostics are employed to explore topics like hereditary diseases, performance traits, and immune responses in equines. This page assembles peer-reviewed research studies and scholarly articles that investigate the molecular biology of horses, focusing on genetic research, molecular techniques, and their applications in equine science.
Laminin receptor 1 is differentially expressed in thoracic and limb wounds in the horse. Healing of wounds located on the distal limbs of horses is often complicated by retarded epithelialization and the development of exuberant granulation tissue (proud flesh). Treatments that definitively resolve this pathological process are still unavailable. Molecular studies of the repair mechanism might contribute to the development of new therapeutic strategies. The study presented herein aimed to clone the full length cDNA and to study the spatio-temporal expression profile of mRNA and protein for LAMR1, previously attributed a role in wound epithelialization, during the repair of body an...
Sharpea azabuensis gen. nov., sp. nov., a Gram-positive, strictly anaerobic bacterium isolated from the faeces of thoroughbred horses. Four bacterial strains, designated ST18(T), HM244, HM250 and DI49, were isolated from the fresh faeces of four thoroughbred horses in Japan. Cells were Gram-positive, strictly anaerobic, catalase-negative, non-spore-forming, non-motile rods that occurred in chains. They were placed in the same subcluster based on 16S rRNA gene sequence analysis, phenotypic characteristics and levels of DNA-DNA relatedness. Their DNA G+C content ranged from 36 to 38 mol%. Lactobacillus catenaformis, Lactobacillus vitulinus and Catenibacterium mitsuokai belong to cluster XVII of the Clostridium subphylum. Strain...
Isolation, establishment, and characterization of ex vivo equine melanoma cell cultures. Gray horses spontaneously develop metastatic melanomas that resemble human disease, and this is often accompanied with metastasis to other organs. Unlike in other species, the establishment of primary equine melanoma cultures that could be used to develop new therapeutic approaches has remained a major challenge. The purpose of the study was to develop a protocol for routine isolation and cultivation of primary equine melanocytes. Melanoma tissues were excised from 13 horses under local anesthesia, mainly from the perianal area. The melanoma cells were isolated from the melanoma tissue by seri...
Development of a real-time duplex TaqMan-PCR for the detection of Equine rhinitis A and B viruses in clinical specimens. Equine rhinitis A and B viruses (ERAV and ERBV) are respiratory viruses of horses belonging to the family Picornaviridae. Although these viruses are considered to cause respiratory disease in horses and are potentially infectious for humans, little is known about their prevalence and pathogenesis. Virus isolation is often unsuccessful due to their inefficient growth and lack of cytopathic effect in cell cultures. Therefore, molecular assays should be considered as the method of choice to detect infection in symptomatic or apparently healthy horses. In the present study, a novel real-time duple...
Different mechanisms of spinal fusion using equine bone protein extract, rhBMP-2 and autograft during the process of anterior lumbar interbody fusion. To elucidate the molecular mechanisms of spinal fusion with different graft materials during an anterior lumbar interbody fusion, we examined the gene-expression profiles after implantation of equine bone protein extract, rhBMP-2 and autograft using microarray technology and data analysis, including hierarchical clustering, self-organizing maps (SOM), KEGG pathway and Biological process GO analyses in a porcine model. The results suggest that equine bone protein extract exhibited a more similar expression pattern with autograft than that of rhBMP-2. rhBMP-2 recruits progenitor cells, prolifera...
Cloning and expression of ADAM-related metalloproteases in equine laminitis. Equine laminitis is a debilitating disease affecting the digital laminae that suspend the distal phalanx within the hoof. While the clinical progression of the disease has been well documented, the molecular events associated with its pathogenesis remain largely unknown. Using real time quantitative PCR (RT-qPCR), we have investigated the expression of genes coding for proteins containing a Disintegrin and Metalloprotease domain (ADAM), as well as genes encoding the natural inhibitors of these enzymes (tissue inhibitor of metalloprotease; TIMP) in horses with naturally-acquired (acute, chronic...
Phenotypic and genotypic characterization of Arcanobacterium haemolyticum isolates from infections of horses. The present study was designed to characterize phenotypically and genotypically seven Arcanobacterium haemolyticum strains obtained from infections of six horses. All seven strains showed the cultural and biochemical properties typical of A. haemolyticum and were susceptible to most of the antibiotics tested. The species identification could be confirmed by amplification and sequencing of the 16S rRNA gene and the 16S-23S rRNA intergenic spacer region and by PCR amplification of species-specific parts of the gene encoding phospholipase D in A. haemolyticum. Use of the latter could possibly imp...
A glycosylated peptide in the West Nile virus envelope protein is immunogenic during equine infection. Using a monoclonal antibody directed to domain I of the West Nile virus (WNV) envelope (E) protein, we identified a continuous (linear) epitope that was immunogenic during WNV infection of horses. Using synthetic peptides, this epitope was mapped to a 19 aa sequence (WN19: E147-165) encompassing the WNV NY99 E protein glycosylation site at position 154. The inability of WNV-positive horse and mouse sera to bind the synthetic peptides indicated that glycosylation was required for recognition of peptide WN19 by WNV-specific antibodies in sera. N-linked glycosylation of WN19 was achieved through ...
The involvement of beta-1,4-Galactosyltransferase and N-Acetylglucosamine residues in fertilization has been lost in the horse. In human and rodents, sperm-zona pellucida binding is mediated by a sperm surface Galactosyltransferase that recognizes N-Acetylglucosamine residues on a glycoprotein ZPC. In large domestic mammals, the role of these molecules remains unclear: in bovine, they are involved in sperm-zona pellucida binding, whereas in porcine, they are not necessary. Our aim was to clarify the role of Galactosyltransferase and N-Acetylglucosamine residues in sperm-zona pellucida binding in ungulates. For this purpose, we analyzed the mechanism of sperm-zona pellucida interaction in a third ungulate: the horse, si...
RNA interference protects horse cells in vitro from infection with Equine Arteritis Virus. Equine Arteritis Virus (EAV) belongs to the Arteriviridae and causes viral arteritis in horses. In an attempt to develop novel and save therapies against the infection it was tested whether EAV is susceptible to RNA interference (RNAi) in an equine in vitro system. Horse cells were transfected with chemically synthesized small interfering RNA oligonucleotides (siRNAs) and challenged with EAV. Application of these siRNAs led to a significant protection of the cells, and virus titers decreased drastically. siRNAs derived from DNA plasmids expressing small hairpin RNAs (shRNAs) were also effectiv...
Molecular phylogeny of equine herpesvirus 1 isolates from onager, zebra and Thomson’s gazelle. Viruses related to equine herpesvirus type 1 (EHV-1) were isolated from an aborted fetus of an onager (Equus hemionus) in 1984, an aborted fetus of Grevy's zebra (Equus grevyi) in 1984 and a Thomson's gazelle (Gazella thomsoni) with nonsuppurative encephalitis in 1996, all in the USA. The mother of the onager fetus and the gazelle were kept near plains zebras (Equus burchelli). In phylogenetic trees based on the nucleotide sequences of the genes for glycoproteins B (gB), I (gI), and E (gE), and teguments including ORF8 (UL51), ORF15 (UL45), and ORF68 (US2), the onager, Grevy's zebra and gazell...
Role of p38 MAPK in LPS induced pro-inflammatory cytokine and chemokine gene expression in equine leukocytes. Endotoxemia occurs when bacterial lipopolysaccharide (LPS) in the blood induces a dysregulated inflammatory response, resulting in circulatory shock and multi-organ failure. Laminitis is a common complication in endotoxemic horses and is frequently the reason for humane euthanasia of these cases. Blood leukocytes are a principal target of LPS in endotoxemia leading to activation of multiple signal transduction pathways involved in the induction of a number of pro-inflammatory genes. In other animal models, the p38 mitogen activated protein kinase (MAPK) pathway has been associated with induced...
Application of a Reverse Line Blot hybridisation assay for the species-specific identification of cyathostomins (Nematoda, Strongylida) from benzimidazole-treated horses in the Slovak Republic. Five horse farms located in eastern Slovakia were investigated for the presence of benzimidazole-resistant strongyles by faecal egg count reduction test and egg hatch assay. Coprocultures were prepared for each farm from faecal samples taken pre- and post-treatment and harvested larvae were molecularly examined with a Reverse Line Blot assay. Faecal egg count reduction values ranged from 0 to 52.5% and all farms were positive for benzimidazole-resistant cyathostomins. Seven benzimidazole-resistant cyathostomin species were molecularly identified on farms before and also after treatment. These ...
Characterization of an equine macrophage cell line: application to studies of EIAV infection. EIAV is a monocyte/macrophage tropic virus. To date, even though EIAV has been under investigation for numerous years, very few details have been elucidated about EIAV/macrophage interactions. This is largely due to the absence of an equine macrophage cell line that would support viral replication. Herein we describe the spontaneous immortalization and generation of a clonal equine macrophage-like (EML) cell line with the functional and immunophenotype characteristics of differentiated equine monocyte derived macrophage(s) (eMDM(s)). These cells possess strong non-specific esterase (NSE) activ...
Glial cells missing homologue 1 is induced in differentiating equine chorionic girdle trophoblast cells. The objective of this study was to identify transcription factors associated with differentiation of the chorionic girdle, the invasive form of equine trophoblast. The expression patterns of five transcription factors were determined on a panel of conceptus tissues from early horse pregnancy. Tissues from Days 15 through 46 were tested. Eomesodermin (EOMES), glial cells missing homologue 1 (GCM1), heart and neural crest derivatives expressed transcript 1 (HAND1), caudal type homeobox 2 (CDX2), and distal-less homeobox 3 (DLX3) were detected in horse trophoblast, but the expression patterns for...
Expression microarrays in equine sciences. Microarrays have become an important research tool for life science researchers. Expression microarrays are capable of profiling the gene expression pattern of tens of thousands of genes in a single experiment. It appears to be the platform of choice for parallel gene expression profiling. Various equine-specific gene expression microarrays have been generated and used. However, homologous microarrays are not yet commercially available for the horse. An alternative is the use of heterologous microarrays, mainly microarrays specific for mice or humans. Although the use of microarrays in equine ...
Development of a bead-based multiplex assay for simultaneous quantification of cytokines in horses. The detection and quantification of equine cytokines has been hampered by the lack of antibodies for many years. With the development of antibody pairs for equine cytokines during the past years, the quantification of these essential regulators of the immune response became possible. After being successfully tested by enzyme-linked immunosorbent assays (ELISA), three of these anti-cytokine reagents were used here to establish the first cytokine multiplex assay for equine IL-4, IL-10 and IFN-alpha. A fluorescent bead-based system was used as matrix for this assay that allows the simultaneous de...
Age-related changes in cytokine expression by neutrophils of foals stimulated with virulent Rhodococcus equi in vitro. Although evidence exists that neutrophils play a vital role in resistance to infection with Rhodococcus equi, the means by which neutrophils exert their effects have not been clearly defined. In the present study we evaluated differences in cytokine expression by unstimulated and R. equi-stimulated neutrophils obtained from newborn foals and subsequently at 2-, 4-, and 8-weeks of age. Stimulation with virulent R. equi induced significantly (P<0.05) greater expression of IFNgamma, TNFalpha, IL-6, IL-8, IL-12p40, IL-12p35, and IL-23p19 mRNA relative to expression by unstimulated neutrophils, ...
Sequence heterogeneity in the 18S rRNA gene within Theileria equi and Babesia caballi from horses in South Africa. A molecular epidemiological survey of the protozoal parasites that cause equine piroplasmosis was conducted using samples collected from horses and zebra from different geographical locations in South Africa. A total of 488 samples were tested for the presence of Theileria equi and/or Babesia caballi using the reverse line blot hybridization assay. Ten percent of the samples hybridized to the Theileria/Babesia genus-specific probe and not to the B. caballi or T. equi species-specific probes, suggesting the presence of a novel species or genotype. The small subunit of rRNA gene (18S; approximat...
A unique method to produce transgenic embryos in ovine, porcine, feline, bovine and equine species. Transgenesis is an essential tool in many biotechnological applications. Intracytoplasmic sperm injection (ICSI)-mediated gene transfer is a powerful technique to obtain transgenic pups; however, most domestic animal embryos do not develop properly after ICSI. An additional step in the protocol, namely assistance by haploid chemical activation, permits the use of ICSI-mediated gene transfer to generate transgenic preimplantation embryos in a wide range of domestic species, including ovine, porcine, feline, equine and bovine. In the present study, spermatozoa from five species were coincubated ...
Species-specific identification of equine cyathostomes resistant to fenbendazole and susceptible to oxibendazole and moxidectin by macroarray probing. Cyathostome populations in horses on two farms located in central Italy with a history of fenbendazole (FBZ) resistance were investigated with the Faecal Egg Count Reduction Test to evaluate the susceptibility to oxibendazole and moxidectin. Faecal eggs were collected pre- and post-treatment on each farm and molecularly examined with a Reverse Line Blot (RLB) assay able to unequivocally detect and identify 13 cyathostome species. Resistance to FBZ was confirmed on both farms, while oxibendazole and moxidectin demonstrated 97% and 100% efficacy, respectively. Overall eight species of cyathostom...
A survey of equine abortion and perinatal foal losses in Hungary during a three-year period (1998-2000). Cases of equine abortion and perinatal foal losses were investigated in Hungary during a three-year period (1998-2000). Samples from aborted equine fetuses and newborn foals (total n = 96) were examined using bacteriological, virological, pathological, immunohistochemical (IHC), molecular biological and serological methods. The cause of abortion and perinatal foal loss was identified in 67/96 cases (70%); viral infection was found in 22 (23%), viral and bacterial coinfection in 1 (1%), bacterial infection in 23 (24%), protozoan infection in 1 (1%) and fungal infection in 2 cases (2%). Morpholo...
Effect of two synthetic peptides mimicking conserved regions of equine infectious anemia virus proteins gp90 and gp45 upon cytokine mRNA expression. Gp90 and gp45 synthetic peptides, which mimic conserved sequences of native viral proteins, are recognized by antibodies to equine infectious anemia virus (EIAV) in asymptomatic carrier horses and generate humoral and cellular responses in immunized mice. Cytokine mRNA levels were evaluated in equine peripheral blood mononuclear cells (PBMCs) after in vitro stimulation with gp90 and gp45 with the aim of determining the cytokine profile associated with the proliferative response. Stimulation index (SI) values indicate that 100 and 60% of EIAV-infected horses recognized gp90 and gp45, respective...
Molecular identification of Streptococcus equi subsp. equi and Streptococcus equi subsp. zooepidemicus in nasal swabs samples from horses suffering respiratory infections in Iran. The objective of this study was to evaluate the existence of Streptococcus equi subsp. equi and Streptococcus equi subsp. zooepidemicus as probable agents associated with naturally occurring infection of the equine upper respiratory disease in Mashhad area. Nasal swabs samples from thirty horses with upper respiratory tract infections were collected. The bacteria isolated and identified were Streptococcus equi subsp. equi (1 isolate), Streptococcus equi subsp. zooepidemicus (25 isolates), Pasteurella sp. (11 isolates), Staphylococcus sp. (17 isolates), Bacillus sp. (4 isolates), Pseudomonas sp...
Trypanosoma evansi: A comparison of PCR and parasitological diagnostic tests in experimentally infected mice. Trypanosoma evansi is the causative agent of equine trypanosomosis, disease that affects horse's productivity and health. Parasitological and molecular methods are mostly used to detect the infection. The aim of this work was evaluate PCR sensitivity to detect T. evansi using the primers 21/22-mer, ITS1, ESAG 6/7 and TBR 1/2 designed from repetitive (multicopies) genomic sequences. The results were compare with two parasitological tests in mice, micro-haematocrite centrifugation technique and direct microscopic examination. The results shows (a) that the minimum amount of DNA from blood of hig...
Equine infectious anemia virus resists the antiretroviral activity of equine APOBEC3 proteins through a packaging-independent mechanism. Equine infectious anemia virus (EIAV), uniquely among lentiviruses, does not encode a vif gene product. Other lentiviruses, including human immunodeficiency virus type 1 (HIV-1), use Vif to neutralize members of the APOBEC3 (A3) family of intrinsic immunity factors that would otherwise inhibit viral infectivity. This suggests either that equine cells infected by EIAV in vivo do not express active A3 proteins or that EIAV has developed a novel mechanism to avoid inhibition by equine A3 (eA3). Here, we demonstrate that horses encode six distinct A3 proteins, four of which contain a single copy o...
The structure of the polysaccharide of the lipopolysaccharide produced by Taylorella equigenitalis type strain (ATCC 35865). Taylorella equigenitalis is a Gram-negative bacterium that causes venereally transmitted contagious equine metritis (CEM), and its identification and differentiation from other bacteria and Taylorella species is an important requirement for the control of CEM infection. Based on the results of NMR and MS analysis, the antigenic O-polysaccharide (O-PS) component of the lipopolysaccharide (LPS) produced by the type strain T. equigenitalis (ATCC 35865) was found to be a linear polymer composed of a repeating disaccharide unit, containing partially amidated 2,3-diacetamido-2,3-dideoxy-alpha-L-gulu...
Equine herpesvirus 1 entry via endocytosis is facilitated by alphaV integrins and an RSD motif in glycoprotein D. Equine herpesvirus 1 (EHV-1) is a member of the Alphaherpesvirinae, and its broad tissue tropism suggests that EHV-1 may use multiple receptors to initiate virus entry. EHV-1 entry was thought to occur exclusively through fusion at the plasma membrane, but recently entry via the endocytic/phagocytic pathway was reported for Chinese hamster ovary cells (CHO-K1 cells). Here we show that cellular integrins, and more specifically those recognizing RGD motifs such as alphaVbeta5, are important during the early steps of EHV-1 entry via endocytosis in CHO-K1 cells. Moreover, mutational analysis revea...
Missense mutation in exon 2 of SLC36A1 responsible for champagne dilution in horses. Champagne coat color in horses is controlled by a single, autosomal-dominant gene (CH). The phenotype produced by this gene is valued by many horse breeders, but can be difficult to distinguish from the effect produced by the Cream coat color dilution gene (CR). Three sires and their families segregating for CH were tested by genome scanning with microsatellite markers. The CH gene was mapped within a 6 cM region on horse chromosome 14 (LOD = 11.74 for theta = 0.00). Four candidate genes were identified within the region, namely SPARC [Secreted protein, acidic, cysteine-rich (osteonectin)], SL...
Sensitive and specific detection of Cryptosporidium species in PCR-negative samples by loop-mediated isothermal DNA amplification and confirmation of generated LAMP products by sequencing. 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...
