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Topic:Cell Culture
Cell culture in horses involves the in vitro cultivation of equine cells under controlled conditions. This technique is employed to study various cellular processes, including growth, differentiation, and response to external stimuli, in an isolated environment. Equine cell cultures can be derived from various tissues, such as skin, muscle, or bone, and are used in a range of research applications, including genetic studies, drug testing, and disease modeling. These cultures provide a valuable platform for understanding cellular mechanisms and developing therapeutic strategies. This page compiles peer-reviewed research studies and scholarly articles that explore the methodologies, applications, and findings related to cell culture in equine research.
Cultivation of Plasmodium falciparum using animal serum (horse, calf and bovine) as human serum substitute. Horse, calf and bovine serum were successfully used as human serum substitutes in the in vitro cultivation of Plasmodium falciparum. Positive results were obtained only after gradually adapting the parasites to the substitute serum. Adapted lines were established within 4-5 weeks. 10% horse serum was observed to be the best substitute with growth rates comparable or even surprising that obtained in human serum. Pure calf or bovine serum supported stable growths of 20-30% less which was enhanced to comparable levels after addition of 1% glucose-peptone to the medium. Direct transfers of adapted...
[Concentration of the Venezuelan equine encephalomyelitis virus in a 2-phase system of water-soluble polymers]. A three-step concentration of Venezuelan equine encephalomyelitis (VEE) virus from tissue culture fluid was carried out in a two-phase system of polyethyleneglycol (PEG)--sodium dextran sulphate (SDS). The concentration method was based on the dependence of virus distribution coefficient upon NaCl content in the system which allowed alternating transfer of the virus from one phase of the system into the other. The infectious activity of the virus increased approximately 100-fold after the first step, 190-fold after the second, and 300-fold after the third step. The process of concentration was...
Comparative studies of the binding and growth-supportive ability of mammalian transferrins in human cells. The ability of human-derived cells in culture to bind, remove iron from, and grow in the presence of transferrins (Tf) isolated from the sera of species commonly included in tissue culture medium was investigated. Kinetic studies on HeLa cells reveal apparent first-order association rate constants of 0.43 min-1 for human Tf and 0.15 min-1 for equine Tf. Labeled chicken ovo-Tf and fetal bovine Tf were not recognized by the HeLa cells. Competition experiments with HeLa cells that use either isolated Tf or parent serum confirm these findings. Equilibrium binding experiments performed on HeLa cell...
Equine arteritis virus-induced polypeptide synthesis. Intracellular virus-specific proteins induced by equine arteritis virus (EAV) have been compared with in vitro translation products of virion and intracellular EAV RNAs. In infected BHK-21 cells, the two major virion proteins (C and E1) and polypeptides with mol. wt. of 60,000 (p60), 42,000 (p42) and 30,000 (p30) were found. There were no indications that the viral proteins were processed from a larger precursor as shown by pulse-chase, amino acid analogue and protease inhibitor experiments. The six polyadenylated RNAs that occur in EAV-infected cells were isolated and translated in an mRNA-de...
Equine endothelial cells in vitro. Certain in vitro culture conditions were determined for equine endothelial cells obtained from the aorta and pulmonary arteries. Cells were enzymatically isolated from the vessel lumen, using clostridial collagenase (2.5 mg/ml of Hanks's balanced salt solution) incubated at 37 C for 30 minutes. Cells were cultured in alpha minimum essential medium supplemented with plasma-derived and nonplasma-derived bovine fetal sera, endothelial cell-growth supplement, heparin, and antibiotics. Smooth muscle cell growth was not inhibited with nonplasma-derived animal sera, plasma-derived equine serum, or he...
In vitro interference between equine herpesvirus types 1 and 2. Interference between equine herpesvirus types 1 (EHV-1) and 2 (EHV-2) was studied in equine dermis (ED) monolayer cell cultures and equine lymphocyte cultures. Cell cultures were infected with EHV-2, and after a short incubation period, the cultures were superinfected with EHV-1. At various intervals, different measurements of EHV-1 expression in dually infected cultures, compared with those in cultures infected with EHV-1 alone, were studied. In dually infected ED cell cultures, the EHV-1 cytopathic effect, EHV-1 titer, and EHV-1 enzyme-linked immunosorbent assay antigen titer were maximally ...
Characteristics of cells derived from the girdle region of the pre-implantation blastocyst of the donkey. The establishment of a monolayer culture of cells derived from the girdle region of a 34-day-old donkey conceptus is described. These cells have had over 100 repeated passages in culture. Low levels of pregnant mares' serum gonadotrophin (PMSG, eCG) could be detected in the cells by indirect immunofluorescence using some monoclonal anti-eCG antibodies, but the cells did not secrete eCG as measured by radioimmunoassay or inhibition of haemagglutination. There was marked nuclear polymorphism with binucleate and occasional multinucleate cells. The cells were strongly reactive with wheatgerm agglu...
Spontaneous expression of an endogenous retrovirus by the equine sarcoid-derived MC-1 cell line. A retrovirus is spontaneously released into the culture medium of the equine sarcoid-derived MC-1 cell line. The MC-1 virus did not exhibit in vitro transforming activity or replication when tested on equine fibroblasts or a variety of other mammalian cell cultures. Complementary DNA, synthesized using detergent-activated MC-1 virus RNA-dependent DNA polymerase, detected homologous sequences in the DNA of an established equine dermal cell line and in the DNA of primary equine dermal fibroblasts. Iododeoxyuridine or azacytidine induced a replication-deficient endogenous retrovirus in the normal...
[Creation of an avirulent and immunogenic mutant from the rhinopneumonitis virus]. An avirulent immunogenic virus strain mutant of the causative agent of rhinopneumonia was found to cause abortions and respiratory diseases in horses. The mutant was obtained with the use of a virulent strain that induced strongly manifested clinical symptoms of the disease, and was cultured in cell media containing 5-iodine-2-desoxiuridine as an antimetabolite, following a definite pattern. It was found that the mutant completely lost its virulence, however, it retained its immunogenicity. It likewise retained these newly acquired biologic properties with regard to its being stable and irreve...
Nitrogen utilization in bacterial isolates from the equine cecum. A total of 114 bacterial isolates were obtained from the cecal contents of two mature cecally fistulated horses on a habitat-simulating medium containing 40% energy-depleted cecal fluid. Of these isolates, 108 were maintained in pure cultures and were tentatively grouped on the basis of cell morphology and physiological characteristics. Gram-negative rods (50.9%), gram-positive rods (22.8%), and gram-positive cocci (21.9%) represented the largest groups isolated from these animals. Fifty isolates were tested for their ability to grow in media containing urea, ammonia, peptones, or amino acids ...
Horse kidney neutral alpha-D-glucosidase: purification of the detergent-solubilized enzyme; comparison with the proteinase-solubilized forms. Neutral alpha-D-glucosidase (alpha-D-glucoside glucohydrolase, EC 3.2.1.20) from horse kidney brush-border membranes was solubilized using Emulphogene BC 720 and purified by an affinity chromatography technique. The enzyme preparation (390-fold purified), which was free of other known microvillus hydrolases, exhibited one precipitate line in crossed immunoelectrophoresis and migrated as a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Several criteria (charge-shift crossed immunoelectrophoresis and hydrophobic chromatography) revealed the purified detergent form of t...
High resolution R-bands produced in equine chromosomes after incorporation of bromodeoxyuridine. Cell synchronization was used to obtain an adequate percentage of very long chromosomes in equine mitotic spreads. Reported here is our variation, adapted to horse chromosomes, of a method using excess thymidine followed by bromodeoxyuridine incorporation. This technique routinely yields excellent quality cells, predominantly in prometaphase and prophase. Among other differences with the standard technique, this method does not use Colcemid, which, in addition to inhibiting spindle fiber formation, also increases chromosome contraction resulting in thicker and thus fewer bands. Consequently, h...
In vitro development of Strongylus edentatus to the fourth larval stage with notes on Strongylus vulgaris and Strongylus equinus. Strongylus edentatus was successfully cultured in vitro to the fourth larval stage (L4). Some growth continued for periods of 40-50 days at which time reductions in viability were observed in some of the culture systems tested. Various combinations of media, sera, buffers and organ explant cultures were tested. All cultures were incubated at 37 C in an atmosphere of 95% air and 5% CO2. Larvae underwent growth and differentiation to the L4 in all medium-serum combinations with and without organ explant cultures. Development and growth did occur but viability was reduced to insignificant levels ...
Experimental reproduction of Potomac horse fever in horses with a newly isolated Ehrlichia organism. Potomac horse fever, a recently recognized disease of equines, characterized by high fever, leukopenia, and a profuse diarrhea, was studied for its etiology. An Ehrlichia organism was isolated in equine macrophage-fibroblast cell cultures and mouse macrophage cell cultures from the mononuclear cells of blood of infected horses. The agent was continuously propagated in mouse macrophage cell cultures. The organism multiplied in the cytoplasm of mouse macrophage cells and was identified by Giemsa staining, acridine orange staining, and by indirect immunofluorescence with convalescent sera from in...
Estrogen metabolites in equine ovarian follicles: gas chromatographic-mass spectrometric determinations in relation to follicular ultrastructure and progestin content. Equine follicular fluid was aspirated at various developmental stages (viable, preovulatory and atretic) determined by ultrastructural study. Estrogens and progestins were analyzed by gas chromatography-mass spectrometry associated with stable isotope dilution. Progesterone and 17-hydroxyprogesterone were the principal progestins of the preovulatory and viable follicles. Among the catechol estrogens, 2-hydroxy-estradiol was particularly abundant in the preovulatory follicle and its definitive identification was made by the scan of a full mass spectrum.
Transformation of cultured equine fibroblasts with a bovine papillomavirus. Fetal equine fibroblasts exposed to bovine papillomavirus became transformed by the criteria of morphological alterations and the acquisition of an increased life span, although they failed to grow in soft agar. Papillomavirus genome persisted in the transformed fibroblasts and was apparently not integrated with the cellular genome. These findings support the notion that bovine papillomaviruses are involved in the production of equine sarcoids.
Endotoxin-induced eicosanoid production by equine vascular endothelial cells and neutrophils. Dispersed equine vascular endothelial cells grown in tissue culture, and freshly isolated neutrophils were used to determine direct effects of endotoxin on cyclooxygenase and lipoxygenase products. Endothelial cells (10(7)/ml) or neutrophils (2 X 10(6)/ml) were incubated with (a) buffer, (b) endotoxin (10 micrograms/ml), (c) endotoxin + flunixin meglumine (10 micrograms/ml), or (d) calcium ionophore, A23187 (10 micrograms/ml). Thromboxane (TxB2), prostacyclin (6-keto-PGF1 alpha), and leukotriene C4 (LTC4) were determined in the incubation fluid by radioimmunoassay. Thromboxane and prostacyclin...
A new surface marker on equine peripheral blood lymphocytes. I. Subpopulations of lymphocytes with receptors for Helix pomatia A hemagglutinin (HP). Untreated and neuraminidase-treated equine peripheral blood lymphocytes were analysed for binding of the A hemagglutinin of the snail Helix pomatia (HP). For optimal staining by direct immunofluorescence, the concentration of neuraminidase had to be increased as compared to that needed for other species. Moreover, higher concentrations of HP were required for optimal staining of equine lymphocytes as compared to lymphocytes from other species. Even so, the maximal number of equine lymphocytes exhibiting positive staining was only about 20%. No, or very few, HP-positive lymphocytes were seen wh...
A new surface marker on equine peripheral blood lymphocytes. II. Characterization and separation of purified blood lymphocytes with receptors for Helix pomatia A hemagglutinin (HP). In a preceding report we have shown that the lectin Helix pomatia A hemagglutinin (HP) binds to two subpopulations of neuraminidase-treated equine peripheral blood lymphocytes (PBL), constituting about 20% and 75% of PBL, respectively. The aim of the present study was to further characterize these HP+ cells in regard to other surface markers such as receptors for guinea pig erythrocytes (GPR+ cells), membrane-bound immunoglobulins (sIg+ cells), receptors for activated complement (C3R+ cells) and receptors for IgG (Fc alpha R+ cells). This was done by double marker analysis and by lymphocyte fr...
Equine cell-mediated immune response to Rhodococcus (Corynebacterium) equi. A lymphocyte blastogenic assay was developed to serve as an in vitro correlate of cell-mediated immunity to Rhodococcus (Corynebacterium) equi (R equi) in the equine species. Lymphocytes obtained from a group of experimental ponies showed no response in cell culture to R equi heat extract or lysozyme extract antigens. Ponies were assigned to groups for experimental inoculation. Three ponies were inoculated subcutaneously with live R equi, 3 were given live R equi by intranasal and intratracheal routes, and 4 ponies were left untreated. Lymphocytes from all inoculated ponies had a mitogenic res...
Continuous cultivation of equine lymphocytes: evidence for occasional T cell-like maturation events in horses with hereditary severe combined immunodeficiency. Peripheral blood mononuclear cells (PBMC) from 14 foals with hereditary severe combined immunodeficiency (SCID) were studied to determine the extent of lymphocyte differentiation that occurs in this disorder. PBMC from all 14 horses had the morphologic characteristics of large granular lymphocytes (LGL). Cells from only one of 14 horses were responsive to phytolectin stimulation in a standard blastogenesis assay; however, PBMC from all 14 horses proliferated in continuous culture in the presence of partially purified interleukin 2. Furthermore, there were differences in the growth patterns of ...
Growth kinetics of equine respiratory tract viruses in cell and organ cultures. Growth kinetics of equine influenza virus-A1, equine herpesvirus-1, and equine rhinovirus-1 were determined in susceptible cell monolayers and in organ cultures of equine fetal tracheal and nasal turbinate epithelium. Equine influenza virus-A1 was replicated in cell and organ cultures and was released more readily and for longer periods from nasal turbinate epithelium than from tracheal epithelium. Equine herpesvirus-1 was also replicated in cell and organ cultures. During the first 24 hours after inoculation, equine herpesvirus-1 was released more readily from tracheal epithelium than from na...
Microneutralization test in PK(15) cells for assay of antibodies to louping ill virus. A microneutralization test in PK(15) cells was developed to measure the neutralizing antibody response of a group of ponies experimentally challenged with louping ill virus. Viral cytopathic effect was maximal after 6 days of incubation, at which point titration endpoints were clear-cut and readily determinable. The assay compared favorably with the mouse neutralization test for accuracy and ease of performance.
Culture characteristics and tumorigenicity of the equine sarcoid-derived MC-1 cell line. MC-1 is an equine sarcoid-derived cell line which spontaneously releases a retrovirus possessing genomic sequence homology with an inducible endogenous retrovirus of normal equine cells. A complete characterization of MC-1 tumor cells was undertaken, including morphology, growth kinetics, and saturation density, selective growth in semisolid media, uptake of 2-deoxyglucose, and tumorigenicity in athymic nude mice. MC-1 cells, in contrast to normal equine dermal fibroblasts, exhibit all of the characteristics of malignantly transformed cells.
Ultrastructural comparison of Oncovirinae (type C), Spumavirinae, and Lentivirinae: three subfamilies of Retroviridae found in farm animals. The successive steps of maturation of seven retroviruses from five species of farm animals and one retrovirus from a mouse were compared in cell cultures. The viruses included three type C oncoviruses, one spumavirus, and three lentiviruses. Although members of the 3 subfamilies shared some gross morphologic features such as budding on plasma membranes, core, and surface projections, differences were noted in the ultrastructural detail of these features. Type C oncoviruses did not show any structural differentiation in identifiable form in the cytoplasm as opposed to characteristic features ob...
Xenogeneic monoclonal antibodies to cell surface antigens of equine lymphocytes. Monoclonal antibodies to equine lymphocyte antigens were produced, using normal peripheral blood lymphocytes as the immunogen and standard hybridoma techniques. Antibody producing hybridomas were detected by a solid-phase enzyme-linked immunosorbent assay. Antibodies produced by 6 cloned hybrids were characterized further by microlymphocytotoxicity, indirect immunofluorescence, and agglutination assays on peripheral blood lymphocytes, platelets, and erythrocytes. Reaction patterns on leukocytes indicated that these antibodies may recognize at least 3 different cell-surface antigens: (1) an ant...
Plaque assay of equine influenza virus. ESK cells, a stable cell line derived from a swine embryo kidney, were found to be a good medium for plaque formation of the Prague and Miami strains of equine influenza virus. Factors influencing the plaque formation were investigated and a plaque assay for these viruses was worked out. The method is not only simple enough for routine use, but also is as sensitive as the egg inoculation method. The method was readily adapted for a neutralization test.
Enzyme-linked immunosorbent assay for detection of equine infectious anemia virus p26 antigen and antibody. A sensitive specific enzyme-linked immunosorbent assay utilizing purified p26 antigen was developed for the detection of antibodies to equine infectious anemia virus in naturally and experimentally infected horses. Generally, antibodies to the virus could be detected by the enzyme-linked immunosorbent assay 3 to 4 days earlier than by the standard agar gel immunodiffusion test, and they could be detected more reliably in horses with weak or equivocal agar gel immunodiffusion test reactions. The enzyme-linked immunosorbent assay was also successfully applied to the detection of p26 antigen in t...
