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Topic:Antigen

Antigens are substances that can induce an immune response in horses, typically by being recognized as foreign by the immune system. These substances can include proteins, polysaccharides, or lipids, and are often components of pathogens such as bacteria, viruses, or parasites. In horses, antigens are essential for the activation of both the innate and adaptive immune responses, leading to the production of antibodies and the activation of immune cells. The study of antigens in equines encompasses understanding their structure, the mechanisms by which they are recognized by the immune system, and their role in vaccine development. This page compiles peer-reviewed research studies and scholarly articles that explore the identification, characterization, and immunological impact of antigens in equine health and disease.
Immunological reagents: catalysts for research progress.
Equine veterinary journal    January 5, 2002   Volume 33, Issue 7 628-629 doi: 10.2746/042516401776249390
McGuire TC.No abstract available
IgG antibody responses to an inhaled antigen in horses with “heaves” (recurrent airway obstruction).
Veterinary immunology and immunopathology    January 5, 2002   Volume 84, Issue 3-4 169-180 doi: 10.1016/s0165-2427(01)00400-7
Ainsworth DM, Appleton JA, Antczak DF, Santiago MA, Aviza G.A controlled experimental system for the evaluation of pulmonary immune responses in horses with "heaves" (recurrent airway obstruction) has been developed. We hypothesized that the humoral immune response to an inhaled antigen in diseased horses would be different from that of healthy horses and that chronic pulmonary inflammation would bias the production of IgG isotypes in diseased horses as compared to healthy horses. Healthy and affected horses were housed in a natural challenge environment (stabled, fed dusty hay) and exposed by inhalation, to a nebulized solution of keyhole limpet hemoc...
Detection of IgG and IgE serum antibodies to Culicoides salivary gland antigens in horses with insect dermal hypersensitivity (sweet itch).
Equine veterinary journal    January 5, 2002   Volume 33, Issue 7 707-713 doi: 10.2746/042516401776249363
Wilson AD, Harwood LJ, Björnsdottir S, Marti E, Day MJ.We postulated that all horses exposed to the bites of Culcoides (midges) would have an antibody response to the antigen secreted in Culcoides saliva, but that IgE antibody would be restricted to allergic individuals. Using immunohistology on sections of fixed Culicoides, we have demonstrated the presence of antibodies in horse serum which recognise Culicoides salivary glands. Antibodies were detected in the serum of horses with insect dermal hypersensitivity and in the serum of normal horses exposed to Culicoides bites. In contrast, no antibodies were detected in serum from native Icelandic po...
Prevalence of virulent Rhodococcus equi in soil from five R. equi-endemic horse-breeding farms and restriction fragment length polymorphisms of virulence plasmids in isolates from soil and infected foals in Texas. Takai S, Chaffin MK, Cohen ND, Hara M, Nakamura M, Kakuda T, Sasaki Y, Tsubaki S, Martens RJ.Rhodococcus equi isolates (462) obtained from 64 soil samples collected on 5 R. equi-endemic horse-breeding farms and isolates from 100 infected foals in Texas were examined to determine the prevalence and genotypic diversity of virulence-associated plasmids. Isolates were tested for the presence of 15-17-kDa virulence-associated protein antigens (VapA) by immunoblotting and virulence-associated plasmids by PCR. Plasmid DNAs were isolated and analyzed by digestion with restriction endonucleases for estimation of size and comparison of polymorphisims. Rhodococcus equi were isolated from soil of...
Diagnosis of equine piroplasmosis in Brazil by serodiagnostic methods with recombinant antigens.
The Journal of veterinary medical science    November 21, 2001   Volume 63, Issue 10 1159-1160 doi: 10.1292/jvms.63.1159
Xuan X, Nagai A, Battsetseg B, Fukumoto S, Makala LH, Inoue N, Igarashi I, Mikami T, Fujisaki K.Serum samples from horses in the States of Sao Paulo and Mato Grosso do Sul, Brazil were examined for diagnosis of equine piroplasmosis by both the latex agglutination test (LAT) and enzyme-linked immunosorbent assay (ELISA) with recombinant antigens. Of the 47 samples analyzed, 38 (81%) and 42 (90%) samples were positive for B. equi infection and B. caballi infection, respectively. In addition, 35 (75%) samples were positive for both B. equi and B. caballi infections. These results indicate that equine piroplasmosis is widespread and therefore a cause for serious concern in the States of Sao ...
Specific antibody levels and antigenic recognition of Wistar rats inoculated with distinct isolates of Trypanosoma evansi.
Memorias do Instituto Oswaldo Cruz    October 31, 2001   Volume 96, Issue 7 965-972 doi: 10.1590/s0074-02762001000700014
Queiroz AO, Legey AP, Xavier SC, Jansen AM."Mal de Cadeiras", an enzootic disease caused by Trypanosoma evansi, is one of the most important trypanosomiases in the Brazilian Pantanal region. The disease affects mainly horses, which are widely used in extensive cattle production, an activity of greatest economical significance for the region. The parasite also infects sylvan (coatis and capybaras) and domestic (dogs) animals, respectively considered wild and domestic reservoirs of T. evansi. For a better understanding of the interaction of T. evansi with its rodent host, we evaluated the differences in the specific antibody level patter...
Prevalence of virulent Rhodococcus equi in isolates from soil collected from two horse farms in South Africa and restriction fragment length polymorphisms of virulence plasmids in the isolates from infected foals, a dog and a monkey.
The Onderstepoort journal of veterinary research    October 5, 2001   Volume 68, Issue 2 105-110 
Takai S, Henton MM, Picard JA, Guthrie AJ, Fukushi H, Sugimoto C.The prevalence of virulent Rhodococcus equi in soil isolates from two horse farms in South Africa and nine clinical isolates from six foals, a foal foetus, a dog, and a monkey was investigated. The isolates were tested for the presence of virulence plasmid DNA and 15- to 17-kDa antigens by immunoblotting. Rhodococcus equi was isolated from almost all of the soil samples obtained from the two farms with 5.0 x 10(1) to 3.3 x 10(4) colony forming units per gram of soil. Virulent R. equi was isolated from three soil samples from one of the farms and appeared in 3.8% (three of 80 isolates), but not...
Sequence conservation and antigenic variation of the structural proteins of equine rhinitis A virus.
Journal of virology    October 3, 2001   Volume 75, Issue 21 10550-10556 doi: 10.1128/JVI.75.21.10550-10556.2001
Varrasso A, Drummer HE, Huang JA, Stevenson RA, Ficorilli N, Studdert MJ, Hartley CA.The nucleotide and deduced amino acid sequences of the P1 region of the genomes of 10 independent equine rhinitis A virus (ERAV) isolates were determined and found to be very closely related. A panel of seven monoclonal antibodies to the prototype virus ERAV.393/76 that bound to nonneutralization epitopes conserved among all 10 isolates was raised. In serum neutralization assays, rabbit polyclonal sera and sera from naturally and experimentally infected horses reacted in a consistent and discriminating manner with the 10 isolates, which indicated the existence of variation in the neutralizatio...
Two new variants of the Rhodococcus equi virulence plasmid, 90 kb type III and type IV, recovered from a foal in Japan.
Veterinary microbiology    August 17, 2001   Volume 82, Issue 4 373-381 doi: 10.1016/s0378-1135(01)00398-4
Takai S, Murata N, Kudo R, Narematsu N, Kakuda T, Sasaki Y, Tsubaki S.This report describes the discovery of two new virulence plasmid types from a crossbred foal with Rhodococcus equi pneumonia in Kumamoto died with severe R. equi pneumonia and ulcerative enteritis. R. equi was isolated in large numbers and isolates from the foal were investigated for the presence of virulence-associated 15-17 kDa antigens (VapA) by colony blotting, using the monoclonal antibody 10G5, and by gene coding for VapA by PCR. Plasmid DNAs extracted from the isolates were digested with restriction endonucleases BamHI, EcoRI, EcoT22I, and HindIII. The digestion patterns that resulted d...
Borna disease virus-specific circulating immune complexes, antigenemia, and free antibodies–the key marker triplet determining infection and prevailing in severe mood disorders.
Molecular psychiatry    July 10, 2001   Volume 6, Issue 4 481-491 doi: 10.1038/sj.mp.4000909
Bode L, Reckwald P, Severus WE, Stoyloff R, Ferszt R, Dietrich DE, Ludwig H.Borna disease virus (BDV), a unique genetically highly conserved RNA virus (Bornaviridae; Mononegavirales), preferentially targets neurons of limbic structures causing behavioral abnormalities in animals. Markers and virus in patients with affective disorders and schizophrenia have raised worldwide interest. A persistent infection was suggestive from follow-up studies, but inconstant detectability weakened a possible linkage.This study for the first time discloses that detection gaps are caused by BDV-specific circulating immune complexes (CIC), and their interplay with free antibodies and pla...
Neonatal isoerythrolysis involving the Qc and Db antigens in a foal.
Journal of the American Veterinary Medical Association    July 7, 2001   Volume 219, Issue 1 79-50 doi: 10.2460/javma.2001.219.79
MacLeay JM.In 1992, a multiparous 13-year-old Thoroughbred mare and her 48-hour-old colt were examined because of possible neonatal isoerythrolysis (NI). Supportive treatment was administered, and the foal recovered without requiring a transfusion. According to the owners, the mare had delivered foals without incident during 1987 and 1991. The mare was barren during 1993, but in 1994, delivered a filly that developed severe NI. The foal was given 3 transfusions and eventually recovered without complications. Blood typing analysis of the mare and its foals indicated that all 4 foals were positive for the ...
Culicoides antigen extract stimulates equine blood mononuclear (BMN) cell proliferation and the release of eosinophil adherence-inducing factor(s).
Research in veterinary science    May 18, 2001   Volume 70, Issue 2 115-122 doi: 10.1053/rvsc.2000.0452
Mckelvie J, Foster AP, Hamblin AS, Cunningham FM.Intradermal injection of a Culicoides antigen extract (CAgX) induces T lymphocyte and eosinophil accumulation in the skin of horses with sweet itch. Blood mononuclear (BMN) cells from normal ponies proliferate when stimulated by mitogen (phytohaemagglutinin, PHA) or antigen (tetanus toxoid, TT) and, as shown here, release soluble factor(s) that induce eosinophil adherence. CAgX also caused concentration dependent proliferation of BMN cells from sweet itch and normal ponies [stimulation index: 29 (13) and 17 (7) for BMN cells from sweet itch and normal ponies, respectively during the active pha...
Preliminary evaluation of diagnostic tests using horses experimentally infected with trypanosoma evansi.
Veterinary journal (London, England : 1997)    May 16, 2001   Volume 161, Issue 3 287-300 doi: 10.1053/tvjl.2000.0560
Wernery U, Zachariah R, Mumford JA, Luckins T.Seven surra negative horses were intravenously inoculated with 3 x 10(6)Trypanosoma evansi parasites derived from a camel. One horse was maintained as an uninfected negative control. Three antigen and three antibody detection tests were evaluated for diagnosis of infection in horses. The microhaematocrit centrifugation test (MHCT) was the most sensitive, first detecting parasites between one and three days (x 2.4) post infection (p.i.). The antigen (ag)-ELISA detected antigen between three and ten days (x 6.6) p.i. The latex agglutination test (LAT) first gave positive results on day 3 (x 3.0)...
West Nile virus recombinant DNA vaccine protects mouse and horse from virus challenge and expresses in vitro a noninfectious recombinant antigen that can be used in enzyme-linked immunosorbent assays.
Journal of virology    April 5, 2001   Volume 75, Issue 9 4040-4047 doi: 10.1128/JVI.75.9.4040-4047.2001
Davis BS, Chang GJ, Cropp B, Roehrig JT, Martin DA, Mitchell CJ, Bowen R, Bunning ML.Introduction of West Nile (WN) virus into the United States in 1999 created major human and animal health concerns. Currently, no human or veterinary vaccine is available to prevent WN viral infection, and mosquito control is the only practical strategy to combat the spread of disease. Starting with a previously designed eukaryotic expression vector, we constructed a recombinant plasmid (pCBWN) that expressed the WN virus prM and E proteins. A single intramuscular injection of pCBWN DNA induced protective immunity, preventing WN virus infection in mice and horses. Recombinant plasmid-transform...
Response to immunotherapy in six related horses with urticaria secondary to atopy.
Journal of the American Veterinary Medical Association    March 31, 2001   Volume 218, Issue 5 753-755 doi: 10.2460/javma.2001.218.753
Rees CA.Urticaria secondary to atopy may be a familial problem in some horses. Immunotherapy using a vaccine containing antigens selected on the basis of history and results of intradermal testing can be an effective method of managing atopy in horses; a response to therapy may be seen within 2 months.
Antigenic variation among equine H 3 N 8 influenza virus hemagglutinins.
The Japanese journal of veterinary research    March 30, 2001   Volume 48, Issue 4 177-186 
Ozaki H, Shimizu-Nei A, Sugita S, Sugiura T, Imagawa H, Kida H.To provide information on the antigenic variation of the hemagglutinins (HA) among equine H 3 influenza viruses, 26 strains isolated from horses in different areas in the world during the 1963-1996 period were analyzed using a panel of monoclonal antibodies recognizing at least 7 distinct epitopes on the H 3 HA molecule of the prototype strain A/equine/Miami/1/63 (H 3 N 8). The reactivity patterns of the virus strains with the panel indicate that antigenic drift of the HA has occurred with the year of isolation, but less extensively than that of human H 3 N 2 influenza virus isolates, and diff...
Cross reaction of recombinant equine infectious anemia virus antigen to heterologous strains and application for serological survey among horses in the field.
Microbiology and immunology    March 29, 2001   Volume 45, Issue 1 45-50 doi: 10.1111/j.1348-0421.2001.tb01273.x
Sentsui H, Inoshima Y, Murakami K, Akashi H, Purevtseren B, Pagmajav O, Sugiura T.Cross reactivity of equine infectious anemia virus (EIAV) antigen prepared using a recombinant baculovirus containing the p26 gene of strain P337-V70 was examined by the agar gel immunodiffusion (AGID) test and enzyme-linked immunosorbent assay (ELISA). Serum samples serially collected from 13 horses experimentally infected with six different EIAV strains (two or three horses per strain) were subjected to the test. Positive reactions were observed in the AGID test and ELISA before or soon after the first feverish period and continued persistently in most of the horses. The results with recombi...
An enzyme-linked immunosorbent assay for the convenient serodiagnosis of contagious equine metritis in mares. Katz J, Geer P.An enzyme-linked immunosorbent assay (ELISA) was developed for the serodiagnosis of contagious equine metritis (CEM), a sexually transmitted disease caused by Taylorella equigenitalis. Antigen preparation was simple, and antigens derived from both classical and atypical forms of T. equigenitalis enabled detection of antibody responses elicted in horses experimentally exposed to either form of the bacterium. Sera serially obtained from these horses from 0 to 63 days postexposure were tested by the traditional complement fixation test (CFT) for CEM and with the ELISA, using both antigens separat...
Characterization of a Sarcocystis neurona isolate from a Missouri horse with equine protozoal myeloencephalitis.
Veterinary parasitology    February 27, 2001   Volume 95, Issue 2-4 143-154 doi: 10.1016/s0304-4017(00)00386-1
Marsh AE, Johnson PJ, Ramos-Vara J, Johnson GC.Little information is available about antigenic variation of Sarcocystis neurona isolated from horses with equine protozoal myeloencephalitis, nor is there much information available on the specific antibody pattern to S. neurona antigens of horses from different geographic regions where S. neurona isolates have been obtained. This communication reports on the characterization of a new S. neurona isolate, SN-MU1. The isolate was obtained from a 3-year old Thoroughbred that had asymmetrical neurological signs and localized skeletal muscle atrophy. This S. neurona isolate is similar to other S. ...
Evaluation of a polyvalent enzyme-linked immunosorbent assay incorporating a recombinant p44 antigen for diagnosis of granulocytic ehrlichiosis in dogs and horses.
American journal of veterinary research    February 24, 2001   Volume 62, Issue 1 29-32 doi: 10.2460/ajvr.2001.62.29
Magnarelli LA, Ijdo JW, Van Andel AE, Wu C, Fikrig E.To develop and evaluate a polyvalent ELISA incorporating a highly specific recombinant antigen (p44) for diagnosis of granulocytic ehrlichiosis in dogs and horses. Methods: 32 dogs and 43 horses. Methods: Results of the ELISA were compared with results of indirect fluorescent antibody (IFA) staining and western immunoblotting incorporating whole-cell antigen. Results: For the canine and equine samples, percentages of samples with positive IFA staining, western immunoblotting, and ELISA results were similar. For 29 (91 %) canine samples and 30 (70%) equine samples, results of IFA staining, west...
Expression of Babesia equi merozoite antigen 1 in insect cells by recombinant baculovirus and evaluation of its diagnostic potential in an enzyme-linked immunosorbent assay.
Journal of clinical microbiology    February 7, 2001   Volume 39, Issue 2 705-709 doi: 10.1128/JCM.39.2.705-709.2001
Xuan X, Larsen A, Ikadai H, Tanaka T, Igarashi I, Nagasawa H, Fujisaki K, Toyoda Y, Suzuki N, Mikami T.The gene encoding the entire Babesia equi merozoite antigen 1 (EMA-1) was inserted into a baculovirus transfer vector, and a recombinant virus expressing EMA-1 was isolated. The expressed EMA-1 was transported to the surface of infected insect cells, as judged by an indirect fluorescent-antibody test (IFAT). The expressed EMA-1 was also secreted into the supernatant of a cell culture infected with recombinant baculovirus. Both intracellular and extracellular EMA-1 reacted with a specific antibody in Western blots. The expressed EMA-1 had an apparent molecular mass of 34 kDa that was identical ...
Immunohistochemical detection of virulence-associated Rhodococcus equi antigens in pulmonary and intestinal lesions in horses.
Journal of comparative pathology    October 18, 2000   Volume 123, Issue 2-3 186-189 doi: 10.1053/jcpa.2000.0392
Mariotti F, Cuteri V, Takai S, Renzoni G, Pascucci L, Vitellozzi G.Two horses with Rhodococcus equi infection were examined post mortem by an immunohistochemical method (peroxidase-antiperoxidase; PAP) with a monoclonal antibody (Mab 10G5) to the 15-17 kDa antigen of R. equi. One of the horses was also examined bacteriologically, R. equi being isolated in culture. Immunolabelling with this Mab was marked and widespread. On the other hand, the immunohistochemical reactivity of infected macrophages with a polyclonal antibody specific for lysozyme was slight. Thus, Mab 10G5 would appear to be a useful diagnostic reagent in R. equi infection, with or without cult...
Induction of mucosal and systemic antibody specific for SeMF3 of Streptococcus equi by intranasal vaccination using a sucrose acetate isobutyrate based delivery system.
Vaccine    October 12, 2000   Volume 19, Issue 4-5 492-497 doi: 10.1016/s0264-410x(00)00187-0
Nally JE, Artiushin S, Sheoran AS, Burns PJ, Simon B, Gilley RM, Gibson J, Sullivan S, Timoney JF.Streptococcus equi causes equine strangles, a highly contagious disease of the upper respiratory tract. The antiphagocytic surface protein SeM is strongly immunogenic and evokes mucosal and systemic antibodies during convalescence. The present study investigated the potential of sucrose acetate isobutyrate (SAIB); a high viscosity excipient that provides controlled release of biologically active substances, to enhance antibody responses following intranasal immunization of horses with a 108 a.a. peptide of SeM (SeMF3). SeMF3-SAIB was administered intranasally to each of the 11 adult horses on ...
Detection of biological threat agents by immunomagnetic microsphere-based solid phase fluorogenic- and electro-chemiluminescence.
Biosensors & bioelectronics    August 17, 2000   Volume 14, Issue 10-11 829-840 doi: 10.1016/s0956-5663(99)00068-8
Yu H, Raymonda JW, McMahon TM, Campagnari AA.This article reviews the recent development of two solid-phase chemiluminescence-based techniques, fluorogenic-chemiluminescence (FCL) and electro-chemiluminescence (ECL) for detection of biological threat agents. Both techniques entail a labeled sandwich immunoassay. The objectives of this work are to develop advanced techniques for sensitive and effective detection of a target analyte, particularly in cases where the analysis includes complex samples containing multiple contaminating factors. Other important considerations in developing such detection techniques include the ease of use, the ...
Utilisation of bacteriophage display libraries to identify peptide sequences recognised by equine herpesvirus type 1 specific equine sera.
Journal of virological methods    August 2, 2000   Volume 88, Issue 1 89-104 doi: 10.1016/s0166-0934(00)00183-x
Birch-Machin I, Ryder S, Taylor L, Iniguez P, Marault M, Ceglie L, Zientara S, Cruciere C, Cancellotti F, Koptopoulos G, Mumford J, Binns M....Three filamentous phage random peptide display libraries were used in biopanning experiments with purified IgG from the serum of a gnotobiotic foal infected with equine herpesvirus-1 (EHV-1) to enrich for epitopes binding to anti-EHV-1 antibodies. The sequences of the amino acids displayed were aligned with protein sequences of EHV-1, thereby identifying a number of potential antibody binding regions. Presumptive epitopes were identified within the proteins encoded by genes 7 (DNA helicase/primase complex protein), 11 (tegument protein), 16 (glycoprotein C), 41 (integral membrane protein), 70 ...
Equine influenza vaccine efficacy: the significance of antigenic variation.
Veterinary microbiology    May 9, 2000   Volume 74, Issue 1-2 173-177 doi: 10.1016/s0378-1135(00)00177-2
Yates P, Mumford JA.To investigate the level of cross-protection induced by equine influenza H3N8 vaccines derived from different lineages, two studies have been carried out with ponies vaccinated with 'American-like' and 'European-like' vaccines and experimentally challenged with a European-like strain. The results demonstrated that equine influenza vaccines clearly protect against challenge with homologous virus if serum antibody titres are sufficiently high. On the other hand, protection is incomplete even when animals vaccinated with heterologous strains have comparative antibody levels. Nevertheless, the pro...
DNA vaccination against influenza viruses: a review with emphasis on equine and swine influenza.
Veterinary microbiology    May 9, 2000   Volume 74, Issue 1-2 149-164 doi: 10.1016/s0378-1135(00)00175-9
Olsen CW.The influenza virus vaccines that are commercially-available for humans, horses and pigs in the United States are inactivated, whole-virus or subunit vaccines. While these vaccines may decrease the incidence and severity of clinical disease, they do not consistently provide complete protection from virus infection. DNA vaccines are a novel alternative to conventional vaccination strategies, and offer many of the potential benefits of live virus vaccines without their risks. In particular, because immunogens are synthesized de novo within DNA transfected cells, antigen can be presented by MHC c...
Consideration of the role of antigenic keratan sulphate reacting to a 1/14/16H9 antibody as a molecular marker to monitor cartilage metabolism in horses.
The Journal of veterinary medical science    April 19, 2000   Volume 62, Issue 3 281-285 doi: 10.1292/jvms.62.281
Okumura M, Tagami M, Fujinaga T.The role of keratan sulphate (KS) as a marker of cartilage metabolism was evaluated by using an in vitro model of equine articular cartilage. Articular cartilage was harvested from clinically healthy 6-month-old foals (n=3). Chondrocytes were centrifuged and cultured as pellets. Chondrocyte pellets were stimulated by insulin-like growth factor (IGF)-Ialpha or interleukin (IL)-1alpha for 2 weeks. The sulfated glycosaminoglycans (GAG) and antigenic KS concentrations in the culture media were measured by a 1,9-dimethyl-methylene blue (DMMB) colorimetric assay and an inhibition ELISA using a 1/14/...
Inflammation in horses.
The Veterinary clinics of North America. Equine practice    April 7, 2000   Volume 16, Issue 1 15-v doi: 10.1016/s0749-0739(17)30116-5
MacKay RJ.After inflammation is initiated by detection of antigen, plasma components and activated leukocytes are concentrated at the inflammatory site. Cellular and chemical effectors of inflammation are focused on the offending antigen, usually resulting in its destruction and elimination. Activation of endogenous counter-regulatory systems damps down the inflammatory process and is the first stage of repair. In addition to local effects, the inflammatory focus may initiate a continuum of systemic acute phase responses ranging from the systemic inflammatory response syndrome (SIRS) to generalized immu...
[Demonstration of Chlamydia from an equine abortion].
DTW. Deutsche tierarztliche Wochenschrift    April 1, 2000   Volume 107, Issue 2 49-52 
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 ...
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