African horse sickness in Portugal: a successful eradication programme.
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African horse sickness (AHS) was diagnosed for the first time in southern Portugal in autumn 1989, following outbreaks in Spain. AHS virus presence was confirmed by virus isolation and serotyping. An eradication campaign with four sanitary zones was set up by Central Veterinary Services in close collaboration with private organizations. Vaccination began on 6 October. In February 1990, vaccination was extended to all Portuguese equines (170000 animals). There were 137 outbreaks on 104 farms: 206 of the equidae present died (16%) or were slaughtered (14%); 81.5% were horses, 10.7% were donkeys and 7.8% were mules. Clinical AHS occurred more frequently in horses than donkeys and mules. In the vaccinated population, 82 animals (62.2% horses and 37.8% mules and donkeys), died or were slaughtered due to suspected or confirmed AHS. One year after ending vaccination, December 1991, Portugal was declared free of AHS. Cost of eradication was US$1955513 (US$11.5/Portuguese equine). (+info)
Identification and differentiation of the nine African horse sickness virus serotypes by RT-PCR amplification of the serotype-specific genome segment 2.
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This paper describes the first RT-PCR for discrimination of the nine African horse sickness virus (AHSV) serotypes. Nine pairs of primers were designed, each being specific for one AHSV serotype. The RT-PCR was sensitive and specific, providing serotyping within 24 h. Perfect agreement was recorded between the RT-PCR and virus neutralization for a coded panel of 56 AHSV reference strains and field isolates. Serotyping was achieved successfully with live and formalin-inactivated AHSVs, with isolates of virus after low and high passage through either tissue culture or suckling mouse brain, with viruses isolated from widely separated geographical areas and with viruses isolated up to 37 years apart. Overall, this RT-PCR provides a rapid and reliable method for the identification and differentiation of the nine AHSV serotypes, which is vital at the start of an outbreak to enable the early selection of a vaccine to control the spread of disease. (+info)
Effects of chlorine, iodine, and quaternary ammonium compound disinfectants on several exotic disease viruses.
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The effects of three representative disinfectants, chlorine (sodium hypochlorite), iodine (potassium tetraglicine triiodide), and quaternary ammonium compound (didecyldimethylammonium chloride), on several exotic disease viruses were examined. The viruses used were four enveloped viruses (vesicular stomatitis virus, African swine fever virus, equine viral arteritis virus, and porcine reproductive and respiratory syndrome virus) and two non-enveloped viruses (swine vesicular disease virus (SVDV) and African horse sickness virus (AHSV)). Chlorine was effective against all viruses except SVDV at concentrations of 0.03% to 0.0075%, and a dose response was observed. Iodine was very effective against all viruses at concentrations of 0.015% to 0.0075%, but a dose response was not observed. Quaternary ammonium compound was very effective in low concentration of 0.003% against four enveloped viruses and AHSV, but it was only effective against SVDV with 0.05% NaOH. Electron microscopic observation revealed the probable mechanism of each disinfectant. Chlorine caused complete degeneration of the viral particles and also destroyed the nucleic acid of the viruses. Iodine destroyed mainly the inner components including nucleic acid of the viruses. Quaternary ammonium compound induced detachment of the envelope of the enveloped viruses and formation of micelle in non-enveloped viruses. According to these results, chlorine and iodine disinfectants were quite effective against most of the viruses used at adequately high concentration. The effective concentration of quaternary ammonium compound was the lowest among the disinfectants examined. (+info)
Transmission patterns of African horse sickness and equine encephalosis viruses in South African donkeys.
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African horse sickness (AHS) and equine encephalosis (EE) viruses are endemic to southern Africa. AHS virus causes severe epidemics when introduced to naive equine populations, resulting in severe restrictions on the movement of equines between AHS-positive and negative countries. Recent zoning of South Africa has created an AHS-free zone to facilitate equine movement, but the transmission dynamics of these viruses are not fully understood. Here, we present further analyses of serosurveys of donkeys in South Africa conducted in 1983-5 and in 1993-5. Age-prevalence data are used to derive estimates of the force of infection, A. For both viruses, A was highest in the northeastern part of the country and declined towards the southwest. In most of the country, EE virus had a higher transmission rate than AHS. The force of infection increased for EE virus between 1985 and 1993, but decreased for AHS virus. Both viruses showed high levels of variation in transmission between districts within the same province, particularly in areas of intermediate transmission. These data emphasize the focal nature of these viruses, and indicate areas where further data will assist in understanding the geographical variation in transmission. (+info)
A first full outer capsid protein sequence data-set in the Orbivirus genus (family Reoviridae): cloning, sequencing, expression and analysis of a complete set of full-length outer capsid VP2 genes of the nine African horsesickness virus serotypes.
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The outer capsid protein VP2 of African horsesickness virus (AHSV) is a major protective antigen. We have cloned full-length VP2 genes from the reference strains of each of the nine AHSV serotypes. Baculovirus recombinants expressing the cloned VP2 genes of serotypes 1, 2, 4, 6, 7 and 8 were constructed, confirming that they all have full open reading frames. This work completes the cloning and expression of the first full set of AHSV VP2 genes. The clones of VP2 genes of serotypes 1, 2, 5, 7 and 8 were sequenced and their amino acid sequences were deduced. Our sequencing data, together with that of the published VP2 genes of serotypes 3, 4, 6 and 9, were used to generate the first complete sequence analysis of all the (sero)types for a species of the Orbivirus genus. Multiple alignment of the VP2 protein sequences showed that homology between all nine AHSV serotypes varied between 47.6 % and 71.4 %, indicating that VP2 is the most variable AHSV protein. Phylogenetic analysis grouped together the AHSV VP2s of serotypes that cross-react serologically. Low identity between serotypes was demonstrated for specific regions within the VP2 amino acid sequences that have been shown to be antigenic and play a role in virus neutralization. The data presented here impact on the development of new vaccines, the identification and characterization of antigenic regions, the development of more rapid molecular methods for serotype identification and the generation of comprehensive databases to support the diagnosis, epidemiology and surveillance of AHS. (+info)
Expression of the major core antigen VP7 of African horsesickness virus by a recombinant baculovirus and its use as a group-specific diagnostic reagent.
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The major core protein, VP7, of African horsesickness virus serotype 4 (AHSV-4), the aetiological agent of a recent outbreak of the disease in southern Europe, was expressed in insect cells infected with a recombinant baculovirus containing a cloned copy of the relevant AHSV gene (S7). Analyses of its biochemical and antigenic properties confirmed the authenticity of the protein expressed. The high-level expression of VP7 under the control of the strong polyhedrin promoter of Autographa californica nuclear polyhedrosis virus induced disc-shaped crystals in infected insect cells. This enabled us to purify the protein by a one-step ultracentrifugation procedure and to utilize it for the detection of antibodies raised in horses to various serotypes of AHSV. A serological relationship between AHSV and two other orbiviruses, bluetongue virus and epizootic haemorrhagic disease virus, was also demonstrated. (+info)
A large semi-synthetic single-chain Fv phage display library based on chicken immunoglobulin genes.
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BACKGROUND: Antibody fragments selected from large combinatorial libraries have numerous applications in diagnosis and therapy. Most existing antibody repertoires are derived from human immunoglobulin genes. Genes from other species can, however, also be used. Because of the way in which gene conversion introduces diversity, the naive antibody repertoire of the chicken can easily be accessed using only two sets of primers. RESULTS: With in vitro diagnostic applications in mind, we have constructed a large library of recombinant filamentous bacteriophages displaying single chain antibody fragments derived from combinatorial pairings of chicken variable heavy and light chains. Synthetically randomised complementarity determining regions are included in some of the heavy chains. Single chain antibody fragments that recognise haptens, proteins and virus particles were selected from this repertoire. Affinities of three different antibody fragments were determined using surface plasmon resonance. Two were in the low nanomolar and one in the subnanomolar range. To illustrate the practical value of antibodies from the library, phage displayed single chain fragments were incorporated into ELISAs aimed at detecting African horsesickness and bluetongue virus particles. Virus antibodies were detected in a competitive ELISA. CONCLUSION: The chicken-derived phage library described here is expected to be a versatile source of recombinant antibody fragments directed against a wide variety of antigens. It has the potential to provide monoclonal reagents with applications in research and diagnostics. For in vitro applications, naive phage libraries based on avian donors may prove to be useful adjuncts to the selectable antibody repertoires that already exist. (+info)
Molecular detection of Culicoides spp. and Culicoides imicola, the principal vector of bluetongue (BT) and African horse sickness (AHS) in Africa and Europe.
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Bluetongue (BT) and African Horse Sickness (AHS) are infectious arthropod-borne viral diseases affecting ruminants and horses, respectively. Culicoides imicola Kieffer, 1913, a biting midge, is the principal vector of these livestock diseases in Africa and Europe. Recently bluetongue disease has re-emerged in the Mediterranean Basin and has had a devastating effect on the sheep industry in Italy and on the islands of Sicily, Sardinia, Corsica and the Balearics, but fortunately, has not penetrated onto mainland France and Spain. To survey for the presence of C. imicola, an extensive light-trap network for the collection of Culicoides, was implemented in 2002 in southern mainland France. The morphological identification of Culicoides can be both tedious and time-consuming because its size ranges from 1.5 to 3 mm. Therefore, an ITS1 rDNA polymerase chain reaction (PCR)-based diagnostic assay was developed to rapidly and reliably identify Culicoides spp. and C. imicola. The aim of this work was to set up a rapid test for the detection of C. imicola amongst a pool of insects collected in areas at risk for BT. The sequence similarity of the rDNA (nuclear ribosomal DNA), which is greater within species than between species, is the foundation of its utilisation in species-diagnostic assays. The alignment of the 11 ITS1 sequences of Culicoides obtained from Genbank and EMBL databases helped us to identify one region in the 5' end and one in the 3' end that appear highly conserved. PCR primers were designed within these regions to amplify genus-specific fragments. In order to set up a C. imicola-specific PCR, another forward primer was designed and used in combination with the previously designed reverse primer. These primers proved to be highly specific and sensitive and permitted a rapid diagnostic separation of C. imicola from Culicoides spp. (+info)