Biomedical subjects
M M Binns
Publications and source records attributed to M M Binns.
Use of transabdominal ultrasound-guided amniocentesis for detection of equid herpesvirus 1-induced fetal infection in utero.
OBJECTIVE: To evaluate transabdominal ultrasound-guided amniocentesis for detection of equid herpes-virus 1 (EHV-1)-induced fetal infection in utero. ANIMALS: 4 Welsh Mountain mares. PROCEDURE: Pregnant mares were inoculated intranasally with EHV-1 during the ninth month of gestation. Amniocentesis was initiated on postinoculation day (PID) 12, and was performed at 2- to 3-day intervals in standing mares under deep sedation. Amniotic fluid samples were tested by virus isolation (VI), polymerase chain reaction (PCR), and immunoperoxidase cytologic examination (IC) for detection of EHV-1. RESULTS: Exposure to EHV-1 in the ninth month of gestation resulted in nasal shedding of infective virus, establishment of cell-associated viremia, and seroconversion. Equid herpesvirus 1 was detected by VI, PCR, and IC in amniotic fluid collected on PID 14 from 1 mare and on PID 16 and 17 from a second mare. Specimens of amniotic fluid from a third mare were VI negative until PID 18, when collections ceased, although this mare subsequently aborted an EHV-1-infected fetus on PID 28. The fourth mare aborted an EHV-1 infected fetus on PID 14. The 2 mares with VI-positive amniotic fluid were each carrying an EHV-1 infected fetus in utero, confirmed by examination of the uterus, placenta, and fetus, using specific immunohistochemistry and in situ hybridization. Endothelial cells in the endometrium and allantochorion were often virus-infected, with accompanying vascular lesions. The fetus had been infected via the chorionic vasculature in the first and fourth mares, and by inhalation of infected amniotic fluid in the second mare. CONCLUSION: Amniocentesis permits specific detection of EHV-1-induced fetal infection in utero. CLINICAL RELEVANCE: Amniocentesis may have a clinical role in the specific identification and isolation of mares carrying virus-infected fetuses during EHV-1-induced abortion epizootics.
Chromosome-specific paints from a high-resolution flow karyotype of the dog.
Using peripheral blood lymphocyte cultures and dual-laser flow cytometry, we have routinely obtained high-resolution bivariate flow karyotypes of the dog in which 32 peaks are resolved. To allow the identification of the chromosome types in each peak, chromosomes were flow sorted, amplified and labelled by polymerase chain reaction with partially degenerate primers and hybridized onto metaphase spreads of a male dog. The chromosome paints from 22 of the 32 peaks each hybridized to single homologue pairs and eight peaks each hybridized to two pairs. Paints from the remaining two peaks hybridized to only one homologue each in the male metaphase spread, thus corresponding to the sex chromosomes X and Y. All of the 38 pairs of autosomes and the two sex chromosomes of the dog could be accounted for in these painting experiments. The positions of chromosomes 1-21 were assigned to the flow karyotype (only chromosomes 1-21 have as yet been officially designated). The high-resolution flow karyotype and the chromosome paints will facilitate further standardization of the dog karyotype. The ability to sort sufficient quantities of dog chromosomes for the production of chromosome-specific DNA libraries has the potential to accelerate the physical and genetic mapping of the dog genome.
The application of FISH techniques for physical mapping in the dog (Canis familiaris).
The abundance of CA/GT repeats in the DNA of the dog (Canis familiaris) has established the importance of polymorphic microsatellites in the development of a low density map of the canine genome. The assignment of linkage groups of markers to chromosomes by physical mapping requires reliable cytogenetic techniques for routine production of metaphase cells. The dog has 78 chromosomes, many of which are smaller and more contracted than those of other mammals. Although the molecular study of inherited disease in dogs has important implications for both improved welfare in dogs and the provision of animal models for human diseases, the small size and large number of chromosomes in the canine genome has discouraged the inclusion of cytogenetic analysis in the planning of relevant research protocols. In this report, Fluorescence In Situ Hybridization (FISH) techniques have been optimized for the physical mapping of probes in C. familiaris. A method to obtain a good yield of early and midmetaphases from short-term peripheral blood cultures and the optimal conditions for hybridization and detection of probes is described. Thirteen microsatellite-containing cosmid probes from a canine genomic library in pWE15, a highly repetitive probe (human ribosomal DNA pHr14E3), and a human X Chromosome (Chr) paint have been mapped. Six microsatellites, two ribosomal sites, and the human paint have been assigned to specific chromosomes.
Replication of equid herpesvirus-1 (EHV-1) in the testes and epididymides of ponies and venereal shedding of infectious virus.
Six Welsh Mountain pony colts were infected intranasally with the Ab4 isolate of EHV-1. Clinical and virological monitoring demonstrated mild upper respiratory tract disease, with nasal shedding of virus and establishment of a cell-associated viraemia. Detailed pathological examination of the urogenital tract was performed post mortem on days 4-9 post-infection (PI). EHV-1 was isolated from the epididymis on day 8 and the testis on day 9 PI, with viral replication in endothelial cells of these organs and an associated necrotizing vasculitis and thrombosis. Productive viral infection of germinal epithelium was not observed. In a further study, three Welsh Mountain pony stallions were infected intranasally with Ab4, which again resulted in mild upper respiratory tract disease and the establishment of a cell-associated viraemia. Semen samples were collected up to day 60 PI. Two stallions showed a decrease in the proportion of morphologically normal sperm. Significant numbers of inflammatory cells were observed in the sperm-rich fraction of ejaculates collected from one stallion between days 16 and 28 PI; infectious virus was recovered from the semen of this animal between days 17 and 25 PI, after the cessation of viraemia. The affected stallion appeared clinically normal over the period of venereal EHV-1 shedding.
Antigenic and genetic evolution of equine H3N8 influenza A viruses.
Evolution of equine influenza a H3N8 viruses was examined by antigenic and genetic analysis of a collection isolates from around the world. It was noted that antigenic and genetic variants of equine H3N8 viruses cocirculate, and in particular that variants currently circulating in Europe and the USA are distinguishable from one another both in terms of antigenic reactivity and genetic structure of the HA1 portion of the haemagglutinin (HA) molecule. Whilst the divergent evolution of American and European isolates may be due to geographical isolation of the two gene pools, some mixing is believed to occur as 'American-like' viruses have been isolated during outbreaks of equine influenza in the UK. The cocirculation of two antigenically and genetically distinct lineages of equine influenza H3N8 viruses has serious implications for vaccine strain selection.
Von Wille-brand's disease in UK dobermanns: possible correlation of a polymorphic DNA marker with disease status.
Ninety-one dobermanns have been typed for a polymorphic microsatellite DNA marker situated within an intron of the von Willebrand factor gene and the alleles correlated with von Wille-brand's disease status. Two alleles were identified, one associated only with the normal gene and the other with both normal and disease genes.
Development of PCR assays to detect genetic variation amongst equine herpesvirus-1 isolates as an aid to epidemiological investigation.
A search for variable restriction sites has been carried out for equine herpesvirus-1 (EHV-1) in an attempt to develop markers which can be used to group epidemiologically related viruses into groups, and to learn more about the dynamics of EHV-1 disease. Crude viral DNA extracts of EHV-1, prepared by Hirt extraction, were digested with AluI, HaeIII, or RsaI, and Southern blotted following electrophoresis. DNA fingerprints, produced by probing the Southern blots with the EHV-1 EcoR1-I fragment, separated 56 isolates into 16 groups. The variable sites within the EcoR1-I fragment were mapped approximately using fragments from within EcoR1-I, and the precise location of the variable sites determined from the DNA sequence of this fragment. Oligonucleotide primers flanking the variable sites were synthesized, and used in PCR assays to detect variable fragments. The AluI variable fragment was found to result from the presence or absence of a single AluI site. In contrast, the variable bands seen with HaeIII and RsaI, resulted from variation in the copy number of two tandemly repeated sequences, one of which had not previously been recognized. In addition, HaeIII digests of EHV-1 isolates probed with the glycoprotein B (gB) gene of EHV-1 also separated isolates into two groups. The variable HaeIII site was mapped towards the 5'-end of the gB gene and a PCR assay established. The distribution of the variable AluI site within the EcoR1-I fragment and the HaeIII site within the gB gene were estimated on a large number of clinical isolates using PCR on unpurified viral tissue culture medium. Both sites had a good distribution and together with additional variable sites should provide the basis for the rapid DNA fingerprinting of EHV-1 isolates.
Replication of equid herpesvirus 4 in endothelial cells and synovia of a field case of viral pneumonia and synovitis in a foal.
Equid herpesvirus 4 (EHV-4) infection was diagnosed as the cause of interstitial pneumonia in a 6-week-old conventionally reared Welsh pony foal, by cocultivation and immunolabelling with specific monoclonal antibodies, EHV-4 specific amplification of viral DNA, and immunohistological examination of infected tissues. The case was novel in that replication of the EHV-4 isolate in endothelial cells and in the synovial epithelium was a feature. Restriction digests of this isolate were compared with those of seven respiratory and one abortigenic EHV-4 isolate, and no differences in restriction pattern were evident. The implications of these findings for the pathogenesis of EHV-4 infection are discussed.
Eighteen canine microsatellites.
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Dog parentage testing using canine microsatellites.
Canine microsatellites, comprising one to four base pair repeated sequences, were identified as part of a project to generate a genetic linkage map of the dog. They have been used to assign parentage in a range of doubtful paternity cases, for example, all the pups in a litter from a bitch mated on different days to two different stud dogs were identified by DNA analysis to come from one dog only. DNA typing in another case was able to exclude a male dog as the father of a litter. DNA analysis can be of relevance by allowing pedigree dogs, which would have previously been excluded on the basis of unknown paternity, to be registered with the appropriate kennel club.
Identical triplets in a thoroughbred mare.
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Comparison of M and N gene sequences distinguishes variation amongst equine arteritis virus isolates.
cDNA copies of the M and N genes of equine arteritis virus (EAV) isolates were synthesized by reverse transcription followed by polymerase chain reaction amplification. The cDNA was subjected to a cycle sequencing strategy using Taq polymerase, and the nucleotide and derived amino acid sequences of 10 virus isolates were compared. The M and N genes of all isolates had the same initiation and termination sites as the prototype Bucyrus strain and the encoded proteins were conserved between viruses. Comparison of nucleotide sequence homologies and phylogenetic tree analysis implied the existence of three EAV variants originating from the U.S.A. (Bucyrus), Austria (Vienna) and Switzerland (Bibuna), and suggested that RNA recombination between EAV isolates may have occurred.
Deletion of fowlpox virus homologues of vaccinia virus genes between the 3 beta-hydroxysteroid dehydrogenase (A44L) and DNA ligase (A50R) genes.
A fragment of 4156 bp of fowlpox virus (FPV) genomic DNA contains homologues of vaccinia virus 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD; A44L) and DNA ligase (A50R) genes. The FPV locus has clearly been rearranged relative to that of vaccinia virus as homologues of genes A45R to A49R, including the thymidylate kinase and a gene with homology to superoxide dismutase, are deleted. The deleted genes are replaced by two open reading frames: for a serine proteinase inhibitor with homology to vaccinia virus gene K2L and for a protein with no significant homology to proteins in the databases. In addition, the FPV homologues of A44L and A50R are in the same polarity in FPV whereas they are in opposite polarities in vaccinia virus. Increased 3 beta-HSD activity has been demonstrated in cells infected with either of two different strains of FPV or with canarypox virus.
Three polymorphic canine microsatellites.
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Characterization of canine microsatellites.
Canine DNA was cloned in M13 and screened for the presence of (dC-dA)n.(dG-dT)n repeats. Oligonucleotide primers were synthesised to the microsatellite flanking sequences and used in the polymerase chain reaction to amplify those loci from genomic DNA. The polymorphism of each microsatellite was estimated in a set of unrelated dogs.
Genetic and antigenic analysis of an equine influenza H3 isolate from the 1989 epidemic.
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Genetic and antigenic analysis of an equine influenza H 3 isolate from the 1989 epidemic.
The haemagglutinin (HA) gene from the equine influenza H3N8 isolate Suffolk/89 has been cloned by reverse transcription and polymerase chain reaction amplification. The nucleotide sequence of the HA gene was determined from two independently cloned copies of the gene and was found to be most closely related to recent American isolates supporting the idea that most isolates of equine H3N8 are evolving as a single lineage. When the predicted amino acid sequence of the Suffolk/89 HA was examined, changes had taken place in at least four of the major antigenic sites, A, B, C, and D when compared to the sequences of the isolates used in the current vaccines (Miami/63 and Fontainebleau/79). Surprisingly, when the Suffolk/89 isolate was tested in haemagglutination inhibition (HI) assays with a panel of six mouse monoclonal antibodies, no differences were observed between the Suffolk/89 and the Fontainebleau/79 isolates, suggesting that this panel of monoclonal antibodies may recognise a limited subset of the major antigenic sites. Three anti-HA horse heterohybridoma monoclonals were able to distinguish between the Suffolk/89 and Fontainebleau/79 viruses, demonstrating that the horse does recognise these isolates as being antigenically different. The results of the work suggest that the isolates used in current equine influenza vaccines may need updating.