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Joe Brownlie

Publications and source records attributed to Joe Brownlie.

15 recordsLinked to original sources
PubMed2006

Isolation and sequence analysis of canine respiratory coronavirus.

Canine respiratory coronavirus (CRCoV) has frequently been detected in respiratory samples from dogs by RT-PCR. In this report the first successful isolation of CRCoV from a dog with respiratory disease is described. The isolate CRCoV-4182 was cultured in HRT-18 cells but failed to replicate in a number of other cell lines. The nucleotide sequence of the 3'-terminal portion of the CRCoV genome was determined including all open reading frames from the NS2 gene to the N gene. Comparison with other coronavirus sequences showed a high similarity to bovine coronavirus (BCoV). The region between the spike and the E gene was found to be the most variable and was used for phylogenetic analysis of several CRCoV strains. CRCoV-4182 showed a mutation within the non-structural protein region downstream of the S gene leading to the translation of an 8.8 kDa putative protein comprising a fusion of the equivalent of the BCoV 4.9 kDa protein to a truncated version of the BCoV 4.8 kDa protein. The culture of CRCoV will enable analysis of the expression and function of this and other CRCoV proteins as well as allowing the study of the role of CRCoV in the aetiology of canine infectious respiratory disease.

Amino Acid Sequence↗
PubMed2006

Serological prevalence of canine respiratory coronavirus.

Canine respiratory coronavirus (CRCoV) has recently been detected in dogs; it is a group 2 coronavirus showing similarity to bovine coronavirus (BCoV) but is distinct from canine enteric coronavirus (CECoV). CRCoV may play an important role in canine infectious respiratory disease (CIRD) either by predisposing to further and potentially more serious viral and bacterial infections or possibly as a primary pathogen. The prevalence of serum antibodies to CRCoV, in a population of dogs in the south east of England, has been shown previously to be 30.1% on the first day of entry to a rehoming kennel [Erles, K., Toomey, C., Brooks, H.W., Brownlie, J., 2003. Detection of a group 2 coronavirus in dogs with canine infectious respiratory disease. Virology 310, 216-223]. The purpose of this study was to establish the prevalence of CRCoV in the general canine population within as well as outside the UK. An ELISA, used to test for the presence of antibodies to CRCoV in canine serum samples, identified seropositive dogs in UK, USA, Canada, Republic of Ireland and Greece. The development of an ELISA based on CRCoV antigen and immunofluorescence assay are described here. 54.7% (547/1000) of North American and 36.0% (297/824) of United Kingdom dogs were seropositive for CRCoV. The age and geographical distribution of seropositive dogs was also assessed. The cross-reactivity demonstrated between CRCoV antibodies from different countries and a UK viral isolate suggests immunological similarity. The overall prevalence of this virus in both North America and the UK suggests that CRCoV has international significance and that further epidemiological studies are required.

Age Factors↗
PubMed2005

Ability to differentiate between cp and ncp BVDV by microarrays: towards an application in clinical veterinary medicine?

Microarray expression profiling provides a comprehensive portrait of the transcriptional world enabling us to view the organism as a 'system' that is more than the sum of its parts. The vigilance of cells to environmental change, the alacrity of the transcriptional response, the short half-life of cellular mRNA and the genome-scale nature of the investigation collectively explain the power of this method. These same features pose the most significant experimental design and execution issues which, unless surmounted, predictably generate a distorted image of the transcriptome. Conversely, the expression profile of a properly conceived and conducted microarray experiment can be used for hypothesis testing: disclosure of the metabolic and biosynthetic pathways that underlie adaptation of the organism to infectious processes; the identification of co-ordinately regulated genes; the regulatory circuits and signal transduction systems that mediate the adaptive response; and temporal features of developmental programmes. The study of viral pathogenesis by microarray expression profiling poses special challenges and opportunities. Although the technical hurdles are many, obtaining expression profiles of an organism growing in tissue will probably reveal strategies for growth and survival of the virus in the host's cells. Here, we show data obtained using a tailored microarray system based on synthetic polynucleotides derived from human sequences (SIRS-Lab GmbH, Jena, Germany) to study the effect of cytopathogenic (cpe) and non-cytopathogenic (ncp) bovine viral diarrhoea virus (BVDV) infection of bovine macrophages, focusing on intracellular signalling molecules. Of the 575 genes present on the array, more than 70% showed a reaction with the oligonuleotides spotted on the array, and 26 genes were differentially expressed comparing cDNA derived from cpe and ncp infected cells. These data will help to further understand our knowledge regarding BVDV infection, and will especially help to understand differences in cellular responses to cpe and ncp biotypes.

Animals↗
PubMed2004

Development of a polymerase chain reaction for the detection of Mycoplasma felis in domestic cats.

Mycoplasma felis is associated with conjunctivitis and respiratory disease in domestic cats. Currently no rapid diagnostic test is available for the detection of M. felis in clinical samples that does not rely on prior cultivation of the organism. The objective of this study was to determine whether a polymerase chain reaction (PCR) based upon the 16S/23S rRNA intergenic spacer sequence is suitable for the identification of M. felis directly in feline clinical samples. The high conservation between the 16S/23S rRNA intergenic spacers (IGS) of differing isolates of M. felis was established by sequence analysis and a PCR was developed to this region by comparison to IGS of other mycoplasmas. The PCR was found to be highly specific for M. felis and further PCR analysis on clinical samples showed the PCR to be highly sensitive and more rapid than the other methods of identification currently available.

Animals↗
PubMed2004

Taxonomy of the canine Mollicutes by 16S rRNA gene and 16S/23S rRNA intergenic spacer region sequence comparison.

The taxonomy of canine Mollicutes is described, based on phylogenetic analysis of 16S rRNA gene and 16S/23S rRNA intergenic spacer (IGS) region sequences. The nucleotide sequences of the 16S rRNA gene of two untyped mycoplasmas and the IGS region of 11 Mycoplasma species were determined and used for phylogenetic analysis. The two untyped Mycoplasma strains, HRC 689 and VJC 358, were found to be distinct from all known canine mycoplasmas and all published mycoplasma 16S rRNA gene sequences.

Animals↗
PubMed2004

Mycoplasmas associated with canine infectious respiratory disease.

Canine infectious respiratory disease (CIRD) is a complex infection that occurs worldwide predominantly in kennelled dogs, and several bacterial and viral micro-organisms have been associated with outbreaks of CIRD. However, few studies have comprehensively examined the species of mycoplasma present in healthy dogs and those with CIRD. As part of an extensive study investigating the micro-organisms involved in CIRD, the species of mycoplasma present throughout the respiratory tract of dogs with and without CIRD were determined. Mycoplasmas were cultured from tonsillar, tracheal and bronchial lavage samples, and identified to the species level by PCR and sequencing. Mycoplasma cynos was demonstrated on the ciliated tracheal epithelium by in situ hybridization and was the only mollicute found to be associated with CIRD, but only in the lower respiratory tract. Isolation of M. cynos was correlated with an increased severity of CIRD, younger age and a longer time in the kennel.

Animals↗
PubMed2004

Longitudinal study of viruses associated with canine infectious respiratory disease.

In this investigation a population of dogs at a rehoming center was monitored over a period of 2 years. Despite regular vaccination of incoming dogs against distemper, canine adenovirus type 2 (CAV-2), and canine parainfluenza virus (CPIV), respiratory disease was endemic. Tissue samples from the respiratory tract as well as paired serum samples were collected for analysis. The development of PCR assays for the detection of CPIV, canine adenovirus types 1 and 2, and canine herpesvirus (CHV) is described. Surprisingly, canine adenovirus was not detected in samples from this population, whereas 19.4% of tracheal and 10.4% of lung samples were positive for CPIV and 12.8% of tracheal and 9.6% of lung samples were positive for CHV. As reported previously, a novel canine respiratory coronavirus (CRCoV) was detected in this population (K. Erles, C. Toomey, H. W. Brooks, and J. Brownlie, Virology 310:216-223, 2003). Infections with CRCoV occurred mostly during the first week of a dog's stay at the kennel, whereas CPIV and CHV were detected at later time points. Furthermore, the evaluation of an enzyme-linked immunosorbent assay for detection of antibodies to CPIV and an immunofluorescence assay for detection of antibodies to CHV is described. This study shows that CPIV is present at kennels despite vaccination. In addition, other agents such as CHV and CRCoV may play a role in the pathogenesis of canine respiratory disease, whereas CAV-2 and canine distemper virus were not present in this population, indicating that their prevalence in the United Kingdom is low due to widespread vaccination of dogs.

Adenoviruses, Canine↗
PubMed2003

The association of Streptococcus equi subsp. zooepidemicus with canine infectious respiratory disease.

Canine infectious respiratory disease (CIRD) is a multi-factorial infection that affects many kennelled dogs despite the wide use of vaccination. Current vaccines aim to protect against viral agents and a single bacterial agent, Bordetella bronchiseptica. We sought to examine the role of streptococcal species in CIRD. The isolation and identification of streptococci in the lower respiratory tract of clinically healthy dogs and those with CIRD were used to correlate the presence of specific streptococcal species with respiratory disease. In this study we report that the presence of S. equi subsp. zooepidemicus is associated with increasing severity of disease in a population of kennelled dogs with endemic CIRD.

Animals↗
PubMed2003

Detection of a group 2 coronavirus in dogs with canine infectious respiratory disease.

An investigation into the causes of canine infectious respiratory disease was carried out in a large rehoming kennel. Tissue samples taken from the respiratory tract of diseased dogs were tested for the presence of coronaviruses using RT-PCR with conserved primers for the polymerase gene. Sequence analysis of four positive samples showed the presence of a coronavirus with high similarity to both bovine and human coronavirus (strain OC43) in their polymerase and spike genes, whereas there was a low similarity to comparable genes in the enteric canine coronavirus. This canine respiratory coronavirus (CRCV) was detected by RT-PCR in 32/119 tracheal and 20/119 lung samples, with the highest prevalence being detected in dogs with mild clinical symptoms. Serological analysis showed that the presence of antibodies against CRCV on the day of entry into the kennel decreased the risk of developing respiratory disease.

Animals↗
PubMed2003

DNA vaccination against bovine viral diarrhoea virus induces humoral and cellular responses in cattle with evidence for protection against viral challenge.

The immune response induced by a DNA construct expressing the E2 envelope glycoprotein of bovine viral diarrhoea virus (BVDV) was studied in cattle. Four groups of five calves, were immunised by intradermal injection with a total of 1mg of plasmid DNA on each of two occasions, with a 3-week dose interval. Group 1 received non-coding plasmid DNA only (control), group 2 received the E2 coding plasmid (0.5mg) plus non-coding plasmid DNA (0.5mg) and groups 3 and 4 received the E2 coding plasmid plus plasmid encoding either bovine interleukin 2 (IL-2) or granulocyte macrophage colony stimulating factor (GM-CSF) respectively. Two weeks after the final immunisation, all calves were challenged by intranasal inoculation with 5 x 10(6) TCID(50) of homologous virus. On the day of challenge, neutralising antibodies were detectable in 13 of 15 vaccinated calves (one animal in each of groups 3 and 4 remained seronegative at this point). Thereafter, a strong anamnestic serological response was evident in all vaccinated animals. Furthermore, T-cell proliferation following in vitro re-stimulation with BVDV antigen was significantly elevated in the cytokine adjuvanted groups. This enhancement of BVDV specific immune responses in vaccinated animals was reflected in the clinical responses observed post-challenge. In particular, reduced febrile responses provided evidence of a disease sparing effect of vaccination. Significantly, whilst a transient viraemia was detected in all control animals following challenge, no virus was isolated from the leucocytes from 8 out of the 15 vaccinated animals. In groups 2 and 4, three animals remained virus free, although virus was isolated from two animals in each group at a single time point, while in group 3, three out of five animals had detectable viraemia. In summary, the administration of a DNA vaccine encoding only the E2 glycoprotein of BVDV induced a disease sparing effect in vaccinated calves following challenge and protected more than half of the vaccinated animals from detectable viraemia.

Animals↗
PubMed2003

Differences in cytokine synthesis by the sub-populations of dendritic cells from afferent lymph.

Two phenotypically distinct subpopulations of dendritic cells (SIRPalpha+ CC81Ag- DC and SIRPalpha- CC81Ag+ DC) have previously been identified in bovine afferent lymph which show functional differences when assayed in vitro. The purpose of this study was to investigate whether differences in cytokine production between the two subpopulations might occur which could influence the bias of the immune response they stimulate. Qualitative and quantitative polymerase chain reactions were used to detect specific mRNA transcripts and flow cytometry and enzyme-linked immunosorbent assays were used to detect protein production. The SIRPalpha- CC81Ag+ DC produced considerably more interleukin-12 (IL-12) mRNA transcripts and protein than the SIRPalpha+ CC81Ag- DC. Conversely, SIRPalpha+ CC81Ag- DC contained more of both transcripts and protein for IL-1 and of transcripts for IL-6. A small percentage of both subpopulations produced interferon-gamma (IFN-gamma) as evidenced by cytoplasmic staining. Stimulation of DC by culture with CD40L+ cells increased the production of IL-1, IL-6 and IL-12 but quantitative differences between the subpopulations remained. Production of IL-10 was also evident following culture with CD40L+ cells or lipopolysaccharide primarily by the SIRPalpha+ CC81Ag- DC. No evidence was found for type 1 IFN production, and hence plasmacytoid DC, by DC in afferent lymph; both subpopulations appear to be myeloid in origin. These different cytokine repertoires of the two subpopulations of ex vivo DC isolated from afferent lymph imply functional differences that could influence the presentation of antigen to T cells and bias of the immune response following vaccination or infection.

Animals↗
PubMed2003

Respiratory disease in kennelled dogs: serological responses to Bordetella bronchiseptica lipopolysaccharide do not correlate with bacterial isolation or clinical respiratory symptoms.

The role of Bordetella bronchiseptica in a natural outbreak of canine infectious respiratory disease was investigated both by culture and serological analysis. B. bronchiseptica was found in the lungs of a large proportion of clinically healthy dogs and in a greater proportion of dogs with respiratory disease. Using a lipopolysaccharide (LPS) antigen-based enzyme-linked immunosorbent assay, we analyzed the serological responses of a large number of dogs. Dogs with high antibody levels showed no protection from disease, and there was no correlation between the development of disease and rising antibody titer. Similarly, there was no difference in antibody levels in dogs with and without B. bronchiseptica in the lungs. Antibodies to LPS have no predictive value in determining which animals will contract respiratory disease, how severe the disease will be, or which dogs will have B. bronchiseptica colonizing the lungs.

Animals↗
PubMed2002

Cellular sequences in pestivirus genomes encoding gamma-aminobutyric acid (A) receptor-associated protein and Golgi-associated ATPase enhancer of 16 kilodaltons.

The presence of cellular protein coding sequences within viral RNA genomes is a unique and particularly interesting feature of cytopathogenic (cp) pestiviruses. Here we report the identification and characterization of two novel cellular sequences in the genomes of cp bovine viral diarrhea virus (BVDV) strains. In BVDV strain CP X604, we detected a duplication of the genomic region encoding NS3, NS4A, and part of NS4B, together with an insertion of sequences that code for cellular gamma-aminobutyric acid (A) receptor-associated protein [GABA(A)-RAP]. Transient-expression studies showed that the GABA(A)-RAP sequence leads to additional processing of the viral polyprotein and thereby to the expression of nonstructural protein NS3. Transfection of bovine cells with RNA transcribed from an infectious cDNA clone revealed that the GABA(A)-RAP-encoding insertion together with the duplicated viral sequences constitutes the genetic basis for the cytopathogenicity of strain CP X604. Surprisingly, molecular analysis of another cp BVDV strain (CP 721) resulted in the identification of a cellular Golgi-associated ATPase enhancer of 16 kDa (GATE-16)-encoding insertion together with duplicated viral sequences. To our knowledge, the genomes of CP X604 and CP 721 are the first viral RNAs found with cellular sequences encoding GABA(A)-RAP and GATE-16, respectively. Interestingly, the two cellular proteins belong to a family of eukaryotic proteins involved in various intracellular trafficking processes. Processing after the C-terminal glycine residue of GABA(A)-RAP and GATE-16 by cellular proteases is essential for covalent attachment to target molecules. Accordingly, it can be assumed that these cellular proteases also recognize the cleavage sites in the context of the respective viral polyproteins and thereby lead to the generation of NS3, the marker protein of cp BVDV.

Adaptor Proteins, Signal Transducing↗