Kerstin Erles

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

Abstract 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.

Longitudinal Study of Viruses Associated with Canine Infectious Respiratory Disease

ABSTRACT 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 dogs 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.

Streptococcus zooepidemicus: an emerging canine pathogen.

Abstract Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) has caused several outbreaks of haemorrhagic pneumonia in dogs in recent years. This highly contagious and often fatal disease is characterised by sudden onset of clinical signs including pyrexia, dyspnoea and haemorrhagic nasal discharge. Post mortem examination typically reveals pulmonary haemorrhage and pleural effusion. Histopathology demonstrates fibrino-suppurative, necrotising and haemorrhagic pneumonia in most cases. The pathogenesis of S. zooepidemicus infection in dogs is incompletely understood. Bacterial virulence factors as well as host factors may contribute to the severe outcome. S. zooepidemicus occasionally causes zoonotic infections with potentially serious consequences. Canine vaccines for S. zooepidemicus are currently not available and prevention of the disease therefore relies on limiting bacterial spread by implementing stringent control measures in kennels. Further research, particularly sequence analysis of canine strains, is required to gain insights into epidemiology and pathogenesis of this emerging disease.

Identification of Three Novel Superantigen-Encoding Genes in Streptococcus equi subsp. zooepidemicus, szeF, szeN, and szeP

ABSTRACT The acquisition of superantigen-encoding genes by Streptococcus pyogenes has been associated with increased morbidity and mortality in humans, and the gain of four superantigens by Streptococcus equi is linked to the evolution of this host-restricted pathogen from an ancestral strain of the opportunistic pathogen Streptococcus equi subsp. zooepidemicus. A recent study determined that the culture supernatants of several S. equi subsp. zooepidemicus strains possessed mitogenic activity but lacked known superantigen-encoding genes. Here, we report the identification and activities of three novel superantigen-encoding genes. The products of szeF, szeN, and szeP share 59%, 49%, and 34% amino acid sequence identity with SPEH, SPEM, and SPEL, respectively. Recombinant SzeF, SzeN, and SzeP stimulated the proliferation of equine peripheral blood mononuclear cells, and tumor necrosis factor alpha (TNF-α) and gamma interferon (IFN-γ) production, in vitro. Although none of these superantigen genes were encoded within functional prophage elements, szeN and szeP were located next to a prophage remnant, suggesting that they were acquired by horizontal transfer. Eighty-one of 165 diverse S. equi subsp. zooepidemicus strains screened, including 7 out of 15 isolates from cases of disease in humans, contained at least one of these new superantigen-encoding genes. The presence of szeN or szeP, but not szeF, was significantly associated with mitogenic activity in the S. equi subsp. zooepidemicus population (P < 0.000001, P < 0.000001, and P = 0.104, respectively). We conclude that horizontal transfer of these novel superantigens from and within the diverse S. equi subsp. zooepidemicus population is likely to have implications for veterinary and human disease.

Serological prevalence of canine respiratory coronavirus.

Abstract 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.

Investigation into the causes of canine infectious respiratory disease: antibody responses to canine respiratory coronavirus and canine herpesvirus in two kennelled dog populations

Summary.Two training centres for working dogs were monitored for one year to determine the presence of viruses and viral antibodies and their association with canine infectious respiratory disease (CIRD). Tonsillar swabs and serum were obtained from dogs on entry into the kennels and in regular intervals thereafter. Additional samples were collected during outbreaks of CIRD. The swabs were examined by virus culture and PCR for canine parainfluenza virus, canine adenovirus, canine herpesvirus (CHV) and canine respiratory coronavirus (CRCoV). Furthermore the prevalence of antibodies to CHV and CRCoV was determined. During this study CIRD was reported mainly in one of the two kennels investigated. In that kennel antibody responses to CRCoV indicated a seasonal occurrence of the virus, which coincided with two outbreaks of respiratory disease. CHV antibody responses were detected throughout the year. In the other kennel, which reported few cases of CIRD a high prevalence of antibodies to CRCoV was detected on entry but only sporadic seroconversions to CRCoV or CHV. By PCR three dogs were found positive for CRCoV in one kennel whereas all PCR tests for other viruses were negative for both kennels. Virus culture failed to detect any viruses in either kennel.

New and Emerging Pathogens in Canine Infectious Respiratory Disease

Canine infectious respiratory disease is a common, worldwide disease syndrome of multifactorial etiology. This review presents a summary of 6 viruses (canine respiratory coronavirus, canine pneumovirus, canine influenza virus, pantropic canine coronavirus, canine bocavirus, and canine hepacivirus) and 2 bacteria (Streptococcus zooepidemicus and Mycoplasma cynos) that have been associated with respiratory disease in dogs. For some pathogens a causal role is clear, whereas for others, ongoing research aims to uncover their pathogenesis and contribution to this complex syndrome. Etiology, clinical disease, pathogenesis, and epidemiology are described for each pathogen, with an emphasis on recent discoveries or novel findings.

Isolation and sequence analysis of canine respiratory coronavirus

Abstract 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 8.8kDa putative protein comprising a fusion of the equivalent of the BCoV 4.9kDa protein to a truncated version of the BCoV 4.8kDa 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.

Characterization of Pneumonia Due to Streptococcus equi subsp. zooepidemicus in Dogs

ABSTRACT Streptococcus equi subsp. zooepidemicus has been linked to cases of acute fatal pneumonia in dogs in several countries. Outbreaks can occur in kenneled dog populations and result in significant levels of morbidity and mortality. This highly contagious disease is characterized by the sudden onset of clinical signs, including pyrexia, dyspnea, and hemorrhagic nasal discharge. The pathogenesis of S. equi subsp. zooepidemicus infection in dogs is poorly understood. This study systematically characterized the histopathological changes in the lungs of 39 dogs from a large rehoming shelter in London, United Kingdom; the dogs were infected with S. equi subsp. zooepidemicus. An objective scoring system demonstrated that S. equi subsp. zooepidemicus caused pneumonia in 26/39 (66.7%) dogs, and most of these dogs (17/26 [65.4%]) were classified as severe fibrino-suppurative, necrotizing, and hemorrhagic. Three recently described superantigen genes (szeF, szeN, and szeP) were detected by PCR in 17/47 (36.2%) of the S. equi subsp. zooepidemicus isolates; however, there was no association between the presence of these genes and the histopathological score. The lungs of S. equi subsp. zooepidemicus-infected dogs with severe respiratory signs and lung pathology did however have significantly higher mRNA levels of the proinflammatory cytokines tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), and interleukin 8 (IL-8) than in uninfected controls, suggesting a role for an exuberant host immune response in the pathogenesis of this disease.

Sequence analysis of divergent canine coronavirus strains present in a UK dog population.

Abstract Forty faecal samples were tested by RT-PCR using coronavirus consensus primers to determine faecal shedding of canine coronavirus (CCoV) and canine respiratory coronavirus (CRCoV) in a dog population housed at a rescue centre. Seven samples were positive for CCoV while all samples were negative for CRCoV. Sequence analysis of five CCoV strains showed a high similarity with transmissible gastroenteritis virus (TGEV) at the N-terminus of the spike protein. All strains contained an open reading frame for the nonstructural protein 7b, which is not present in TGEV, indicating that the strains were related to the previously described CCoV strain UCD-1. Two samples contained CCoV strains with 5′ spike sequences most similar to type II CCoV while one sample was found to contain type I CCoV. Primers directed to the N gene allowed specific detection of all CCoV strains analysed in this study. This investigation shows that CCoV strains containing spike proteins similar to TGEV are present in the UK dog population. PCR primers directed to conserved regions of the CCoV genome are recommended for detection of CCoV in clinical samples due to high genetic variability.