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.     

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.     

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.     

Detection of Canine Pneumovirus in Dogs with Canine Infectious Respiratory Disease

Canine pneumovirus (CnPnV) was recently identified during a retrospective survey of kenneled dogs in the United States. In this study, archived samples from pet and kenneled dogs in the United Kingdom were screened for CnPnV to explore the relationship between exposure to CnPnV and the development of canine infectious respiratory disease (CIRD). Within the pet dog population, CnPnV-seropositive dogs were detected throughout the United Kingdom and Republic of Ireland, with an overall estimated seroprevalence of 50% (n = 314/625 dogs). In the kennel population, there was a significant increase in seroprevalence, from 26% (n = 56/215 dogs) on the day of entry to 93.5% (n = 201/215 dogs) after 21 days (P <0001). Dogs that were seronegative on entry but seroconverted while in the kennel were 4 times more likely to develop severe respiratory disease than those that did not seroconvert (P < 0.001), and dogs with preexisting antibodies to CnPnV on the day of entry were significantly less likely to develop respiratory disease than immunologically naive dogs (P < 0.001). CnPnV was detected in the tracheal tissues of 29/205 kenneled dogs. Detection was most frequent in dogs with mild to moderate respiratory signs and histopathological changes and in dogs housed for 8 to 14 days, which coincided with a significant increase in the risk of developing respiratory disease compared to the risk of those housed 1 to 7 days (P < 0.001). These findings demonstrate that CnPnV is present in the United Kingdom dog population; there is a strong association between exposure to CnPnV and CIRD in the kennel studied and a potential benefit in vaccinating against CnPnV as part of a wider disease prevention strategy.     

Infectious causes of equine respiratory disease on Ontario standardbred racetracks.

Upper respiratory disease has been a serious problem in Standardbred horses on racetracks in Ontario, with outbreaks occurring once or twice annually in late winter and early spring seasons. To determine the causes of these epidemics, a 3-year investigation was carried out in which nasal swabs and serum samples were obtained at intervals from apparently healthy horses and from horses suffering from upper respiratory disease. The nasal swabs were used to isolate bacteria and viruses. The serum samples were examined for the presence and level of antibodies to equine influenza viruses and equine herpesvirus 1. None of the bacteria isolated were associated with the outbreaks of disease. Equine herpesvirus 2 was isolated 72 times from both diseased and apparently healthy horses. Equine herpesvirus 1 was isolated 10 times from horses with respiratory disease, both during and between epidemics. Influenza equine/1 virus was isolated seven times and influenza equine/2 was isolated once during severe outbreaks of upper respiratory disease. Serological evidence confirmed that influenza viruses were the causes of the major epidemics, with the equine/1 strain being involved most often.     

Analysis of canine herpesvirus gB, gC and gD expressed by a recombinant vaccinia virus

Summary.  The genes encoding the canine herpesvirus (CHV) glycoprotein B (gB), gC and gD homologues have been reported already. However, products of these genes have not been identified yet. Previously, we have identified three CHV glycoproteins, gp145/112, gp80 and gp47 using a panel of monoclonal antibodies (MAbs). To determine which CHV glycoprotein corresponds to gB, gC or gD, the putative genes of gB, gC, and gD of CHV were inserted into the thymidine kinase gene of vaccinia virus LC16mO strain under the control of the early-late promoter for the vaccinia virus 7.5-kilodalton polypeptide. We demonstrated here that gp145/112, gp80 and gp47 were the translation products of the CHV gB, gC and gD genes, respectively. The antigenic authenticity of recombinant gB, gC and gD were confirmed by a panel of MAbs specific for each glycoprotein produced in CHV-infected cells. Immunization of mice with these recombinants produced high titers of neutralizing antibodies against CHV. These results suggest that recombinant vaccinia viruses expressing CHV gB, gC and gD may be useful to develop a vaccine to control CHV infection. Accepted November 20, 1996 Received October 10, 1996     

Glycoprotein-specific immune response in canine herpesvirus infection

Summary Sera from dogs which were infected with canine herpesvirus (CHV) were analyzed for their serological reactivities against virus-specific glycoproteins (gps). By sequential immunoblot analysis using sera from experimentally infected dogs, it was found that the antibody response to gp 145/112 appeared first followed by responses to gp 47 and gp 80. In addition, all sera from naturally infected dogs which showed neutralizing activity to CHV reacted with gp 145/112, whereas 77% and 70% reacted with gp 47 and gp 80, respectively. Furthermore, some of the sera also cross-neutralized feline herpesvirus type 1 (FHV-1) and reacted with gp 143/108 of FHV-1, indicating that gp 145/112 of CHV induced cross-neutralizing antibody response to FHV-1.     

Evaluation and clinical validation of an alcohol-based transport medium for preservation and inactivation of respiratory viruses

The clinical and public health importance of influenza and other respiratory viruses has accelerated the development of highly sensitive molecular diagnostics, but data are limited regarding preanalytical stages of diagnostic testing. We evaluated CyMol, an alcohol-based transport medium, for its ability to maintain specimen integrity for up to 21 days of storage at various temperatures; for its ability to inactivate virus; and for its compatibility with antigen- or nucleic acid-based diagnostics for respiratory viruses in clinical samples. In mock-infected samples, both universal transport medium (UTM-RT) and CyMol maintained equivalent viral quantities for at least 14 days at room temperature or colder, whereas a dry swab collection maintained viral quantities only if refrigerated or frozen. CyMol inactivated influenza virus within 5 min of sample immersion. UTM-RT- and CyMol-collected nasal swab specimens from 73 symptomatic students attending a campus health clinic were positive for a respiratory virus in 56.2% of subjects by multiplex PCR testing, including influenza A and B viruses, rhinovirus/enteroviruses, coronaviruses, respiratory syncytial virus, parainfluenza viruses, metapneumovirus, and adenovirus. Detection by PCR was equivalent in UTM-RT- and CyMol-collected specimens and in self- and staff-collected swabs. Direct fluorescent antibody (DFA) testing was substantially less sensitive (23.3%) than multiplex PCR, and DFA testing from UTM-RT-collected swabs was more sensitive than that from CyMol-collected swabs. These data indicate that an alcohol-based transport medium such as CyMol preserves respiratory virus integrity, rapidly inactivates viruses, and is compatible with PCR-based respiratory diagnostics.     

Detection of canine distemper virus in dogs by real-time RT-PCR

Canine distemper virus is the etiological agent of a severe disease in dogs and many other carnivores. Clinical diagnosis of canine distemper is difficult due to the broad spectrum of signs that may be confounded with other respiratory and enteric diseases of dogs. Accordingly, a laboratory confirmation is required for suspected cases. In this study a real-time RT-PCR assay was developed for detection and quantitation of canine distemper virus. The assay exhibited high specificity as all the negative controls (no-template-controls and samples from healthy sero-negative dogs) and other canine pathogens were not misdetected. Up to 1 x 10(2) copies of RNA were detected by the TaqMan assay, thus revealing a high sensitivity. Quantitative TaqMan was validated on clinical samples, including various tissues and organs collected from dogs naturally infected by canine distemper virus. Urines, tonsil, conjunctival swabs and whole blood were found to contain high virus loads and therefore proved to be suitable targets for detection of canine distemper virus RNA.     

Nasal virome of dogs with respiratory infection signs include novel taupapillomaviruses

Using viral metagenomics, we characterized the mammalian virome of nasal swabs from 57 dogs with unexplained signs of respiratory infection showing mostly negative results using the IDEXX Canine Respiratory Disease RealPCR? Panel. We identified canine parainfluenza virus 5, canine respiratory coronavirus, carnivore bocaparvovirus 3, canine circovirus and canine papillomavirus 9. Novel canine taupapillomaviruses (CPV21-23) were also identified in 3 dogs and their complete genome sequenced showing L1 nucleotide identity ranging from 68.4 to 70.3% to their closest taupapillomavirus relative. Taupapillomavirus were the only mammalian viral nucleic acids detected in two affected dogs, while a third dog was coinfected with low levels of canine parainfluenza 5. A role for these taupapillomavirues in canine respiratory disease remains to be determined.

     

Nosocomial Outbreak of Serious Canine Infectious Tracheobronchitis (Kennel Cough) Caused by Canine Herpesvirus Infection

Canine herpesvirus (CHV; Canid herpesvirus 1) is principally a perinatal pathogen of pregnant bitches and newborn pups and secondarily a respiratory tract pathogen of older pups and dogs. Infectious disease of the canine respiratory tract frequently occurs among dogs in groups, in which it is called “ infectious tracheobronchitis” (ITB). Mortality from ITB is generally negligible, and the clinical importance of CHV as an ITB pathogen is considered to be low. The present report describes a novel ITB outbreak accompanied by death among aged dogs in an animal medical center. Most inpatient dogs had received medications that could induce immunosuppression. CHV was the only pathogen identified, and several CHV isolates were recovered in cell culture. No other viral pathogens or significant bacterial pathogens were found. Molecular and serological analyses revealed that the causative CHV isolates were from a single source but that none was a peculiar strain when the strains were compared with previous CHV strains. The virus had presumably spread among the dogs predisposed to infection in the center. The present results serve as a warning to canine clinics that, under the specific set of circumstances described, such serious CHV outbreaks may be expected wherever canine ITB occurs.Canine herpesvirus (CHV; Canid herpesvirus 1) is classified in the Varicellovirus genus of the Alphaherpesvirinae subfamily of the Herpesviridae. CHV was first described in 1965 as a pathogen responsible for a fatal generalized hemorrhagic disease of newborn pups (5). The host range of CHV is generally restricted to domestic and wild Canidae (11), and the worldwide distribution of CHV infection in domestic dog populations has been shown by virus isolation (2, 7, 10, 12, 24, 27) and seroepidemiological studies (6, 25, 28, 30, 31, 34). It is now well recognized that the pathogenic potential of CHV is mostly influenced by the age of the host (11). Pups infected in the postnatal period show the typical fatal hemorrhagic syndrome. Older pups manifest less severe clinical syndromes upon infection, and the respiratory disease called “infectious tracheobronchitis” (ITB) or “kennel cough” may be the most frequent clinical disorder in the field. Ocular disorders such as conjunctivitis and keratitis, either with or without ulceration, were also observed in young pups (17). Although the pathogenic potential of CHV for older dogs is apparently low and adult dogs often do not show any clinical signs following CHV infection, CHV may become an important perinatal pathogen for pregnant bitches, causing papulovesicular genital lesions and reproductive disorders such as embryonic resorption, abortion, and stillbirth (11).CHV is spread mainly by the oronasal and venereal transmission of viruses in the respiratory and genital secretions of acutely infected dogs, and fetuses are infected in utero (13, 14). Following the initial productive infection, in most cases CHV is not fully cleared by acquired immunity so that latency becomes established in several tissues, including the sensory ganglia (4, 19), and may persist for life. Latent viruses are sometimes reactivated by factors that alter immunity, such as stress, immunosuppressive therapy, or pregnancy, with the virus subsequently being excreted.The significance of CHV in the etiology of canine ITB has been considered to be rather low compared with that of other agents, such as canine parainfluenza virus (CPIV), canine adenovirus type 2 (CAV-2), and Bordetella bronchiseptica (9). Experimental infection of older pups or adult dogs with CHV isolates often resulted in either no clinical signs or mild upper respiratory symptoms (1, 15, 24, 32). Consequently, CHV has not generally been regarded to be a primary cause of canine ITB (9, 11). On the other hand, CHV has repeatedly been detected in dogs with ITB (3, 16, 24, 36), and a longitudinal study on the respiratory diseases of dogs housed in a rehoming kennel in the United Kingdom (8) indicated that CHV should be reevaluated as a significant agent responsible for ITB.This report is an account of a novel outbreak of canine ITB which was accompanied by deaths among dogs hospitalized in an animal referral medical center near Tokyo, Japan. It was concluded that CHV alone was responsible for the nosocomial outbreak. All of the dogs had been fully vaccinated before they entered the hospital, and during the hospitalization, most of the dogs had received steroid medication, surgery, or radiation therapy. It was considered that such medical treatments, in addition to hospitalization itself, might induce stress in the patients, leading to a condition of low-level immune resistance, and cause the recrudescence of latent CHV. Canine herpesvirus might then have spread contagiously among the dogs in the same clinic. The results of the present study suggest the need for caution, because CHV, which was previously thought to be an agent of low pathogenicity for adult dogs, should not be overlooked in specific circumstances.     

Non-invasive sample collection for respiratory virus testing by multiplex PCR

Background

Identifying respiratory pathogens within populations is difficult because invasive sample collection, such as with nasopharyngeal aspirate (NPA), is generally required. PCR technology could allow for non-invasive sampling methods.

Objective

Evaluate the utility of non-invasive sample collection using anterior nare swabs and facial tissues for respiratory virus detection by multiplex PCR.

Study design

Children aged 1 month–17 years evaluated in a pediatric emergency department for respiratory symptoms had a swab, facial tissue, and NPA sample collected. All samples were tested for respiratory viruses by multiplex PCR. Viral detection rates were calculated for each collection method. Sensitivity and specificity of swabs and facial tissues were calculated using NPA as the gold standard.

Results

285 samples from 95 children were evaluated (92 swab-NPA pairs, 91 facial tissue-NPA pairs). 91% of NPA, 82% of swab, and 77% of tissue samples were positive for ≥ 1 virus. Respiratory syncytial virus (RSV) and human rhinovirus (HRV) were most common. Overall, swabs were positive for 74% of virus infections, and facial tissues were positive for 58%. Sensitivity ranged from 17 to 94% for swabs and 33 to 84% for tissues. Sensitivity was highest for RSV (94% swabs and 84% tissues). Specificity was ≥95% for all viruses except HRV for both collection methods.

Conclusions

Sensitivity of anterior nare swabs and facial tissues in the detection of respiratory viruses by multiplex PCR varied by virus type. Given its simplicity and specificity, non-invasive sampling for PCR testing may be useful for conducting epidemiologic or surveillance studies in settings where invasive testing is impractical or not feasible.     

Detection of respiratory viruses by molecular methods

SUMMARY: Clinical laboratories historically diagnose seven or eight respiratory virus infections using a combination of techniques including enzyme immunoassay, direct fluorescent antibody staining, cell culture, and nucleic acid amplification tests. With the discovery of six new respiratory viruses since 2000, laboratories are faced with the challenge of detecting up to 19 different viruses that cause acute respiratory disease of both the upper and lower respiratory tracts. The application of nucleic acid amplification technology, particularly multiplex PCR coupled with fluidic or fixed microarrays, provides an important new approach for the detection of multiple respiratory viruses in a single test. These multiplex amplification tests provide a sensitive and comprehensive approach for the diagnosis of respiratory tract infections in individual hospitalized patients and the identification of the etiological agent in outbreaks of respiratory tract infection in the community. This review describes the molecular methods used to detect respiratory viruses and discusses the contribution that molecular testing, especially multiplex PCR, has made to our ability to detect respiratory viruses and to increase our understanding of the roles of various viral agents in acute respiratory disease.     

Disclosing respiratory co-infections: a broad-range panel assay for avian respiratory pathogens on a nanofluidic PCR platform

Respiratory syndromes (RS) are among the most significant pathological conditions in edible birds and are caused by complex coactions of pathogens and environmental factors. In poultry, low pathogenic avian influenza A viruses, metapneumoviruses, infectious bronchitis virus, infectious laryngotracheitis virus, Mycoplasma spp. Escherichia coli and/or Ornithobacterium rhinotracheale in turkeys are considered as key co-infectious agents of RS. Aspergillus sp., Pasteurella multocida, Avibacterium paragallinarum or Chlamydia psittaci may also be involved in respiratory outbreaks. An innovative quantitative PCR method, based on a nanofluidic technology, has the ability to screen up to 96 samples with 96 pathogen-specific PCR primers, at the same time, in one run of real-time quantitative PCR. This platform was used for the screening of avian respiratory pathogens: 15 respiratory agents, including viruses, bacteria and fungi potentially associated with respiratory infections of poultry, were targeted. Primers were designed and validated for SYBR green real-time quantitative PCR and subsequently validated on the Biomark high throughput PCR nanofluidic platform (Fluidigm©, San Francisco, CA, USA). As a clinical assessment, tracheal swabs were sampled from turkeys showing RS and submitted to this panel assay. Beside systematic detection of E. coli, avian metapneumovirus, Mycoplasma gallisepticum and Mycoplasma synoviae were frequently detected, with distinctive co-infection patterns between French and Moroccan flocks. This proof-of-concept study illustrates the potential of such panel assays for unveiling respiratory co-infection profiles in poultry.     

Antibiotic resistance among fecal indicator bacteria from healthy individually owned and kennel dogs

Escherichia coli and Enterococcus faecalis strains isolated from anal swabs of clinically healthy dogs were examined for the presence of acquired antimicrobial resistance. The strains originated from dogs of 92 different owners and from eight breeding kennels. The purpose of the present study was to evaluate the resistance situation in the intestinal flora of the dog to assess the possible role of the dog flora as a reservoir of antimicrobial resistance. Multiple resistance was rarely found in E. coli strains collected from individually owned dogs, in contrast with strains from kennel dogs. Resistance to ampicillin, trimethoprim, and sulfamethoxazole was significantly less prevalent in E. coli from privately owned dogs than in strains from kennel dogs. Resistance rates against tetracycline and macrolides were unexpectedly high in E. faecalis strains. Two and three E. faecalis strains from individually owned dogs and kennel dogs, respectively, were resistant to gentamicin, an antibiotic often used for treating enterococcal infections in humans. This study demonstrates that resistance percentages may fluctuate with the choice of dog population. The observed antimicrobial resistance percentages indicate that the flora of healthy dogs may act as a reservoir of resistance genes.     

Simple tests for rapid detection of canine parvovirus antigen and canine parvovirus-specific antibodies

Canine parvovirus (CPV) is the number one viral cause of enteritis, morbidity, and mortality in 8-week-old young puppies. We have developed twin assays (slide agglutination test [SAT] for CPV antigen and slide inhibition test [SIT] for CPV antibody) that are sensitive, specific, cost-effective, generic for all genotypes of CPV, and provide instant results for CPV antigen detection in feces and antibody quantification in serum. We found these assays to be useful for routine applications in kennels with large numbers of puppies at risk. The results of these assays are available in 1 min and do not require any special instrumentation. SAT-SIT technology will find applications in rapid screening of samples for other hemagglutinating emerging viruses of animals and humans (influenza virus and severe acute respiratory syndrome coronavirus).     

Prevalence of Leishmania infantum infection in dogs living in an area of canine leishmaniasis endemicity using PCR on several tissues and serology

We studied and compared the prevalence of Leishmania infection and the seroprevalence and the prevalence of canine leishmaniasis in an area where canine leishmaniasis is endemic. One hundred dogs living on the island of Mallorca (Spain) were studied. In this study, we clinically examined each dog for the presence of symptoms compatible with leishmaniasis, determined the titer of anti-Leishmania antibodies, and investigated the presence of Leishmania DNA by PCR in skin, conjunctiva, and bone marrow samples of each dog. The prevalence of the disease and the seroprevalence were 13 and 26%, respectively. In 63% of the dogs, Leishmania DNA could be detected by PCR in at least one of the tissues studied. The results of positive PCR in the bone marrow, the conjunctiva, and the skin were 17.8, 32, and 51%, respectively. The prevalence of the infection, 67%, was calculated using all animals that were seropositive and/or positive by PCR with any tissue. The results showed that the majority of dogs living in an area where canine leishmaniasis is endemic are infected by Leishmania and that the prevalence of infection is much greater than the prevalence of overt Leishmania-related disease.     

Comparison of nasopharyngeal aspirate with flocked swab for PCR‐detection of respiratory viruses in children

Fast‐ and high‐throughput molecular workflows require sample matrices to be suitable for automation. Respiratory swabs are better suited for this purpose compared to the more viscous nasopharyngeal aspirates. Samples collected by nasopharyngeal aspiration and nasopharyngeal flocked swab from 81 children were compared for detection and recovery of respiratory viruses. Using real‐time RT‐PCR, no statistically significant differences in virus detection between the two sample types were found, supporting the use of flocked swabs in children aged one month to two years.     

Use of monoclonal antibodies for rapid diagnosis of respiratory viruses in a community hospital

An indirect fluorescence antibody (IFA) procedure was used for the rapid detection of respiratory viruses in direct clinical specimens and for determining the epidemiology of viruses in a community hospital setting. Viral respiratory diseases were monitored for 10 consecutive respiratory seasons. The Bartels Viral Respiratory Screening and Identification Kit is an IFA method that contains pooled and individual monoclonal antibodies for seven common respiratory viruses. Compared with 8,670 conventional tube cell cultures, IFA staining of direct patient specimens had an overall sensitivity of 84.2% and a specificity of 87.7%. Yearly epidemics of respiratory syncytial virus were seen with alternating short and long intervals between successive periods when virus was isolated. Epidemics following short intervals were more severe. Influenza A virus epidemics occurred yearly, and influenza B virus activity was seen generally every other year. When influenza A and influenza B viruses were cocirculating in a given season, the months of peak activity of one virus were always within 1 month of the peak activity of the other virus. Parainfluenza virus type 1 was detected in the autumn of odd-numbered years, and parainfluenza type 2 virus was seen usually in the autumn of even-numbered years. Parainfluenza type 3 virus and adenovirus were the most ubiquitous agents, with peak incidence occurring in the late winter to spring.