Involvement of the skin during bluetongue virus infection and replication in the ruminant host.

Bluetongue virus (BTV) is a double stranded (ds) RNA virus (genus Orbivirus; family Reoviridae), which is considered capable of infecting all species of domestic and wild ruminants, although clinical signs are seen mostly in sheep. BTV is arthropod-borne ("arbovirus") and able to productively infect and replicate in many different cell types of both insects and mammalian hosts. Although the organ and cellular tropism of BTV in ruminants has been the subject of several studies, many aspects of its pathogenesis are still poorly understood, partly because of inherent problems in distinguishing between "virus replication" and "virus presence".BTV replication and organ tropism were studied in a wide range of infected sheep tissues, by immuno-fluorescence-labeling of non-structural or structural proteins (NS2 or VP7 and core proteins, respectively) using confocal microscopy to distinguish between virus presence and replication. These results are compared to gross and microscopic pathological findings in selected organs from infected sheep. Replication was demonstrated in two major cell types: vascular endothelial cells, and agranular leukocytes which morphologically resemble lymphocytes, monocytes/macrophages and/or dendritic cells. Two organs (the skin and tonsils) were shown to support relatively high levels of BTV replication, although they have not previously been proposed as important replication sites during BTV infection. The high level of BTV replication in the skin is thought to be of major significance for the pathogenesis and transmission of BTV (via biting insects) and a refinement of our current model of BTV pathogenesis is discussed.

Development of a quantitative real-time PCR for the detection of canine respiratory coronavirus.

Canine respiratory coronavirus (CRCoV) has been detected recently in dogs with canine infectious respiratory disease and is involved in the clinical disease complex. CRCoV is a group 2 coronavirus most closely related to bovine coronavirus and human coronavirus OC43. A real-time PCR assay was developed for the detection of CRCoV. The assay was validated against cell culture grown virus and shown to have a high level of sensitivity. A range of tissue samples were collected from dogs at a re-homing centre with a history of endemic respiratory disease. The samples were tested using a conventional nested PCR assay and CRCoV was quantitated by real-time PCR. CRCoV was detected most frequently in the nasal mucosa, nasal tonsil and trachea. It was also detected in the lung, and bronchial lymph node. Of the enteric tissues, only one mesenteric lymph node sample was positive. In addition two colon samples were positive for CRCoV by nested PCR only. In conclusion, CRCoV appears to infect the upper respiratory tract preferentially. The CRCoV real-time PCR assay has proved to be a highly specific and sensitive assay that can be applied for diagnostic purposes as well as to investigate further the tissue tropism of CRCoV.

Quantification of mRNA encoding cytokines and chemokines and assessment of ciliary function in canine tracheal epithelium during infection with canine respiratory coronavirus (CRCoV).

One of the first lines of defence against viral infection is the innate immune response and the induction of antiviral type I interferons (IFNs). However some viruses, including the group 2 coronaviruses, have evolved mechanisms to overcome or circumvent the host antiviral response. Canine respiratory coronavirus (CRCoV) has previously been shown to have a widespread international presence and has been implicated in outbreaks of canine infectious respiratory disease (CIRD). This study aimed to quantify pro-inflammatory cytokine mRNAs following infection of canine air-interface tracheal cultures with CRCoV. Within this system, immunohistochemistry identified ciliated epithelial and goblet cells as positive for CRCoV, identical to naturally infected cases, thus the data obtained would be fully transferable to the situation in vivo. An assay of ciliary function was used to assess potential effects of CRCoV on the mucociliary system. CRCoV was shown to reduce the mRNA levels of the pro-inflammatory cytokines TNF-alpha and IL-6 and the chemokine IL-8 during the 72 h post-inoculation. The mechanism for this is unknown, however the suppression of a key antiviral strategy during a period of physiologic and immunological stress, such as on entry to a kennel, could potentially predispose a dog to further pathogenic challenge and the development of respiratory disease.

Foot-and-mouth disease virus infection in young lambs: pathogenesis and tissue tropism.

Foot-and-mouth disease (FMD) in adult sheep usually causes milder clinical signs than in cattle or pigs, and is often subtle enough to go undiagnosed. In contrast, FMD in lambs has been reported to cause high mortality during field outbreaks. In order to investigate the pathogenesis of FMD in lambs, two groups, aged 10-14 days, were infected with foot-and-mouth disease virus (FMDV) type O UKG. One group of lambs (n=8) was inoculated with FMDV in the coronary band, while the other (n=4) was infected by direct contact with FMDV-inoculated ewes. Daily serum samples and temperature measurements were taken. Lambs were killed sequentially and tissue samples taken for analysis. Using real-time RT-PCR, viral RNA levels in tissue samples and serum were measured, and a novel strand-specific real-time RT-PCR assay was used to quantify viral replication levels in tissues. Tissue sections were examined for histopathological lesions, and in situ hybridisation (ISH) was used to localise viral RNA within histological sections. The contact-infected lambs became infected approximately 24h after the ewes were inoculated. Vesicular lesions developed on the feet of all lambs and on the caudo-lateral part of the tongues of six of the eight inoculated lambs and three of the four contact-infected lambs. Although no lambs developed severe clinical signs, one of the contact-infected lambs died acutely at 5 days post-exposure. Histological examination of the heart from this lamb showed multi-focal areas of lymphocytic-plasmacytic myocarditis; similar lesions were also observed in the hearts of three of the inoculated lambs. Using ISH, viral RNA was localised within cardiac and skeletal muscle cells from the lamb which had died, and also from vesicular lesions on the coronary band and tongue of inoculated lambs. These results provide a detailed description of the pathogenesis of the disease in lambs.

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.

Chronic pleuropulmonary fibrosis and elastosis of aged donkeys: similarities to human pleuroparenchymal fibroelastosis.

BACKGROUND: Donkey pulmonary fibrosis (DPF) is a spontaneous syndrome of aged donkeys with a high prevalence (35%). No previous detailed characterization of DPF has been performed. We sought to determine the similarities between DPF and recognized patterns of human pulmonary fibrosis. METHODS: Whole lungs were collected from 32 aged donkeys at routine necropsy. Gross examination revealed pulmonary fibrosis in 19 donkeys (DPF cases), whereas 13 (control cases) had grossly normal lungs. Eighteen whole inflated ex vivo lungs (11 DPF cases, seven control cases) were imaged with high-resolution CT (HRCT) scan, whereas the remainder were sectioned and photographed. Tissue samples were collected from all lungs for histopathologic evaluation using a standardized protocol. HRCT images and histology sections underwent independent blinded review. Lung tissue was analyzed for herpes virus, fungal hyphae, mycobacteria, and dust content. RESULTS: Ten of 19 DPF lungs were categorized as being consistent with pleuroparenchymal fibroelastosis (PPFE) according to previously defined histologic and imaging criteria. All 10 PPFE-like lungs had marked pleural and subpleural fibrosis, predominantly within the upper lung zone, with accompanying intraalveolar fibrosis and elastosis. Asinine herpesvirus was ubiquitously expressed within control and DPF lung tissue. No other etiologic agents were identified. CONCLUSIONS: Many cases of DPF share key pathologic and imaging features with human PPFE, a rare interstitial pneumonia. Consequently, further study of DPF may help to elucidate the etiopathogenesis of human PPFE.

Tropism and pathological findings associated with canine respiratory coronavirus (CRCoV).

Canine infectious respiratory disease (CIRD) occurs frequently in densely housed dog populations. One of the common pathogens involved is canine respiratory coronavirus (CRCoV), however little is known regarding its pathogenesis and the role it plays in the development of CIRD. The pathogenesis of five geographically unrelated canine respiratory coronavirus (CRCoV) isolates was investigated. Following experimental infection in dogs, all five CRCoV isolates gave rise to clinical signs of respiratory disease consistent with that observed during natural infection. The presence of CRCoV was associated with marked histopathological changes in the nares and trachea, with loss and damage to tracheal cilia, accompanied by inflammation. Viral shedding was readily detected from the oropharynx up to 10 days post infection, but there was little or no evidence of rectal shedding. The successful re-isolation of CRCoV from a wide range of respiratory and mucosal associated lymphoid tissues, and lung lavage fluids demonstrates a clear tropism of CRCoV for respiratory tissues and fulfils the final requirement for Koch's postulates. By study day 14 dogs had seroconverted to CRCoV and the antibodies raised were neutralising against both homologous and heterologous strains of CRCoV in vitro, thus demonstrating antigenic homogeneity among CRCoV strains from the two continents. Defining the role that CRCoV and other agents play in CIRD is a considerable, but important, challenge if the disease is to be managed, treated and prevented more successfully. Here we have successfully developed a model for studying the pathogenicity and the role of CRCoV in CIRD.

Congenital large intestinal hypoganglionosis in a domestic shorthair kitten.

UNLABELLED: CLINICAL PRESENTATION AND INVESTIGATIONS: An 11-week-old female domestic shorthair kitten presented with a history of constipation since weaning. Abdominal distension was noted on physical examination and abdominal radiographs revealed a large volume of faeces in the descending and transverse colon. Colonoscopy was unremarkable and euthanasia was performed at the owners' request. DIAGNOSIS: The histological diagnosis of large intestinal hypoganglionosis was made post mortem. SIGNIFICANCE: This appears to be the first reported case of congenital hypoganglionosis in a kitten with histopathological confirmation of the diagnosis. It may potentially be a more common cause of constipation in kittens and young cats than has previously been suspected.

Characterization of pneumonia due to Streptococcus equi subsp. zooepidemicus in dogs.

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.

The effects of inoculation of Mannheimia haemolytica into the teat of lactating ewes.

The objectives of the work described in this paper were: (i) to study the outcome of challenging ewes with Mannheimia haemolytica, at different sites of their teats, (ii) to compare the effects of two different isolates of the organism and (iii) to describe the features of the resulting lesions. Thirty-two ewes were used in the study and allocated into one of two groups (A or B, n = 16); they were challenged with one of two isolates of M. haemolytica, respectively, strain ES26L of known pathogenicity or strain VSM08L from the teat duct of a healthy ewe. Each group was further divided into four equal subgroups: the ewes in the A1/B1 subgroups were intramammarily challenged; one teat of the ewes in the A2/B2 subgroups was immersed into a broth-culture of the organisms; one teat of the ewes in the A3/B3 subgroups was inoculated 2 mm-deep, whilst one teat of the ewes in the A4/B4 subgroups was inoculated 6 mm-deep. The animals were monitored clinically, bacteriologically and cytologically before and after challenge; one animal in each subgroup was euthanised 2, 4, 7 and 11 days after challenge. All ewes in the A1/B1 subgroups developed clinical mastitis, whilst of the other animals, only one ewe in each of the A4/B4 subgroups did. Neither of the two strains used was associated with more positive bacteriological or CMT results; the A2/B2 subgroups were associated with less positive results than the A3/B3 and A4/B4 subgroups. In some ewes of the A2/B2 subgroups, mild leucocytic infiltration in the teat was evident; in the ewes of the A3/B3 subgroups, leucocytic infiltration (neutrophils, lymphocytes, plasma cells) was seen, as well as a lymphoid hyperplasia at the border between the teat duct and teat cistern; in ewes of the A4/B4 subgroups, intense subepithelial leucocytic infiltration was the salient feature. No differences were found in the severity of lesions between the two strains used or the three treatments carried out. Although strain VSM08L had been isolated from the teat duct of a healthy ewe, it caused mastitis when inoculated intramammarily; although strain ES26L is of known pathogenicity for the mammary gland, it did not cause clinical mastitis when deposited 2 mm-deep into the teat. These findings point to a protective role of the teat of ewes, which appear to limit bacterial penetration from the teat duct or cistern to the mammary gland. The lymphoid tissue, at the border between the teat duct--teat cistern, may play a significant protective role.

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.

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.

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.

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.