Gail Leeming

Chemokine binding protein M3 of murine gammaherpesvirus 68 modulates the host response to infection in a natural host.

Murine γ-herpesvirus 68 (MHV-68) infection of Mus musculus-derived strains of mice is an attractive model of γ-herpesvirus infection. Surprisingly, however, ablation of expression of MHV-68 M3, a secreted protein with broad chemokine-binding properties in vitro, has no discernable effect during experimental infection via the respiratory tract. Here we demonstrate that M3 indeed contributes significantly to MHV-68 infection, but only in the context of a natural host, the wood mouse (Apodemus sylvaticus). Specifically, M3 was essential for two features unique to the wood mouse: virus-dependent inducible bronchus-associated lymphoid tissue (iBALT) in the lung and highly organized secondary follicles in the spleen, both predominant sites of latency in these organs. Consequently, lack of M3 resulted in substantially reduced latency in the spleen and lung. In the absence of M3, splenic germinal centers appeared as previously described for MHV-68-infected laboratory strains of mice, further evidence that M3 is not fully functional in the established model host. Finally, analyses of M3s influence on chemokine and cytokine levels within the lungs of infected wood mice were consistent with the known chemokine-binding profile of M3, and revealed additional influences that provide further insight into its role in MHV-68 biology.

Equine multinodular pulmonary fibrosis in horses in the UK

EQUINE multinodular pulmonary fibrosis (EMPF) is a relatively recently described condition in horses, characterised by a loss of functional pulmonary parenchyma due to extensive nodular to coalescing interstitial fibrosis; the remaining alveoli are lined by cuboidal epithelium and are filled with inflammatory cells (Williams and others 2007). EMPF was described first in the USA (Williams and others 2007, Hart and others 2008, Wong and others 2008), but more recently has been reported in Europe, in Germany and Austria (Poth and others 2009, Niedermaier and others 2010). A strong association between EMPF and equine herpesvirus type 5 (EHV-5) has been established, although the pathogenesis of the condition is still unclear (Williams and others 2007). EHV-5 is a DNA gammaherpesvirus, which has been detected in the USA, Australia, New Zealand and Europe in nasal swabs and peripheral blood lymphocytes (PBLs) from both healthy horses and those with respiratory signs, at highly variable rates (Agius and Studdert 1994, Reubel and others 1995, Franchini and others 1997, Borchers and others 1999, Dunowska and others 1999, Nordengrahn and others 2002, Bell and others 2006, Wang and others 2007, Torfason and others 2008, Fortier and others 2009). EHV-5 has also been identified in horses in the UK, where PCR yielded a positive result in PBLs from five of 21 healthy adult horses (Nordengrahn and others 2002). This short communication describes two cases of EMPF in the UK, and their association with EHV-5. The first case, an 11-year-old thoroughbred gelding, was presented in February 2010 with a two-week history of lethargy, listlessness and an increased respiratory rate. Radiographic and ultrasonographic examinations (Fig 1a, b) revealed nodular lesions throughout both lungs. The animal was euthanased. At postmortem examination, gross findings in the lungs were consistent …

Equine rhinosporidiosis: an exotic disease in the uk

Rhinosporidium seeberi is the infective, waterborne agent that causes rhinosporidiosis, which commonly presents as polypoid masses of the nasal mucosa or conjunctiva in both human beings and animals (Arseculeratne and Atapattu 2004). Previously classified as a fungus, R seeberi has recently been grouped into the class Mesomycetozoa (order Dermocystida), which comprises 10 different parasitic and saprophytic microbes (Herr and others 1999). R seeberi is primarily a human pathogen, and rhinosporidiosis is predominantly seen in the tropics. The disease is endemic in southern India and Sri Lanka, and sporadic cases have been reported in Africa, the Americas and Europe (Fredricks and others 2000). Cases in the UK and other parts of Europe are rare and predominantly involve persons native to, or having travelled from, endemic areas (Mears and Amerasinghe 1992, van der Coer and others 1992). One outbreak was reported in northern Serbia (Vukovic and others 1995). Rhinosporidiosis has been reported occasionally in equids in South America (Londero and others 1977), southern USA (Smith and Frankson 1961, Myers and others 1964) and South Africa (Zschokke 1913), but also in other mammals, including cats (Moisan and Baker 2001, Wallin and others 2001), dogs (Mosier and Creed 1984, Easley and others 1986, Jimenez and others 1986), cattle (Moses and Balachandran 1987) and a pink river dolphin (Armed Forces Institute of Pathology [AFIP] 1998); it has also been reported in captive swans (Kennedy and others 1995). A surgical biopsy was taken from one of several, focally ulcerated, polypoid lesions (Fig 1) in the nasal mucosa of a six-year-old polo pony gelding recently imported from Argentina. The lesion was not associated with clinical signs. Tissue was fixed in formalin and routinely embedded in paraffin wax; 3 to 5 μm sections were stained with haematoxylin and eosin and periodic acid-Schiff (PAS). For transmission electron microscopy (TEM), fixed tissue was post-fixed in 2·5 per cent glutaraldehyde and routinely processed and embedded in epoxy resin. Semithin and ultrathin sections were examined. Multiple, 6 μm paraffin sections were treated with xylene, ethanol and proteinase K, and DNA was extracted using the DNeasy Tissue Kit, according to the manufacturer’s protocol (Qiagen). DNA extracted from the skin of an unaffected horse was used as a negative control. R seeberispecific primers for the 18S rDNA sequence was used in a 40 μl PCR, modified from Fredricks and others (2000). The PCR product was purified using the QIAquick PCR Purification Kit (Qiagen), and forward and reverse products were sequenced (Lark Technologies). Histologically, focal ulceration and multifocal hyperplasia of the nasal mucosa were seen. The mucosa and submucosa were expanded by spherical to polygonal organisms. The smaller (up to 100 μm in diameter), predominantly spherical structures had an eosinophilic and PAS-positive wall, enclosing eosinophilic to basophilic fibrillar material (juvenile sporangia; Fig 2). The larger (up to 300 μm in diameter), spherical to polygonal structures had a thin, eosinophilic wall with closely opposed basophilic stippled material and basophilic and eosinophilic ovoid structures in the central lumen (Fig 3). These are mature sporangia, containing endospores, which are known to be released via a pore (Fig 4) (Mendoza and others 1999). An intermediate stage (immature sporangia; Fig 3), in which the endospores were observed in the periphery, was also present. Surrounding these structures was a moderate, multifocal, lymphoplasmacellular, inflammatory infiltrate. Within some areas of tissue, free endospores were seen, associated with a pyogranulomatous inflammation (Fig 3). Ultrastructurally, juvenile sporangia with a nucleus, lipid bodies and laminated bodies (Fig 5), as well as mature endospores with electron-dense bodies (Fig 6) were observed. PCR yielded a product of the expected size (377 base pairs [bp]; Fredricks and others 2000), the sequences of which represented an exact match with 369 bp of the sequence for R seeberi (Herr and others 1999, Fredricks and others 2000). The diagnosis of rhinosporidiosis in this case was based on the characteristic morphological features of R seeberi (Gardiner and others 1998), and was confirmed by sequence analysis (Fredricks and others 2000). However, morphologically, fungal infections with Coccidioides immitis or Chrysosporium parvum (the causative agent of adiaspiromycosis, a rare pulmonary disorder), should be considered (Kennedy and others 1995, AFIP 1998). The natural habitat for R seeberi appears to be stagnant or lacustrine waters, although this has not been confirmed due to the failure to isolate R seeberi from such habitats (Arseculeratne 2002, 2005). It is current opinion that ‘elec-

Influenza A Virus Challenge Models in Cynomolgus Macaques Using the Authentic Inhaled Aerosol and Intra-Nasal Routes of Infection

Non-human primates are the animals closest to humans for use in influenza A virus challenge studies, in terms of their phylogenetic relatedness, physiology and immune systems. Previous studies have shown that cynomolgus macaques (Macaca fascicularis) are permissive for infection with H1N1pdm influenza virus. These studies have typically used combined challenge routes, with the majority being intra-tracheal delivery, and high doses of virus (> 107 infectious units). This paper describes the outcome of novel challenge routes (inhaled aerosol, intra-nasal instillation) and low to moderate doses (103 to 106 plaque forming units) of H1N1pdm virus in cynomolgus macaques. Evidence of virus replication and sero-conversion were detected in all four challenge groups, although the disease was sub-clinical. Intra-nasal challenge led to an infection confined to the nasal cavity. A low dose (103 plaque forming units) did not lead to detectable infectious virus shedding, but a 1000-fold higher dose led to virus shedding in all intra-nasal challenged animals. In contrast, aerosol and intra-tracheal challenge routes led to infections throughout the respiratory tract, although shedding from the nasal cavity was less reproducible between animals compared to the high-dose intra-nasal challenge group. Intra-tracheal and aerosol challenges induced a transient lymphopaenia, similar to that observed in influenza-infected humans, and greater virus-specific cellular immune responses in the blood were observed in these groups in comparison to the intra-nasal challenge groups. Activation of lung macrophages and innate immune response genes was detected at days 5 to 7 post-challenge. The kinetics of infection, both virological and immunological, were broadly in line with human influenza A virus infections. These more authentic infection models will be valuable in the determination of anti-influenza efficacy of novel entities against less severe (and thus more common) influenza infections.

Experimental infection of laboratory-bred bank voles (Myodes glareolus) with murid herpesvirus 4

MuHV-4 is a natural pathogen of rodents of the genus Apodemus (e.g., wood mice, yellow-necked mice) and Myodes glareolus (bank voles). We report experimental MuHV-4 infection of bank voles in comparison with infection of A. sylvaticus (wood mice) and BALB/c mice. Like in wood mice, the level of productive replication in the lungs of bank voles was significantly lower than in BALB/c mice. In contrast to other hosts, however, the level of latent infection in the lung and spleen of bank voles was extremely low. These findings, together with those of previous studies, suggest that bank voles are an occasional and inefficient host for MuHV-4.

Equine rhinosporidiosis in United Kingdom.

We report 4 cases of equine rhinosporidiosis in the United Kingdom. These cases provide evidence of spread of infectious agents from rhinosporidiosis-endemic areas to nonendemic areas by increased international movement of livestock. Surveillance should continue for this infective agent of potential relevance for numerous species, including humans.

Alveolar macrophages are the main target cells in feline calicivirus-associated pneumonia

Feline calicivirus (FCV) is a pathogen of felids and one of the most common causative agents of feline upper respiratory disease (URD). Reports of natural FCV pneumonia in the course of respiratory tract infections are sparse. Therefore, knowledge on the pathogenesis of FCV-induced lung lesions comes only from experimental studies. The aim of the present study was to assess the type and extent of pulmonary involvement in natural respiratory FCV infections of domestic cats and to identify the viral target cells in the lung. For this purpose, histology, immunohistochemistry and RNA-in situ hybridisation for FCV and relevant cell markers were performed on diagnostic post-mortem specimens collected after fatal URD, virulent systemic FCV or other conditions. All groups of cats exhibited similar acute pathological changes, dominated by multifocal desquamation of activated alveolar macrophages (AM) and occasional type II pneumocytes with fibrin exudation, consistent with diffuse alveolar damage (DAD). In fatal cases, this was generally seen without evidence of epithelial regeneration. In cats without clinical respiratory signs, type II pneumocyte hyperplasia was present alongside the other changes, consistent with the post-damage proliferative phase of DAD. FCV infected and replicated in AM and, to a lesser extent, type II pneumocytes. This study shows that lung involvement is an infrequent but important feature of FCV-induced URD. AM are the main viral target cell and pulmonary replication site, and their infection is associated with desquamation and activation, as well as death via apoptosis.

Dermoid sinus type IV associated with spina bifida in a young Victorian bulldog.

DERMOID sinus (also termed pilonidal sinus, pilonidal cyst or dermoid cyst) is a developmental anomaly characterised by incomplete separation of the skin from the neural tube during embryonic development (Lord and others 1957, Mann and Stratton 1966, Antin 1970). Six different types of dermoid sinus exist and classification is based on their relationship to the supraspinous ligament (Mann and Stratton 1966, Kiviranta and others 2011). This short communication describes the clinical, MRI and histopathological features of a type IV dermoid sinus and spina bifida in a young Victorian bulldog. An 11-month-old male Victorian bulldog was referred for investigation of progressive paraparesis, moderate hindlimb ataxia and moderate proprioceptive deficits in both hindlimbs. At physical examination, it was noted that the hair was growing in a spiral fashion on the dorsal midline in the mid-thoracic region. A small, fluctuating mass was palpable subcutaneously and evoked considerable pain for the dog; the mass was continuous with a firm fistulous cord attached to the underlying tissues. A round-shaped opening in the skin (diameter 0.5 cm) containing no exudates was found after clipping of the hair. Radiographs of the thoracic and thoracolumbar spine were unremarkable. MRI scans (Vet-MR Grande; Esaote) of the dogs thoracic region were obtained in three planes of orientation before and after the intravenous administration of 0.1 mmol/kg gadolinium (Magnevist; Schering). Transverse T1-weighted and T2-weighted images revealed an invagination of the epidermal and dermal tissues towards the spinous process of T8. A fistular tract was visible connecting the spinal cord with the opened skin layers. The normal morphology of T8 was lost: this vertebra lacked a normal vertebral arch and the spinous process …

Salmonella Virchow Infection of the Chicken Elicits Cellular and Humoral Systemic and Mucosal Responses, but Limited Protection to Homologous or Heterologous Re-Challenge

Salmonella enterica serovar Virchow usually causes mild gastroenteritis in humans; however, it is frequently invasive and many isolates are resistant to a broad-range of therapeutic antimicrobials. Poultry meat is considered a major source of human infection. In this study, we characterize the infection biology and immune response to S. Virchow in chickens and determine protection against homologous and heterologous re-challenge, with S. Virchow or S. Typhimurium. Following oral infection of 7-day-old chickens, S. Virchow colonized the gastrointestinal tract and the spleen. Infection elicited an increase in specific IgA, IgG, and IgM antibodies and relative quantitative changes in several leukocyte populations, including CD3, CD4, CD8α, CD8β, MHC II, KuL01, and γδ TCR positive cells, both in the gastrointestinal tract and systemically. Increased expression of pro-inflammatory cytokines IL-1β and IL-6 and the chemokine CXCLi2 was also found. Primary infection with S. Virchow offered limited systemic protection against re-challenge with S. Virchow or S. Typhimurium, but no protection against cecal colonization. In conclusion, S. Virchow exhibits similar infection biology and immune responses in the chicken to that previously described for S. Typhimurium. Unlike S. Typhimurium, S. Virchow infection is poorly protective to homologous and heterologous re-challenge. These findings suggest that S. Virchow is capable of colonizing the chicken well and therefore, presents a risk of entering the food chain in meat production. Furthermore, the development of vaccines that protect effectively against S. Virchow and indeed multivalent vaccines that protect across all Salmonella serogroups in the chicken would appear to remain a challenging proposition.

An innate defense peptide BPIFA1/SPLUNC1 restricts influenza A virus infection

The airway epithelium secretes proteins that function in innate defense against infection. Bactericidal/permeability-increasing fold-containing family member A1 (BPIFA1) is secreted into airways and has a protective role during bacterial infections, but it is not known whether it also has an antiviral role. To determine a role in host defense against influenza A virus (IAV) infection and to find the underlying defense mechanism, we developed transgenic mouse models that are deficient in BPIFA1 and used these, in combination with in vitro three-dimensional mouse tracheal epithelial cell (mTEC) cultures, to investigate its antiviral properties. We show that BPIFA1 has a significant role in mucosal defense against IAV infection. BPIFA1 secretion was highly modulated after IAV infection. Mice deficient in BPIFA1 lost more weight after infection, supported a higher viral load and virus reached the peripheral lung earlier, indicative of a defect in the control of infection. Further analysis using mTEC cultures showed that BPIFA1-deficient cells bound more virus particles, displayed increased nuclear import of IAV ribonucleoprotein complexes, and supported higher levels of viral replication. Our results identify a critical role of BPIFA1 in the initial phase of infection by inhibiting the binding and entry of IAV into airway epithelial cells.