Simon L. Priestnall

Evaluation of “Helicobacter heilmannii” Subtypes in the Gastric Mucosas of Cats and Dogs

ABSTRACT Infection with candidatus “Helicobacter heilmannii” is associated with gastritis and mucosa-associated lymphoid tissue lymphoma in people. Infection with “H. heilmannii” type 1 predominates (80%) and is thought to be acquired from dogs, cats, or pigs. We further examined the zoonotic potential of dogs and cats by amplifying gastric DNA from cats (n = 45) and dogs (n = 10) with primers against “H. heilmannii” ureB and 16S rRNA genes and sequencing the products. Fluorescence in situ hybridization (FISH) with eubacterial and “H. heilmannii”-specific probes was employed to directly visualize “H. heilmannii” types and their intragastric distribution. ureB sequences of “H. heilmannii” amplicons clustered with human and feline isolates of “H. heilmannii” and were distinct from the “H. heilmannii”-like organisms (HHLO) H. felis, H. salomonis, and H. bizzozeronii. 16S ribosomal DNA sequences in 20 “H. heilmannii”-infected cats and dogs were distinct from “H. heilmannii” type 1 and “H. suis” and clustered with “H. heilmannii” types 2 and 4. FISH confirmed the presence of “H. heilmannii” types 2 and 4 in dogs but failed to definitively characterize the “H. heilmannii” types present in cats. In infected dogs, “H. heilmannii” inhabited the gastric mucus and glands, and in dogs coinfected with other HHLO it shared the same gastric niche. The results indicate that dogs and cats are predominantly colonized by “H. heilmannii” bacteria that are distinct from type 1 and from “H. suis.” As “H. heilmannii” type 1 predominates in people, the zoonotic risk posed by dogs and cats is likely small.

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.

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.

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.

Extracorporeal liver assist device to exchange albumin and remove endotoxin in acute liver failure: Results of a pivotal pre-clinical study

Background & Aims In acute liver failure, severity of liver injury and clinical progression of disease are in part consequent upon activation of the innate immune system. Endotoxaemia contributes to innate immune system activation and the detoxifying function of albumin, critical to recovery from liver injury, is irreversibly destroyed in acute liver failure. University College London-Liver Dialysis Device is a novel artificial extracorporeal liver assist device, which is used with albumin infusion, to achieve removal and replacement of dysfunctional albumin and reduction in endotoxaemia. We aimed to test the effect of this device on survival in a pig model of acetaminophen-induced acute liver failure. Methods Pigs were randomised to three groups: Acetaminophen plus University College London-Liver Dialysis Device (n = 9); Acetaminophen plus Control Device (n = 7); and Control plus Control Device (n = 4). Device treatment was initiated two h after onset of irreversible acute liver failure. Results The Liver Dialysis Device resulted in 67% reduced risk of death in acetaminophen-induced acute liver failure compared to Control Device (hazard ratio = 0.33, p = 0.0439). This was associated with 27% decrease in circulating irreversibly oxidised human non-mercaptalbumin-2 throughout treatment (p = 0.046); 54% reduction in overall severity of endotoxaemia (p = 0.024); delay in development of vasoplegia and acute lung injury; and delay in systemic activation of the TLR4 signalling pathway. Liver Dialysis Device-associated adverse clinical effects were not seen. Conclusions The survival benefit and lack of adverse effects would support clinical trials of University College London-Liver Dialysis Device in acute liver failure patients.

A reproducible, clinically relevant, intensively managed, pig model of acute liver failure for testing of therapies aimed to prolong survival

A clinically relevant, translational large animal model of acute liver failure (ALF) is required for testing of novel therapies to prolong survival in acute liver failure, to permit spontaneous liver recovery or to act as a bridge to transplantation.

Association between fecal S100A12 concentration and histologic, endoscopic, and clinical disease severity in dogs with idiopathic inflammatory bowel disease

Idiopathic inflammatory bowel disease (IBD) in dogs can be challenging to diagnose and fecal markers of disease that correlate with its severity could potentially be clinically useful. Surrogate inflammatory markers, such as the concentration of fecal S100A12, are used to detect active IBD in humans. The aim of this study was to determine the relationship between fecal canine S100A12 concentrations and clinical, endoscopic, and histologic disease severity. Twenty-six dogs with IBD and 90 healthy control dogs were enrolled. Spot fecal samples were collected and fecal canine S100A12 concentrations measured by an in-house ELISA. The correlation of fecal canine S100A12 concentrations with clinical disease activity (using the canine chronic enteropathy clinical activity index scoring system) and with endoscopic and histologic disease severity (using semi-quantitative grading systems) was assessed in dogs with IBD. Concentrations of fecal canine S100A12 were significantly higher in dogs with IBD (median [interquartile range]: 223 [21-3477]ng/g) than in healthy controls (median [interquartile range]: 9 [5-31]ng/g; P<0.0001). Fecal canine S100A12 concentrations correlated with the CCECAI score (ρ=0.4778; P=0.0408) and the severity of endoscopic lesions in the duodenum (ρ=0.4703; P=0.0354) and colon (ρ=0.9747; P=0.0144), but not with the severity of histopathologic changes except for inflammatory lesions in the colon (ρ=0.8669; P=0.0230). A concentration of 273ng fecal canine S100A12/g feces or greater distinguished (a) dogs with moderate to severe endoscopic disease in any GI section from dogs with at most mild endoscopic disease, and (b) dogs with very severe clinical disease (i.e., a CCECAI score of ≥12) from dogs with a CCECAI score of <12, with a sensitivity of 71% and 90%, respectively, and a specificity of 89% and 75%, respectively. This study showed that fecal canine S100A12 concentrations are increased in dogs with IBD. Further, this study showed that fecal canine S100A12 is associated with the clinical disease activity, the severity of endoscopic lesions, and the severity of colonic inflammation in dogs with IBD. Fecal S100A12 concentrations are potentially useful as a biomarker of inflammation in dogs with IBD.

CD11c+ Cells are Significantly Decreased in the Duodenum, Ileum and Colon of Dogs with Inflammatory Bowel Disease

CD11c serves as a marker for human and murine dendritic cells (DCs) and cells expressing this marker have been shown to have similar morphological and functional characteristics in the canine immune system. The aim of this study was to quantify CD11c(+) cells in the duodenum, ileum and colon of healthy dogs and dogs with inflammatory bowel disease (IBD). Endoscopic biopsies from the duodenum (n=12 cases), ileum (n=8 cases) and colon (n=12 cases) were obtained from dogs diagnosed with IBD. Intestinal tissue from 10 healthy beagle dogs was used as control. Immunofluorescence microscopy was carried out using an anti-canine CD11c monoclonal antibody. Labelled cells were recorded as cells per 120,000 μm(2). The canine chronic enteropathy clinical activity index (CCECAI) was calculated for all dogs with IBD. In addition, sections from all dogs with IBD were evaluated according to the guidelines of the World Small Animal Veterinary Association Gastrointestinal Standardization Group. The number of CD11c(+) cells in the duodenum, ileum and colon of dogs with IBD was significantly reduced compared with controls (P<0.01, P<0.01 and P<0.05, respectively). There was a significant negative correlation between the number of CD11c(+) cells in the colon of dogs with IBD and the CCECAI (P=0.044, r(2)=-0.558). Chronic inflammation in canine IBD appears to involve an imbalance in the intestinal DC population. Future studies will determine whether reduced expression of CD11c could be a useful marker for the diagnosis and monitoring of canine IBD.