The innate antiviral immune system of the cat: molecular tools for the measurement of its state of activation

The innate immune system plays a central role in host defence against viruses. While many studies portray mechanisms in early antiviral immune responses of humans and mice, much remains to be discovered about these mechanisms in the cat. With the objective of shedding light on early host-virus interactions in felids, we have developed 12 real-time TaqMan(?) qPCR systems for feline genes relevant to innate responses to viral infection, including those encoding for various IFNα and IFNω subtypes, IFNβ, intracellular antiviral factor Mx, NK cell stimulator IL-15 and effectors perforin and granzyme B, as well as Toll-like receptors (TLRs) 3 and 8. Using these newly developed assays and others previously described, we measured the relative expression of selected markers at early time points after viral infection in vitro and in vivo. Feline embryonic fibroblasts (FEA) inoculated with feline leukemia virus (FeLV) indicated peak levels of IFNα, IFNβ and Mx expression already 6h after infection. In contrast, Crandell-Rees feline kidney (CrFK) cells inoculated with feline herpes virus (FHV) responded to infection with high levels of IFNα and IFNβ only after 24h, and no induction of Mx could be detected. In feline PBMCs challenged in vitro with feline immunodeficiency virus (FIV), maximal expression levels of IFNα, β and ω subtype genes as well as IL-15 and TLRs 3, 7 and 8 were measured between 12 and 24h after infection, whereas expression levels of proinflammatory cytokine gene IL-6 were consistently downregulated until 48h post inoculation. A marginal upregulation of granzyme B was also observed within 3h after infection. In an in vivo experiment, cats challenged with FIV exhibited a 2.4-fold increase in IFNα expression in blood 1 week post infection. We furthermore demonstrate the possibility of stimulating feline immune cells in vitro with various immune response modifiers (IRMs) already known for their immunostimulatory properties in mice and humans, namely Poly IC, Resiquimod (R-848) and dSLIM?, a synthetic oligonucleotide containing several unmethylated CpG motifs. Stimulation of feline PBMCs with dSLIM? and R-848 effectively enhanced expression of IFNα within 12h by factors of 6 and 12, respectively, and Poly IC induced an increase in Mx mRNA expression of 28-fold. Altogether, we describe new molecular tools and their successful use for the characterization of innate immune responses against viruses in the cat and provide evidence that feline cells can be stimulated by synthetic molecules to enhance their antiviral defence mechanisms.     

First morphological characterization of 'Candidatus Mycoplasma turicensis' using electron microscopy

At least three haemotropic mycoplasmas have been recognized in cats: Mycoplasma haemofelis (Mhf), 'Candidatus Mycoplasma haemominutum' (CMhm) and 'Candidatus M. turicensis' (CMt). The latter was originally identified in a Swiss pet cat with haemolytic anaemia and shown to be prevalent in domestic cats and wild felids worldwide using molecular methods. So far, there has been no confirmatory morphological evidence of the existence of CMt presumably due to low blood loads during infection while CMhm has only been characterized by light microscopy with discrepant results. This study aimed to provide for the first time electron microscopic characteristics of CMt and CMhm and to compare them to Mhf. Blood samples from cats experimentally infected with CMt, CMhm and Mhf were used to determine copy numbers in blood by real-time PCR and for transmission and scanning electron microscopy. High resolution scanning electron microscopy revealed CMt and CMhm to be discoid-shaped organisms of 0.3 μm in diameter attached to red blood cells (RBCs). In transmission electron microscopy of CMt, an oval organism of about 0.25 μm with several intracellular electron dense structures was identified close to the surface of a RBC. CMhm and CMt exhibited similar morphology to Mhf but had a smaller diameter. This is the first study to provide morphological evidence of CMt thereby confirming its status as a distinct haemoplasma species, and to present electron microscopic features of CMhm.     

Real-time PCR-based prevalence study, infection follow-up and molecular characterization of canine hemotropic mycoplasmas

Hemotropic mycoplasmas (hemoplasmas) have been reported in several mammalian species including dogs. Infections may lead to hemolytic anemia, but investigations in the dog had been hampered by the lack of adequate diagnostic methods. Only recently sensitive PCR-based assays were reported for the two canine hemoplasmas, Mycoplasma haemocanis and Candidatus Mycoplasma haematoparvum. By applying these assays, 15.4% of 460 dogs from the south of France tested hemoplasma positive. It was hypothesized that this high prevalence may be associated with the presence of Rhipicephalus sanguineus, a proposed vector for canine hemoplasmas. To address this hypothesis and expand the PCR-based knowledge on canine hemoplasmosis, we investigated dogs in a climatic zone that does not allow for the permanent establishment of R. sanguineus. Blood samples were collected throughout a year from 889 dogs in Switzerland: 1.2% of the dogs tested real-time PCR positive. The infection status was not significantly associated with anemia, age or gender. Phylogenetic analyses of Candidatus M. haematoparvum and M. haemocanis isolates revealed > or =99.8% identity to published sequences. All samples collected from three infected dogs throughout a follow-up period of < or =13 months tested PCR positive. Interestingly, the majority of the infected dogs either had been imported from or had visited regions where R. sanguineus is indigenous. Thus, canine hemoplasma prevalence was found to be low in a country with a climate incompatible with frequent occurrence of R. sanguineus. Nonetheless, veterinarians may expect hemoplasma infections in dogs with a travel history and/or after potential tick vector exposure.     

Nasopharyngeal turbinates in brachycephalic dogs and cats

This retrospective study reports the presence and incidence of nasal turbinates in the nasopharynx (nasopharyngeal turbinates) in a population of brachycephalic dogs and cats exhibiting signs of upper respiratory disease. Medical records were reviewed for 53 brachycephalic dogs and 10 brachycephalic cats undergoing upper airway endoscopy. Nasopharyngeal turbinates were identified in 21% of brachycephalic animals, including 21% of dogs and 20% of cats. Pugs accounted for 32% of all dogs in the study population and 82% of dogs with nasopharyngeal turbinates. The presence of nasopharyngeal turbinates may play a role in upper airway obstruction in the brachycephalic airway syndrome.     

Splenic torsion in an alpaca

OBJECTIVE: To describe the clinical signs, diagnostic evaluation and surgical management of an alpaca with splenic torsion. ANIMALS: Six-year-old female alpaca. RESULTS: Splenic torsion and uterine torsion were the inciting cause for persistent abdominal discomfort in this alpaca. Rectal examination, abdominocentesis, and transabdominal ultrasonographic findings were suggestive of a splenic lesion. Surgical management involved splenectomy of a necrotized spleen. CONCLUSIONS: Although rare in occurrence, splenic torsion should be considered as a potential cause of abdominal discomfort in alpacas. Splenectomy is a reasonable and successful method of treatment for a devitalized spleen secondary to splenic torsion in alpacas. CLINICAL RELEVANCE: Splenic torsion causes persistent abdominal discomfort in camelids and may be associated with uterine torsion. Rectal examination, transabdominal ultrasound and abdominocentesis are useful diagnostic tools to differentiate splenic torsion from other causes of abdominal discomfort. Splenectomy is an uncomplicated procedure in camelids and has a favorable prognosis.     

Comparative spatiotemporal analysis of the intrathecal immune response in natural listeric rhombencephalitis of cattle and small ruminants

This study examined the spatiotemporal immune response in listeric rhombencephalitis of ruminants in situ. Our data support the view that astrocytes facilitate the containment of infectious lesions. Results on the natural disease recapitulate observations in experimental rodent models and suggest that the mounted adaptive lymphocytic response of ruminants is effective in eliminating Listeria monocytogenes (LM). However, our data indicate earlier participation of the adaptive immune response, a stronger B lymphocyte contribution and a more protracted macrophage infiltration in the natural disease than it has been deduced from experimental models. Therefore, such models should be complemented by studies in natural host systems. Various macrophage and microglia subsets are involved in listeric rhombencephalitis and their differential contribution may account for species differences in clinical course and outcome of infection as might species differences in the B-cell response. Future functional ex vivo and in vitro studies are necessary to further investigate the findings obtained in the present study.