Diagnosis of non-effusive feline infectious peritonitis by reverse transcriptase quantitative PCR from mesenteric lymph node fine-needle aspirates.

OBJECTIVES: The aim of this study was to evaluate a feline coronavirus (FCoV) reverse transcriptase quantitative PCR (RT-qPCR) on fine-needle aspirates (FNAs) from mesenteric lymph nodes (MLNs) collected in sterile saline for the purpose of diagnosing non-effusive feline infectious peritonitis (FIP) in cats. METHODS: First, the ability of the assay to detect viral RNA in MLN FNA preparations compared with MLN biopsy preparations was assessed in matched samples from eight cats. Second, a panel of MLN FNA samples was collected from a series of cats representing non-effusive FIP cases (n = 20), FCoV-seropositive individuals (n = 8) and FCoV-seronegative individuals (n = 18). Disease status of the animals was determined using a combination of gross pathology, histopathology and/or 'FIP profile', consisting of serology, clinical pathology and clinical signs. RESULTS: Viral RNA was detected in 18/20 non-effusive FIP cases; it was not detected in two cases that presented with neurological FIP. Samples from 18 seronegative non-FIP control cats and 7/8 samples from seropositive non-FIP control cats contained no detectable viral RNA. Thus, as a method for diagnosing non-effusive FIP, MLN FNA RT-qPCR had an overall sensitivity of 90.0% and specificity of 96.1%. CONCLUSIONS AND RELEVANCE: In cases with a high index of suspicion of disease, RT-qPCR targeting FCoV in MLN FNA can provide important information to support the ante-mortem diagnosis of non-effusive FIP. Importantly, viral RNA can be reliably detected in MLN FNA samples in saline submitted via the national mail service. When applied in combination with biochemistry, haematology and serological tests in cases with a high index of suspicion of disease, the results of this assay may be used to support a diagnosis of non-effusive FIP.

Evaluation of cytokine mRNA expression in bronchoalveolar lavage cells from horses with inflammatory airway disease.

Inflammatory airway disease (IAD) is a common disorder of performance horses and is associated with poor performance and accumulation of mucus and inflammatory cells in lower airway secretions. Horses with IAD frequently have increased relative counts of neutrophils in bronchoalveolar lavage fluid (BALF); less commonly relative counts of eosinophils and/or mast cells may be increased. The aetiopathogenesis of IAD is unknown and may involve innate and/or acquired immune responses to various factors including respirable dust constituents, micro-organisms, noxious gases and unconditioned air. The molecular pathways and role of the immune system in the pathogenesis of IAD remain poorly defined and it is unknown whether polarised T cell responses occur in the disease, as have been reported to occur in equine recurrent airway obstruction and asthma in humans. Elucidating cytokine responses that develop in horses with IAD may allow a greater understanding of the possible aetiopathological pathway(s) involved and could contribute to development of novel treatments. We compared the mRNA expression of tumour necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), interleukin (IL)-1ß, IL-2, IL-4, IL-8, IL-13, IL-17 and IL-23 in cell pellets extracted from BALF of horses with IAD (n=21) and horses free of respiratory tract disease (n=17). Horses with IAD had significantly increased levels of TNF-α, IL-1ß and IL-23 mRNA; no significant differences in the other cytokine mRNAs were detected. The results of this study indicate that IAD of horses is associated with increased mRNA expression of pro-inflammatory cytokines in BALF cells, which may reflect stimulation of the innate immune responses to inhaled antigens. There was no evidence of a polarised T-cell cytokine response suggesting hypersensitivity responses may not be involved in the aetiopathogenesis of IAD.

Double-blind placebo-controlled study with interleukin-18 and interleukin-12-encoding plasmid DNA shows antitumor effect in metastatic melanoma in gray horses.

Melanoma is a disease with high incidence in gray horses and has limited therapeutic options in metastatic disease. Gene therapy has shown some success in animal models and human patients. A randomized double-blind, placebo-controlled study was conducted to investigate 2 treatment options using cytokine-encoding plasmid DNA in horses with metastatic melanoma to induce immunologic antitumor effects. Adult gray horses with spontaneously occurring metastatic melanoma (n=26) were included in the study. Treatment of 26 gray horses with metastatic melanoma consisted of interleukin-18-encoding plasmid DNA, interleukin-12-encoding plasmid DNA, or empty plasmid DNA (control group), injected intratumorally, respectively. Tumor response was assessed using ultrasound and caliper measurements and histologic assessment of tumor biopsies. Significant tumor regression could be shown in both the treatment groups receiving IL-18 and IL-12-encoding plasmid DNA whereas placebo-treated control patients showed tumor growth over the course of the treatment. In addition, 7 of 10 tumors from horses treated with IL-18 or IL-12 showed peritumoral and/or intratumoral inflammatory infiltrates after treatment compared with 1 of the 6 in the control group. The treatment as assessed by serial blood draws and clinical investigation, was safe and well tolerated. These data suggest that the intratumoral treatment with IL-18 and IL-12-encoding plasmid DNA has antitumor effects, which is well tolerated and thus holds promise for the treatment of patients with metastatic melanoma.

A vector expressing feline mature IL-18 fused to IL-1beta antagonist protein signal sequence is an effective adjuvant to a DNA vaccine for feline leukaemia virus.

DNA vaccination using vectors expressing the gag/pol and env genes of feline leukaemia virus (FeLV) and plasmids encoding feline interleukin-12 (IL-12) and IL-18 completely protected cats from viraemia following challenge [Hanlon L, Argyle D, Bain D, Nicolson L, Dunham S, Golder MC, et al. Feline leukaemia virus DNA vaccine efficacy is enhanced by coadministration with interleukin-12 (IL-12) and IL-18 expression vectors. J Virol 2001;75:8424-33]. However, the relative contribution of each cytokine gene towards protection is unknown. This study aimed to resolve this issue. IL-12 and IL-18 constructs were modified to ensure effective expression, and bioactivity was demonstrated using specific assays. Kittens were immunised intramuscularly with FeLV DNA and various cytokine constructs. Together with control kittens, these were challenged oronasally with FeLV and monitored for 15 weeks. All six kittens given FeLV, IL-12 and IL-18 were protected from the establishment of persistent viraemia and four from latent infection. Of six kittens immunised with FeLV DNA and IL-18, all were protected from viraemia and five from latent infection. In contrast, three of five kittens given FeLV DNA and IL-12 became persistently viraemic. Therefore, the adjuvant effect on the FeLV DNA vaccine appears to reside in the expression of IL-18.

Facilitation of antibody forming responses to viral vaccine antigens in young cats by recombinant baculovirus-expressed feline IFN-gamma.

We assessed the effect of recombinant feline IFN-gamma as vaccine adjuvant for in vivo antibody responses of young 3-month-old kittens to inactivated antigens of rabies and calicivirus, both natural pathogens for cats. When compared to responses following immunization with antigen alone co-administration of baculovirus-expressed cat IFN-gamma significantly enhanced serum antibody titers to both viral antigens; to levels comparable with responses evoked by commonly known saponin and alum adjuvants. Adjuvanticity by feline IFN-gamma was dose-dependent and all doses tested were well tolerated. We conclude that, when further optimized for in vivo delivery, feline IFN-gamma may represent a safe and efficient natural vaccine adjuvant for certain antigens in cats.

Generation of E3-deleted canine adenoviruses expressing canine parvovirus capsid by homologous recombination in bacteria.

E3-deleted canine adenovirus type 1 (CAV-1) was generated by homologous recombination in bacterial cells, using an antibiotic resistance marker to facilitate the recovery of recombinants. This marker was flanked by unique restriction endonuclease sites, which allowed its subsequent removal and the insertion of cassettes expressing the canine parvovirus capsid at the E3 locus. Infectious virus was recovered following transfection of canine cells and capsid expression was observed by RT-PCR from one of the virus constructs. A second construct, containing a different promoter, showed delayed growth and genome instability which, based on the size difference between these inserts, suggests a maximum packaging size of 106 to 109% wild-type genome size for CAV-1.