Development of immunochromatographic strip assay to detect recent infection of Japanese encephalitis virus in swine population.

Japanese Encephalitis (JE) is a mosquito borne re-emerging viral zoonotic disease. Sero-conversion in swine occurs 2-3 weeks before human infection, thus swine act as a suitable sentinel for predicting JE outbreaks in humans. The present study was undertaken with the objective of developing immunochromatographic strip (ICS) assay to detect recent infection of Japanese Encephalitis virus (JEV) in swine population. The two formats of ICS assay were standardized. In the first format, gold nanoparticles (GNP) were conjugated with goat anti-pig IgM (50 µg/ml) followed by spotting of recombinant NS1 protein (1 mg/ml) of JEV on NCM as test line and protein G (1 mg/ml) as control line. In the format-II, GNP were conjugated with rNS1 protein (50 µg/ml) followed by spotting of Goat anti-pig IgM (1 mg/ml) as test line and IgG against rNS1 (1 mg/ml) as control line. To decrease the non- specific binding, blocking of serum and nitrocellulose membrane (NCM) was done using 5% SMP in PBS-T and 1% BSA, respectively. Best reaction conditions for the assay were observed when 10 µl of GNP conjugate and 50 µl of 1:10 SMP blocked sera was reacted on BSA blocked NCM followed by reaction time of 15 mins. Samples showing both test and control line were considered positive whereas samples showing only control line were considered negative. A total of 318 field swine sera samples were screened using indirect IgM ELISA and developed ICS assay. Relative diagnostic sensitivity and specificity of format-I was 81.25% and 93.0% whereas of format-II was 87.50% and 62.93%, respectively. Out of 318 samples tested, 32 were positive through IgM ELISA with sero-positivity of 10.06% while sero-positivity with format-I of ICS was 8.1%. Owing to optimal sensitivity and higher specificity of format-I, it was validated in three different labs and the kappa agreement ranged from 0.80 to 1, which signifies excellent repeatability of the developed assay to test field swine sera samples for detecting recent JEV infection.

Effect of systemic inflammatory response syndrome on thrombocytogram, acute phase proteins, electrolytes, acid-base indices and cytokine expression in naturally canine parvovirus infected dogs.

Systemic inflammatory response syndrome (SIRS) in canine parvoviral enteritis (CPVE) is associated with high mortality in young puppies. Changes in acute phase response, thrombocytogram, inflammatory cytokine profiles, and disturbances in electrolyte and acid-base homeostasis are thought to have a significant impact on the development of SIRS. However, the mechanisms causing these perturbations have not been well described in CPVE puppies, especially with SIRS. The purpose of this study was to assess the changes of electrolytes, acid-base indices using strong ion model, acute phase proteins and thrombocytogram in blood and expressions of inflammatory cytokines in blood mononuclear cells of CPVE puppies with or without SIRS at admission. Additionally, the positive predictive value (PPV) and cut-off value with specificity and sensitivity of the biomarkers were determined by receiver operating characteristic (ROC) curve analysis to predict the development of SIRS in CPVE puppies at admission. A case-controlled, prospective and observational study was conducted on fifteen SIRS-positive CPVE, twenty-one SIRS-negative CPVE and six healthy puppies. Our data showed marked hyponatremia, hypokalemia, hypoalbuminemia and hypoproteinemia, decreased ATot-albumin and ATot-total protein and increased mean platelet volume (MPV), platelet distribution width (PDW) and C-reactive protein (CRP) concentration and up-regulation of TNF-α, IL-8 and IL-10 expressions in SIRS-positive CPVE puppies as compared to SIRS-negative CPVE puppies at admission. Based on sensitivity, specificity and AUC from ROC curve analysis and PPV, the CRP concentration in serum at a cut-off value of 141.9 mg/L and TLC of blood at a cut-off value of 3.355 × 103/µL were identified as potential prognostic biomarkers followed by ATot-total protein and total protein at a cut-off value of 11.80 and 4.72 g/dL, respectively to predict the development of SIRS in CPVE puppies at admission. In conclusion, the findings of the current study will help the canine practitioners to institute the time-sensitive and need based interventions to disrupt progression along the continuum of shock and multi-organ dysfunction syndrome in CPVE puppies that develop SIRS at admission.