Cytokine expression in caspase-6 deficient mice 6 hours after infection with <i>B</i>. <i>pseudomallei</i>.

<p>Quantitative real-time PCR analysis of inflammatory parameters in the spleens of C57BL/6 WT (n = 8) and <i>caspase6</i>^<i>-/-</i> mice (n = 8) 6 hours after infection with 5 × 10⁴ CFU <i>B</i>. <i>pseudomallei</i> strain E8. Data were analysed using Student’s <i>t</i> test. Values are means ± standard deviations from two independent experiments.</p

Diagnostic validation of a rapid and field-applicable PCR-lateral flow test system for point-of-care detection of cyprinid herpesvirus 3 (CyHV-3).

Koi herpesvirus disease (KHVD) is a highly infectious disease leading to outbreaks and mass mortality in captive and free-ranging common carp and koi carp. Outbreaks may result in high morbidity and mortality which can have a severe economic impact along the supply chain. Currently, control and prevention of KHVD relies on avoiding exposure to the virus based on efficient hygiene and biosecurity measures. An early diagnosis of the disease is crucial to prevent its spread and to minimize economic losses. Therefore, an easy-to-handle, sensitive, specific and reliable test prototype for a point-of-care detection of KHV was developed and evaluated in this study. We used a multiplex-endpoint-PCR followed by a specific probe hybridization step. PCR-products/hybridization-products were visualized with a simple and universal lateral flow immunoassay (PCR-LFA). Fifty-four gill tissue samples (KHV-positive n = 33, KHV-negative n = 21) and 46 kidney samples (KHV-positive n = 24, KHV-negative n = 22) were used to determine diagnostic sensitivity and specificity of the PCR-LFA. In addition, the usability of PCR-LFA to detect CyHV-3-DNA in gill swabs taken from 20 perished common carp during a KHVD-outbreak in a commercial carp stock was examined. This assay gave test results within approximately 60 min. It revealed a detection limit of 9 KHV gene copies/μl (95% probability), a diagnostic specificity of 100%, and diagnostic sensitivity of 94.81% if samples were tested in a single test run only. PCR inhibition was noticed when examining gill swab samples without preceding extraction of DNA or sample dilution. Test sensitivity coud be enhanced by examining samples in five replicates. Overall, our PCR-LFA proved to be a specific, easy-to-use and time-saving point-of-care-compatible test for the detection of KHV-DNA. Regarding gill swab samples, further test series using a higher number of clinical samples should be analyzed to confirm the number of replicates and the sample processing necessary to reveal a 100% diagnostic sensitivity

Invasion and replication of <i>B</i>. <i>pseudomallei</i> in caspase-6 deficient macrophages.

<p>Intracellular bacterial burden of C57BL/6-WT and C57BL/6-<i>caspase6</i>^<i>-/-</i> macrophages after infection with <i>B</i>. <i>pseudomallei</i> strain E8 at an MOI of ~ 25. The CFU data were logarithmized to achieve normal distribution and compared using Student’s t-test. Values are means ± standard deviations from triplicate determinations. The experiment was repeated three times.</p

Effect of IL-10 in macrophages during <i>B</i>. <i>pseudomallei</i> infection.

<p>(A) Quantitative real-time PCR analysis of IL-10 expression in macrophages from C57BL/6 WT and <i>caspase6</i>^<i>-/-</i> mice 6 h after infection with <i>B</i>. <i>pseudomallei</i> strain E8 (MOI ~ 50). The data were logarithmized to achieve normal distribution and compared using Student’s <i>t</i>-test. Values are means ± standard deviations from three independent experiments. (B) Intracellular bacterial burden of IL-10-treated and non-treated C57BL/6 macrophages after infection with <i>B</i>. <i>pseudomallei</i> strain E8 (MOI ~ 25). Data were analysed using Student’s <i>t</i>-test. Values are means ± standard deviations from triplicate determinations. The experiment was repeated three times.</p

Cytokine expression in caspase-6 deficient mice 24 hours after infection with <i>B</i>. <i>pseudomallei</i>.

<p>Quantitative real-time PCR analysis of inflammatory parameters in the spleens of C57BL/6 WT and <i>caspase6</i>^<i>-/-</i> mice. Mice were infected with 5 × 10⁴ CFU of <i>B</i>. <i>pseudomallei</i> strain E8 i.v. for 24 h. Uninfected control animals received PBS. Each of the 4 groups contained 9 mice from 3 replicates (n = 36). Data were analysed using Student’s <i>t</i> test. Values are means ± standard deviations from three independent experiments.</p

Cell death induction in caspase-6 deficient macrophages after infection with <i>B</i>. <i>pseudomallei</i>.

<p>Course of cell damage in C57BL/6 WT and <i>caspase6</i>^<i>-/-</i> macrophages after infection with <i>B</i>. <i>pseudomallei</i> strain E8. (A) For the LDH release assay cells were infected with an MOI of ~10. Data were analysed using Student’s <i>t</i> test. Values are means ± standard deviations from triplicate determinations. (B) Real-time cell status analysis of C57BL/6 WT (blue, triangles) and <i>caspase6</i>^<i>-/-</i> macrophages (red, circles). Infected cells are represented by brighter lines (top-down triangle, small circle) and received an MOI of ~5. Uninfected controls are represented by darker lines (upright triangle, big circle). The y-axis is a relative scale for the cell status measured by the XCelligence system. The experiments were repeated twice.</p

Caspase-6 mediates resistance against <i>Burkholderia pseudomallei</i> infection and influences the expression of detrimental cytokines

<div><p>Caspase-6 is a member of the executioner caspases and known to play a role in innate and adaptive immune processes. However, its role in infectious diseases has rarely been addressed yet. We here examined the impact of caspase-6 in an <i>in vivo</i> infection model using the Gram-negative rod <i>Burkholderia pseudomallei</i>, causing the infectious disease melioidosis that is endemic in tropical and subtropical areas around the world. <i>Caspase-6</i>^-/- and C57BL/6 wild type mice were challenged with <i>B</i>. <i>pseudomallei</i> for comparing mortality, bacterial burden and inflammatory cytokine expression. Bone-marrow derived macrophages were used to analyse the bactericidal activity in absence of caspase-6. Caspase-6 deficiency was associated with higher mortality and bacterial burden <i>in vivo</i> after <i>B</i>. <i>pseudomallei</i> infection. The bactericidal activity of <i>caspase-6</i>^-/- macrophages was impaired compared to wild type cells. <i>Caspase-6</i>^-/- mice showed higher expression of the IL-1β gene, known to be detrimental in murine melioidosis. Expression of the IL-10 gene was also increased in <i>caspase-6</i>^-/- mice as early as 6 hours after infection. Treatment with exogenous IL-10 rendered mice more susceptible against <i>B</i>. <i>pseudomallei</i> challenge. Thus, caspase-6 seems to play a crucial role for determining resistance against the causative agent of melioidosis. To our knowledge this is the first report showing that caspase-6 is crucial for mediating resistance in an <i>in vivo</i> infection model. Caspase-6 influences the expression of detrimental cytokines and therefore seems to be important for achieving a well-balanced immune response that contributes for an efficient elimination of the pathogen.</p></div