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Culicoides biting midges transmit multiple ruminant viruses, including bluetongue virus and epizootic hemorrhagic disease virus, causing significant economic burden worldwide. To further enhance current control techniques, understanding vector-virus interactions within the midge is critical. We developed previously a double-stranded RNA (dsRNA) delivery method to induce RNA interference (RNAi) for targeted gene knockdown in adult Culicoides sonorensis Wirth & Jones. Here, we confirm the C. sonorensis inhibitor of apoptosis 1 (CsIAP1) as an anti-apoptotic functional ortholog of IAP1 in Drosophila, identify the ortholog of the Drosophila initiator caspase DRONC (CsDRONC), and demonstrate that injection of dsRNA into the hemocoel can be used for targeted knockdown in the midgut in C. sonorensis. We observed CsIAP1 transcript reduction in whole midges, with highest transcript reduction in midgut tissues. IAP1knockdown (kd) resulted in pro-apoptotic caspase activation in midgut tissues. In IAP1kd midges, midgut tissue integrity and size were severely compromised. This phenotype, as well as reduced longevity, was partially reverted by co-RNAi suppression of CsDRONC and CsIAP1. Therefore, RNAi can be directed to the midgut of C. sonorensis, the initial site of virus infection, using dsRNA injection into the hemocoel. In addition, we provide evidence that the core apoptosis pathway is conserved in C. sonorensis and can be experimentally activated in the midgut to reduce longevity in C. sonorensis. This study thus paves the way for future reverse genetic analyses of midgut-virus interactions in C. sonorensis, including the putative antiviral properties of RNAi and apoptosis pathways., (© The Authors 2017. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Oils from cold-water fish are rich in (n-3) polyunsaturated fatty acids, in particular eicosapentaenoic acid (20:5) and docosahexaenoic acid (22:6). Although those fatty acids are beneficial in the prevention of cardiac disease and have anti-inflammatory properties, they can also decrease survival rates of mice during challenges with food-borne pathogens. This study was designed to determine dietary fat effects on Kupffer cells and splenocytes during a Salmonella typhimurium challenge. Mice were fed a low corn oil diet (3%, control), high corn oil diet (20%, HCO), or a menhaden fish oil diet (17% + 3% corn oil, FO) for 28 days and then orally given 3.1 x 10(8) colony-forming units of S. typhimurium. Kupffer cells and splenocytes were separated immediately prior to and on days 6, 10, and 14 postchallenge. Fish oil decreased Kupffer cell phagocytosis and oxidative burst early in the infection and adhesion molecule (CD18) expression at the end of the infection. In splenocytes, fish oil affected Ia expression prior to and late in the infection and depressed CD18 expression late in the infection. These data suggest that the diet affected Kupffer cells most early in the infection but affected splenocytes primarily later in the infection. Therefore, because the greatest death rate during an S. typhimurium infection occurs early, the reduced function of the Kupffer cells is probably a major factor.

Cluster of differentiation antigen 14 (CD14) functions as a receptor for lipopolysaccharide (LPS) LPS-binding protein (LBP) complexes. Because LPS has varying effects on CD14 expression in vitro, we evaluated CD14 expression in response to LPS with a fully differentiated macrophage phenotype, the alveolar macrophage. By using flow microfluorometric analysis and a radioimmunoassay with an anti-human CD14 monoclonal antibody (My4) that cross-reacts with porcine CD14, we found that macrophages stimulated with LPS for 24 h exhibited a two- to fivefold increase in CD14-like antigen compared with unstimulated cells. At low concentrations of LPS, up-regulation of the CD14-like antigen was dependent on serum; at higher concentrations of LPS, serum was not required. In the absence of serum a 10-fold higher dose of LPS (10 ng/ml) was required to increase CD14-like expression. In addition, LPS-induced CD14-like up-regulation correlated with secretion of tumor necrosis factor-alpha, regardless of serum concentration. Blockade with My4 antibody significantly inhibited LPS-induced tumor necrosis factor-alpha secretion at 1 ng/ml of LPS. However, inhibition decreased as we increased the LPS concentration, suggesting the existence of CD14-independent pathways of macrophage activation in response to LPS. Alternatively, My4 may have a lower affinity for the porcine CD14 antigen than LPS, which may have only partially blocked the LPS-LBP binding site at high concentrations of LPS. Therefore, these data suggest that LPS activation of porcine alveolar macrophages for 24 h increased CD14-like receptor expression. The degree of CD14-like up-regulation was related to LPS concentration, however, activation did not require the presence of serum at high concentrations of LPS.

Nonspecific immunity is usually lower in neonates than adults. Consequently, enhancing the neonate's nonspecific immune capability may be beneficial for the health and growth performance of young animals. We conducted two experiments in which neonatal pigs were injected with recombinant bovine interleukin-1 beta (rBoIL-1 beta) at 9 to 11 days of age. Three consecutive daily injections of rBoIL-1 beta increased neutrophil and monocyte numbers, which remained elevated until the animals were challenged with Streptococcus suis at 19 days of age. Neutrophil bactericidal activity was greater in interleukin-1-treated pigs than in saline-injected controls. At lower ratios of effector to target cells, neutrophil-mediated, antibody-dependent cellular cytotoxicity was increased in neonates treated with IL-1. However, natural killer cell activity and neutrophil production of superoxide anion were not affected by treatment with IL-1. Expression of CD18 was increased transiently on neutrophils from IL-1-treated pigs at 15 days of age. Severity of the streptococcal infection was less in pigs that were treated with IL-1 at 9 to 11 days of age. These data suggest that IL-1 treatment in neonates may augment nonspecific immune function and disease resistance.

The purpose of this study was to determine whether antiorthostatic suspension of C3HeB/FeJ mice for a period of 11 days affected macrophage and spleen cell function. We found that antiorthostatic suspension did not alter macrophage secretion of prostaglandin E2, tumor necrosis factor alpha, and interleukin-1. Antiorthostatic suspension also did not affect macrophage-mediated contact-dependent cytotoxicity, TNF-mediated cytotoxicity, expression of class II histocompatibility molecules, or concanavalin A and Bandeiraea simplicifolia lectin binding sites. The proliferative response of splenic T cells in response to mitogens and staphylococcal exotoxins was significantly enhanced in antiorthostatically suspended mice. We detected significantly higher concentrations of corticosterone in the plasma of antiorthostatically suspended mice. Therefore, there did not appear to be any direct immunosuppressive effects of corticosterone on the parameters tested.