Bacteria isolated from the gut of different developmental stages of Phlebotomus duboscqi Neveu-Lemaire, 1906 belonged almost all to aerobic or facultatively anaerobic gram-negative rods. In females, the highest bacterial counts were observed two days after bloodfeeding; seven days after bloodfeeding the bacterial counts returned to pre-feeding levels. Most isolates were identified phenotypically as Ochrobactrum sp. The distinctiveness and homogeneity of the phenotypic and genotypic characteristics of Ochrobactrum isolates indicated that they belonged to a single strain (designated AK). This strain was acquired by larvae from food and passaged transtadially: it was isolated from the guts of fourth-instar larvae shortly before pupation, from pupae as well from newly emerged females. Most other bacteria found in females were acquired from the sugar solution fed to adults. To determine if the midgut lectin activity may serve as antibacterial agent females were membrane-fed on blood with addition of inhibitory carbohydrates. No significant differences in bacterial infections were found between experimental and control groups and we suppose that the lectin activity has no effect on gram-negative bacteria present in sandfly gut.
Physico-chemical properties and carbohydrate-binding specificity of hemagglutination activity (HA) were compared in tissue lysates and haemolymph of unfed and bloodied females of five sandfly species. Sandfly gut lectins were found to be heat-labile, sensitive to dithiotreitol treatment, freezing/thawing procedures and were affected by divalent cations. The pH optimum of HA ranged between 7.0-7.5. Specificity of gut HA of all species studied was directed towards aminosugars and some glycoconjugates, mainly lipopolysaccharide from Escherichia coli K-235, heparin and fetuin. Gut HA of Phlebotomus papatasi (Scopoli, 1786) was strongly inhibited by lipophosphoglycan (LPG) from Leishmania major promastigotes. In females, that took blood, the HA was higher but the carbohydrate-binding specificity remained the same; this suggests that the same lectin molecule was present, at different levels, both in unfed and fed flies. High HA was found in ovaries of fed females of Lutzomyia longipalpis (Lutz et Nieva, 1912), P. papatasi and P. duhoscqi Neveu-Lemaire, 1906. In P. papatasi and P. duboscqi the HA was present also in the haemolymph and head lysates of both fed and unfed females. Carbohydrate-binding specificity of HA present in these tissues was similar with the gut lectin.
Haemagglutination activity (HA) was found and characterized in a midgut homogenate of Ixodes ricinus (L.). HA was induced by tick feeding; it was not detected in starved ticks. In a haemagglutination inhibition test, HA showed an affinity for some carbohydrates (N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, rhamnose, and dulcit) and glycoconjugates (especially lipopolysaccharides). Midgut protein components of 37, 60, 65, and 73 kDa were identified by immunoblotting as potential structural subunits of the new agglutinin.
In the framework of a stochastic optimization problem, it is assumed that the stochastic characteristics of optimized system are estimated from randomly right-censored data. Such a case is frequently encountered in time-to-event or lifetime studies. The analysis of precision of such a solution is based on corresponding theoretical properties of estimated stochastic characteristics. The main concern is to show consistency of optimal solution even in the random censoring case. Behavior of solutions for finite data sizes is studied with the aid of randomly generated example.
Sandfly females, while feeding on the host, excrete urine to concentrate proteins of the bloodmeal and restore weight and water balance. This process, analogous to prediuresis in mosquitoes, was observed in 100% of Phlebotomus papatasi (Scopoli) and 85% of P. duboscqi Neveu-Lemaire females studied. Individual females, however, differed in duration of prediuresis and in the number of ejected urine droplets. In both species the prediuresis generally started 1-2 min after the commencement of feeding and the variation in urine production was positively correlated with the length of feeding. The first one or two droplets were opaque whitish while the remaining ones were clear. Erythrocytes were found sporadically in first droplets of some females. Representative prediuresis in P. duboscqi included 27 droplets, i.e., about 325 nl urine in total, ejected during 8 min of feeding. The study revealed prediuresis in P. papatasi and P. duboscqi as a regular physiological process which may have consequences in transmission of infective diseases.
The feeding success of sand flies (Diptera: Phlebotominae) is linked to the vast array of pharmacological substances in their saliva, which interferes with the host haemostasis and immune response. Modification of feeding site plays also an important role in Leishmania transmission. In naive hosts, co-inoculation of saliva and Leishmania parasites increases the chance of successful transmission. Disease exacerbation seems to be associated with enhanced production of type 2 cytokines and selective inhibition of some macrophage functions including the production of NO and H2O2. On the other hand, hosts repeatedly exposed to sand fly bites develop anti-saliva immune response that results in a protection against Leishmania infection. This led to a new interesting approach to anti-Leishmania vaccine - using salivary components to block parasite transmission. The review is therefore focused on the interactions that run between immunomodulatory molecules in sand fly saliva and host immune response, with the impact on Leishmania infection development. Recent studies revealed that saliva-based vaccine for leishmaniasis might be effective and feasible, however, several questions still require to be solved. The knowledge based on experimental mouse model cannot be fully extrapolated to dogs or humans and due to differences in salivary antigens between sand fly species the protective effect is species-specific. On the other hand, the specificity of salivary antigens enables the use of anti-saliva antibodies for monitoring the exposure of hosts to sand fly bites and might be used as a marker of risks for Leishmania transmission in endemic areas.
The Leishmania metalloproteinase GP63 has been reported to play important roles mainly in resistance of promastigotes to complement-mediated lysis and in interaction with macrophage receptors. On the other hand, its function in insect vectors is still unclear. We compared the structure and dosage of gp63 genes and the activity of GP63 in Leishmania major Yakimoff et Schokhor strains and lines differing in virulence for mice and ability to develop in sand flies. The results demonstrate considerable variability in amount and proteolytical activity of GP63 among L. major strains although genomic changes in the gp63 locus were not found. Attenuated LV561/AV line showed low amount and low enzymatic activity of GP63. Serial passages of attenuated parasites through either Phlebotomus duboscqi Neveu-Lemaire or through mice led to a recovery of GP63 proteolytical activity to the level present in virulent LV561/V line. Overexpression of GP63 was found in two L. major strains (L119, Neal) with defective lipophosphoglycan (LPG); both these strains were capable to cause mice infection but unable to survive and multiply in sand flies. Differences were found also in karyotypes and in amount of minichromosomes amplified in some lines of the LV561 strain. The results suggest that parasite virulence is not simply correlated with the activity of GP63; however, this enzyme plays a significant role in association with other surface molecules, especially LPG. Overexpression of GP63 can compensate LPG defect in the vertebrate host but in sand flies both molecules fulfil quite different functions and the defect in LPG is lethal for the parasite. On the other hand, linear minichromosomes of about 200 kb found in some lines of the LV561 strain are associated with development in vitro and in the vector but they are not essential for the infection of the vertebrate host.