Andreas Krueger

Manipulation of Host Hepatocytes by the Malaria Parasite for Delivery into Liver Sinusoids

The merozoite stage of the malaria parasite that infects erythrocytes and causes the symptoms of the disease is initially formed inside host hepatocytes. However, the mechanism by which hepatic merozoites reach blood vessels (sinusoids) in the liver and escape the host immune system before invading erythrocytes remains unknown. Here, we show that parasites induce the death and the detachment of their host hepatocytes, followed by the budding of parasite-filled vesicles (merosomes) into the sinusoid lumen. Parasites simultaneously inhibit the exposure of phosphatidylserine on the outer leaflet of host plasma membranes, which act as “eat me” signals to phagocytes. Thus, the hepatocyte-derived merosomes appear to ensure both the migration of parasites into the bloodstream and their protection from host immunity.

The liver stage of Plasmodium berghei inhibits host cell apoptosis

Plasmodium berghei is the causative agent of rodent malaria and is widely used as a model system to study the liver stage of Plasmodium parasites. The entry of P. berghei sporozoites into hepatocytes has extensively been studied, but little is known about parasite–host interaction during later developmental stages of the intracellular parasite. Growth of the parasite far beyond the normal size of the host cell is an important stress factor for the infected cell. Cell stress is known to trigger programmed cell death (apoptosis) and we examined several apoptotic markers in P. berghei‐infected cells and compared their level of expression and their distribution to that of non‐infected cells. As none of the apoptotic markers investigated were found altered in infected cells, we hypothesized that parasite infection might confer resistance to apoptosis of the host cell. Treatment with peroxide or serum deprivation induced apoptosis in non‐infected HepG2 cells, whereas P. berghei‐infected cells appeared protected, indicating that the parasite interferes indeed with the apoptotic machinery of the host cell. To prove the physiological relevance of these results, mice were infected with high numbers of P. berghei sporozoites and treated with tumour necrosis factor (TNF)‐α/d‐galactosamine to induce massive liver apoptosis. Liver sections of these mice, stained for degraded DNA, confirmed that infected cells containing viable parasites were protected from programmed cell death. However, in non‐treated control mice as well as in TNF‐α‐treated mice a small proportion of dead intracellular parasites with degraded DNA were detected. Most hepatocytes containing dead parasites provoked an infiltration of immunocompetent cells, indicating that these cells are no longer protected from cell death.

Taxonomy and inventory of the cytospecies and cytotypes of the Simulium damnosum complex (Diptera: Simuliidae) in relation to onchocerciasis

We provide an inventory of all named cytoforms of the Simulium damnosum complex (including those which are now considered invalid), along with all inversions that have been recorded (including synonyms and homonyms). There are 55 valid and distinct cytoforms known from the S. damnosum complex making it the largest sibling species complex of any vectors, and probably of any insect or other animal. All cytoforms are listed along with their fixed and diagnostic inversions and country distribution. There are 183 inversions known from the complex as a whole, of which 49% are fixed and/or diagnostic between cytoforms, and the fixed/diagnostic inversions seem to occur disproportionately on chromosome arm 2L.

Mice Deficient in Interleukin-4 (IL-4) or IL-4 Receptor α Have Higher Resistance to Sporozoite Infection with Plasmodium berghei (ANKA) than Do Naive Wild-Type Mice

ABSTRACT BALB/c interleukin-4 (IL-4−/−) or IL-4 receptor-α (IL-4rα−/−) knockout (KO) mice were used to assess the roles of the IL-4 and IL-13 pathways during infections with the blood or liver stages of plasmodium in murine malaria. Intraperitoneal infection with the blood-stage erythrocytes of Plasmodium berghei (ANKA) resulted in 100% mortality within 24 days in BALB/c mice, as well as in the mutant mouse strains. However, when infected intravenously with the sporozoite liver stage, 60 to 80% of IL-4−/− and IL-4rα−/− mice survived, whereas all BALB/c mice succumbed with high parasitemia. Compared to infected BALB/c controls, the surviving KO mice showed increased NK cell numbers and expression of inducible nitric oxide synthase (iNOS) in the liver and were able to eliminate parasites early during infection. In vivo blockade of NO resulted in 100% mortality of sporozoite-infected KO mice. In vivo depletion of NK cells also resulted in 80 to 100% mortality, with a significant reduction in gamma interferon (IFN-γ) production in the liver. These results suggest that IFN-γ-producing NK cells are critical in host resistance against the sporozoite liver stage by inducing NO production, an effective killing effector molecule against Plasmodium. The absence of IL-4-mediated functions increases the protective innate immune mechanism identified above, which results in immunity against P. berghei infection in these mice, with no major role for IL-13.

Short communication: First record of Aedes albopictus in Gabon, Central Africa

The Asian tiger mosquito Aedes albopictus is an important arbovirus vector. Originating in East Asia, the species has been introduced to the Americas, the Indo‐Pacific and Australasian regions as well as Europe and Africa, mostly during the past 30 years and probably by transportation in used tires. We report Ae. albopictus for the first time from Gabon (Libreville). In addition, the yellow fever mosquito Ae. aegypti ssp. formosus and 16 other culicid species were detected throughout the city, four of which are also new records for Gabon.

Cytogenetic and PCR‐based identification of S. damnosum‘Nkusi J’ as the anthropophilic blackfly in the Uluguru onchocerciasis focus in Tanzania

A combined molecular and cytogenetic analysis was used to identify the local blackfly vectors of onchocerciasis in the Uluguru Mts. focus, Morogoro region, central Tanzania. Cytotaxonomic identification revealed the occurrence of three cytospecies of the Simulium damnosum complex, along altitudinal gradients. The cytoform ‘Nkusi’ was found breeding at low to mid‐altitudes (100–500 m), while S. kilibanum was found at mid‐ and high altitudes (300–1260 m), being sympatric with ‘Sebwe’ only in the highlands. The analyses further revealed the cytoform ‘Nkusi’ from the nearby Kilosa focus. Simulium kilibanum and ‘Nkusi’ are potential vectors of onchocerciasis, while the ‘Sebwe’ form is considered non‐anthropophilic and therefore not a vector. By means of molecular typing of wild‐caught females, ‘Nkusi’ was incriminated as the man‐biting fly at all catching sites, and consequently, is the assumed vector of onchocerciasis over the full altitude range. In the high altitude zones, however, S. neavei group species may act as secondary vectors at the edge of the forest reserve, if transmission actually occurs there at all. Our results also suggest that vector control could be feasible because of the vector species breeding in the foothills below 500 m in relatively accessible open country.

Investigations into the isolation of the Tukuyu focus of onchocerciasis (Tanzania) from S. damnosum s.l. vector re-invasion.

As part of the feasibility study for an onchocerciasis vector elimination project we investigated the isolation of the Tukuyu focus in Tanzania from possible vector re-invasion. This was achieved by examining the distribution of the Simulium damnosum complex vector cytospecies outside the focus to look for potential sources of re-invasion. Besides cytotaxonomic identifications of the aquatic stages, we applied morphotaxonomic and molecular techniques to identify S. thyolense and confirm it as the anthropophilic species in both the Tukuyu and the neighbouring Ruvuma foci. We detected significant differences in chromosome inversion frequencies between the Tukuyu populations and those breeding to the southwest in the adjacent Songwe river basin and in northern Malawi (where there is no man-biting and no onchocerciasis), suggesting that there is not normally a great deal of migration in either direction. By contrast, populations of S. thyolense from the Tukuyu and Ruvuma foci (150km southeast of Tukuyu) were much more similar in terms of their chromosomal polymorphisms, indicating a higher possibility of re-invasion, although migration is still restricted to some extent, as indicated by some differences in chromosome polymorphisms between the two foci. Future migratory events which might be associated with vector control operations can be monitored by vector cytospecies identification, the frequency of polymorphic inversions which characterise the different vector populations, and the identification of accompanying non-vector cytospecies (e.g. S. plumbeum and cytotype Kasyabone occur exclusively in the two foci, and hence their re-appearance in Tukuyu could have only one outside source). The morphology of the scutal pattern of neonate males may act as a quick test for vector species identification where chromosome squashes are unavailable.

Molecular systematics of Simulium squamosum, the vector in the Kinsuka onchocerciasis focus (Kinshasa, Democratic Republic of Congo)

Human onchocerciasis (‘river blindness’) is a vector-borne, anthroponotic disease that is predominantly endemic to sub-Saharan Africa, where about 99% of all cases occur. Its causative agent, the spirurid filaria Onchocerca volvulus, is transmitted by haematophagous Simulium blackflies, mainly by S. damnosum s.l. (Diptera: Simuliidae). Simulium damnosum s.l. comprises a complex of about 50 sibling species, roughly half of which are vectors (Vajime and Gregory, 1990; www.nhm.ac.uk/research-curation/ projects/blackflies). So far, the discrimination and identification of these species have mostly relied on the cytotaxonomic analysis of larval polytene chromosomes, although morphotaxonomic and molecular markers have also been found useful (Krueger, 2006). One of the known vector sibling species is Simulium squamosum. This has a wide distribution in the forest and savannah zones of West and Central Africa and has also been identified from onchocerciasis foci near the River Congo and some of its tributaries (Elsen et al., 1988; Henry and Meredith, 1990) in Kinshasa (Democratic Republic of the Congo) and Brazzaville (Republic of the Congo). Further east can be found a distinct subspecies — S. s. kitetense, from the eastern Democratic Republic of the Congo (Elsen and Post, 1989) — and the related ‘Kagera’ cytoform, on the Ugandan–Tanzanian frontier (Dunbar, 1966). To the north, S. squamosum occurs in Cameroon (Traore-Lamizana et al., 2001; Mustapha et al., 2004) and, represented by the ‘Bioko’ cytoform of the closely related species S. yahense, on the island of Bioko (Post et al., 2003). Other potential vectors of the S. damnosum complex have, however, been identified, by morphology, in and around Kinshasa, namely S. damnosum s.s. and S. sirbanum (Elsen et al., 1988). In addition, several morphospecies were described in former Zaire, most of which, unfortunately, do not have cytological correlates (Fain and Elsen, 1980; Elsen et al., 1983). In their short overview of the onchocerciasis focus at the village of Kinsuka, on the western outskirts of Kinshasa, Henry and Meredith (1990) provided the first and, as yet, only conclusive data on the identity of the local vector, which were based on cytotaxonomy. Until the 1940s, the biting nuisance caused by blackflies in this area was immense and sufficient to trigger some of the first blackfly-control measures ever implemented. Since then, for unknown reasons, the biting-fly population has never reached such a nuisance level. Although the Simulium species causing the biting nuisance 60 or 70 years ago was never identified, Henry and Meredith (1990) unequivocally identified S. squamosum in the Kinshasa region. Certain sex-differentiating features of the Kinshasa flies resembled, however, those of the cytotypes A, B, C, and D that were subsequently described from Cameroon and Nigeria (Traore-Lamizana et al., 2001; Mustapha et al., 2004). The primary goal of the present study was to re-establish the cytotaxonomic background for the Kinsuka population of S. squamosum. In a second step, based on the studies of Annals of Tropical Medicine & Parasitology, Vol. 101, No. 3, 275–279 (2007)