Walter A. Maier

Identification of six sibling species of the Anopheles maculipennis complex (Diptera: Culicidae) by a polymerase chain reaction assay

Abstract Until the eradication of malaria from Europe, members of the Anopheles maculipennis complex had been the major vectors for plasmodial parasites. With the possible reintroduction of Plasmodium species due to climate change and increased travel to and from countries where malaria is endemic, accurate identification of mosquito species will be essential for preventive studies. For this purpose, a diagnostic PCR system to differentiate between six of the seven A. maculipennis sibling species occurring in Europe was developed. The second internal transcribed spacer (ITS2) of the ribosomal DNA was amplified and sequenced for all six species. Based on differences in the nucleotide sequences, species-specific primers were constructed for PCR amplification of mosquito DNA that in combination with a universal primer generate amplification products of different length, each unique for one species.

Individual cases of autochthonous malaria in Evros Province, northern Greece: serological aspects.

Abstract. Prompted by four autochthonous cases of malaria in 1994 and 1995 in Evros Province, northern Greece, we conducted an entomological study between 1997 and 1999 in Nipsa and Chandras, rural locations where two of the four cases had occurred, and in Feres where two additional autochthonous malaria cases had been diagnosed in 1998. In Nipsa and Chandras, we identified 29 Anopheles breeding sites and characterized them by physicochemical parameters. Larvae were collected both at these sites and in a brackish water breeding site near Feres in the Evros River delta. Adults were caught in sheds at all three locations. Morphology was used to classify larvae and adults as A. superpictus or as species belonging to the A. claviger or A. maculipennis species complexes. The latter were further identified by PCR as being A. maculipennis s.s., A. melanoon and A. sacharovi. Of the A.maculipennis complex larvae collected inland, approximately 94% were A. maculipennis s.s. and 6% A. melanoon, whereas all larvae collected in the coastal region were A. sacharovi. In contrast, the A.maculipennis adults were A. maculipennis s.s. and A. melanoon (both 47%), and A. sacharovi (6%). In the coastal region, no A. maculipennis s.s. adults were caught. The ratio of A. melanoon adults collected to A. sacharovi was about 3:1. As shown by a bloodmeal ELISA, only 5 of 266 fed females (1.9%) had ingested human blood, whereas 232 (87%) had fed on goats. Of the mosquitoes containing human blood, two were A. melanoon, one A. sacharovi and one A. maculipennis s.s. One human blood specimen could no longer be assigned to a particular mosquito.

Substantial rise in the prevalence of Lyme borreliosis spirochetes in a region of western Germany over a 10-year period.

ABSTRACT More than a decade after a study on the transmission cycle of Borrelia burgdorferi sensu lato in the Siebengebirge, a nature reserve near Bonn, Germany, questing nymphal and adult Ixodes ricinus ticks were collected again in three selected areas of the same low mountain range and examined for infection with B. burgdorferi sensu lato. Between May and October 2001, a total of 1,754 ticks were collected by blanket dragging; 374 ticks were analyzed for B. burgdorferi sensu lato by both an immunofluorescence assay (IFA) and at least two different PCR tests, whereas 171 ticks were analyzed by PCR only. By combining all assays, an average of 14% of the ticks tested positive for B. burgdorferi sensu lato, 5.5, 15.8, and 21.8% in the three collection areas. Of the nymphs and adults examined, 12.9 and 21.1%, respectively, were found to be spirochete infected. A lower total infection prevalence was obtained by IFA (14.4%) than by a nested PCR approach (16.5%), but both were higher than that obtained by a simple PCR approach (11.9%). Compared with data collected over a decade ago, the mean infection prevalence of B. burgdorferi sensu lato in the ticks was significantly higher for all three biotopes, whereas a similar pattern of habitat-specific infection prevalence was observed. Genotyping of B. burgdorferi sensu lato revealed high relative prevalences of B. valaisiana (identified in 43.1% of infected ticks) and B. garinii (32.3%), whereas B. afzelii (12.3%) and B. burgdorferi sensu stricto (1.5%) were relatively rare. We conclude that B. burgdorferi sensu lato infection has increased in this region over the last 15 years due to presently unknown changes in ecological conditions, perhaps related to climate change or wildlife management.

Opportunistic properties of Nosema algerae (Microspora), a mosquito parasite, in immunocompromised mice.

ABSTRACT. In the last ten years microspordia have been recognized as opportunistic pathogens in AIDS patients. The sources of infection and the mechanisms of transmission of these organisms in humans are mostly uncertain. Transmission of invertebrte microsporidia to mammals is normally considered impossible, temperature being a limiting factor for development. Mice treated with cortisone acetate and with cyclosporin A, respectively, as well as athymic mice were injected intravenously, intranasally, perorally and subcutaneously with spores of Nosema algerae, a microsporidian species of culicine mosquitoes. No infection could be detected in tissue samples of cortisone acetate and cyclosporin A treated mice. However, the experimental inoculation of spores into the tail and foot of athymic mice caused severe infection in skeletal muscles and the connective tissue. In some tails, nerve tissue and bone marrow were also infected. Vegetative stages and spores were seen in direct contact to host cell cytoplasma. For the first time the prolonged and progressive development of an invertebrate microsporidium in a mammalian host is shown. The possibility of invertebrate microsporidia as a source of human microsporidiosis should now be taken into consideration.

Concomitant infections of anopheles stephensi with plasmodium berghei and serratia marcescens: Additive detrimental effects*

The mortality rate of Anopheles stephensi increased after infection with Plasmodium berghei and correlated negatively with temperature. Development of oocysts is inhibited at temperatures above 21 degrees C. We tested the hypothesis that microorganisms were involved in killing the mosquitoes. In fact we were able to demonstrate that in our A. stephensi colony great numbers of Serratia marcescens could be found in the midgut of the insects. The highest value was 2.3 x 10(7) cfu/ml. Other bacteria were rarely seen (1 out of 30 females had flavobacteria). Serratia was neither found in larvae and pupae nor in the water of the breeding dishes. Moderate numbers were detectable in glucose solutions (for feeding of adult mosquitoes) as well as in jars where pupae emerged. Isolated Serratia strains grew faster at 25 degrees C than at 21 degrees C. In glucose solutions alone growth rates were low but they rose rapidly after the addition of blood. -In experimental infections of A. stephensi with S. marcescens (1 x 10(7) bacteria/ml glucose solution) the mortality increased at 25 degrees C. At 21 degrees C the effect of Serratia was insignificant whereas in P. berghei-infected A. stephensi the damaging effects of migrating ookinetes were obvious. Additive detrimental effects were observed at 25 degrees C in mosquitoes infected with P. berghei and Serratia concomitantly.

In Vitro Replication of Nosema algerae (Microsporidia), a Parasite of Anopheline Mosquitoes, in Human Cells above 36° C

Microsporidia form a large and ubiquitous group of obligately intracellular parasitic eukaryotes, increasingly recognized as pathogens in humans. Transmission of invertebrate microsporidia to mammals has been considered impossible because temperature seemed to be a limiting factor for development. Nosema algerae, a microsporidian of anopheline mosquitoes, was cultured in human muscle fibroblasts at temperatures of 31° C and 38° C. This is the first record of an invertebrate microsporidian developing in human cells at a temperature above 36° C. The ultrastructure of N. algerue growing in human muscle fibroblasts is similar to that of Bruchiola vesicularum, a microsporidian species previously described in the muscle of an AIDS patient.

Pathology ofAnopheles stephensi after infection withPlasmodium berghei berghei

SummaryThe mortality ofP. berghei-infectedAnopheles stephensi females can be about 30% higher during the first three days than in normal blood-fed mosquitoes. As expected the mortality is higher after feeding on highly infected mice but also depends on the date of feeding and the temperature. Infected mosquitoes kept at 25° C die more often than those kept at 21° C. On the other hand sporozoite production needs the low temperature of 21°C. So the sporozoite production rate falls with increasing temperature, and the mortality rate increases.ZusammenfassungEin Vergleich der Mortalitätsrate derAnopheles-Mücken während der Plasmodien-Entwicklung zeigt, daß die Sterblichkeit in den ersten drei Tagen um ca. 30% höher sein kann als bei Kontrolltieren, die mit normalem Blut gefüttert worden waren. Sie ist erwartungsgemäß um so höher, je größer die Parasitämie im Blut der Spendermaus war. Allerdings spielt neben dem Fütterungszeitpunkt die Temperatur eine wichtige Rolle. Infizierte Mücken sterben bei 25°C weit häufiger als bei 21°C; Sporozoitenbildung findet aber bei 21°C statt. Die Fähigkeit zur Sporozoitenbildung nimmt also mit steigender Temperatur ab, die Mortalität zu.

Neotrombicula autumnalis (Acari, Trombiculidae) as a vector for Borrelia burgdorferi sensu lato?

Larvae of the trombiculid mite Neotrombicula autumnalis were collected at 18 sites in and around Bonn, Germany, to be screened for infection with Borrelia burgdorferi s.l. by means of PCR. Questing larvae numbering 1380 were derived from the vegetation and 634 feeding ones were removed from 100 trapped micromammals including voles, mice, shrews and hedgehogs. In a laboratory infection experiment, a further 305 host-seeking larvae from the field were transferred onto Borrelia-positive mice and gerbils, and examined for spirochete infection at various intervals after repletion. In three cases borrelial DNA could be amplified from the mites: (1) from a larva feeding on a wild-caught greater white-toothed shrew (Crocidura russula), (2) from a pool of four larvae feeding on a B. garinii-positive laboratory mouse, and (3) from a nymph that had fed on a B. afzelii-positive laboratory gerbil as a larva. In the first case, borrelial species determination by DNA hybridization of the PCR product was only possible with a B. burgdorferi complex-specific probe but not with a species-specific one. In the second case, probing showed the same borrelial genospecies (B. garinii) as the laboratory host had been infected with. In the latter case, however, DNA hybridization demonstrated B. valaisiana while the laboratory host had been infected with B. afzelii. Subsequent DNA sequencing confirmed much higher similarity of the PCR product to B. valaisiana than to B. afzelii indicating an infection of the mite prior to feeding on the laboratory host. The negligible percentage of positive mites found in this study suggests that either the uptake of borrelial cells by feeding trombiculids is an extremely rare event or that ingested spirochetes are rapidly digested. On the other hand, the results imply a possible transstadial and transovarial transmission of borreliae once they are established in their trombiculid host. However, unless the transmission of borreliae to a given host is demonstrated, a final statement on the vector competence of trombiculid mites is not possible.

Morphologic changes in Nosema algerae (Microspora) during extrusion

Abstract As a member of the phylum Microspora, Nosema algerae is a small obligate intracellular parasite. Its free invasive stage is a spore with a characteristic cellular organization, including an apically anchored polar tube that serves as a tool for the transmission of genetic material into the host cell. By detailed electron micrographic documentation of the spore ultrastructure we present the aspects related to the biologic process of spore extrusion. Our ultrastructure findings confirm that the extrusion process of microsporidian spores is based on extreme changes in their organization. This study is the first complete ultrastructural documentation of N. algerae concerning the extrusion process, which can be subdivided into different stages: the breakdown of the microsporidian cellular compartmentation; the filling of a preformed polar tube with modified sporoplasm; the uncoiling of the polar tube, which in this stage has reached its final length; and, finally, its extrusion and screw-like movement.

Über die Mortalität von Culex pipiens fatigans nach Infektion mit Plasmodium cathemerium

SummaryThe registration of the rate of mortality of Culex pipiens after feeding them with the blood of healthy and with Plasmodium cathemerium infected canary birds showed that the death rate of infected mosquitoes was higher under specific conditions. The period when the ookinetes entered the midgut epithelium appears to be decisive. Histological studies showed that the destruction of the midgut epithelium is likely to responsible for the death. A correlation appears between the number of mosquitoes which have sucked the infected blood and their mortality. When a small number of mosquitoes suck on infected birds, their life expectancy is shortened compared to those which suck in larger number. It can be explained, if we assume that in the first case the smaller number of mosquitoes take up a larger amount of infected blood and thereby a larger dosis of infection. An opposite correlation appears if the mosquitoes are allowed to suck healthy birds. — In the discussion we have also refered to the importance the damage on the mosquitoes has for the following generation and thereby for the evolution of resistant strains.ZusammenfassungEin Vergleich der Mortalität von Culex pipiens nach Fütterung an gesunden und mit P. cathemerium infizierten Kanarienvögeln ergab, daß unter bestimmten Bedingungen die Sterberate bei den infizierten Mücken höher lag. Als entscheidender Termin erwies sich der Zeitpunkt des Durchtritts der Ookineten durch das Darmepithel um den 2. und 3. Tag. Nach den vorliegenden lichtoptischen Befunden dürfte die Zerstörung des Mitteldarmepithels für den Tod verantwortlich sein. Eine positive Korrelation konnte nachgewiesen werden zwischen der Anzahl Moskitos, die bei der geschilderten Versuchsanordnung gesogen hatten, und deren Lebenserwartung. Saugt eine kleine Anzahl von Mücken an infizierten Vögeln, so ist ihre Lebenserwartung verkürzt gegenüber denen, die in größerer Zahl gesogen haben. Saugen die Mücken an gesunden Vögeln, so ergibt sich ebenfalls eine Korrelation zwischen der Zahl der Mücken und der Überlebensrate jedoch mit negativem Regressionskoeffizienten. — In der Diskussion wird auf die Bedeutung hingewiesen, die die Schädigung der Moskitos für die Folgegeneration und damit auch für die Evolution übertragungsfähiger Stämme haben kann.