Anopheles plumbeus (Diptera: Culicidae) in Europe: a mere nuisance mosquito or potential malaria vector?
© Schaffner et al.; licensee BioMed Central Ltd. 2012
Received: 21 October 2012
Accepted: 22 November 2012
Published: 26 November 2012
Anopheles plumbeus has been recognized as a minor vector for human malaria in Europe since the beginning of the 20th century. In recent years this tree hole breeding mosquito species appears to have exploited novel breeding sites, including large and organically rich man-made containers, with consequently larger mosquito populations in close vicinity to humans. This lead to investigate whether current populations of An. plumbeus would be able to efficiently transmit Plasmodium falciparum, the parasite responsible for the most deadly form of malaria.
Anopheles plumbeus immatures were collected from a liquid manure pit in Switzerland and transferred as adults to the CEPIA (Institut Pasteur, France) where they were fed on P. falciparum gametocytes produced in vitro. Anopheles gambiae mosquitoes served as controls. Development of P. falciparum in both mosquito species was followed by microscopical detection of oocysts on mosquito midguts and by sporozoite detection in the head/thorax by PCR and microscopy.
A total of 293 wild An. plumbeus females from four independent collections successfully fed through a membrane on blood containing P. falciparum gametocytes. Oocysts were observed in mosquito midguts and P. falciparum DNA was detected in head-thorax samples in all four experiments, demonstrating, on a large mosquito sample, that An. plumbeus is indeed receptive to P. falciparum NF54 and able to produce sporozoites. Importantly, the proportion of sporozoites-infected An. plumbeus was almost similar to that of An. gambiae (31 to 88% An. plumbeus versus 67 to 97% An. gambiae). However, the number of sporozoites produced was significantly lower in infected An. plumbeus.
The results show that a sample of field-caught An. plumbeus has a moderate to high receptivity towards P. falciparum. Considering the increased mobility of humans between Europe and malaria endemic countries and changes in environment and climate, these data strongly suggest that An. plumbeus could act as a vector for malaria and thus significantly contribute to increasing the malaria transmission risk in Central-Western Europe. In locations showing high vulnerability to the presence of gametocyte carriers, the risk of transmission of malaria by An. plumbeus should be considered.
Global changes in health of humans and livestock are of concern to policy and decision making bodies. Over the last fifteen years, autochthonous cases and epidemic outbreaks of vector-borne diseases have occurred in Europe (e.g. bluetongue, chikungunya, dengue[3, 4], Schmallenberg disease, Usutu virus infection), including local transmission of malaria in France, Germany, Greece, Italy, and Spain[7–11]. Among the factors that contribute to the emergence or re-emergence of vector-borne diseases are the increased mobility of humans, livestock, and pathogens, as well as environmental modification, and climate change. This emphasizes the need to evaluate or re-evaluate the capability of European Anopheline mosquitoes to allow local transmission of malaria, in particular of Plasmodium falciparum, which is responsible for the most deadly form of the disease.
Several factors have contributed to the decline and disappearance of malaria in Western Europe and most of the Mediterranean countries during the 19th and early 20th centuries[8, 13], including improvements in socio-economic conditions (particularly the separation of human and animal housing), the development and widespread use of efficient anti-malarial drugs, large-scale elimination of mosquito breeding sites through drainage, and improvement in mosquito control activities. Several Anopheles species had contributed to the transmission of malaria parasites in Europe, with Plasmodium vivax presumably being the prevalent species, although this is not fully ascertained. The primary vector species belong to the Maculipennis complex with Anopheles atroparvus acting as the main vector in western, northern and central Europe, and Anopheles labranchiae and Anopheles sacharovi in southern regions. Other species have been considered as minor vectors, including further species of the Maculipennis complex (Anopheles messeae, Anopheles maculipennis s.s., Anopheles melanoon), as well as Anopheles algeriensis, Anopheles claviger s.s., Anopheles cinereus, Anopheles hyrcanus, Anopheles plumbeus, Anopheles sergentii, and Anopheles superpictus[14, 15]. All the above mentioned species are still present in Europe[16, 17]. Following malaria eradication campaigns, the abundance and distribution of the main vectors have been sustainably reduced in some areas but have recovered to initial levels in others[18, 19]. The principal vectors of P. vivax (An. atroparvus, An. labranchiae, An. sacharovi) were refractory or had a low vector competence for P. falciparum in experimental investigations[20–22]. Recent studies of An. labranchiae populations collected in France and Italy confirmed that this species is receptive to P. falciparum, but exhibits a low vector competence[23, 24].
This study shows that An. plumbeus mosquitoes from a field collection from Switzerland, with nearly three hundred females tested, are indeed competent towards P. falciparum NF54. The possibility of An. plumbeus becoming a more important vector of P. falciparum in Western Europe is discussed, considering the large number of malaria cases imported into the European Union every year (10,000 to 12,000) and the occurrences of episodes of higher than normal temperatures during summer[34, 35] which may offer a more favourable environment for autochthonous transmission of P. falciparum in temperate parts of Europe.
Anopheles plumbeus immatures were collected from a liquid manure pit located in Bäretswil, canton of Zurich, in a hilly region of northern Switzerland. A high number of immatures were present in this large breeding site at all visits from May to November, 2010. Immatures were sampled on four occasions (June, July, September and November 2010), reared to the adult stage in the laboratory and maintained on a 10% glucose solution at room temperature (21°C ± 2°C) and 40% ± 10% relative humidity (RH) under a 14 h-10 h day-night photoperiod. From each collection, 200 to 250 An. plumbeus females were sent to the CEPIA (Institut Pasteur, Paris, France) 6 days prior to P. falciparum challenge. Upon arrival, females were maintained under standard conditions (25°C ± 1°C, 75% ± 5% RH and 12 h-12 h day-night photoperiod) and provided a 10% sucrose solution ad libidum before being starved from sugar 24 h prior feeding on blood containing P. falciparum gametocytes. As a control, the An. gambiae Ngousso strain, which is highly receptive to P. falciparum, was fed on the same gametocyte preparations. All An. gambiae females used as control were maintained on 10% sucrose supplemented with a 100 U penicillin-0.01% Streptomycin final concentration solution (Invitrogen, France) from adult emergence until the infection experiment to minimize bacterial flora and thus increase susceptibility to P. falciparum (Thiéry et al., personal communication;). This treatment was not applied to An. plumbeus in order to assess its receptivity under more natural conditions.
Parasites and experimental feeding
Plasmodium falciparum isolate NF54 was cultured using a semi automated tipper table system, and subcultures were produced according to Mitri and colleagues using fresh erythrocytes from anonymous blood donors (7% final haematocrit) provided by ICAReB platform, Institut Pasteur. Fourteen days after initiation of the subculture, parasitemia and gametocyte sex ratio were determined on Giemsa-stained smears, and gametocyte maturity was evaluated by checking for exflagellation of microgametes. Ten ml of 14 day-old gametocyte culture were centrifuged at 800 g for 5 min and the pellet was resuspended in a mixture of fresh red blood cells and AB human serum (EFS, Rungis, France) to a 40% final haematocrit. One ml of parasitized blood was poured in each blood feeder maintained at 37°C. The gametocyte density for the four experiments were 8.3, 4.2, 8.0 and 5.7 × 107/ml, respectively. Mosquitoes were allowed to feed in the dark through a Parafilm® membrane for 15 min. Unengorged females were discarded while fully engorged females were transferred into small cages and provided with 10% sucrose containing 0.05% PABA (4-para-amino benzoic acid, Sigma, France) and 0.01% final concentration penicillin-streptomycin for An. gambiae only. To mimic Anopheles feeding behaviour in nature, additional uninfected fresh blood meals were provided to all mosquitoes 3 and 9 days post- P. falciparum ingestion (p.i.). Mosquitoes were kept at 25°C, 75% RH for the duration of the experiments.
Analysis of infection
The presence of developing oocysts in mosquito midguts and of sporozoites in salivary glands was determined. Microscopic oocyst detection and quantification were performed on dissected midguts, stained with 0.1% mercuro-bromo fluorescein (Fluka, France) in phosphate buffered saline, at days 8 and 15 p.i. When available, at least 30 females per experiment and species were analysed. A female was considered infected if at least one oocyst was seen. At day 15 p.i., oocysts were recorded as either fully developed or ruptured. At this time point, the head-thorax tissue containing the salivary glands were isolated and stored at −20°C for PCR detection of sporozoite DNA as described. In addition, quantification of sporozoites in head-thorax tissue was performed using the Ozaki method.
Results and discussion
Plasmodium falciparum infection and dissemination in field-caught An. plumbeus and laboratory-reared An. gambiae Ngousso
Anopheles gambiae Ngousso
Day of observationa
Prevalence (%) of midgut infection (no. positive / no. analysed)
Intensity of infection: Mean no. of oocysts / positive femaled (range)
8.9 ± 6.3
50.4 ± 56.0
12.0 ± 0
42.4 ± 28.2
71.5 ± 63.3
29.5 ± 23.5
13.7 ± 13.6
5.3 ± 4.9
2.0 ± 0
28.8 ± 14.4
2.4 ± 2.1
22.4 ± 25.2
44.8 ± 36.4
26.4 ± 23.7
10.7 ± 10.1
Prevalence (%) of females with sporozoitese (no. positive / no. analysed)
Intensity of infection: Mean no. sporozoites / positive femalef (no. analysed)
Anopheles plumbeus has been described as a putative vector for P. vivax under central European[29, 42] and Russian climate conditions. Studies have found that it is a more efficient carrier of P. vivax than An. atroparvus and An. claviger. The first experimental demonstration of An. plumbeus competence towards P. vivax and P. falciparum were reported nearly 100 years ago[31, 44, 45]. Anopheles plumbeus competence towards P. vivax has not been experimentally reevaluated since, mainly due to the difficulty of access to P. vivax gametocytes. In contrast, An. plumbeus ability to permit P. falciparum development to the sporozoite stage was recently shown under laboratory conditions on a sample of ten mosquitoes. The present study demonstrates the development of P. falciparum sporozoites, the stage infective to humans, in a wild population of An. plumbeus and for a large number of specimens (n = 293) collected at different times during the yearly natural occurrence of the species. Although these results show that An. plumbeus developed less P. falciparum oocysts than An. gambiae, the proportion of mosquitoes harbouring sporozoites was similar in both species. Associated with its fierce human biting (see below), these data indicate that An. plumbeus could play a significant role for local transmission of P. falciparum where there is imported malaria. In comparison, An. labranchiae, which is believed to have played a significant role in indigenous malaria transmission in parts of Europe, showed a lower prevalence of P. falciparum sporozoite infection (3 to 10%) in two independent sets of infections performed under similar conditions[23, 24].
Parasite transmission depends on multiple factors which define the vectorial capacity C, i.e. the daily rate of future inoculations originating from a currently infective source, in the equation C = b m a2 pt / (−ln p), where b is the vector competence, i.e. the proportion of vectors that develop infective parasite stages; m the vector density in relation to host density; a the vector’s daily blood-feeding rate; p the vector’s daily survival rate; t the duration of the parasite’s extrinsic incubation period in days. Thus, besides being competent to P. falciparum, a mosquito species needs to both sustain parasite development upon natural temperature ranges and have a daily survival rate high enough to transmit the parasite to humans.
In the experimental setting of the presented work, mosquitoes were maintained at a constant temperature of 25°C, which corresponds to the mean temperature range of July in the subtropical Mediterranean climate. Thus, the observed infectivity of P. falciparum can be expected to be similar under natural conditions for southern Europe. At this temperature, the extrinsic incubation period of P. falciparum is 12–14 days, which increases to 22–23 days at 20°C. Considering the longevity of An. plumbeus of up to two months, completion of parasite development to the sporozoite stage under cooler conditions is feasible. Furthermore, recent studies indicate that, at low mean average temperatures, parasite development is faster under realistic daily fluctuating temperatures as compared to corresponding constant temperatures. Altogether this suggests that An. plumbeus could be a competent vector for P. falciparum in a Central European climate.
Additional evidence of a possible role for An. plumbeus in the transmission of malaria comes from its ability to feed on humans (factor a), often in large numbers (factor m), due to the proliferation of this species in large man-made containers such as abandoned manure pits[16, 17, 25]. For example, in Alsace (north-eastern France) during the 1990’s, populations of An. plumbeus expanded leading to the observation of human leg landing rates of up to 365 individuals in 15 minutes (Schaffner, unpublished). At this time, the highest infestation rate was located nearby the international Basel-Mulhouse-Freiburg airport.
The source of the An. plumbeus used in this study was an abandoned manure pit which was discovered during mosquito field surveys in Switzerland in 2007–2010. During these surveys, An. plumbeus was the most frequently collected Anopheles species, being reported in 40 out of 396 mosquito collections (34 larval samplings; 6 adult catches/trapping of which 5 human landing catches, one BG-Sentinel™ trapping) from all urban, suburban, rural, and nature land-use zones from an altitudinal range from 268 to 858 m above sea level. In contrast, An. claviger s.s., An. maculipennis s.s. and An. messeae were reported at 22, 18, and three locations, respectively (Schaffner, unpublished data). Additionally, An. plumbeus was the third most common species after Culex pipiens and Aedes japonicus found in 3,000 vases in Swiss cemeteries, screened for invasive Aedes species such as Aedes albopictus.
Taking into account human and parasite movements and changes in environment and climate, the data presented strongly suggest that An. plumbeus can play the vector role for malaria and can significantly contribute to increase the malaria transmission risk in Central-Western Europe for both P. vivax and P. falciparum, due to (1) its proven vector competence for these two parasites, (2) the occurrence of locally high population densities as a result of the recently established exploitation of man-made breeding habitats, (3) the aggressiveness of this species to humans and (4) its longevity for up to two months enabling the parasites to complete their development to the sporozoite stage. In localities showing high vulnerability for the presence of gametocyte carriers, the risk of transmission of malaria by An. plumbeus should be considered.
We thank the CEPIA technicians for rearing An. gambiae mosquitoes, the ICAReB Platform for providing human blood samples used for the in vitro production of Plasmodium gametocytes and the farmer for providing access to the larval breeding site. We highly acknowledge the Swiss Federal Veterinary Office for funding FS as collaborator of the Swiss Reference Laboratory for Epizootic Vectors, and the Swiss Tropical and Public Health Institute for co-funding this study.
- Wilson AJ, Mellor PS: Bluetongue in Europe: past, present and future. Phil Trans R Soc B. 2009, 364: 2669-2681. 10.1098/rstb.2009.0091.PubMed CentralView ArticlePubMed
- Rezza G, Nicoletti L, Angelini R, Romi R, Finarelli AC, Panning M, Cordioli P, Fortuna C, Boros S, Magurano F, Silvi G, Angelini P, Dottori M, Ciufolini MG, Majori GC, Cassone A, for the CHIKV study group: Infection with chikungunya virus in Italy: an outbreak in a temperate region. Lancet. 2007, 370: 1840-1846. 10.1016/S0140-6736(07)61779-6.View ArticlePubMed
- Gould EA, Gallian P, De Lamballerie X, Charrel RN: First cases of autochthonous dengue fever and chikungunya fever in France: from bad dream to reality!. Clin Microbiol Infect. 2010, 16: 1702-1704. 10.1111/j.1469-0691.2010.03386.x.View ArticlePubMed
- Gjenero-Margan I, Aleraj B, Krajcar D, Lesnikar V, Klobučar A, Pem-Novosel I, Kurečić-Filipović S, Komparak S, Martić R, Đuričić S, Betica-Radić L, Okmadžić J, Vilibić-Čavlek T, Babić-Erceg A, Turković B, Avšić-Županc T, Radić I, Ljubić M, Šarac K, Benić N, Mlinarić-Galinović G: Autochthonous dengue fever in Croatia, August–September. Euro Surveill. 2011, 16: pii=19805-
- Rasmussen LD, Kristensen B, Kirkeby C, Rasmussen TB, Belsham GJ, Bødker R, Bøtner A: Culicoids as vectors of Schmallenberg virus. Emerg Infect Dis. 2012, 18: 1204-1206.PubMed CentralView ArticlePubMed
- Steinmetz HW, Bakonyi T, Weissenbock H, Hatt JM, Eulenberger U, Robert N, Hoop R, Nowotny N: Emergence and establishment of Usutu virus infection in wild and captive avian species in and around Zurich, Switzerland - Genomic and pathologic comparison to other central European outbreaks. Vet Microbiol. 2011, 148: 207-212. 10.1016/j.vetmic.2010.09.018.View ArticlePubMed
- Krüger A, Rech A, Su XZ, Tannich E: Two cases of autochthonous Plasmodium falciparum malaria in Germany with evidence for local transmission by indigenous Anopheles plumbeus. Trop Med Int Health. 2001, 6: 983-985. 10.1046/j.1365-3156.2001.00816.x.View ArticlePubMed
- Alten B, Kampen H, Fontenille D: Malaria in Southern Europe: resurgence from the past?. Emerging pests and vector-borne diseases in Europe. Edited by: Takken W, Knols BG. 2007, Wageningen: Wageningen Academic Publishers, 35-57.
- Zoller T, Naucke TJ, May J, Hoffmeister B, Flick H, Williams CJ, Frank C, Bergmann F, Suttorp N, Mockenhaupt FP: Malaria transmission in non-endemic areas: case report, review of the literature and implications for public health management. Malar J. 2009, 8: 71-10.1186/1475-2875-8-71.PubMed CentralView ArticlePubMed
- Santa-Olalla Peralta P, Vazquez-Torres MC, Latorre-Fandos E, Mairal-Claver P, Cortina-Solano P, Puy-Azón A, Adiego Sancho B, Leitmeyer K, Lucientes-Curdi J, Sierra-Moros MJ: First autochthonous malaria case due to Plasmodium vivax since eradication, Spain, October 2010. Euro Surveill. 2010, 15: pii=19684-
- Danis K, Baka A, Lenglet A, Van Bortel W, Terzaki I, Tseroni M, Detsis M, Papanikolaou E, Balaska A, Gewehr S, Dougas G, Sideroglou T, Economopoulou A, Vakalis N, Tsiodras S, Bonovas S, Kremastinou J: Autochthonous Plasmodium vivax malaria in Greece, 2011. Euro Surveill. 2011, 16: pii=19993-
- Randolph SE, Rogers DJ: The arrival, establishment and spread of exotic diseases: patterns and predictions. Nat Rev Microbiol. 2010, 8: 361-371. 10.1038/nrmicro2336.View ArticlePubMed
- Bruce-Chwatt LJ, De Zulueta J: The rise and fall of malaria in Europe: A historico-epidemiological study. 1980, Oxford: Oxford University Press
- Hackett LW: Malaria in Europe. An ecological study. 1937, London: Oxford University Press
- Zahar AR: Vector bionomics in the epidemiology and control of malaria. Part II. The WHO European Region & the WHO Eastern Mediterranean Region. Vol II: Applied field studies. 1990, Geneva: World Health Organisation
- Schaffner F, Angel G, Geoffroy B, Hervy J-P, Rhaiem A, Brunhes J: The Mosquitoes of Europe. An identification and training programme. 2001, Montpellier: IRD Editions & EID Méditerranée, CD-Rom
- Becker N, Petrić D, Zgomba M, Boase C, Madon M, Dahl C, Kaiser A: Mosquitoes and their control. 2010, Heidelberg: Springer-Verlag, 2View Article
- Blazquez J, De Zulueta J: The disappearance of Anopheles labranchiae from Spain. Parassitologia. 1980, 22: 161-163.PubMed
- Romi R, Pierdominici G, Severini C, Tamburro A, Cocchi M, Menichetti D, Pili E, Marchi A: Status of malaria vectors in Italy. J Med Entomol. 1997, 34: 263-271.View ArticlePubMed
- Daškova NG, Rasnicyn SP: Review of data on susceptibility of mosquitoes in the USSR to imported strains of malaria parasites. Bull World Health Organ. 1982, 60: 893-897.PubMed CentralPubMed
- Marchant P, Eling W, van Gemert GJ, Leake CJ, Curtis CF: Could British mosquitoes transmit Falciparum malaria?. Parasitol Today. 1998, 14: 344-345. 10.1016/S0169-4758(98)01274-5.View ArticlePubMed
- Sousa C: PhD thesis. Malaria vectorial capacity and competence of Anopheles atroparvus Van Thiel, 1927 (Diptera, Culicidae): Implications for the potential re-emergence of malaria in Portugal. 2008, Lisbon: Universidade Nova de Lisboa
- Toty C, Barré H, Le Goff G, Larget-Thiéry I, Rahola N, Couret D, Fontenille D: Malaria risk in Corsica, former hot spot of malaria in France. Malar J. 2010, 9: 231-10.1186/1475-2875-9-231.PubMed CentralView ArticlePubMed
- Romi R, Boccolini D, Vallorani R, Severini F, Toma L, Cocchi M, Tamburro A, Messeri G, Crisci A, Angeli L, Costantini R, Raffaelli I, Pontuale G, Thiéry I, Landier A, Le Goff G, Fausto AM, Di Luca M: Assessment of the risk of malaria re-introduction in the Maremma plain (Central Italy) using a multi-factorial approach. Malar J. 2012, 11: 98-10.1186/1475-2875-11-98.PubMed CentralView ArticlePubMed
- Dekoninck W, Hendrickx F, Van Bortel W, Versteirt V, Coosemans M, Damien D, Hance T, De Clercq EM, Hendrickx G, Schaffner F, Grootaert P: Human induced expanded distribution of Anopheles plumbeus, experimental vector of West Nile virus and a potential vector of human malaria in Belgium. J Med Entomol. 2011, 48: 924-928. 10.1603/ME10235.View ArticlePubMed
- Schaffner F: Mosquitoes in used tyres in Europe: species list and larval key. Eur Mosq Bull. 2003, 16: 7-12.
- Schaffner F, Kaufmann C, Hegglin D, Mathis A: The invasive mosquito Aedes japonicus in Central Europe. Med Vet Entomol. 2009, 23: 448-451. 10.1111/j.1365-2915.2009.00825.x.View ArticlePubMed
- Bueno-Marí R, Jiménez-Peydró R: Anopheles plumbeus Stephens, 1828: a neglected malaria vector in Europe. Malaria Reports. 2011, 1: e2-View Article
- Shute PG, Maryon M: Malaria in England past, present and future. Roy Soc Health J. 1974, 94: 23-29. 10.1177/146642407409400111.View Article
- Sokolova MI, Snow KR: Malaria vectors in European Russia. Eur Mosq Bull. 2002, 12: 1-6.
- Blacklock B, Carter HF: Further experiments with Anopheles plumbeus Stephens, its infection with P. falciparum in England; Also notes on the apparatus and technique employed. Ann Trop Med Par. 1920, 13: 275-283.
- Eling W, van Gemert G-J, Akinpelu O, Curtis J, Curtis CF: Production of Plasmodium falciparum sporozoites by Anopheles plumbeus. Eur Mosq Bull. 2003, 15: 12-13.
- World Health Organization: From malaria control to elimination in the WHO European Region 2006–2015. Regional Strategy. 2006, Copenhagen: World Health Organization, Regional Office for Europe
- Luterbacher J, Dietrich D, Xoplaki E, Grosjean M, Wanner H: European seasonal and annual temperature variability, trends, and extremes since 1500. Science. 2004, 303: 1499-1503. 10.1126/science.1093877.View ArticlePubMed
- Xoplaki E, Luterbacher J, Paeth H, Dietrich D, Steiner N, Grosjean MHW: European spring and autumn temperature variability and change of extremes over the last half millennium. Geophys Res Lett. 2005, 32: L15713-View Article
- Mitri C, Jacques J-C, Thiéry I, Riehle MM, Xu J, Bischoff E, Morlais I, Nsango SE, Vernick KD, Bourgouin C: Fine pathogen discrimination within the APL1 gene family protects Anopheles gambiae against human and rodent malaria species. PLoS Pathog. 2009, 5: e1000576-10.1371/journal.ppat.1000576.PubMed CentralView ArticlePubMed
- Rodrigues J, Brayner FA, Alves LC, Dixit R, Barillas-Mury C: Hemocyte differentiation mediates innate immune memory in Anopheles gambiae mosquitoes. Science. 2010, 239: 1353-1355.View Article
- Ponnudurai T, Meuwissen JHET, Leeuwenberg ADEM, Verhave JP, Lensen AHW: The production of mature gametocytes of Plasmodium falciparum in continuous cultures of different isolates infective to mosquitoes. Trans R Soc Trop Med Hyg. 1982, 76: 242-250. 10.1016/0035-9203(82)90289-9.View ArticlePubMed
- Mitri C, Thiéry I, Bourgouin C, Paul REL: Density-dependent impact of the human malaria parasite Plasmodium falciparum gametocyte sex ratio on mosquito infection rates. Proc R Soc Biol Sci B. 2009, 276: 3721-3726. 10.1098/rspb.2009.0962.View Article
- Padley D, Moody AH, Chiodini PL, Saldanha J: Use of a rapid, single-round, multiplex PCR to detect malarial parasites and identify the species present. Ann Trop Med Parasitol. 2003, 97: 131-137. 10.1179/000349803125002977.View ArticlePubMed
- Ozaki LS, Gwadz RW, Godson GN: Simple centrifugation method for rapid separation of sporozoites from mosquitoes. J Parasitol. 1984, 70: 831-833. 10.2307/3281779.View ArticlePubMed
- Shute PG: Indigenous P. vivax malaria in London believed to have been transmitted by Anopheles plumbeus. Mon Bull Minist Health Publ Health Lab Serv. 1954, 13: 48-51.
- James SP: The disappearance of malaria from England. Proc R Soc Med. 1929, 23: 1-15.
- Blacklock B, Carter HF: The experimental infection, in England, of Anopheles plumbeus with Plasmodium vivax (sporozoites in salivary glands). Preliminary note. Ann Trop Med Parasitol. 1919, 13: 187-188.
- Blacklock B, Carter HF: The experimental infection in England, of Anopheles plumbeus Stephens and Anopheles bifurcatus with Plasmodium vivax. Ann Trop Med Parasitol. 1920, 13: 413-420.
- Scott TW, Takken W: Feeding strategies of anthropophilic mosquitoes result in increased risk of pathogen transmission. Trends Parasitol. 2012, 28: 114-121. 10.1016/j.pt.2012.01.001.View ArticlePubMed
- Anonymous: Manual on practical entomology in malaria. Part II: Methods and Techniques. 1975, Geneva: World Health Organization
- Paaijmans KP, Blanford S, Bell AS, Blanford JI, Read AF, Thomas MB: Influence of climate on malaria transmission depends on daily temperature variation. Proc Natl Acad Sci USA. 2010, 107: 15135-15139. 10.1073/pnas.1006422107.PubMed CentralView ArticlePubMed
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