Immunoassays of malaria sporozoites in mosquitoes

ParasitologyToday June 1986

Immunoassays of Malaria Sporozoites in Mosquitoes T.FL Burkot* and R.A. Wirtz** *PapuaNew Guinea Inst3tuteof Medical Research, PO Box 378, Madang, PapuaNew Guinea **Department of Entomology, Walter ReedArmy In~tute of Research, Washington, DC 20307, USA

Fig. I. Enzyme-linked immunasorbent assay detecting P. falciparum sporozoite antigen in mosquitoes. Sporozo/teinfected mosquitoes give dark reactions (wells 4C, 7E and 8A). Serial dilutJons of known numbersof sporozo/tesare assayed in the right and left columns so that the numbers of sporozoites in wild-caught mosquitoes can be quanti~ed.

Malaria transmission from mosquito to man occurs when an Anopheles mosquito infected with sporozoites of one of the four species of human malaria takes a blood meal from man. During the feeding process, malaria sporozoites, the initial stage of the parasite that infects man, are inoculated from the mosquito's salivary glands into the blood stream. These sporozoites make their way to the liver where they divide and undergo further development to produce schizoms. Eventually merozoites are released from the liver schizonts which then invade red blood cells to begin the blood stage of the infection. Determination of sporozoite rates or the percentage of mosquitoes whose salivary glands contain sporozoites is an important component of epidemiological investigations of malaria (see Box 1). The incrimination of a malaria vector requires the discovery of sporozoites in the mosquito's salivary glands. The determination of sporozoite rates then defines which vectors are primary vectors and which are only of secondary importance. Finally, the time interval during which sporozoites are found

in mosquitoes marks the transmission season and therefore the period when protection from mosquitoes is required to prevent new infections. The measurement of sporozoite rates is therefore essential to determine the efficacy of intervention strategies directed against either the vector or the sporozoite stage.

Problems of Sporozoite Rates The traditional method for incriminating malaria vectors requires dissection of the salivary glands of individual anopheline mosquitoes for sporozoites. Such dissections are labour intensive but manageable when investigating anophelines with high sporozoite rates. However, in many endemic areas the vectors have very low sporozoite rates (less than 0.1%) 1and therefore the number of dissections required to establish a sporozoite rate or even to incriminate the vector species becomes very high. A second disadvantage of dissecting salivary glands to look for sporozoites is the difficulty of determining the species of malaria parasite. Not only are the sporozoites of the four human species of malaria impossible to distinguish from each other morphologically, but they also cannot be differentiated from the sporozoites of more than 100 other malaria species which infect rodents, birds, reptiles and nonhuman primates.

Cireumsporozoite Proteins Circumsporozoite proteins of mahria parasites are hot-stable proteins which cover the external surface of the sporozoite. These proteins, found on all species of sporozoites so far studied, consist of a signal sequence, a charged region, a central region of repeated amino acid units, two other charged regions and an anchor sequenceZ,3. The most important characteristic of circumsporozoite proteins, as far as the )1986, Elsevier Science Publisl~ers B.V., Amsterdam 0169-4758t86,~02,00

For technical reasons we are unable to reproduce this figure in colour. See the June issue of Parasitology Today for full colour illustration.

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Parasitology Today, vol. Z no. 6, 1986

Box 1. The Basic Reproductive Rate of Malaria The basic reproduction rate of malaria (%) is defined as the average number of new cases of malaria arising from a single primary case in a large popuiadon of susceptible hosts. It follows that if % is less than 1 then any initial transmission will die out, while ifzois greater than 1 then mmsmission will proceed. The classical deterministic formulation of Zois defined as follows. If infected humans recover at a rate r per day, then each case will be infective for an average of l/r days. On each day, the infective person will be bitten by ma mosquitoes (where m is the mosquito density per person, and a is the proportion that bite man). The probability that a mosquito will survive for the n days required for malaria parasites to develop in the mosquito (the extrinsic cycle) will be p~, after which the expected time of further survival is 1/(-logp). On each day of this survival, the mosquito will bite a times on average, and a proportion b will be infective. Writing these terms together gives MacDonald's formula for the basic reproductive rate: - r log p In this expression, b is measured as the proportion of mosquitoes with infective sporozoites in their salivary glands (see text).

development of immunoassays for their detection is concerned, is the presence of the immunodominant central region made up of repeated amino acid sequences4. The length and amino acid sequence which makes up the repeats are unique for each species of malaria. The repeat sequence for P. know/es/sporozoites, a monkey mahria, consists of 12 amino acids repeated 12 times5,6. The human malaria, P. falcipamm, has repeats consisting of four amino acids repeated up to 41 fimesT,s, while P. vivax has 9 amino acids repeated 19 times9. The amino acid sequence comprising these repeats is unique for each species of sporozoite, and the sequence seems to be conserved worldwide for both P. falciparum and P. ¢)/¢)ax10.

lmmtmoassays The produ~ion of high affinity monoclonal antibodies directed against the species-specific repeat region of the drFig. 2. S~-ozo/te-infected

mosquitoes from different geograph/c areas can be detected using a mon~

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from Brazil (168) and Thailand but does not crass-rea~ w/th uninfected rnosqu/toes (An. freeborni and An. dirus) or w/th mosqu/toes iaF.-cted w ~ other s'pec/esofspotozoites (P. berghei, P. knowlesi

and P. viva~ (From Ref. 13J

cumsporozoite proteins has led to the development of immunoassays which circumvent the need for dissecting mosquitoes to detect sporozoites. The first such assay to be developed was a direct immunoradiometric assayn. Using triturated mosquitoes as antigen, this assay was able to detect, identify and quantify the sporozoites. However, the number of mosquitoes which could be processed simultaneously was limited by the antigen binding capacity of the plastic (i.e. the larger the pool size of mosquitoes assayed, the greater the competition for binding sites on the plastic plate and therefore the less sensitive the assay). Developmem of 'sandwich' assays allowed for easier quantification of the number of sporozoites present in larger pools of mosquitoes because the initial antibody used to coat the plate selectively binds the circumsporozoite protein, and thereby eliminates the competition with mosquito proteins for binding sites on the plate. Since the circumsporozoite proteins contain a repetitive epitope, sandwich assays can employ a single monoclonal antibody directed against this epitope. Such antibodies can be used as the capture antibody and can also be linked to a marker as a conjugate. The presence of the repeats acts as a built in amplification system for detection by providing many sites for antibody recognition on the same protein. Because the circumsporozoite proteins are membrane-bound surface antigens, a non-ionic detergent is used to solub'flize the proteins and thereby increase the efficiency of the extraction process and the sensitivity of the assay. These sandwich assays are able to detect a single sporozoite-infected mosquito in a pool with up to 19 uninfected mosquitoes without suffering a loss in sensitivity. Immunoradiometric assays specific for simian and rodent sporozoites have now been developed, as well as assays for the human malarias P. falciluman and P. Z~/~3~¢11.

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However, immunoradiometric assays depend on short-lived radioactive reagents and are therefore limited in their ability to be applied in the field. Since enzyme-linked immunosorbant assays (ELISA) use stable reagents, they are better suited for use in field laboratories. Sandwich ELISAs have now been developed for the simian rnalada, P. knog0/eg/12, as well as the human mahrias P. falciparum 13and P. v/vax14(Fig. 1). The sensitivity of these assays has been increased to detect as few as 15 sporozoites per infected mosquito (R.A. Wirtz, unpublished). Quantification of sporozoites from field collected mosquitoes has indicated that

Parasitology Today, voL Z no. 6, 1986

more than 90% of infected P. fa/c/panon sporozoite-infected mosquitoes contain greater than 500 sporozoites15, so the sensitivity of these assays is sufficient to detect virtually all positive mosquitoes. Limitations Although the sandwich assays represent a vast improvement over dissecting mosquitoes in terms of sensitivity, specificity and time required for determination of sporozoite rates, they do have some drawbacks which must be taken into consideration. The assays measure sporozoite antigen and therefore are measuring infected mosquitoes, not necessarily infectious o n e s an important epidemiological consideration. The 'sporozoite rates' determined by these assays are slight exaggerations over true sporozoite rates because sporozoite antigen can be detected in mature oocysts on the mosquito's stomach, and in sporozoites from ruptured oocysts which have not made their way to the salivary glands. The assays cannot be considered as a substitute for dissections in the incrimination of new vectors because some species of anopheline will s u p p o r t t h e g r o w t h o f m a l a r i a parasites to the sporozoite stage yet the sporozoites are incapable of entering the salivary glands 16. An anopheline only becomes a malaria vector when sporozoites are found in the salivary glands. Although the repetitive epitope of the circumsporozoite protein of P. falciparura and P. vivax is similar throughout the world 10 (Fig. 2), the circumsporozoite protein of the monkey malaria, P. cynomo/g/, exhibits variation in the repeats from different isolates 17. It is not unreasonable to expect that antigenic variation does exist in the sporozoites of human malarias. Even though such variation is probably a relatively rare occurrence, such variation may increase when selective pressure is applied against the sporozoite stage, for example by anti-sporozoite vaccines. Poss~ilities Since the assays can use dried mosquitoes for sporozoite antigen detection, weeks or months worth of field collected mosquitoes can be held in the laboratory at room temperature until ready to be processed. The ability of the assays to detect single infected mosquitoes in pools of up to 20 mosquitoes means that two technicians in one day can process almost 6000 anophelines for sporozoite antigen detection and identification. By dissecting, the same two technicians might process 150 mosquitoes in a

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day - with the assay, two months work can now be done in a day! Intensive epidemiological studies can now be performed on large numbers of mosquitoes to study the transmission of several species of malaria simultaneously. As an example, in Papua New Guinea, we have now analyzed over 70 000 anophelines from 12 villages for the presence of sporozoite antigens of P. falciparum and P. vivax in a single year. We are thus able to study simultaneously the transmission of malaria under a variety of conditions, including monitoring the efficacy of intervention strategies. 1 Boyd, M.F. (1949) Ma/ar/o/o~, pp. 668-677 2 Nardin, E.H. etal. (1982)J. Exp. Med. 156, 20-30 3 Santoro, F. etal. (1983)J. Biol. Chem. 258, 3341-3345 4 Zavala, F. et al. (1983)J. Exp. Med. 157, 1947-1957 5 FAils, J. et al. (1983) N a ~ e 302, 536-538 6 Godson, G.N. et al. (1983) Nature 305, 29-33 7 Dame, J.B. et al. (1984) Science 225, 593-599 8 Enea, V. et aL (1984) Sc/¢nce 225, 628--629 9 Amot, D.E. et al. (1985) Science 230, 815-818 10 Zavala, F. et aL (1985)J. Iramunol. 135, 2790-2793 11 Zavala, F. et aL (1982) Nature 299, 737-738 12 Burkot, T.R. et aL (1984) Am. J. Trop. Med. Hyg. 33, 227-23 l 13 Burkot, T.R., Williams, J.L. and Schneider, I. (1984) Am. J. Trop. Med. Hyg. 33, 783-788 14 Wirtz, R.A. et al. (1985) Am. J. Trop. Med. Hyg. 34, 1048-1054 15 Collins, F.H. et aL (1984) Am. J. Trop. Meal. Hyg. 33, 538-543 16 Rosenberg, R. (1985) Am. J. Trop. Med. Hyg. 34, 687691 17 Cochrane, A.H. et al. (1985) Mo/. B/ochem. Paras/to/. 14, 111-124

Parasitology in Australia The s~xth International Congress of Parasitology (ICOPA VI) will be held in August atthe University of Queensland, Brisbane, Austraha (see DIARY). To coincide with this event, the July issue of Parasitology Today will feature a special supplement on ParQsitologyin Australia, collated by Dr Graham Mitchell of the Walter and Eliza Hall Institute, Melbourne. One of the main themes of this supplement is the contribution made by Austrahan research workers towards vaccines against bovine babesiosps, cutaneous myiasis, cysticercosls, gastro-intestinal helminths and malaria. The scientific programme of ICOPA Vl shows a broad coverage, though with some emphasis on veterinary parasites. O f the six main 'streams', one is devoted to molecular parasitology and one to parasite evolution, The remaining four, however, have a welcome bias towards solving parasitic problems in human and animal health and in pisciculture, with a clear attempt to integrate new research techniques in biochemistry, immunology and population biology, with the economics and practical problems of parasite control. This is the first time that this prestigious six-yearly congress has gone to the southern hemisphere, but the venue has aroused some concern amongst parasitologists working in developing countries - especially in Latin America. O f the invited speakers at ICOPA VI, relatively few are drawn from developing countries, But the main question, strongly debated at the recent Latin American Congress of Parasitology in Ecuador, asks 'why aren't international parasitology congresses held in tropical countries?' After all, these are the countries with the greatest burden of parasitic diseases. A proposal to hold the next ICOPA in Brazil is currently under consideration. Such a venue could not only stimulate those working in tropical countries, but might also lead more scientists from temperate laboratories to understand the full context of their research - and look further than the proverbial laboratory mouse!