Detection of malaria sporozoites by standard ELISA and VecTestTM dipstick assay in field-collected anopheline mosquitoes from a malaria endemic site in Ghana

We compared the VecTestTM dipstick assay for detection of Plasmodium sporozoites in Anopheles vectors of malaria with standard circumsporozoite (CS) microplate ELISA for detection of Plasmodium falciparum circumsporozoite protein (PfCSP) in Anopheles mosquitoes. Mosquitoes were collected from a malaria endemic site (Kassena Nankana district) in northern Ghana. Of 2620 randomly sampled mosquitoes tested, the standard CS-ELISA gave a sporozoite rate of 10.8% compared with 11.2% by VecTestTM, which was not statistically different (P = 0.66). Visual reading of the CS-ELISA results gave a sporozoite rate of 13.4%, which was higher than the other tests (P > 0.05). To allow a more objective evaluation of the sensitivity of the dipstick, an additional 136 known CS-ELISA-positive specimens were analysed. The prevalence of the test (including the additional samples) was 14.6% and 14.7% for CS-ELISA and dipstick, respectively (P > 0.05). The estimated prevalence by visual assessment of the CS-ELISA results was 17.5%. The relative specificity and sensitivity of the VecTestTM dipstick and visually read ELISA were estimated based on the CS-ELISA as a gold standard. The specificities of the dipstick and visual ELISA were high, 98.0% and 96.6%, respectively. However, the sensitivities of the two assays were 88.8% for VecTest and 100% for visual ELISA (P < 0.01). Concordance between VecTest and CS-ELISA was good (kappa = 0.86). Similarly, there was a good concordance between the dipstick and the visually read ELISA (kappa = 0.88). Extrapolating from PfCSP controls (titrated quantities of P. falciparum sporozoites), mean sporozoite loads of CS-ELISA-positive An. gambiae (286 +/- 28.05) and An. funestus (236 +/- 19.32) were determined (P = 0.146). The visual dipstick grades showed high correlation with sporozoite load. The more intense the dipstick colour, the higher the mean sporozoite load (+ = 108, ++ = 207, +++ = 290, r = 0.99, r2 = 1). The VecTest dipstick offers practical advantages for field workers needing rapid and accurate means of detection of sporozoites in mosquitoes.     

抗蚊中肠抗体对斯氏按蚊体内约氏疟原虫卵囊发育的影响

[目的 ]研究抗斯氏按蚊中肠抗体对约氏疟原虫蚊期的抑制作用。 [方法 ]在感染血中加入抗蚊中肠抗体 ,羊膜饲蚊 ,在蚊虫血餐后 14h、9和 12d ,分别检查蚊虫中肠动合子、卵囊及唾液腺中子孢子 ;并观察抗体浓度和蚊虫血餐抗体次数对卵囊的抑制作用。 [结果 ]抗蚊中肠抗体可降低蚊体内卵囊的感染率和子孢子的进腺率 ,尚可降低动合子的感染度和卵囊指数 (P <0 0 5 ) :且抗体浓度越高、蚊虫血餐抗体次数越多 ,卵囊数量越少。 [结论 ]抗蚊中肠抗体对疟原虫的动合子、卵囊发育有抑制作用 ,但对卵囊的抑制作用最明显 ,抗体的浓度高和维持时间长 ,抑制作用越明显。     

Identification of malaria species by ELISA in sporozoite and oocyst infected Anopheles from western Kenya

Enzyme-linked immunosorbent assays (ELISAs) for the circumsporozoite (CS) antigens of Plasmodium falciparum, P. malariae, and P. ovale were used to identify species of sporozoite and oocyst infections detected by dissection in Anopheles gambiae s.1. and An. funestus collected in western Kenya. ELISAs identified 92.5% of 1,113 salivary gland infections; Plasmodium species infections included 79.4% P. falciparum, 3.2% P. malariae, 1.7% P. ovale, and 2 or more Plasmodium species were detected in 15.7% of the Anopheles in which the species of parasite was identified. Identification was more likely with greater numbers of sporozoites observed in dissections, increasing from 65% ELISA positivity in mosquitoes with 1-10 sporozoites in their salivary glands to 96% in mosquitoes with over 1,000 sporozoites. ELISAs detected CS antigen in 66% of 294 Anopheles that by dissection had oocysts but uninfected salivary glands. Of 112 Anopheles with a single species of Plasmodium detected in the salivary glands, 29 (25.9%) had 1 or more additional species detected in the midgut, indicating a high potential for multiple infections. Similar proportions of Plasmodium species were found in An. gambiae s.1. and An. funestus.     

Seasonal density, sporozoite rates and entomological inoculation rates of Anopheles gambiae and Anopheles funestus in a high-altitude sugarcane growing zone in western kenya

Summary An entomological study was conducted on vectors of malaria and their relative contribution to Plasmodium falciparum transmission in Mumias, a high-altitude site and large-scale sugarcane growing zone in Kakamega district, western Kenya. Anopheles gambiae s. l ., the predominant vector species, represented 84% ( n = 2667) of the total Anopheles mosquitoes collected with An. funestus comprising only 16%. Polymerase chain reaction (PCR) identified all 600 specimens of the An. gambiae complex tested as An. gambiae sensu stricto , an indication that it is the only sibling species represented in the high-altitude sites in western Kenya. Plasmodium falciparum sporozoite rates of 6.3% (133/2118) for An. gambiae s.l . and 9.5% (38/402) for An. funestus by ELISA were obtained in Mumias. None of 1600 mosquitoes tested for P. malariae sporozoites was positive. ELISA tests of mosquito blood meals indicated a high tendency of anthropophagy, a behaviour contributing significantly to malaria transmission by the vector species, with 95.9%, 4.86% and 0.2% having taken at least one blood meal on human, bovine and avian hosts, respectively.  Malaria transmission intensity was low as revealed by the low entomological inoculation rates (EIR) recorded. The EIR values for An. gambiae s.l . were 29.2 infective bites per person per year (ib/p/year) and 17.5 ib/p/year for An. funestus in Mumias. The highest inoculation rate for both vector species was 7.0 ib/p/month in July. Plasmodium falciparum parasite rate among asymptomatic children was 55.4% and 44% in the wet (July–September) and dry (December–February) seasons, respectively. These results indicate that malaria transmission intensity in the high-altitude site is low but perennial, with transmission being maintained by An. gambiae s.s . and An. funestus .     

Plasmodium coatneyi: observations on periodicity, mosquito infection, and transmission to Macaca mulatta monkeys.

Plasmodium coatneyi has adapted well to experimental studies with Macaca mulatta monkeys and Anopheles dirus mosquitoes. Studies were made to determine 1) the course of asexual parasitemia, 2) periods when infective gametocytes were produced, 3) the laboratory-reared mosquitoes susceptible to infection, 4) the mosquito most capable of transmitting the infection to monkeys via bite, 5) the pattern of recrudescence, and 6) the prepatent periods following the bites of infected An. dirus mosquitoes. The period when infective gametocytes are produced is concentrated primarily in the first week when parasitemia exceeds 1,000/microl. Mosquitoes were more heavily infected on days when the asexual parasite counts were highest. Gametocyte counts were generally low. Mature forms of the parasite markedly sequestered giving a pattern of high-low periodicity. Anopheles dirus and An. freeborni mosquitoes were nearly equal in terms of their ability to support oocyst development. Other species (An. stephensi, An. maculatus, and An. gambiae.) were less supportive. High sporozoite densities in the salivary glands were frequently produced in An. dirus and sporozoite transmission was obtained via the bites of these mosquitoes after 12-18 days of extrinsic incubation. Prepatent periods ranged from 10 to 15 days. The presence of frequent parasitic recrudescences suggests mechanisms similar to that seen in human infections with P. falciparum. It is proposed that P. coatneyi in M. mulatta monkeys can be a suitable model for studies on cerebral pathology, vaccine efficacy, and the testing of antimalarial drugs.     

Aotus nancymaae as a potential model for the testing of anti-sporozoite and liver stage vaccines against Plasmodium falciparum

The Santa Lucia strain of Plasmodium falciparum was transmitted to Aotus lemurinus griseimembra, A. azarae boliviensis, A. vociferans, and A. nancymaae monkeys by bite and by intravenous inoculation of sporozoites dissected from Anopheles freeborni, An. stephensi, An. gambiae, An. albimanus, and An. maculatus mosquitoes. The data obtained from these infections indicate that A. nancymaae can be considered a suitable host model when combined with the Santa Lucia strain of P. falciparum for the testing of candidate anti-sporozoite and liver stage vaccines.     

发作期与间歇期间日疟原虫在不同按蚊体内发育差异的研究

目的  比较临床发作期与间歇期间日疟原虫在中华按蚊和嗜人按蚊体内发育的差异。方法在我国间日疟流行区分别采集临床发作期与间歇期间日疟病例的血样，采用体外人工膜饲感染系统在实验室同时体外人工感染中华按蚊和嗜人按蚊，在感染后7～9 d和14 d分别解剖蚊胃和唾液腺，并检测蚊体内的卵囊和子孢子数。结果临床发作期间日疟原虫感染中华按蚊的卵囊阳性率和阳性蚊比率、子孢子阳性蚊比率和感染度均低于间歇期间日疟原虫感染，临床发作期间日疟原虫感染嗜人按蚊的子孢子阳性率、阳性蚊比率和卵囊阳性蚊比率均低于间歇期间日疟原虫感染，而子孢子感染度则高于间歇期疟原虫感染。结论临床发作期与间歇期间日疟原虫体外人工感染中华按蚊、嗜人按蚊卵囊和子孢子有差异     

硝喹对斯氏按蚊体内不同时期约氏疟原虫发育的影响

目的　观察硝喹对斯氏按蚊体内不同时期约氏疟原虫发育的影响。方法　在感染约氏疟原虫1 d前给予常规蔗糖水或硝喹蔗糖水（含100 μmol/L硝喹）供斯氏按蚊吸食,停糖水24 h后用感染约氏疟原虫的昆明鼠血餐,观察硝喹处理后约氏疟原虫在蚊胃内的卵囊数量变化。感染6、14 d后停常规蔗糖水24 h,随后给予常规蔗糖水或硝喹蔗糖水供斯氏按蚊吸食,观察斯氏按蚊血淋巴及唾液腺中的约氏疟原虫子孢子数量变化。结果　感染前1 d将斯氏按蚊暴露于硝喹蔗糖水后,感染第7天蚊胃中约氏疟原虫卵囊数量[（119.2 ± 16.1）只]较常规蔗糖水组[（207.3 ± 21.8）只]显著降低（t = 3.207,P < 0.05）。感染第6天停常规蔗糖水24 h,将斯氏按蚊暴露于硝喹蔗糖水后,按蚊血淋巴中约氏疟原虫子孢子数量峰值[（952.3 ± 22.7）只]在感染第14天出现,常规蔗糖水组按蚊血淋巴中子孢子数量峰值[（1 287.0 ± 39.0）只]在感染第12天出现;感染第17天,硝喹蔗糖水和常规蔗糖水组按蚊唾液腺中子孢子数量分别为（9 467.0 ± 1 304.0）只和（10 533.0 ± 758.7）只,差异无统计学意义（t = 0.707,P = 0.506）。感染第14天停常规蔗糖水24 h,将斯氏按蚊暴露于硝喹蔗糖水后,按蚊唾液腺中约氏疟原虫子孢子数量[（21 900.0 ± 2 613.0）只]较常规蔗糖水处理组[（10 533.0 ± 732.3）只]显著增加（t = 4.188,P < 0.05）。结论　在斯氏按蚊感染疟原虫不同时期给予硝喹处理对其体内约氏疟原虫发育影响不一,未感染斯氏按蚊经硝喹处理后可减少疟原虫传播。     

Increased intradermal probing time in sporozoite-infected mosquitoes

Because malaria sporozoites destroy segments of the salivary glands of vector mosquitoes, we determined whether salivary function is impaired. Such pathology would result in a prolonged intradermal probing phase of feeding behavior, because the role of saliva is to help locate blood vessels. Indeed, non-infected Aedes aegypti mosquitoes probed for a shorter period than did either sporozoite-infected or saliva-deprived mosquitoes. Salivary apyrase activity is reduced to a third following maturation of sporozoites. Apyrase activity, normally, is confined to those regions invaded by sporozoites. Sporozoite-infected and non-infected mosquitoes produced equal volumes of saliva. We conclude that sporozoite infection impairs the vector's ability to locate blood vessels by affecting the quality of salivary product, thereby increasing potentially infective host contacts.     

Re-ingestion of Plasmodium berghei sporozoites after delivery into the host by mosquitoes

Malaria-infected mosquitoes feeding on a mammalian host inject sporozoites into the skin to induce a malaria infection. The numbers of sporozoites ultimately able to reach the liver may be important determinants of the characteristics of the ensuing blood infection. Because feeding mosquitoes not only inject sporozoites into the host but concomitantly ingest blood to obtain their bloodmeal, some sporozoites are re-ingested by the feeding mosquito. We studied transmission of fluorescent Plasmodium berghei sporozoites injected into mice by Anopheles stephensi mosquitoes and found that the numbers of sporozoites re-ingested by mosquitoes are comparable to numbers previously reported to be delivered directly into mice. Thus, re-ingestion of sporozoites likely plays a significant role in transmission dynamics of malaria by mosquitoes, and may account for the failure of some sporozoite-infected mosquitoes to induce a blood infection.     

Field evaluation of an enzyme-linked immunosorbent assay (ELISA) for Plasmodium falciparum sporozoite detection in anopheline mosquitoes from Kenya

An enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody that recognizes a repetitive epitope on the circumsporozoite protein of Plasmodium falciparum was used in Kenya to assess malaria infections in Anopheles gambiae s.l. and An. funestus. The ELISA confirmed that 88% of 44 sporozoite-positive gland dissections were P. falciparum. The ELISA infection rate of 18.6% (n = 736) for individually tested mosquitoes for both species was significantly higher than the 10.4% (n = 537) salivary gland sporozoite rate determined by dissection. This difference was due to ELISA detection of medium and large sized oocysts on the midguts of infected mosquitoes which did not contain salivary gland sporozoites. From a series of 379 Anopheles that were cut at the thorax, ELISA tests on "head" and "body" portions showed that 29.5% of 95 positive mosquitoes contained circumsporozoite antigen in the body portion in the absence of salivary gland infections. This field evaluation demonstrates that the ELISA can most accurately be used to estimate sporozoite rates by cutting mosquitoes at the thorax and testing anterior portions.     

Short report: failure to select for chloroquine- or mefloquine-resistant Plasmodium berghei through drug pressure in Anopheles stephensi mosquitoes

We investigated whether chloroquine- or mefloquine-resistant Plasmodium berghei could be selected through drug pressure applied during continuous cyclical transmission in Anopheles stephensi mosquitoes. Mosquitoes were infected by feeding them on mice previously inoculated with a drug-sensitive clone of P. berghei ANKA. Mosquitoes ingested mefloquine or chloroquine with the infectious blood-meal, or by feeding on a drug-treated (uninfected) mouse 4 or 10 days after the infectious blood-meal. Twenty-two days after being infected, mosquitoes transmitted sporozoites to uninfected mice. Blood from these animals was used to infect naive mice that were then used to reinitiate the mouse/mosquito/mouse cycle. A total of 20 passages through mosquitoes were completed while under drug pressure. Drug-resistance levels were assessed in the initial clone and after 20 passages through mosquitoes. None of 18 "sub-clones" of parasites showed significant increases in chloroquine or mefloquine resistance, suggesting that exposure of sporogonic stage Plasmodium to chloroquine or mefloquine will not result in the development of drug resistance.     

Transmission blocking antibody of the Plasmodium falciparum zygote/ookinete surface protein Pfs25 also influences sporozoite development

The Plasmodium falciparum zygote/ookinete surface protein, Pfs25, persists in the oocyst wall throughout its development. Anti-25 kD transmission blocking antibody, given to infected Anopheles stephensi or A. gambiae mosquitoes in an additional bloodmeal, 3-6 days after being fed gametocyte infected blood, penetrated the oocyst and reacted with the 25 kD protein within it. This reaction caused a significant reduction in the number of developing sporozoites. Mouse serum containing antibodies raised by immunization with a recombinant 25 kD yeast product showed a similar effect.     

A scanning electron microscopic study of the sporogonic development of Plasmodium falciparum in Anopheles stephensi.

The full development of Plasmodium falciparum in Anopheles stephensi mosquitoes was studied by scanning electron microscopy. Ookinetic development was described from in vitro cultures. Growing oocysts beneath the basal lamina of the midgut wall mechanically stretch this lamina until it is torn and displaced by day 7. In young oocysts the wall appears smooth. In older oocysts wrinkles in the wall are visible after routine fixation. Osmium tetroxide postfixation greatly reduced the occurrence of these wrinkles. Intracapsular development of sporozoites was visualized after mechanical manipulation of the oocysts during sample preparation. In contrast to P. berghei, no ectopic development was seen in P. falciparum in the mosquito midgut. The mechanism of sporozoite escape from the oocyst appears to be similar to that described for rodent malaria. Fracturing of salivary glands provided the first view by scanning electron microscopy of sporozoites located in proximal and distal gland cells and in the draining duct.     

First field trial of an immunoradiometric assay for the detection of malaria sporozoites in mosquitoes

An immunoradiometric assay (IRMA) using a monoclonal antibody to the major surface protein of Plasmodium falciparum sporozoites was used to assess the P. falciparum sporozoite rate in a West African population of Anopheles gambiae (s.1.). Unlike current dissection techniques, the IRMA could detect sporozoite antigen in dried as well as fresh mosquitoes. In a controlled comparison, the sensitivity of the IRMA was comparable that of the dissection technique. Additionally, the IRMA was species specific and quantitative. Sensitivity of the assay was sufficient to detect sporozoite infections resulting from the development of a single oocyst.     

An immune-responsive serpin, SRPN6, mediates mosquito defense against malaria parasites

We have functionally analyzed the orthologous SRPN6 genes from Anopheles stephensi and Anopheles gambiae using phylogenetic, molecular, reverse genetic, and cell biological tools. The results strongly implicate SRPN6 in the innate immune response against Plasmodium. This gene belongs to a mosquito-specific gene cluster including three additional Anopheles serpins. SRPN6 expression is induced by Escherichia coli and both rodent and human malaria parasites. The gene is specifically expressed in midgut cells invaded by Plasmodium ookinetes and in circulating and attached hemocytes. Knockdown of SRPN6 expression by RNA interference in susceptible An. stephensi leads to substantially increased parasite numbers, whereas depletion in susceptible An. gambiae delays progression of parasite lysis without affecting the number of developing parasites. However, the An. gambiae SRPN6 knockdown increases the number of melanized parasites in the L3-5 refractory strain and in susceptible G3 mosquitoes depleted of CTL4. These results indicate that AsSRPN6 is involved in the parasite-killing process, whereas AgSRPN6 acts on parasite clearance by inhibiting melanization and/or promoting parasite lysis. We propose that these observed phenotypic differences are due to changed roles of the respective target serine proteases in the two mosquito species.     

Reproductive success in Anopheles arabiensis and the M and S molecular forms of Anopheles gambiae: Do natural sporozoite infection and body size matter?

Malaria parasites stages prior to sporozoite formation are known to affect the fecundity of several species of mosquitoes in the laboratory, but little is known about this phenomenon in natural conditions especially with sporozoite-infected anophelines. The reproductive success of wild-caught Anopheles arabiensis and the M and S molecular forms of Anopheles gambiae was investigated by comparing females infected with Plasmodium falciparum sporozoites to females free of sporozoites. Association between sporozoite-infected females' body size and their egg batch size was also measured. There was no significant reduction in egg production due to sporozoite infection among wild females An. arabiensis and the M and S form of An. gambiae. The infected groups and the controls laid similar numbers of eggs. A positive association was found between body size of females infected with P. falciparum and mean egg production. Infected females of the molecular forms of An. gambiae and their sibling species An. arabiensis invest similarly in egg batch size regardless of their body size although the expected egg batch size may differ among them because of differences in their mean body size. A reduction of egg production related to infection status was not observed among females harboring sporozoites. Therefore for the gonotrophic cycles that occur once sporozoites are present, natural infection of all three vectors we studied has no or minimal effect on their densities or their reproductive outputs.     

斯氏按蚊转录因子Relish基因克隆、定位及不同食源条件下表达

目的 对斯氏按蚊转录因子Relish基因进行克隆、定位和差异分析，初步探讨转录因子Relish在前酚氧化酶（PPO）级联与疟原虫卵囊黑化中的信号调控作用。 方法 设计简并引物，对全蚊cDNA模板进行RT-PCR扩增，产物纯化、克隆、测序和鉴定，利用特异引物分别从血细胞或中肠扩增目的基因，于不同食源条件下进行半定量分析和原位核酸分子杂交定位。 结果 获得了1种斯氏按蚊Relish cDNA部分序列，与冈比亚按蚊Relish基因很相似，并存在于中肠和血细胞，半定量分析显示，感染约氏疟原虫6、12、24和48 h或吸硝喹糖水12和24 h，于诱导约氏疟原虫卵囊黑化之前表达明显增强，原位核酸分子杂交证实血细胞和中肠有表达。 结论 斯氏按蚊转录因子Relish可能在疟原虫感染或/和约氏疟原虫卵囊黑化调控中发挥重要作用。     

Detection and anatomical localization of Plasmodium falciparum circumsporozoite protein and sporozoites in the afrotropical malaria vector Anopheles gambiae s.l

Salivary glands from Anopheles gambiae s.l. collected in Burkina Faso, West Africa, were analyzed by both microscopic examination and immunoradiometric assay to determine the Plasmodium falciparum sporozoite rates. Using the same mosquito samples, the immunoassay revealed positive salivary glands with low sporozoite loads, which were frequently missed by microscopy. A closer agreement between both techniques was found using salivary glands with high sporozoite loads. We also found a number of mosquitoes with uninfected salivary glands which harbored the circumsporozoite antigen in their thoraces. In a particular village these mosquitoes represented 43.5% of all sporozoite antigen carrying specimens.     

伯氏疟原虫红外期体外培养的研究

本文报告在国内首次建成了胚肺细胞-伯氏疟原虫红外期体外培养系统.用胰酶消化法自人工流产胎儿分离出胚肺细胞后,建立Elu 8801细胞株。斯氏按蚊叮咬伯氏疟原虫ANKA株感染的昆明小鼠18～21d后,在无菌条件下解剖出唾腺,制备子孢子悬液,接种于单层培养的胚肺细胞。培养48h后,可见红外期裂殖体。72h后,部分裂殖体内已形成成熟的裂殖子,将培养上清经腹腔接种健康小鼠,可使之感染疟疾并可经血传感染其它小鼠。用第2代感染小鼠血饲喂按蚊,蚊体内可产生子孢子。表明本室建立的人胚肺细胞林对P.b.ANKA株易感染,并能支持其红外期发育成熟,产生有感染力的红外期裂殖子。