First molecular detection of Plasmodium relictum in Anopheles sinensis and Armigeres subalbatus.

Background: Plasmodium relictum is one of the most important avian malaria species, which is mainly seen in wild birds, with infections reported in more than 70 different species and at high prevalence. Aim: The aim of this study was to determine the molecular prevalence of Plasmodium spp. in mosquitoes collected in China. Method: A Plasmodium -specific fluorescence resonance energy transfer (FRET) polymerase chain reaction (PCR) was established in this study to analyze five species of mosquitoes (1,620 Culex pipiens pallens, 806 Aedes albopictus, 377 Armigeres subalbatus, 168 Anopheles sinensis, and 80 Culex tritaeniorhynchus) collected in hand nets from homes in 25 provinces of China.
Results: Only females originated from six provinces were determined to be positive (0.6%, 10/1,809). Plasmodium species were detected in three mosquito species, such as C. pipiens pallens (0.5%, 8/1,620), A. sinensis (0.6%, 1/168), and A. subalbatus (0.3%, 1/377). Of the three mosquito species positive for P. relictum, only C. pipiens pallens is known to feed on birds and is recognized as the natural vector of P. relictum.
Conclusion: This is the first time that P. relictum has been detected in A. sinensis and A. subalbatus. P. relictum, the agent of avian malaria, was present in mosquitoes in China, including mosquito species not previously thought to be the vectors.

Introduction

Plasmodium, the mosquito-borne agent of malaria, belongs to the phylum Apicomplexa which is a taxonomic group of single-celled parasites with characteristic secretory organelles (de Koning-Ward ). The genus Plasmodium contains over 200 species which can be divided into 14 subgenera based on morphology and host range (Martinsen and Perkins, 2013; Perkins, 2014). Plasmodium parasites are the most common in tropical areas, such as India, Australia, and Southeast Asia (Schoener ), and have been described in a broad array of vertebrate hosts. In particular, over 150 Plasmodium species can be found infecting a variety of birds (Sylvie ) with Plasmodium relictum, Plasmodium elongatum, Plasmodium vaughani, and Plasmodium sp. lineage LINN1 being the most common in birds and mosquitoes in Europe (Schoener ). With its size and multiple geographies, China has a wide diversity of mosquito species (Guo ), many of which have been reported to transmit Plasmodium species elsewhere in the world. Four human malarial species have been reported in China, such as Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, and Plasmodium ovale (Li ), but the cases of human malaria have decreased dramatically recently, from the more than 30 million cases a year reported in the 1940s to 1,380 cases in 2018 (Zhang ; Zhou ). Mosquito control programs have resulted in near elimination of the historically most important mosquito vectors of malaria in people in China, Anopheles lesteri (synonym: An. anthropophagus) and Anopheles dirus s.l. This, however, has led to other species such as Anopheles sinensis now becoming important vectors (Zhang ). There are only limited data on bird malaria in China with infection rates of up to 8% in wild species (Zhang ), and a variety of species, such as P. relictum and Plasmodium homonucleophilum, and their lineages were described in wild and captive birds (Huang ; Jia ). To the best of our knowledge, there are no data on the mosquitoes carrying Plasmodium species infecting birds in China. In this report, we describe the development of a Plasmodium-specific FRET-qPCR and its use in detecting Plasmodium species in mosquitoes collected from across China.

Materials and Methods

To establish the Plasmodium-specific FRET-qPCR, we obtained 18S rRNA sequences for representative Plasmodium species from GenBank: P. falciparum (M19172, CP016997, JQ627152, U07367), P. malariae (AF487999, AF488000, M54897), Plasmodium inui (FN256230, FN430725, XR_606809), Plasmodium cynomolgi (L08241, AB287289), P. ovale (KF018655, L48987), P. vivax (X13926, JQ627154), Plasmodium knowlesi (U83876, DQ350263), Plasmodium berghei (AJ243513), Plasmodium vinckei (XR_552296), Plasmodium cathemerium (AY625607), Plasmodium gallinaceum (M61723), Plasmodium lophurae (X13706), Plasmodium juxtanucleare (AF460507), Plasmodium reichenowi (Z25819), Plasmodium gaboni (LVLB01000008), Plasmodium brasilianum (AF130735), Plasmodium gonderi (AB287270), Plasmodium fieldi (AB287284), and Plasmodium fragile (M61722). The Clustal multiple alignment program was used to identify a conserved region of the 18S rRNA common to all the species. Primers and probes were selected to amplify a 234-242 bp target (forward: 5′-TAAGGATAACTACGGAAAAGCTGTA-3′; reverse: 5′-CGTTACCCGTCATAGCCATGT-3′; FAM-probe: 5′-TAGGCCAATACCCTAACATCAAAAG-6-FAM-3′; and LCRed640-probe: 5′-LCred640-TGATAGGTCAGAAACTCGATTGATACAC-phos-3′) and synthesized by Integrated DNA Technologies (Coralville, IA).

The Plasmodium-specific FRET-qPCR reaction and high-resolution melting curve analysis were performed on the LightCycler 480II Real-time PCR platform (Roche, Basal, Switzerland) under previously described conditions (Zhang ), except that the hybridization temperature was 53°C. The specificity and sensitivity of the FRET-qPCR were determined using DNA of four plasmids manufactured with the pUC57 cloning vector (GenScript, Nanjing, Jiangsu, China) containing an appropriate portion of the 18S rRNA gene of P. falciparum, P. malariae, P. ovale, and P. vivax. Specificity was also tested, with DNA of Babesia canis, Hepatozoon americanum, Theileria equi, Hepatocystis kochi, and Dirofilaria immitis obtained as described before (Zhang , 2015). The Plasmodium-specific FRET-qPCR proved to be highly sensitive detecting two copies of the Plasmodium 18S rRNA per 20 µl reaction system. Further, it was highly specific, not detecting the closely related organisms which were included in the study.

The validated Plasmodium-specific FRET-qPCR was used to analyze five species of mosquitoes [Culex pipiens pallens (n = 1,620), Aedes albopictus (806), Armigeres subalbatus (377), A. sinensis (168), and Culex tritaeniorhynchus (80)] which were collected in hand nets from homes in 25 provinces and identified as described previously (Zhang ). Briefly, between July and September 2014, student volunteers from Yangzhou University used hand nets to collect convenience samples of mosquitoes in their primary homes located in 26 cities in 25 provinces or municipalities in China. Mosquitoes were placed individually in sterile tubes containing 400-µl DNA/RNA Stabilization Reagent (Roche Molecular Biochemicals, Indianapolis, IN, USA). Then, the samples were transported to the Yangzhou University College of Veterinary Medicine at room temperature.

Results and Discussion

Only females were positive (0.6%, 10/1,809) for Plasmodium, and these originated from six provinces, such as Zhejiang (4.2%, 1/24), Gansu (2.4%, 2/82), Shandong (5.4%, 3/56), Jilin (1.7%, 2/115), Guizhou (5.6%, 1/18), and Jiangsu (2.0%, 1/51). Plasmodium species were only detected in three mosquito species, such as C. pipiens pallens (0.5%, 8/1,620), A. sinensis (0.6%, 1/168), and A. subalbatus (0.3%, 1/377) (Table 1). The 18S rRNA sequences of all the positive samples were identical to one another (GenBank accession number: MK061746) and to that of a reference sequence of P. relictum (LN835296) from GenBank (Fig. 1). As far as we know, this is the first time that P. relictum has been detected in A. sinensis and A. subalbatus.

Table 1.

Data on mosquitoes positive for P. relictum identified with a Plasmodium-specific FRET-qPCR.

Province	City	Mosquito Species	Gender	Plasmodium Positivity
Zhejiang	Wenzhou	A. sinensis	F	1/24, 4.2%
Gansu	Jingyuan	Culex p. pallens	F	2/82, 2.4%
Shandong	Heze	Culex p. pallens	F	1/6, 16.7%
Shandong	Liaocheng	Culex p. pallens	F	2/50, 4.0%
Jilin	Changchun	Culex p. pallens	F	2/115, 1.7%
Guizhou	Liupanshui	A. subalbatus	F	1/18, 5.6%
Jiangsu	Yangzhou	Culex p. pallens	F	1/51, 2.0%
Total				10/346, 2.9%

Fig. 1.

Phylogenetic analysis of Plasmodium spp. detected in this study. Distances and groupings of Plasmodium detected from the mosquitoes (bold font) were determined by applying the neighbor-joining method to a matrix of pairwise distances estimated using the maximum composite likelihood (MCL) approach with MEGA version 6 software based on 18S rRNA gene (242 bp). Scale bar indicates a genetic distance of 0.2-nt substitution per position.

Of the three mosquito species we found positive for P. relictum, only C. pipiens pallens is known to feed on birds (Wang ) and recognized as the natural vector of P. relictum (Zele ). A. sinensis and A. subalbatus are found widely in China and other countries in Southeast Asia (Chaves ; Guo ) and feed mainly on cattle (Ramesh ; Zhang ). From the data showing very low infection rates with P. relictum, it seems that they might also infrequently feed on birds. It is of note that all three species which are positive for P. relictum feed on people raising the possibility that humans are exposed to infection. Although it has been reported that host shifts appear to have a common occurrence in the evolution of the genus Plasmodium among avian and reptilian malaria parasites (Rich and Ayala, 2003), what would now be impermissible and unethical experiments have shown potential infections are unlikely in human being (McLendon, 1943).

P. relictum is one of the most important avian malaria species, which is mainly seen in wild birds, with infections reported in more than 70 different species (Garcia-Longoria ) and at high prevalence. The organism has been reported in birds in China previously, such as Beijing (Jia ) and Gansu Province (Jia ; Zehtindjiev ), and we now report its presence in mosquitoes in five further provinces, indicating that the organism is present in China, consistent with the findings in Europe (Schoener ). To date, avian Plasmodium parasites have been found in Aedes vexans, C. pipiens complex, Culex modestus, Culex hortensis, Culiseta annulata, Ochlerotatus caspius, and A. albopictus in Central Europe and Culex torrentium in Australia (Schoener ). The findings of P. relictum in A. sinensis and A. subalbatus (Table 1) expand the possible vector range for the parasite.

Conclusion

In conclusion, this study describes the establishment of a sensitive and specific Plasmodium-specific FRET-qPCR. With this validated PCR, we found that P. relictum, the agent of avian malaria, was present in mosquitoes in China, including species not previously thought to be the vectors.