The infection dynamics, co-infection, multi-gene phylogeny, and molecular screening of plasmodium caprae infection in goats and mosquito species

Abstract

Ungulate malaria parasites, including Plasmodium caprae that infects the domestic goats, have received little attention. There are only few studies about the prevalence and mitochondrial genome analyses. This study, therefore, aimed to investigate the natural infection dynamics of P. caprae and its mosquito vectors. Besides, genome analyses based on nuclear genes of P. caprae and other related haemosporidian parasites were also carried out to obtain an in depth understanding about their evolutionary history and origin. A total of 423 goat blood samples were collected during rainy seasons from 2018 to 2021 to assess the malaria infection status. In addition, 1,019 anopheline and 133 non-anopheline mosquitoes were also captured between 2020 and 2021 for species identification, malaria-carrying status and genome analyses. The parasite P. caprae was screened using nested PCR targeting cytochrome b (cytb) gene, whereas four nuclear genes were amplified using touch down nested PCR. The mosquitoes were morphologically identified under the stereomicroscope, followed by PCR and sequencing of cytochrome c oxidase subunit 1 (cox1), cytochrome c oxidase subunit 2 (cox2) and internal transcribed spacer 2 (ITS2). The parasite load was as low as 4,510 DNA copies per microliter of blood and maintained until day 15 of observation. The phylogeny based on multiple nuclear genes revealed that P. caprae was placed in the ungulate malaria parasite clade and formed a sister group with the bat parasite Polychromophilus. Regarding the mosquitoes, nine distinct species were recorded in the goat farm, belonging to Hyrcanus, Barbirostris, Subpictus, Funestus, Tessellatus, and Annularis groups. The Subpictus (An. subpictus) and Funestus (An. aconitus) groups were found to carry the P. caprae’s DNA by molecular method. The analysis of blood meal sources in the engorged mosquitoes showed that the blood sources came from either cattle, human, dog or goat. Two mosquito species of An. subpictus and An. peditaeniatus were found to carry the goat blood. The population genetic structure of the mosquitoes suggested that genetic variations in four dominant species have mostly existed within populations rather than among populations. Low to moderate FST indicated that gene flow has occurred among An. peditaeniatus, An. subpictus, An. vagus, and An. aconitus mosquito populations without geographic distance between northern and western provinces. This study is the first report about the multiple nuclear genes analyses and possible mosquito vectors of P. caprae in Thailand.