Identification of Avian Malaria Parasites Causing Disease in Ghanaian Birds

Abstract

Avian malaria is a blood parasite disease that occurs in various species of birds worldwide except in Antarctica. The avian malaria parasites of the genus Plasmodium belong to a group of apicomplexan protozoan parasites of the order Haemosporida and are closely related genetically to two other protozoan parasites of the genera Haemoproteus, and Leucocytozoon. Bloodsucking dipteran insects of the families Culicidae, Hippoboscidae, Simuliidae, and Ceratopogonidae transmit the three haemosporidian parasites. These parasites cause malaria, haemosporidiosis, and leucocytozoonosis diseases in wild and domestic birds, and cause asymptomatic to fatal cases in birds. The haemosporidian parasites have been extensively studied in many parts of the world, especially in temperate regions. However, little is known about these parasites in Ghana. This study sought to identify the species of avian malaria and related haemosporidians that prevail in Ghanaian birds and establish their genetic diversity. The study was conducted in Sunyani and Aburi in the Brong Ahafo and Eastern regions of Ghana. Birds were sampled from poultry farms and wild habitats around the poultry farms. One thousand one hundred and ten birds were collected, comprising free-range, confined/caged, and wild birds. Blood samples were collected from the brachial veins of birds by needle puncture. Thin blood smears were prepared and stained with Giemsa solution for microscopy. DNA was extracted from whole blood and molecular screening of haemosporidian parasites was done by qPCR and nested PCR protocols. Sanger sequencing was used to identify haemosporidian parasite genera. Microbial DNA enrichment and whole genome amplification were performed on DNA samples to enrich AT biased genome. Phylogenetic analysis was performed to establish the genetic diversity of the parasites. The results reported an overall haemosporidian prevalence of 21.5% for parasites belonging to all the three genera. At the level of individual genus prevalence, Plasmodium had a higher prevalence (18.9%) than Leucocytozoon (10.4%) and Haemoproteus (2.1%). Genus co-infections were also observed in most categories of birds studied. The study results found a 9.8% Plasmodium and Leucocytozoon co-infection mostly observed among domestic birds. In this study, we observed that most of the samples that tested positive were detected using molecular methods. The morphological identification of the parasites was almost not possible in most of the blood smears examined. Extensive screening of blood smears obtained from known positive samples did not reveal parasite morphology, and this provides evidence suggesting that parasitaemia was extremely low and/or the infections were mostly abortive. Histological preparations obtained from an infected turkey did not reveal parasite tropism in any of the organs studied. This could be a confirmation of abortive infections, or parasite development could be found in organs other than those examined. Our study compared the prevalence of haemosporidian parasites belonging to each of the three genera in three categories of birds: confined, free-range, and wild birds. The prevalence of Plasmodium and Leucocytozoon was higher in free-range and wild birds than confined birds. The results from the targeted Sanger sequencing revealed 24 new mitochondrial cytochrome b lineages of the haemosporidian parasites and we deposited them in the NCBI GenBank. The new lineages are composed of 6 Leucocytozoon, 13 Plasmodium, and 5 Haemoproteus and Parahaemoproteus lineages. Some of the parasite lineages found within all three genera of the parasites were host and habitat generalist or specialist, and lineage spillover was observed among the different bird categories. Phylogenetic analyses revealed close genetic relatedness between our parasite lineages and others from the GenBank. A few of our new lineages were closely related to Plasmodium globularis, Plasmodium juxtanucleare, Plasmodium bennettinia, and Leucocytozoon schoutedeni. Haemoproteus parasites morphologically identified in wild passerine birds of the family Ploceidae were similar to the Haemoproteus nucleofascialis. The most prevailing haemosporidian lineages found in our study sites were the Plasmodium lineage PGHA708, Leucocytozoon lineage LGHA146, and Haemoproteus lineage PHGHA989. For future comparisons of new parasite genomes to the few existing avian malaria genomes, we successfully enriched haemosporidian parasite DNA from naturally infected birds, and amplified the AT-biased genome for further genomic analysis. This study provided evidence that avian malaria and related haemosporidian exist in the Ghanaian poultry industry, and there is a community sharing of the parasites among wild birds, free-range birds, and confined birds. This is the first study reporting Leucocytozoon in domestic birds in Ghana and the first describing haemosporidian parasite interactions among wild birds, free range, and confined birds in Ghana.