Haemosporida is a diverse group of vector-borne parasitic protozoa, ubiquitous in terrestrial vertebrates worldwide. The renewed interest in their diversity has been driven by the extensive use of molecular methods targeting mitochondrial genes. Unfortunately, most studies target a 478 bp fragment of the cytochrome b (cytb) gene, which often cannot be used to separate lineages from different genera found in mixed infections that are common in wildlife. In this investigation, an alignment constructed with 114 mitochondrial genome sequences belonging to four genera (Leucocytozoon, Haemoproteus, Plasmodium and Hepatocystis) was used to design two different sets of primers targeting the cytb gene as well as the other two mitochondrial DNA genes: cytochrome c oxidase subunit 1 and cytochrome c oxidase subunit 3. The design of each pair of primers required consideration of different criteria, including a set for detection and another for differential amplification of DNA from parasites belonging to different avian haemosporidians. All pairs of primers were tested in three laboratories to assess their sensitivity and specificity under diverse practices and across isolates from different genera including single and natural mixed infections as well as experimental mixed infections. Overall, these primers exhibited high sensitivity regardless of the differences in laboratory practices, parasite species, and parasitemias. Furthermore, those primers designed to separate parasite genera showed high specificity, as confirmed by sequencing. In the case of cytb, a nested multiplex (single tube PCR) test was designed and successfully tested to differentially detect lineages of Plasmodium and Haemoproteus parasites by yielding amplicons with different sizes detectable in a standard agarose gel. To our knowledge, the designed assay is the first test for detection and differentiation of species belonging to these two genera in a single PCR. The experiments across laboratories provided recommendations that can be of use to those researchers seeking to standardise these or other primers to the specific needs of their field investigations.

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Avian communities from South America harbor an extraordinary diversity of Leucocytozoon species (Haemosporida, Leucocytozoidae). Here, of 890 birds sampled, 10 (1.2%) were infected with Leucocytozoon parasites. Among them, two new species were discovered and described. Leucocytozoon grallariae sp. nov. and Leucocytozoon neotropicalis sp. nov. were found in non-migratory highland passeriforms belonging to the Grallaridae and Cotingidae, respectively. They both possess gametocytes in fusiform host cells. However, due to combining microscopic examination and molecular detection, it was revealed that these parasites were present in co-infections with other Leucocytozoon species, which gametocytes develop in roundish host cells, therefore exhibiting two highly distant parasite lineages isolated from the same samples. Remarkably, the lineages obtained by cloning the mtDNA genomes were not captured by the classic nested PCR, which amplifies a short fragment of cytochrome b gene. Phylogenetic analyses revealed that the lineages obtained by the classic nested PCR clustered with parasites possessing gametocytes in roundish host cells, while the lineages obtained by the mtDNA genome PCR protocol were closely related to Leucocytozoon parasites possessing gametocytes in fusiform host cells. These findings suggest problems with the sensitivity of the molecular protocols commonly used to detect Leucocytozoon species. A detailed analysis of the primers used in the classic nested PCR revealed a match with DNA sequences from those parasites that possess gametocytes in roundish host cells (i.e., Leucocytozoon fringillinarum ), while they differ with the orthologous regions in the mtDNA genomes isolated from the samples containing the two new species. Since these are mixed infections, none of the lineages detected in this study can be assigned accurately to the new Leucocytozoon morphospecies that develops in fusiform host cells. However, phylogenetic analyses allowed us to hypothesize their most probable associations. This study highlights the need for developing detection methods to assess the diversity of Leucocytozoon parasites accurately.

We describe morphologically unique Leucocytozoon pterotenuis sp. nov. (Haemosporida, Leucocytozoidae), the first reported leucocytozoid species developing in fusiform host cell found in a Neotropical passeriform bird. The type host of this parasite is the Chestnut-crowned Antpitta (Grallaria ruficapilla, Grallariidae), an elusive native passerine bird whose natural history remains, to a large degree, unexplored. This bird was captured in Palacio forest in the damping zone of Chingaza National Natural Park, Cundinamarca, Colombia, at 2900 m above sea level (asl). Gametocytes of the new species develop both in roundish and fusiform host cells. This parasite is readily morphologically distinguishable from the described Leucocytozoon species because its host cells possess the narrow (needle-like) spindle-shaped processes, which length markedly exceeds their width. Additionally, the host cell nucleus markedly extends into the processes. Phylogenetic relationships were constructed based on a fragment of the mitochondrial cytochrome b gene and the complete mitochondrial genome. Phylogenetic analysis placed the lineage of L. pterotenuis in different positions depending on the length of the sequence analyzed that is likely due to poor sampling of Leucocytozoon species, especially from rare or non-passerine hosts, as well as a paucity of complete mitochondrial sequences of these parasites. Available data indicate that Leucocytozoon parasites are distributed mainly in mountain regions of the Neotropics where unique morphological forms have been recently discovered. To a better knowledge of the diversity of Leucocytozoon spp. and their host-vector-parasite interactions in Neotropical countries, additional deep and intensive samplings are needed, particularly in orders different to Passeriformes.