Evaluation of a restriction fragment length enzyme assay for differentiation of Haemoproteus and Plasmodium across a standard region of the mitochondrial genome.

Avian hemosporidian parasites are a genetically diverse group of parasites with a near cosmopolitan distribution. Over the past 2 decades, several PCR protocols have been designed to detect these parasites. The majority of these protocols amplify part of or the entire mitochondrial cytochrome b gene. However, many of these protocols co-amplify 2 genera (Haemoproteus and Plasmodium), making it impossible to determine which genus is amplified without post-PCR analysis. A uniform database (MalAvi), containing sequences amplified with the primers HAEMF and HAEMR2, has been developed to increase comparability across studies. We analyzed sequences from the MalAvi database and new sequences and found that digestion with EcoRV could be used to distinguish Haemoproteus from the majority of Plasmodium sequences. In addition, we tested 220 wild birds from Costa Rica and the United States for avian hemosporidians and assessed the ability of EcoRV to distinguish these 2 genera. Thirty-six positive samples were sequenced to confirm the restriction profiles, and we also analyzed 63 new hemosporidian sequences from ongoing studies in the United States for the restriction site. Among these new samples, all of the 85 Haemoproteus (subgenus Parahaemoproteus) and 14 Plasmodium were distinguishable. Overall, 887 of 898 (98.8%) sequences from our studies and the MalAvi database were assigned to the correct genus. Of these samples, all Haemoproteus samples were correctly identified and all but 11 Plasmodium samples were correctly identified by the EcoRV assay. Overall, this restriction enzyme protocol is able to quickly and efficiently classify these 2 genera of avian malarial parasites and would be useful for researchers interested in identifying parasites to genus-level, studies focused on sequence analysis of only a single genus, or for detecting co-infections that would need cloning prior to sequence analysis.

Searching Before It Is Too Late: A Survey of Blood Parasites in Ctenosaura melanosterna, a Critically Endangered Reptile of Honduras.

For species at risk of extinction, any parasites they have would be expected to face a similar fate. In such cases, time is running out for efforts to identify and study their parasitic fauna before they are gone. We surveyed the hemoparasite fauna of 50 black-chested, spiny-tailed iguanas (Ctenosaura melanosterna), a critically-endangered species, on an island off the coast of Honduras. Blood samples from captured animals were tested for hemoparasites by thin blood smear and molecular analyses. Based on microscopy, two parasites were identified, a Plasmodium sp. in 14% of iguanas and a Hepatozoon sp. in 32%. For both parasites, parasitemia levels were <0.1%. Prevalence and parasitemias of Hepatozoon declined with increasing host size, a pattern differing from most prior studies of saurian reptiles. From a subset of iguanas with microscopy-confirmed Plasmodium infections, sequence analysis of 454 bp of the cytochrome b gene indicated that the Plasmodium species was distinct from known Plasmodium and was most closely related to P. chiricahuae (96.5% similarity) followed by P. mexicanum (95.8% similarity). Efforts to amplify the Hepatozoon parasite using PCR were not successful. Additional surveys and studies of this host-parasite system would be valuable, both to science and to the management of this endangered animal.