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Background The malaria mosquito Anopheles punctipennis, a widely distributed species in North America, is capable of transmitting human malaria and is actively involved in the transmission of the ungulate malaria parasite Plasmodium odocoilei. However, molecular diagnostic tools based on Internal Transcribed Spacer 2 (ITS2) of ribosomal DNA are lacking for this species. Anopheles punctipennis is a former member of the Anopheles maculipennis complex but its systematic position remains unclear. Methods In this study, ITS2 sequences were obtained from 276 An. punctipennis specimens collected in the eastern and midwestern United States and a simple and robust Restriction Fragment Length Polymorphism approach for species identification was developed. The maximum-likelihood phylogenetic tree was constructed based on ITS2 sequences available through this study and from GenBank for 20 species of Anopheles. Results The analysis demonstrated a consistent ITS2 sequence length and showed no indications of intragenomic variation among the samples based on ITS2, suggesting that An. punctipennis represents a single species in the studied geographic locations. In this study, An. punctipennis was found in urban, rural, and forest settings, suggesting its potential broad role in pathogen transmission. Phylogeny based on ITS2 sequence comparison demonstrated the close relationship of this species with other members of the Maculipennis group. Conclusions This study developed molecular tools based on ITS2 sequences for the malaria vector An. punctipennis and clarified the phylogenetic position of the species within the Maculipennis group.

We report, for the first time, the presence of ungulate malaria parasites in South America. We conducted PCR-based surveys of blood samples of multiple deer species and water buffalo from Brazil and detected Plasmodium sequences from pampas deer (Ozotoceros bezoarticus) samples. Phylogenic analysis revealed that the obtained sequences are closely related to the Plasmodium odocoilei clade 2 sequence from North American white-tailed deer (Odocoileus virginianus). Nucleotide differences suggest that malaria parasites in South American pampas deer and North American P. odocoilei clade 2 branched more recently than the Great American Interchange., Graphical abstract Image 1, Highlights • Plasmodium sequence was detected from pampas deer in South America. • It was most similar to the North American Plasmodium odocoilei clade 2 sequence. • Estimated divergence time was much more recent than the Great American Interchange.

A wide array of vertebrates can serve as the intermediate hosts to malaria parasites (Apicomplexa: Haemosporida), such as birds, lizards, and several groups of mammals, including primates, bats, rodents, and ungulates. The latter group of hosts has not been intensively studied since early descriptions of a small set of taxa were published, but new reports of these parasites in both expected and new hosts have recently been published., A wide array of vertebrates can serve as the intermediate hosts to malaria parasites (Apicomplexa: Haemosporida), such as birds, lizards, and several groups of mammals, including primates, bats, rodents, and ungulates. The latter group of hosts has not been intensively studied since early descriptions of a small set of taxa were published, but new reports of these parasites in both expected and new hosts have recently been published. A new paper reports the presence of Plasmodium odocoilei in farmed white-tailed deer in Florida, particularly in animals less than 1 year old, and provides evidence that the parasites may contribute to mortality in fawns. That paper opens new opportunities to study the malaria parasite-mammal interface in North America.

Findings suggest that North American white-tailed deer commonly harbor cryptic infection with the only known New World mammalian Plasmodium., Malaria parasites of the genus Plasmodium are diverse in mammal hosts, infecting five mammalian orders in the Old World, but were long considered absent from the diverse deer family (Cervidae) and from New World mammals. There was a description of a Plasmodium parasite infecting a single splenectomized white-tailed deer (WTD; Odocoileus virginianus) in 1967 but none have been reported since, which has proven a challenge to our understanding of malaria parasite biogeography. Using both microscopy and polymerase chain reaction, we screened a large sample of native and captive ungulate species from across the United States for malaria parasites. We found a surprisingly high prevalence (up to 25%) and extremely low parasitemia of Plasmodium parasites in WTD throughout the eastern United States. We did not detect infections in the other ungulate species nor in western WTD. We also isolated the parasites from the mosquito Anopheles punctipennis. Morphologically, the parasites resemble the parasite described in 1967, Plasmodium odocoilei. Our analysis of the cytochrome b gene revealed two divergent Plasmodium clades in WTD representative of species that likely diverged 2.3 to 6 million years ago, concurrent with the arrival of the WTD ancestor into North America across Beringia. Multigene phylogenetic analysis placed these clades within the larger malaria parasite clade. We document Plasmodium parasites to be common in WTD, endemic to the New World, and as the only known malaria parasites from deer (Cervidae). These findings reshape our knowledge of the phylogeography of the malaria parasites and suggest that other mammal taxa may harbor infection by endemic and occult malaria parasites.

With the exception of the human malaria parasites, the mammalian species of Plasmodium have a fairly restricted geographical distribution, while the incidence of the infection in the different orders of mammals is patchy. A few species have been described in ungulates in the Old World Tropics, but Plasmodium odocoilei sp. nov. of the white-tailed deer ( Odocoileus virginianus ) of Texas is the first example to be found in the New World. It was discovered in an adult specimen that had had its spleen removed. Only the blood stages of the parasite are known, but these show various remarkable features: the presence of large vacuoles in the cytoplasm, and the enormous distortion and discoloration of the infected red blood cell. The phylogeny of the parasite is discussed in relation to that of the vertebrate host and it is suggested that Cervid stock crossed the Bering Land Bridge in the Pliocene Age, carrying a plasmodial infection that was the common source of malaria in the Old World Tragulidae and in the New World Odocoileus .