The order Piroplasmida contains a diverse group of intracellular parasites, many of which can cause significant disease in humans, domestic animals, and wildlife. Two piroplasm species have been reported from raccoons ( Procyon lotor ), Babesia lotori ( Babesia sensu stricto clade) and a species related to Babesia microti (called B. microti -like sp.). The goal of this study was to investigate prevalence, distribution, and diversity of Babesia in raccoons. We tested raccoons from selected regions in the United States and Canada for the presence of Babesia sensu stricto and Babesia microti -like sp. piroplasms. Infections of Babesia microti -like sp. were found in nearly all locations sampled, often with high prevalence, while Babesia sensu stricto infections had higher prevalence in the Southeastern United States (20–45% prevalence). Co-infections with both Babesia sp. were common. Sequencing of the partial 18S rRNA and cytochrome oxidase subunit 1 ( cox1 ) genes led to the discovery of two new Babesia species, both found in several locations in the eastern and western United States. One novel Babesia sensu stricto sp. was most similar to Babesia gibsoni while the other Babesia species was present in the ‘western piroplasm’ group and was related to Babesia conradae . Phylogenetic analysis of the cox1 sequences indicated possible eastern and western genetic variants for the three Babesia sensu stricto species. Additional analyses are needed to characterize these novel species; however, this study indicates there are now at least four species of piroplasms infecting raccoons in the United States and Canada ( Babesia microti -like sp., Babesia lotori, a novel Babesia sensu stricto sp., a novel western Babesia sp.) and a possible fifth species ( Babesia sensu stricto) in raccoons in Japan.

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Trypanosoma cruzi, the aetiological agent of Chagas disease, exists as an anthropozoonosis in Louisiana. Raccoons are an important reservoir, as they demonstrate high prevalence and maintain high parasitaemia longer than other mammals. Given the complex nature of parasite transmission networks and importance of raccoons as reservoirs that move between sylvatic and domestic environments, detailing the genetic diversity of T. cruzi in raccoons is crucial to assess risk to human health. Using a next-generation sequencing approach targeting the mini-exon, parasite diversity was assessed in 2 metropolitan areas of Louisiana. Sequences were analysed along with those previously identified in other mammals and vectors to determine if any association exists between ecoregion and parasite diversity. Parasites were identified from discrete typing units (DTUs) TcI, TcII, TcIV, TcV and TcVI. DTUs TcII, TcV and TcVI are previously unreported in raccoons in the United States (US). TcI was the most abundant DTU, comprising nearly 80% of all sequences. All but 1 raccoon harboured multiple haplotypes, some demonstrating mixed infections of different DTUs. Furthermore, there is significant association between DTU distribution and level III ecoregion in Louisiana. Finally, while certain sequences were distributed across multiple tissues, others appeared to have tissue-specific tropism. Taken together, these findings indicate that ongoing surveillance of T. cruzi in the US should be undertaken across ecoregions to fully assess risk to human health. Given potential connections between parasite diversity and clinical outcomes, deep sequencing technologies are crucial and interventions targeting raccoons may prove useful in mitigating human health risk.

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