Babesia coryicola Shock, Jones, Garrett, Hernandez, Burchfield, Haman, Schwantje, Telford, Cunningham & Yabsley, 2024, sp. nov.

3.1. Description of Babesia coryicola sp. nov. Shock, cunningham, and yabsley Type host: Female Florida puma (Puma concolor coryi) FP222 (Mammalia: Felidae) Type locality: Collier County, Florida. Type material: Hapantotype. A thin-stained blood smear from Florida puma FP222 from Collier County, Florida was deposited in the U.S. National Parasite Collection (USNPC accession number). Parahapantotype. A thin-stained blood smear from Florida puma FP93 was deposited in the U.S. National Parasite Collection (USNPC accession number xxxxx). Vector: Currently unknown. Representative sequences: Genbank DQ329139 and PP852299 (18S rRNA gene); KJ592628 (ITS1 region); KJ592629 (ITS2 region); DQ329139 (β- tubulin gene); PP855655 (COI gene); PP855654 (cytb gene); PP855655 (COX3). Etymology: Babesia coryicola sp. nov. is named after the subspecies name of the only known host, the endangered Florida puma concolor coryi. When the Florida puma was initially described in 1896 by Charles B. Cory it was named Felis floridana and was later renamed Felis coryi because F. floridana was previously used for the bobcat (Felis rufus). Following the taxonomic revision of pumas by Nelson and Goldman (1929), the Florida puma was reclassified as a subspecies. Name: coryi-refers to the subspecies of the Florida puma and -cola is inhabitant of. ZooBank reference: urn:lsid:zoobank.org:pub: F7CD2D01-3B51- 4AAC-A30B-6D2240A07C17. Description: All parasites observed (n = 18) were ring stage trophozoites which were morphologically similar to small Babesia spp., including felid-associated species such as B. leo and B. felis (Fig. 1). Most trophozoites were compact ring forms with small amounts of cytoplasm and a single nucleus, but rare trophozoites with 2–4 nuclei were observed. The overall shape of most trophozoites were round, but rare oval or slightly amoeboid forms were observed. Trophozoites measured 0.85–1.3 μm (0.991 μm average) in length and 0.75–1.2 μm (0.941 μm average) in width with no tetrad (Maltese cross) shapes observed (Fig. 1). 3.2. Natural history studies on host(s) and locations The parasite has been detected in blood samples collected from Florida pumas sampled from 1989 to 2013. A previous survey of Florida pumas reported a prevalence of 95% (37 of 39 pumas) (Yabsley et al., 2006). In the current study, 72% of 100 Florida pumas were PCR positive for B. coryicola sp. nov. To date, only Florida pumas in their southern Florida (Collier, Hendry, Broward, Monroe, and Dale Counties) range have been infected. Five cougars from Texas that were translocated to Florida were negative for B. coryicola sp. nov. before their release and two of these animals resampled after release remained negative (Rotstein et al., 1999; Yabsley et al., 2006). Additionally, 19 pumas from Texas, introduced into northern Florida in 1993 and removed in 1995 for a reintroduction feasibility study, were also blood-smear negative for piroplasms prior to their introduction to Florida (Belden and McCown, 1996; Rotstein et al., 1999). Our results confirm that these pumas were negative for piroplasms; by PCR however, some of the pumas did acquire C. felis infections while in Florida (Rotstein et al., 1999). All pumas tested from Georgia, Louisiana, North Dakota, Canada, and Costa Rica were negative for piroplasms. 3.3. Serologic characteristics Samples from two B. coryicola sp. nov. - positive Florida puma samples displayed fluorescence at a 1:128 dilution for B. bovis and B. canis antigens. Samples from four B. coryicola sp. nov. positive Florida pumas displayed fluorescence at 1:256 dilution for B. odocoilei antigens. No sample displayed cross-reaction for T. equi. 3.4. Molecular analysis 3.4.1. Near full-length 18S rRNA gene The near full-length 18S rRNA gene sequences of B. coryicola sp. nov. were obtained from three Florida pumas (FP40, FP44, FP222). Sequences from FP40 and FP44 were identical and were 99.9% similar to FL222 (1608/1,610bp). These sequences were most similar to a Babesia sp. reported from a tick removed from a dog in Japan (Akita 610) (98.3% (1651/1679), AY191024) and a Babesia sp. from a red panda from China (98.1% (1691/1724), OK524313) (Supplementary File 1 shows distance matrix with other Babesia spp.). Phylogenetically, B. coryicola sp. nov. grouped with other Babesia sensu stricto species as an ancestral group within a large group of Babesia spp. of ungulates (Fig. 2). This large group was a sister group to a clade of carnivore-infecting Babesia spp. 3.4.2. Partial β- tubulin gene Partial β- tubulin gene sequences (1,101bp) of B. coryicola sp. nov. were obtained from three Florida pumas (FP44, FP93, and FP222). The β- tubulin gene sequences from B. coryicola sp. nov. from the Florida pumas were 99.5–99.7% similar to each other. These sequences were 87.3% similar to B. odocoilei (of 938-954bp overlapping with AY144706) and 87.2% similar to a Babesia sp. from a Sika deer (Cervus nippon) (of 940-956bp overlapping with KC465971) (Supplementary File 2 shows distance matrix with other Babesia spp.). Phylogenetically, the three B. coryicola sequences grouped together in a sister clade to Babesia spp. that primarily infect ungulates (Fig. 3). 3.4.3. Partial cytb gene Partial cytb gene sequences (1,213bp) of B. coryicola sp. nov. were obtained from two Florida pumas (FP93 and FP222) and they were 99.8% similar to each other. The cytb gene sequences from B. coryicola sp. nov. from the Florida pumas were 84.6–84.8% similar to B. canis (KC207822) and 84.4% similar to a B. vogeli (KC207825) (Supplementary File 3 shows distance matrix with other Babesia spp.). Phylogenetically, the two B. coryicola sequences grouped as a sister group to a group of Babesia spp. of ungulates and carnivores (Fig. 4). 3.4.4. Partial COI gene Partial COI gene sequences (1,035bp) of B. coryicola sp. nov. were obtained from two Florida pumas (FP93 and FP222) and they were 99.7% similar to each other. The COI gene sequences from B. coryicola sp. nov. from the Florida pumas were 89.2% similar to a B. sp. from a raccoon (Procyon lotor) (KR017882), 88.8% similar to a B. sp. from a captive maned wolf (Chrysocyon brachyurus) (KR017881), and 86.2% similar to B. gibsoni from a domestic dog in Japan (AB685183) (Supplementary File 4 shows distance matrix with other Babesia spp.). Phylogenetically, the two B. coryicola sequences grouped with a clade of Babesia sp. from raccoons (Fig. 5). 3.4.5. Partial COX3 gene Partial COX3 gene sequences (717bp) of B. coryicola sp. nov. were obtained from four Florida pumas (FP93, FP222, FP233, and FP235). Each sequence was unique and were 99.4–99.9% similar to each other. The COX3 gene sequences from B. coryicola sp. nov. from the Florida pumas were most similar to B. canis (83.9–84.4% similar) followed by several strains of B. gibsoni (~81–82.8% similar) (Supplementary File 5 shows distance matrix with other Babesia spp.). Phylogenetically, the two B. coryicola sequences grouped with a clade of B. gibsoni sequences and this group was a sister clade to B. canis and B. vogeli (Fig. 6). 3.4.6. Partial 28S rRNA gene A short region of the 28S rRNA gene (245bp) was obtained for B. coryicola sp. nov. from one Florida pumas (FP93). The sequence was most similar (95.5%) to a Babesia sp. from a red panda (OK524314) followed by B. bigemina (90.3%, LK391709) and B. gibsoni (CP141527). 3.4.7. ITS-1 and ITS-2 regions The ITS-1 rRNA sequences of B. coryicola sp. nov. from 23 Florida pumas were all unique and percent identity ranged from 85.7 to 99.5%. The sequences varied in size (641-657bp) due to numerous 1-15bp insertions/deletions. No apparent association was noted between ITS-1 sequences and county of origin or year of sampling (range from 1992 to 2005). The B. coryicola sp. nov. ITS-1 sequences were 87.2–89.4% and 86.8–87.9% identical to B. odocoilei (AY339751) and B. divergens (EF458168), respectively. A representative sequence was submitted to GenBank (KJ592628, FP40). The ITS-2 rRNA sequence of B. coryicola sp. nov. was obtained from one Florida puma (KJ592629; FP40). It was 244 bp long and contained a single polymorphic base. The ITS-2 sequence was 96% similar to B. bigemina (e.g., MH050926), 92% similar to a B. sp. from a red panda (OK524314) and Babesia sp. RD61 from a captive reindeer (Rangifer tarandus) from California (AY339744), and 91% similar to B. odocoilei from a Minnesota caribou (Rangifer tarandus) (AY339758). 3.5. Assessment of vertical transmission All 24 kittens were PCR negative for piroplasms. During the year of sampling, all mothers were confirmed positive for B. coryicola sp. nov. by PCR. 3.6. Hematological evaluation No hematological differences were detected between Babesia -infected Florida pumas included in this study and the seven groups of pumas from the five other studies (Table 2).

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