In vitro cultivation of Babesia duncani (Apicomplexa: Babesiidae), a zoonotic hemoprotozoan, using infected blood from Syrian hamsters (Mesocricetus auratus).

Human babesiosis, a tick-borne disease similar to malaria, is most often caused by the hemoprotozoans Babesia divergens in Europe, and Babesia microti and Babesia duncani in North America. Babesia microti is the best documented and causes more cases of human babesiosis annually than all other agents combined. Although the agents that cause human babesiosis are considered high-risk pathogens in transfusion medicine, federally licensed diagnostics are lacking for B. duncani in both the USA and Canada. Thus, there has been a need to develop and validate diagnostics specifically for this pathogen. In this study, B. duncani (WA1 isolate) was cultivated in vitro from Syrian hamster (Mesocricetus auratus) infected blood. We hypothesized HL-1 media with supplements would result in B. duncani propagating at higher levels in culture than supplemented M199 similar to the medium the parasite was originally cultivated with in 1994. We were unable to recreate Thomford's cultivation results with the M199 medium but supplemented HL-1 medium was able to successfully establish continuous culture. We further hypothesized that RBC from species other than hamsters would support B. duncani in vitro. However, rat, mouse, horse, and cow RBC did not support continuous culture of the parasite. Culture stocks of B. duncani were deposited at BEI Resources and are now commercially available to the scientific community to further research. The cultured parasite developed in this study was instrumental in the adaptation of B. duncani continuous culture to human RBC.

Genetic variability of cloned Cytauxzoon felis ribosomal RNA ITS1 and ITS2 genomic regions from domestic cats with varied clinical outcomes from five states.

Cytauxzoon felis is a tick-borne hemoparasite that causes cytauxzoonosis in domestic cats in the United States. Historically, feline cytauxzoonosis was reported to be nearly always fatal. However, increasing evidence of cats surviving acute infection and/or harboring a chronic, subclinical infection has suggested the existence of different C. felis strains that may vary in pathogenicity. In this study, the intraspecific variation of the C. felis first and second ribosomal RNA internal transcribed spacer (ITS1, ITS2) regions was assessed for any clinical outcome or geographic associations. Sequence data were obtained for 122C. felis ITS1 and ITS2 clones from 41 domestic cat blood samples from Arkansas, Kansas, Missouri, Oklahoma, and Texas. Seven previously reported ITS1 region sequences were found, and a previously undescribed 23-bp insert was detected in cloned ITS1 sequences from a domestic cat in Missouri and two cats in Oklahoma. Four previously reported ITS2 region sequences were identified, and a 40-bp insert similar to that previously reported in C. felis of a domestic cat from Arkansas and pumas was detected in 18 cloned C. felis sequences from 12 domestic cats. One clone contained both the 23-bp insert and 40-bp insert within the ITS1 and ITS2 regions, respectively. Combined ITS1 and ITS2 sequence genotypes revealed that C. felis sequences from 27 cats (72/122 clones) corresponded to four previously described genotypes, ITSa, ITSc, ITSd, and ITSn. Five clones with the novel 23-bp insert from three cat isolates represented two new genotypes, ITSaa and ITSbb. Genotypes ITScc, ITSdd, ITSee, ITSff, ITSgg, and ITShh denoted 13 clones that matched prior sequences but had no previously assigned genotype. Genotypes ITSii through ITStt comprised 32 clones that were similar to, but did not exactly match, previously described genotypes. Twenty-five cats had C. felis infections with multiple ITS genotypes. Considerable C. felis genetic diversity was revealed with no significant geographic or clinical outcome associations.

Molecular and morphological characterization of a haemogregarine in the alligator snapping turtle, Macrochelys temminckii (Testudines: Chelydridae).

A severely underweight alligator snapping turtle Macrochelys temminckii Troost in Harlan, 1835, was found near Tyler, Texas, and taken to the Caldwell Zoo. Blood films were submitted to Texas A&M University, College Station, Texas, for morphological and molecular identification of haemogregarine-like inclusions in the red blood cells. Intraerythrocytic Haemogregarina sp. forms were found on microscopic examination at a parasitemia of <1 %. The morphology and morphometric data for the forms indicate similarity to Haemogregarina macrochelysi n. sp. Telford et al., 2009, previously reported in alligator snapping turtles in Florida and Georgia, but two characteristic stage forms were not shared between H. macrochelysi n. sp. and the parasite found in this report. The haemogregarine 18S ribosomal RNA gene (1555-bp fragment) was amplified and cloned, and five clones sequenced. The sequences were deposited in the NCBI GenBank database. All five showed ∼96 % identity to Haemogregarina balli Paterson and Desser, 1976, Hepatozoon sp., and Hemolivia stellata Petit et al., 1990. A 774-bp segment shared 98-99 % identity with the corresponding Haemogregarina sp. rDNA sequence (KR006985) from Caspian turtles (Mauremys caspica McDowell, 1964) in Iran. A neighbor-joining phylogenetic tree generated from aligned sequences from the clones, 26 hematozoa, Adelina dimidiata Schneider, 1875, and Cryptosporidium serpentis Levine, 1980, revealed the cloned sequences clustered on their own branch within the Haemogregarina spp. clade. No genetic data are available for H. macrochelysi n. sp. at this time, so it remains unclear if this parasite in a Texas alligator snapping turtle is conspecific with H. macrochelysi n. sp.