Alhaboubi, Amer Rasoolfadhl (2018-08). In Vitro Growth and Molecular Characterization of Vector-Borne Intraerythrocytic Parasites of Domestic Animals and Wildlife. Doctoral Dissertation.
Thesis
Haemogregarines are a group of blood sporozoans that parasitize reptiles, most commonly turtles, or tortoises. Haemogregarine-like inclusions in the red blood cells of a severely underweight alligator snapping turtle Macrochelys temminckii Troost in Harlan were examined in this study. The morphology and morphometric data for intraerythrocytic forms found on microscopic examination were similar to Haemogregarina macrochelysi n. sp. previously reported in the same species. The 18S ribosomal RNA (18S rRNA) gene was cloned and five sequences deposited in the NCBI GenBank(R) database. All five showed ~96 % identity to Haemogregarina balli, Hepatozoon sp., and Hemolivia stellata. A phylogenetic tree generated from the five sequences aligned with 18S rDNA sequences of other hematozoa and two outgroup species revealed the cloned sequences clustered on their own branch within the Haemogregarina spp. clade. There is no genetic data for H. macrochelysi n. sp., so it is unclear if the Texas turtle parasite is conspecific with H. macrochelysi n. sp.
Babesia spp. are intraerythrocytic protozoans that parasitize mammals. Cultured Babesia bovis and Babesia bigemina, parasites of cattle, were recovered from liquid nitrogen (LN?) storage nearly 30 years after cryopreservation. Four cattle were compared as donors of red blood cells (RBC) and serum for microaerophilous stationary phase (MASP) cultures in the recovery of B. bigemina. RBC and serum from only one donor supported the growth of B. bigemina. Two B. bigemina (frozen in 1986 and 1987) and two B. bovis (both frozen in 1986) cryostocks were resuscitated from LN? storage and all four recovered and thrived in the donor bovine RBC and serum. In the 3rd passage after recovery, B. bovis cultures were cryopreserved. Six months later they were successfully recovered from LN? using RBC and serum from the same donor. This study shows that B. bovis and B. bigemina stored nearly 30 years in LN? can be successfully recovered in the MASP system. This study also confirms previous observations that selection of a suitable bovine donor of RBC and serum is critical to the success of the Babesia sp. culture.
Two markers, 18S rRNA gene and rRNA intervening transcribed spacer regions 1 and 2 (ITS), in B. bovis and B. bigemina from Puerto Rico (PR) cattle and archived culture samples from Mexico (B. bovis) and the Virgin Islands (B. bigemina) were PCR amplified, cloned and sequenced. In total, 54 18S rDNA and 21 ITS sequences were deposited in GenBank?. The identity scores among the PR B. bovis 18S rDNA cloned sequences were 92.3% to 100%, and 97.7% to 99.99% among the archived Mexico B. bovis. PR and the Virgin Islands B. bigemina 18S rDNA sequence identity scores ranged from 99.1% to 99.98%. The UPMGA cladogram generated from 18S rDNA sequences shows the clear distinction of B. bovis and B. bigemina (and B. ovis). The PR ITS cloned sequences showed 69.3% to 100% identity among them. In the UPMGA cladogram, the PR sequences fell into seven different groups, except for one outlier that branched separately.
Thirty cloned msa-2b sequences, encoding merozoite surface antigen 2b (MSA-2b), were used to further genotype B. bovis. The identity scores among the deduced MSA-2b amino acid sequences ranged from 41.2% to 100% for the PR isolates, and from 45.4% to 100% among the archived Mexico B. bovis. The UPGMA cladogram based on MSA-2b separated the sequences into two major clades with the PR and archived Mexico B. bovis sequences branching into their own groups within the clades. B-cell epitope predictions showed similar topology between the PR and archived Mexico isolates, although some diversity in sequence was noted. This study revealed heterogeneity in 18S rDNA, ITS1-ITS2 and MSA-2b sequences of the Puerto Rico B. bovis isolates suggesting possible origins from different geographic regions.
