Patricia J. Holman

NUCLEOTIDE SEQUENCE HETEROGENEITY IN THE SMALL SUBUNIT RIBOSOMAL RNA GENE VARIABLE (V4) REGION AMONG AND WITHIN GEOGRAPHIC ISOLATES OF THEILERIA FROM CATTLE, ELK AND WHITE-TAILED DEER

The phylogenetic relationships among fourteen isolates of benign Theileria spp. infecting cattle, elk and white-tailed deer were studied by nucleotide sequence comparisons of the variable (V4) region (200 nucleotides) of the small subunit ribosomal RNA gene. Included were six Korean bovine, one Japanese bovine, three North American bovine, and four North American cervine isolates. The SSU rRNA gene from each isolate was amplified, cloned, and the V4 region fragment sequenced. Seven different nucleotide sequence patterns were obtained and classified. Type A was identical to T. buffeli SSU rRNA gene sequence (GenBank Accession No. Z15106) and was found in Korean, Japanese, and North American bovine isolates. Type B was found in bovine isolates from Korea, Japan and North America. Type C was found only in the Korean bovine isolate from Chungnam. Type D was found in a Korean and in a North American bovine isolate. Type E was found in a bovine isolate from Cheju Island of Korea and a North American cervine (elk) isolate. Types F and G were found only in North American cervine isolates (both white-tailed deer and elk) and appear to represent a species separate from the bovine isolates. The presence of several sequence types observed in most of the bovine Theileria isolates may indicate mixed species (or subspecies) populations and/or multiple genotypes within a single species.

A study of the systematics of Theileria spp. based upon small-subunit ribosomal RNA gene sequences

Abstract The systematics of benign and moderately pathogenic Theileria isolates from cattle and deer originating from different geographic regions was undertaken by small-subunit ribosomal RNA (SSU rRNA) gene nucleotide-sequence analysis. A maximum-likelihood phylogenetic tree constructed from these sequences resulted in two major divisions, each with a common ancestor. One major division branches into four relatively divergent groups, including (1) bovine Theileria sp. Type D (USA and Korea), (2) T. mutans Intona and Theileria sp. MSD (Africa), (3) T. cervi (USA), and (4) well-characterized pathogenic Theileria spp. (Africa). The other major division branches into two groups: (1) T. buffeli Warwick and T. buffeli Marula and (2) a second branch of closely related isolates with SSU rRNA gene Types B, B1, C, E, and H. Putative geographically associated diversity was noted only in the Korean bovine Theileria spp. with SSU rRNA gene types C and H and in African T. mutans Intona and Theileria sp. MSD. The current results show that the United States bovine Theileria isolates are not T. mutans because they have T. buffeli Marula (Type A) and/or Type D (species undesignated) SSU rRNA gene sequences. The taxonomic separation of T. buffeli Warwick from African T. mutans is confirmed in this study.

Restriction-fragment-length polymorphism analysis of small-subunit rRNA genes of Blastocystis hominis isolates from geographically diverse human hosts.

Abstract Genomic diversity among 14 isolates of Blastocystis hominis from 4 different geographic locations was examined by small-subunit rRNA (ssu rRNA) restriction-fragment-length polymorphisms (RFLP) using 5 different restriction endonucleases. On the basis of the observed RFLP patterns among the isolates, a total of 12 genotypes were identified, with 7 isolates exhibiting mixed RFLP genotypes. There was no correlation between B. hominis geographic origin and RFLP banding pattern or genotype.

Antigenic, phenotypic and molecular characterization confirms Babesia odocoilei isolated from three cervids.

Babesia isolates from an elk (Cervus elaphus canadensis) and a caribou (Rangifer tarandus caribou) with fatal infections were compared to Babesia odocoilei (Engeling isolate) from white-tailed deer (Odocoileus virginianus) by experimental infection, serologic, and small subunit ribosomal RNA (SSU rRNA) gene sequence analysis studies. Both the indirect fluorescent antibody test and immunoprecipitation assays demonstrated antigenic variation among the isolates. Experimental infection studies showed no clinical differences among the isolates. Nucleotide sequence analysis showed that the elk and caribou Babesia sp. isolates possessed SSU rRNA genes with identical sequences to that of B. odocoilei. A phylogenetic tree constructed from SSU rRNA gene sequences shows that B. odocoilei is most closely related to Babesia divergens, both of which branch together in the true babesia clade.

Naturally acquired babesiosis in a reindeer (Rangifer tarandus tarandus) herd in Great Britain.

Abstract. A provisional diagnosis of babesiosis was made in a reindeer herd in Scotland when seven animals died during 1997 and 1998. Additional clinical cases occurred, but the animals recovered after treatment. Thirty-one reindeer from the herd were tested for the prevalence of exposure to Babesia by the indirect fluorescent antibody test using a bovine isolate of Babesia divergens that had been passaged through gerbils. Infection rates were determined by Giemsa-stained blood smears. In addition, molecular identification of the infecting Babesia sp. was undertaken using SSU rRNA gene sequence analysis. It is likely that the organism causing babesiosis in this reindeer herd is B. divergens.

Restriction-fragment-length polymorphism analysis of small-subunit rRNA genes of Blastocystis isolates from animal hosts.

Abstract The anaerobic enteric protozoan organism Blastocystis sp. has been identified from mammalian, avian, reptilian, and arthropod hosts. Eight Blastocystis isolates from five animal host species (cow, goat, sheep, guinea pig, and rhea) were compared by small-subunit ribosomal RNA (ssu rRNA) restriction-fragment-length polymorphism (RFLP) analyses using five restriction endonucleases. The isolates sorted into five genotypes. Multiple genotypes were found in isolates from a single animal host species, and multiple host species shared a single genotype. A molecular method such as RFLP analysis of ssu rRNA genes facilitates the characterization of Blastocystis isolates from various host species.

TWO THEILERIA CERVI SSU RRNA GENE SEQUENCE TYPES FOUND IN ISOLATES FROM WHITE-TAILED DEER AND ELK IN NORTH AMERICA

Two Theileria cervi SSU rRNA gene sequence Types, F and G, from white-tailed deer (Odocoileus virginianus) and elk (Cervus elaphus canadensis) isolates in North America were confirmed. Previously, nucleotide sequencing through a single variable (V4) region showed the presence of SSU rRNA gene Types F and G in T. cervi isolates from white-tailed deer and an elk. In this study, both sequence types were found in four T. cervi isolates (two from deer and two from elk). Microheterogeneity only appeared in the Type G gene, resulting in Subtypes G1, G2 and G3. Subtype G1 was found in two elk and one white-tailed deer T. cervi isolate; Subtypes G2 and G3 were found in a white-tailed deer T. cervi isolate. The Type F SSU rRNA genes were identical in nucleotide sequence in both elk and white-tailed deer T. cervi isolates. The high degree of conservation in the Type F variable regions may be exploited to design specific oligonucleotide primers for parasite detection by the polymerase chain reaction in cervine or tick hosts.

New ruminant hosts and wider geographic range identified for Babesia odocoilei (Emerson and Wright 1970).

Babesia odocoilei was found to infect two previously unknown host species, desert bighorn sheep (Ovis canadensis nelsoni) and musk oxen (Ovibos moschatus), both of which are members of the family Bovidae. Previously, B. odocoilei has been reported in only Cervidae hosts. New geographic regions where B. odocoilei infections have not been reported previously include Pennsylvania and New York, where fatal babesiosis has occurred in reindeer (Rangifer tarandus tarandus); New Hampshire, where elk (Cervus elaphus canadensis) have been affected; and California, home of the infected desert bighorn sheep. Infection with B. odocoilei in these hosts was confirmed by parasite small subunit ribosomal RNA gene sequence analysis. A serosurvey for B. odocoilei antibody activity in New Hampshire showed prevalence rates of 100% at two elk farms and 12% at another farm. Control of potential vector ticks, Ixodes scapularis, especially when translocating livestock, is imperative to prevent outbreaks of babesiosis in managed herds of potential host species.

Molecular and serologic evidence for Babesia bovis-like parasites in white-tailed deer (Odocoileus virginianus) in south Texas.

The current study was undertaken to determine if white-tailed deer in south Texas harbor Babesia bovis, a causative agent of bovine babesiosis. Blood samples from free-ranging white-tailed deer (Odocoileus virginianus) on two ranches in LaSalle and Webb Counties were screened for B. bovis and other hemoparasites by the polymerase chain reaction (PCR) to detect the piroplasm 18S rDNA. Serology was conducted on selected samples to detect antibody activity to B. bovis by the immunofluorescent antibody test (IFAT). PCR revealed that 16% of the LaSalle County samples and 4% of the Webb County samples were positive for B. bovis. Five of the LaSalle County and the two Webb County B. bovis 18S rDNA amplicons were cloned and sequenced. The resulting clones shared 99% identity to B. bovis 18S rRNA gene sequences derived from cattle isolates. Weak seroreactivity to B. bovis was shown by the IFAT. The samples were also screened for additional hemoparasites of deer including Theileria cervi, Babesia odocoilei and other Babesia spp. A genotypically unique Theileria sp. was found, along with T. cervi and B. odocoilei. The finding of putative B. bovis in white-tailed deer necessitates further study to determine if deer may act as a transient host or even a reservoir of infection for B. bovis pathogenic to cattle.

Theileria buffeli infection of a Michigan cow confirmed by small subunit ribosomal RNA gene analysis

Theileria buffeli, generally a benign parasite of cattle, has been reported in animals from Texas, Missouri and North Carolina. To date, there have been no reports of the parasite in cattle residing in northern portions of the US. An 8-year-old cow (Maine Anjou x Angus cross-bred) in Michigan presented with hemoglobinuria and a packed cell volume of 9. Blood films stained with Giemsa showed numerous intraerythrocytic parasites morphologically consistent with T. buffeli. The parasite was confirmed to be T. buffeli by SSU rRNA gene sequence analysis (SSU rRNA sequence, Type A). This represents the first report of this parasite in an animal in Michigan.