In vitro cultivation of a zoonotic Babesia sp. isolated from eastern cottontail rabbits (Sylvilagus floridanus) on Nantucket Island, Massachusetts.
ABSTRACT: A Babesia sp. found in eastern cottontail rabbits (Sylvilagus floridanus) on Nantucket Island, Massachusetts, is the same organism that caused human babesiosis in Missouri and Kentucky, on the basis of morphology and identical small-subunit rRNA (SSU rRNA) gene sequences. Continuous cultures of the rabbit parasite were established from infected blood samples collected from two cottontail rabbits livetrapped on Nantucket Island. HL-1 medium or minimal essential medium alpha medium supplemented with 20% human serum best supported in vitro propagation of the parasite in human or cottontail erythrocytes, respectively. Parasite growth was not sustained in domestic-rabbit erythrocytes or in medium supplemented with domestic-rabbit serum. The cultured parasites were morphologically indistinguishable from the Kentucky human isolate. Transmission electron microscopy revealed similar fine structures of the parasite regardless of the host erythrocyte utilized in the cultures. Two continuous lines of the zoonotic Babesia sp. were established and confirmed to share identical SSU rRNA gene sequences with each other and with the Missouri and Kentucky human Babesia isolates.
Project description:Theileria sp.-specific small subunit (SSU) rRNA gene amplification confirmed the presence of the organism in cattle and in Amblyomma americanum and Dermacentor variabilis ticks collected from a cattle herd in Missouri. Blood from the index animal had type A and type D Theileria SSU rRNA genes. The type D gene was also found in blood from two cohort cattle and tick tissues. The type A SSU rRNA gene was previously reported from bovine Theileria isolates from Texas and North Carolina; the type D gene was reported from a Texas cow with theileriosis.
Project description:Lyme disease and human babesiosis are the most common tick-borne zoonoses in the Temperate Zone of North America. The number of infected patients has continued to rise globally, and these zoonoses pose a major healthcare threat. This tick-host-pathogen study was conducted to test for infectious microbes associated with Lyme disease and human babesiosis in Canada. Using the flagellin (flaB) gene, three members of the Borrelia burgdorferi sensu lato (Bbsl) complex were detected, namely a Borrelia lanei-like spirochete, Borrelia burgdorferi sensu stricto (Bbss), and a distinct strain that may represent a separate Bbsl genospecies. This novel Bbsl strain was detected in a mouse tick, Ixodes muris, collected from a House Wren, Troglodytes aedon, in Quebec during the southward fall migration. The presence of Bbsl in bird-feeding larvae of I. muris suggests reservoir competency in three passerines (i.e., Common Yellowthroat, House Wren, Magnolia Warbler). Based on the 18S ribosomal RNA (rRNA) gene, three Babesia species (i.e., Babesia divergens-like, Babesia microti, Babesia odocoilei) were detected in field-collected ticks. Not only was B. odocoilei found in songbird-derived ticks, this piroplasm was apparent in adult questing blacklegged ticks, Ixodes scapularis, in southern Canada. By allowing live, engorged ticks to molt, we confirm the transstadial passage of Bbsl in I. muris and B. odocoilei in I. scapularis. Bbss and Babesia microti were detected concurrently in a groundhog tick, Ixodes cookei, in Western Ontario. In Alberta, a winter tick, Dermacentor albipictus, which was collected from a moose, Alces alces, tested positive for Bbss. Notably, a B. divergens-like piroplasm was detected in a rabbit tick, Haemaphysalis leporispalustris, collected from an eastern cottontail in southern Manitoba; this Babesia species is a first-time discovery in Canada. This rabbit tick was also co-infected with Borrelia lanei-like spirochetes, which constitutes a first in Canada. Overall, five ticks were concurrently infected with Babesia and Bbsl pathogens and, after the molt, could potentially co-infect humans. Notably, we provide the first authentic report of I. scapularis ticks co-infected with Bbsl and B. odocoilei in Canada. The full extent of infectious microorganisms transmitted to humans by ticks is not fully elucidated, and clinicians need to be aware of the complexity of these tick-transmitted enzootic agents on human health. Diagnosis and treatment must be administered by those with accredited medical training in tick-borne zoonosis.
Project description:Human babesiosis, which is caused by infection with the intraerythrocytic malarialike protozoan Babesia microti, has recently been diagnosed with increasing frequency in residents of New England. Diagnosis is difficult because of the small size of the parasite and the sparse parasitemia that is characteristic of most infections with this pathogen. We generated B. microti-specific DNA sequence information by universal primer amplification of a portion of the eukaryotic 16S-like gene; this was followed by direct DNA sequence analysis. Specific primers were synthesized on the basis of this sequence information for use in the polymerase chain reaction (PCR). The PCR-based system demonstrates a strong bias for detection of B. microti as opposed to Babesia gibsoni and does not amplify vertebrate DNA. The analytical sensitivity of the system is approximately three merozoites. Blood specimens from 12 patients with clinically diagnosed and parasitologically confirmed babesiosis from Nantucket Island, Mass., were PCR positive in a blinded test of this procedure. Thus, DNA amplification may provide an adjunct to conventional methods for the diagnosis of human babesiosis and may provide a new means of monitoring therapy or enhancing epidemiological surveillance for this emerging pathogen.
Project description:One unique feature of malaria parasites is the differential transcription of structurally distinct rRNA (rRNA) genes at different developmental stages: the A-type genes are transcribed mainly in asexual stages, whereas the S-type genes are expressed mostly in sexual or mosquito stages. Conclusive functional evidence of different rRNAs in regulating stage-specific parasite development, however, is still absent. Here we performed genetic crosses of Plasmodium yoelii parasites with one parent having an oocyst development defect (ODD) phenotype and another producing normal oocysts to identify the gene(s) contributing to the ODD. The parent with ODD--characterized as having small oocysts and lacking infective sporozoites--was obtained after introduction of a plasmid with a green fluorescent protein gene into the parasite genome and subsequent passages in mice. Quantitative trait locus analysis of genome-wide microsatellite genotypes of 48 progeny from the crosses linked an ~200-kb segment on chromosome 6 containing one of the S-type genes (D-type small subunit rRNA gene [D-ssu]) to the ODD. Fine mapping of the plasmid integration site, gene expression pattern, and gene knockout experiments demonstrated that disruption of the D-ssu gene caused the ODD phenotype. Interestingly, introduction of the D-ssu gene into the same parasite strain (self), but not into a different subspecies, significantly affected or completely ablated oocyst development, suggesting a stage- and subspecies (strain)-specific regulation of oocyst development by D-ssu. This study demonstrates that P. yoelii D-ssu is essential for normal oocyst and sporozoite development and that variation in the D-ssu sequence can have dramatic effects on parasite development.Malaria parasites are the only known organisms that express structurally distinct rRNA genes at different developmental stages. The differential expression of these genes suggests that they play unique roles during the complex life cycle of the parasites. Conclusive functional proof of different rRNAs in regulating parasite development, however, is still absent or controversial. Here we functionally demonstrate for the first time that a stage-specifically expressed D-type small-subunit rRNA gene (D-ssu) is essential for oocyst development of the malaria parasite Plasmodium yoelii in the mosquito. This study also shows that variations in D-ssu sequence and/or the timing of transcription may have profound effects on parasite oocyst development. The results show that in addition to protein translation, rRNAs of malaria parasites also regulate parasite development and differentiation in a strain-specific manner, which can be explored for controlling parasite transmission.
Project description:The genus Euduboscquella is one of a few described genera within the syndinean dinoflagellates, an enigmatic lineage with abundant diversity in marine environmental clone libraries based on small subunit (SSU) rRNA. The region composed of the SSU through to the partial large subunit (LSU) rRNA was determined from 40 individual tintinnid ciliate loricae infected with Euduboscquella sampled from eight surface water sites in the Northern Hemisphere, producing seven distinct SSU sequences. The corresponding host SSU rRNA region was also amplified from eight host species. The SSU tree of Euduboscquella and syndinean group I sequences from environmental clones had seven well-supported clades and one poorly supported clade across data sets from 57 to 692 total sequences. The genus Euduboscquella consistently formed a supported monophyletic clade within a single subclade of group I sequences. For most parasites with identical SSU sequences, the more variable internal transcribed spacer (ITS) to LSU rRNA regions were polymorphic at 3 to 10 sites. However, in E. cachoni there was variation between ITS to LSU copies at up to 20 sites within an individual, while in a parasite of Tintinnopsis spp., variation between different individuals ranged up to 19 polymorphic sites. However, applying the compensatory base change model to the ITS2 sequences suggested no compensatory changes within or between individuals with the same SSU sequence, while one to four compensatory changes between individuals with similar but not identical SSU sequences were found. Comparisons between host and parasite phylogenies do not suggest a simple pattern of host or parasite specificity.
Project description:The intraerythrocytic apicomplexan Babesia microti, the primary causative agent of human babesiosis, is a major public health concern in the United States and elsewhere. Apicomplexans utilize a multiprotein complex that includes a type I membrane protein called apical membrane antigen 1 (AMA1) to invade host cells. We have isolated the full-length B. microti AMA1 (BmAMA1) gene and determined its nucleotide sequence, as well as the amino acid sequence of the AMA1 protein. This protein contains an N-terminal signal sequence, an extracellular region, a transmembrane region, and a short conserved cytoplasmic tail. It shows the same domain organization as the AMA1 orthologs from piroplasm, coccidian, and haemosporidian apicomplexans but differs from all other currently known piroplasmida, including other Babesia and Theileria species, in lacking two conserved cysteines in highly variable domain III of the extracellular region. Minimal polymorphism was detected in BmAMA1 gene sequences of parasite isolates from six babesiosis patients from Nantucket. Immunofluorescence microscopy studies showed that BmAMA1 is localized on the cell surface and cytoplasm near the apical end of the parasite. Native BmAMA1 from parasite lysate and refolded recombinant BmAMA1 (rBmAMA1) expressed in Escherichia coli reacted with a mouse anti-BmAMA1 antibody using Western blotting. In vitro binding studies showed that both native BmAMA1 and rBmAMA1 bind to human red blood cells (RBCs). This binding is trypsin and chymotrypsin treatment sensitive but neuraminidase independent. Incubation of B. microti parasites in human RBCs with a mouse anti-BmAMA1 antibody inhibited parasite growth by 80% in a 24-h assay. Based on its antigenically conserved nature and potential role in RBC invasion, BmAMA1 should be evaluated as a vaccine candidate.
Project description:We report on the first case of human babesiosis in Korea. The intraerythrocytic parasite (KO1) in the patient's blood mainly appeared as paired pyriforms and ring forms; but Maltese cross forms were not seen, and the parasite showed morphological features consistent with those of the genus Babesia sensu stricto. The sequence of the 18S rRNA gene of KO1 was closely related to that of Babesia spp. isolated from sheep in China (similarity, 98%). The present study provides the first evidence of the presence of a hitherto unidentified, new type of Babesia parasite capable of infecting humans.
Project description:Deer tick-transmitted human babesiosis due to Babesia microti appears to be expanding its distribution and prevalence in the northeastern United States. One hypothesis for this emergence is the introduction of parasites into new sites from areas of long-standing transmission, such as Nantucket Island, Massachusetts.We developed a typing system based on variable number tandem repeat loci that distinguished individual B. microti genotypes. We thereby analyzed the population structure of parasites from 11 sites, representing long-standing and newly emerging transmission in southern New England (northeastern United States), and compared their haplotypes and allele frequencies to determine the most probable number of B. microti populations represented by our enzootic collections. We expected to find evidence for a point source introduction across southern New England, with all parasites clearly derived from Nantucket, the site with the most intense longstanding transmission.B. microti in southern New England comprises at least two major populations, arguing against a single source. The Nantucket group comprises Martha's Vineyard, Nantucket and nearby Cape Cod. The Connecticut/Rhode Island (CT/RI) group consists of all the samples from those states along with samples from emerging sites in Massachusetts.The expansion of B. microti in the southern New England mainland is not due to parasites from the nearby terminal moraine islands (Nantucket group), but rather from the CT/RI group. The development of new B. microti foci is likely due to a mix of local intensification of transmission within relict foci across southern New England as well as long distance introduction events.
Project description:Babesiosis in a sable antelope (Hippotragus niger Harris, 1838) was first reported in 1930; the parasite was named Babesia irvinesmithi. Recently, specimens from an adult sable that presented with a sudden onset of disease and that subsequently died during immobilization were submitted for molecular characterization. Microscopic examination of thin blood smears revealed the presence of small piroplasms. DNA was extracted from blood samples; the V4 variable region of the 18S rRNA gene was amplified and analyzed using the reverse line blot (RLB) assay. Amplicons did not hybridize with any of the Babesia or Theileria species-specific probes present on the blot and hybridized only with a Babesia or Theileria genus-specific probe, suggesting the presence of a novel species. The full-length 18S rRNA gene sequence was obtained and aligned with published sequences of related genera, and phylogenetic trees were constructed. Sequence similarity analyses indicated that a Babesia species, designated Babesia sp. (sable), was present. The sequence showed its highest similarity to B. orientalis and to an unnamed Babesia species previously detected in bovine samples. The latter was later established to be Babesia occultans. A Babesia sp. (sable)-specific RLB oligonucleotide probe was designed and used to screen 200 South African sable samples, but so far, no other sample has been found to be positive for the presence of Babesia sp. (sable) DNA. In summary, we identified a novel piroplasm parasite from a sable antelope that died from an unknown illness. While the parasite was observed in blood smears, there is no direct evidence that it was the cause of death.
Project description:Serological studies of cottontail rabbits sampled from Nantucket Island, Mass., have suggested exposure to at least two ehrlichiae. The agent of human granulocytic ehrlichiosis (Anaplasma phagocytophilum) is intensely enzootic in rabbits there, but the identity of the other ehrlichial infection remains undescribed. We sampled rabbits over five transmission seasons and tested their blood and tissues for evidence of infection using PCR targeting an Ehrlichia genus-wide 16S rDNA target. Sequence analysis of positive amplicons revealed the presence of Anaplasma bovis, an agent not known to be present in North America. The average annual prevalence of A. bovis within rabbits, as determined by PCR of blood samples, was 18%. Haemaphysalis leporispalustris appears to serve as vector. The public health (human or veterinary) significance of this finding remains speculative.