Use of random amplified polymorphic DNA (RAPD) analysis for the identification of Giardia intestinalis subtypes and phylogenetic tree construction.

A comparison of random amplified polymorphic DNA (RAPD) was used to investigate genetic polymorphisms among 25 isolates of Giardia intestinalis and to assess the utility of RAPD for subtype detection and genealogical analysis. Using data obtained for six human and 19 animal-derived isolates in polymerase chain reactions using 13 different primers, phylogenetic trees were constructed and bootstrap values computed by the program FreeTree. Three major clades were distinguished, corresponding to previously defined genetic assemblages A, B, and E. The purported specificity of assemblage E genotypes for artiodactyl hosts was supported. Assemblages A and B showed wide host spectra, including human and animal hosts. No correlation was found between the genotype of analyzed isolates and the presence or absence of the double-stranded RNA Giardiavirus. The results indicate that RAPD data provide reliable genetic information that can be used for both "fingerprinting" and genealogical purposes.

Genetic diversity within the morphological species Giardia intestinalis and its relationship to host origin.

A genetic analysis of Giardia intestinalis, a parasitic protozoan species that is ubiquitous in mammals worldwide, was undertaken using organisms derived from a variety of mammalian hosts in different geographical locations. The test panel of 53 Giardia isolates comprised 48 samples of G. intestinalis, including representatives of all known genetic subgroups, plus an isolate of G. ardeae and four isolates of G. muris. The isolates were compared by allozymic analysis of electrophoretic data obtained for 21 cytosolic enzymes, representing 23 gene loci. Neighbour Joining analysis of the allelic profiles supported the monophyly of G. intestinalis but showed that the species encompasses a rich population substructure. Seven major clusters were evident within G. intestinalis, corresponding to lineages designated previously as genetic assemblages A-G. Some genotypes, e.g. those defining assemblage A, are found in divergent host species and may be zoonotic. However other genotypes, e.g. those defining assemblages C-G, appear to be confined to particular hosts or host groups. The findings reinforce other evidence that G. intestinalis, which was defined on the basis of morphological criteria only, is a species complex.

Genetic homogeneity of axenic isolates of Giardia intestinalis derived from acute and chronically infected individuals in Mexico.

Twenty-six axenic isolates of Giardia intestinalis, established in Mexico City over an 11-year period from symptomatic and asymptomatic individuals with acute or chronic infections, were typed genetically. A segment of the glutamate dehydrogenase gene was amplified by PCR and examined by restriction analysis using BspH1 and ApaI to determine the major genetic assemblages to which the isolates belonged. This was coupled with the amplification and analysis of segments of variant-specific surface protein genes to determine genetic subgroupings. Despite their heterogeneous clinical backgrounds, the isolates were found to be genetically homogeneous-all belonging to genetic group I of assemblage A. The results show that type A-I G. intestinalis is ubiquitous in Mexico City and that host factors play an important, if not dominant, role in determining the clinical outcome of Giardia infections in humans.

CD36/fatty acid translocase in rats: distribution, isolation from hepatocytes, and comparison with the scavenger receptor SR-B1.

The new mAb UA009 recognizes an antigen expressed by microvascular endothelium, by lymphatic endothelium, and by some epithelia in a number of organs, including the small intestine, lactating mammary gland, kidney, lung, sebaceous glands, and circumvallate papillae of the tongue. This antigen is also expressed abundantly in the splenic red pulp and marginal zone and by monocytes, macrophages, and erythrocytes (but not by platelets). Among tissues that store or metabolize fatty acids, the antigen is expressed by adipocytes, cardiomyocytes, and red skeletal muscle. Importantly, it is expressed by steroidogenic cells in the adrenal gland, testis, and ovary, whereas in the liver it is expressed by hepatocytes in a pattern that is dependent on gender and genetic background. mAb UA009 immunoprecipitated a mol wt 85-kDa surface protein from detergent extracts of hepatocytes from Dark Agouti female rats. The N-terminal amino acid sequence of this protein was identical to fatty acid translocase (FAT), the rat cluster of differentiation 36 (CD36) ortholog. The mAb also reacted with COS-7 cells transfected with cDNA encoding FAT. cDNAs encoding a CD36/FAT-like polypeptide were prepared from both liver and heart RNA by RT-PCR. The nucleotide sequences obtained from these cDNAs (Dark Agouti rats) revealed identity and 99% similarity, respectively, with the published sequences of Cd36/Fat in rats of the Wistar and Sprague-Dawley strains. The absence of the UA009 antigen in CD36/FAT-deficient SHR/N rats confirmed the identity of the UA009 antigen and CD36/FAT. We suggest that CD36/FAT might function in the liver as a sex-regulated accessory molecule, either in reverse cholesterol transport and/or in fatty acid uptake.

VSP417-6, a variant-specific surface protein encoded at a sixth locus within the vsp417 gene subfamily of Giardia intestinalis.

A sixth locus (vsp417-6) belonging to the vsp417 gene subfamily, a subset of the family of genes that encodes 'variant-specific' surface proteins (VSP) in Giardia, is described. The sequence of vsp417-6(A-I), the ortholog representing the vsp417-6 locus in isolates of the type A-I (Assemblage A, Group I) genotype of Giardia intestinalis, was determined from a cloned 5.5-kb Hind III fragment of genomic DNA derived from isolate Ad-1/C1. The gene encodes a 704 residue polypeptide (VSP417-6(A-I), Mr 71,674) that has 75% identity (92% similarity) over a 718 residue overlap with the prototype of the VSP417 subfamily, VSP417-1(A-I)-encoded by the vsp417-1 (syn. tsa417) locus in type A-I isolates. Alignment of VSP417-6(A-I) with the deduced sequences of other known members of this subfamily identified one polypeptide, encoded by a gene found in type A-II (Assemblage A, Group II) isolates, whose homology with VSP417-6(A-I) (91% identity, 98% similarity over 713-residues) indicated that it was VSP417-6(A-II), the VSP417-6 ortholog in type A-II isolates. Sequence-based phylogenetic analyses of known VSP417 subfamily members defined several loci that predate the emergence of the A-I and A-II sublineages of G. intestinalis. Related sequences that may correspond to additional, uncharacterised vsp417 subfamily genes were identified in genomic DNA by Southern hybridisation using subfamily- and locus-specific probes. Variant-specific expression of vsp417-1 and vsp417-6 within axenic cultures of G. intestinalis was detected by in situ mRNA hybridization, indicating that these genes are functional and that they are expressed in an alternative fashion with other vsp genes in these organisms.

Sequence comparison of an Australian duck hepatitis B virus strain with other avian hepadnaviruses.

The genome of an Australian strain of duck hepatitis B virus (AusDHBV) was cloned from a pool of congenitally DHBV-infected-duck serum, fully sequenced and found by phylogenetic analyses to belong to the 'Chinese' DHBV branch of the avian hepadnaviruses. Sequencing of the Pre-S/S gene of four additional AusDHBV clones demonstrated that the original clone (pBL4.8) was representative of the virus present in the pool, and a head-to-tail dimer of the clone was infectious when inoculated into newly hatched ducks. When the published sequences of 20 avian hepadnaviruses were compared, substitutions or deletions in the polymerase (POL) gene were most frequent in the 500 nt segment encoding the 'spacer' domain that overlaps with the Pre-S domain of the Pre-S/S gene in a different reading frame. In contrast, substitutions and deletions were rare within the adjacent segment that encodes the reverse transcriptase domain of the POL protein and the S domain of the envelope protein, presumably because they are more often deleterious.