Sequence and comparative structural analysis of the murine leukaemia virus amphotropic strain 4070A RNase H domain.

The sequence of a 900-nucleotide segment (encoding part of the reverse transcriptase, including the entire RNase H domain) of the pol gene of the murine leukaemia virus (MLV) amphotropic strain 4070A is presented. Alignment of the inferred 4070A RNase H amino acid sequence (157 residues) with other MLV RNase H sequences revealed only minor differences compared with the divergence between other retroviral and prokaryotic or eukaryotic RNase H sequences. Only 10 residues were invariant across the entire sample set, but secondary structure predictions for the enzymes from E. coli, yeast, human liver and diverse retroviruses (HIV, Rous sarcoma virus, foamy viruses) supported, in every case, the five beta-strands (1 to 5) and four or five alpha-helices (A, B/C, D, E) that have been identified by crystallography in the RNase H domain of HIV-1 reverse transcriptase and in E. coli RNase H. In the case of MLV, analysis of the RNase H domain sequences inferred from 10 different strains (including the amphotropic 4070A) predicted all five alpha-helices (A-E), as well as beta-strands 4 and 5. However, the N-terminal segment (residues 1-40) was predicted, without exception and with high probability, to fold uniquely into one (or two adjacent) alpha-helix(es) encompassing residues 13-37, instead of the three beta-strands known to exist in the HIV-1 and E. coli enzymes. The unerring consistency between the known and predicted structures of the HIV-1 and E. coli enzymes, and the prediction of the same structural elements (including beta-strands 1-3 within the N-terminal segment) for all other (non-MLV) RNase H proteins examined in this study, suggests that the N-terminal segment of the MLV RNase H domain assumes a conformation distinct from that of other retroviral and cellular RNase H molecules. An additional (sixth) beta-strand was also predicted uniquely within the C-terminal region of foamy virus RNase H domains.

Molecular systematics of the parasitic protozoan Giardia intestinalis.

The long-standing controversy regarding whether Giardia intestinalis is a single species prevalent in both human and animal hosts or a species complex consisting of morphologically similar organisms that differ in host range and other biotypic characteristics is an issue with important medical, veterinary, and environmental management implications. In the past decade, highly distinct genotypes (some apparently confined to particular host groups) have been identified by genetic analysis of samples isolated from different host species. The aim of this study was to undertake a phylogenetic analysis of G. intestinalis that were representative of all known major genetic groups and compare them with other Giardia species, viz. G. ardeae, G. muris, and G. microti. Segments from four "housekeeping" genes (specifying glutamate dehydrogenase, triose phosphate isomerase, elongation factor 1 alpha, and 18S ribosomal RNA) were examined by analysis of 0.48-0.69-kb nucleotide sequences determined from DNA amplified in polymerase chain reactions from each locus. In addition, isolates were compared by allozymic analysis of electrophoretic data obtained for 21 enzymes representing 23 gene loci. The results obtained from these independent techniques and different loci were essentially congruous. Analyses using G. ardeae and/or G. muris as outgroups supported the monophyly of G. intestinalis and also showed that this species includes genotypes that represent at least seven deeply rooted lineages, herein designated assemblages A-G. Inclusion of G. microti in the analysis of 18S rRNA sequence data demonstrated the monophyly of Giardia with the same median body morphology but did not support the monophyly of G. intestinalis, instead placing G. microti within G. intestinalis. The findings support the hypothesis that G. intestinalis is a species complex and suggest that G. microti is a member of this complex.

A new locus (vsp417-4) belonging to the tsa417-like subfamily of variant-specific surface protein genes in Giardia intestinalis.

A new variant-specific surface protein gene locus (vsp417-4) of Giardia intestinalis is described. Vsp417-4 represents the fourth member of a gene subfamily that is based on a previously described gene, tsa417 ( =vsp417-1). The new locus was detected by characterising DNA amplified in polymerase chain reactions from the 3' ends of divergent homologues (vsp417-4(A-I), vsp417-4(A-II)) found respectively in isolates belonging to the genetic Assemblage A/Group I ('A-I') and Assemblage A/Group II ('A-II') subtypes of G. intestinalis. The complete vsp417-4(A-I) gene was isolated on a 6.2-kb HindIII fragment by screening a genomic DNA library prepared from a type A-I isolate, Ad-1/C7. The deduced polypeptide (VSP417-4(A-I); 709 amino acids, Mr 72662) has properties characterising it as a Giardia variant-specific surface protein, namely a high cysteine content (11.85 mol%), 29 copies of the four amino-acid 'CXXC' motif, and conserved N-terminal signal peptide and C-terminal hydrophobic (membrane-spanning) segments--the latter terminating with the invariant, hydrophilic motif '-CRGKA'. An extended polyadenylation signal sequence (CTTAGRTAGTAAAY), which appears to be a characteristic feature of VSP genes in Giardia, is situated immediately beyond the stop codon. VSP417-4(A-I) shares 87% sequence identity with VSP417-4(A-II) over its C-terminal 235 amino acids, but only 57-58% identity with VSP417-1, VSP417-2 and VSP417-3 which are encoded by other vsp417 family genes identified in these genotypes. Southern hybridisations, using probes derived from the 5' segment of vsp417-4(A-I), indicated the presence of at least five to six closely related loci in both type A-I and type A-II isolates.

Comparison of tsa417-like variant-specific surface protein (VSP) genes in Giardia intestinalis and identification of a novel locus in genetic group II isolates.

A gene (vsp417-3A-II) encoding a new member of the variant-specific surface protein (VSP) family of Giardia is described. Vsp417-3A-II is detected exclusively in isolates belonging to the genetic Assemblage A/Group II sublineage of G. intestinalis, and it is closely related to a previously characterized VSP gene (tsp11, herein renamed vsp417-2A-I) found in Group I isolates of G. intestinalis. Analysis of DNA amplified from the genomes of Group II isolates using consensus primers also revealed products corresponding to the vsp417-2 locus (vsp417-2A-II allele), indicating that vsp417-2A-II and vsp417-3A-II represent separate loci in these organisms. Southern hybridizations revealed a single genomic copy of vsp417-3A-II in Group II isolates, but efforts to detect this locus in Group I organisms were unsuccessful. Together with the vsp417-1 (tsa417) locus that was first identified in the genetic Group I isolate WB and which has been detected since in all tested genetic Group I isolates (as vsp417-1A-I) and in genetic Group II isolates (as vsp417-1A-II), vsp417-3A-II represents a third member of a stable subset of tsa417-like VSP genes in genetic Group II G. intestinalis. The encoded polypeptide, VSP417-3A-II (667 residues, predicted M(r) 69120) possesses a high cysteine content (12.1 mol%), 28 copies of the metal-binding -CXXC- motif, and a highly conserved C-terminal hydrophobic domain--similar to all other characterized Giardia VSP. A revised nomenclature for Giardia VSP genes is proposed, prompted by the need to describe the homologues found in the various major genotypes of the species complex, G. intestinalis.

Giardia intestinalis: conservation of the variant-specific surface protein VSP417-1 (TSA417) and identification of a divergent homologue encoded at a duplicated locus in genetic group II isolates.

The stability of the gene encoding TSA417, a 72-kDa variant-specific surface protein (VSP) produced by trophozoites of Giardia intestinalis isolate WB-C6, was investigated in isolates of similar (Assemblage A / Group I) or distinct (Assemblage A / Group II) genotype. Using primers specific for the WB-C6 tsa417 gene, DNA amplified in polymerase chain reactions from genomic DNA indicated the presence, in every isolate, of an intact coding sequence possessing conserved restriction sites diagnostic for this locus (herein designated vsp417-1). Sequence analysis of the DNA amplified from the genomes of genetic Group I ("A-I") isolates revealed complete identity with the published WB-C6 tsa417 (vsp417-1(A-I)) sequence. Equivalent products, amplified from the genomes of genetic Group II ("A-II") isolates, similarly yielded an invariant and apparently allelic 2142-bp coding sequence (designated vsp417-1(A-II)) possessing 79% nucleotide identity with vsp417-1(A-I) and polymorphisms unique to Group II organisms. The encoded polypeptides (VSP417-1(A-I) and VSP417-1(A-II)) are identical at 75% of amino acid positions. Substitutions are concentrated within the N-terminal portions of the proteins, but the overall structure of VSP417-1 has changed little during the evolution of the Group I and Group II genotypes from their common clonal ancestor. An additional 0.7-kb DNA, representing a separate locus (vsp417-5) encoding a 22.3-kDa VSP, was amplified from genetic Group II genomes exclusively but only using particular primer combinations. The vsp417-5(A-II) gene exhibits >85% sequence identity with the 5' and 3' segments of vsp417-1(A-I) and vsp417-1(A-II) but it lacks a 1482-bp segment that comprises the central portion of the vsp417-1 locus. Excision of this segment seems to have occurred by intragenic recombination, possibly initiated by a stem loop formed between palindromic sequences which border the 1482-bp segment within vsp417-1 but which are contiguous in vsp417-5(A-II). The detection by Southern hybridization of additional genomic sequences that share homology with these genes reveals the existence in these two genotypes of a distinctive "vsp417" gene subset.

Comparison of the levels of intra-specific genetic variation within Giardia muris and Giardia intestinalis.

The extent of intra-specific genetic variation between isolates of Giardia muris was assessed by allozyme electrophoresis. Additionally, the levels of allozymic variation detected within G. muris were compared with those observed between members of the two major assemblages of the morphologically distinct species Giardia intestinalis. Four isolates of G. muris were analysed. Three (Ad-120, -150, -151) were isolated from mice in Australia, while the fourth (R-T) was isolated from a golden hamster in North America. The 11 isolates of G. intestinalis (Ad-1, -12, -2, -62, representing genetic Groups I and II of Assemblage A and BAH-12, BRIS/87/HEPU/694, Ad-19, -22, -28, -45, -52, representing genetic Groups III and IV of Assemblage B) were from humans in Australia. Intra-specific genetic variation was detected between G. muris isolates at four of the 23 enzyme loci examined. Similar levels of variation were found within the genetic groups that comprise Assemblages A and B of G. intestinalis. These levels of intra-specific variation are similar to those observed within other morphologically-distinct species of protozoan parasites. We suggest that the magnitude of the genetic differences detected within G. muris provides an indication of the range of genetic variation within other species of Giardia and that this can be used as a model to delineate morphologically similar but genetically distinct (cryptic) species within this genus.

Characterization of axenic isolates of Giardia intestinalis established from humans and animals in Germany.

A total of 15 isolates of Giardia intestinalis, the first axenic cultures of this organism to be described from Germany, were established in Bonn from faecal cysts obtained from human and animal stool specimens. Measurement of in vitro growth kinetics for 12 of the isolates revealed 3 phenotypes ('rapid', 'medium-rate' and 'slow' growers) characterized by generation times of 9-11 h (5 isolates), 12-15 h (5 isolates) and > or = 18-20 h (2 isolates), respectively. Cloned sublines exhibited growth rates similar to those of the parent isolates. Genetic analyses involving use of the polymerase chain reaction to amplify segments of genes encoding variant-specific surface proteins or the enzyme glutamate dehydrogenase, coupled with the detection of restriction-fragment-length polymorphisms, identified genotypes belonging to three previously described genetic groups. Seven isolates (from humans, a calf and a chinchilla) were typed to genetic group I--a potentially zoonotic genotype belonging to assemblage A, one of two major genetic lineages defined by analysis of G. intestinalis from humans and animals. Six isolates (all from humans) showed identity with the group II genotype--recovered thus far only from humans and also belonging to assemblage A. Two isolates (one from a human, the other from a monkey housed at the Cologne zoo) were classified as assemblage B genotypes. The in vitro growth rates correlated strongly with genotype, group I or group II (assemblage A) genotypes accounting for all of the 'rapid' and 'medium-rate' cultures and both assemblage B isolates being 'slow growers'. The data indicate that genetically based metabolic differences may determine how rapidly G. intestinalis isolates can grow in axenic culture.

Novel lineages of Giardia intestinalis identified by genetic analysis of organisms isolated from dogs in Australia.

Infection of suckling mice with Giardia trophozoites recovered from the intestines of 11 dogs autopsied in Central and Southern Australia in each case produced an established isolate. In contrast, only 1 isolate was obtained by inoculation of faecal cysts. The organisms grew poorly in comparison with isolates from humans or non-canine animal hosts. Light microscopy revealed that the trophozoites had median bodies with the 'claw hammer' appearance typical of G. intestinalis (syn. G. duodenalis, G. lamblia) but that they differed in shape and nuclear morphology from axenic isolates of human or canine origin. Allozymic analysis of electrophoretic data representing 26 loci and phylogenetic analysis of nucleotide sequences obtained from DNA amplified from the glutamate dehydrogenase locus showed that the 11 isolates examined from Australian dogs were genetically distinct from all isolates of G. intestinalis that have been established previously from humans and animals, and also from G. muris. Both analytical methods placed 10 of the Australian canine isolates into a unique genetic lineage (designated Assemblage C) and the eleventh into a deep-rooted second branch (designated Assemblage D), each well separated from the 2 lineages (Assemblages A and B) of G. intestinalis that encompass all the genotypes known to infect humans. In contrast, 4 axenic isolates derived from dogs in Canada and Europe (the only other isolates to have been established from dogs) have genotypes characteristic of genetic Assemblages A or B. The findings indicate that the novel Giardia identified in these rural Australian dogs have a restricted host range, possibly confined to canine species. The poor success rate in establishing Giardia from dogs in vitro suggests, further, that similar genotypes may predominate as canine parasites world-wide. The absence of such organisms among isolates of Giardia that have been established from humans by propagation in suckling mice indicates that they are unlikely to infect humans. However, infection of humans by those dog-derived genotypes that grow in vitro cannot be excluded.

Nucleotide sequence of the murine leukaemia virus amphotropic strain 4070A integrase (IN) coding region and comparative structural analysis of the inferred polypeptide.

The complete nucleotide sequence of the integrase (IN) protein coding region of the murine leukaemia virus (MLV) amphotropic strain 4070A is presented. The sequence comprises 1,224 nucleotides, encoding a 408-residue polypeptide of M(r) 46,312. Alignment of the inferred 4070A IN amino acid sequence with the IN proteins of other MLV showed that substitutions are confined largely to segments within the N- and C-terminal domains. In the N-terminal domain the majority of substitutions occur as contiguous 2- to 6-residue blocks, whereas in the C-terminal domain they occur as isolated entities except within a short segment characterized by deletions/insertions. Selection appears to act on the C-terminal 19 residues of IN rather than on the N-terminal residues of ENV (encoded by overlapping reading frames), suggesting a functional role for this segment. Phylogenetic analyses grouped the sequences into two clusters, one comprising IN from the amphotropic strain 4070A and three ecotropic MLV (CAS-BR-E, Moloney and Friend), the other consisting of IN from three ecotropic MLV (two radiation-induced viruses and AKV) and a mink cell focus-forming (MCF) MLV virus. The same dichotomy and cluster composition was obtained from analysis of the long terminal repeat (LTR) regions from these viruses (consistent with the functional interrelationship of IN and LTR) but not from analysis of envelope protein sequences (consistent with the functional independence of ENV proteins from both IN and LTR). Secondary structure predictions supported features determined from the catalytic domain of human immunodeficiency virus and avian sarcoma virus IN, and identified probable structures within the relatively long N- and C-terminal domains of MLV IN proteins.

Genetic analysis of Giardia from hoofed farm animals reveals artiodactyl-specific and potentially zoonotic genotypes.

Thirty one Giardia isolates, established from six species of hoofed livestock by axenic culture or growth in suckling mice, were compared genetically by analysis of DNA amplified from loci encoding variant surface proteins or the enzyme glutamate dehydrogenase and by allozyme analysis. The isolates were heterogeneous, but all showed affinity with genetic Assemblage A--one of two major assemblages defined previously by analysis of Giardia from humans. Three distinct genotypes were evident. Ten isolates (eight axenic and two established in suckling mice) from an alpaca, pig, horse, cattle and sheep were indistinguishable from human-derived G. intestinalis belonging to a previously designated genetic group (Group I). This genotype seems to have broad host specificity, including a zoonotic potential for humans. Five isolates (two axenic and three established in suckling mice) from an alpaca, a horse and sheep had close affinity with human-derived Group I and Group II G. intestinalis genotypes. The other 16 isolates (comprising both axenic and suckling mouse-propagated cultures derived from cattle, sheep, alpaca, a goat and pigs in Australia and Europe) differed from all other Giardia with "duodenalis" morphology that have been examined by these methods and they segregated as a highly distinct sublineage (referred to herein as 'Novel livestock') within genetic Assemblage A. The predominance of 'Novel livestock' genotypes in the test panel and their apparent exclusive association with artiodactyl hosts indicates that they may be confined to this group of mammals. Assemblage B genotypes, which are prevalent in humans and some other animal species, were not detected.

Comparison of genetic groups determined by molecular and immunological analyses of Giardia isolated from animals and humans in Switzerland and Australia.

Nine axenic isolates of Giardia originating from four different host species in Switzerland were subjected to genetic analysis using the polymerase chain reaction (PCR) to amplify segments of genes encoding different trophozoite variant surface proteins (VSPs). Three genotypes were identified on the basis of product yield, size, and restriction-fragment-length polymorphisms (RFLPs). Five G. duodenalis isolates (O1, B1, B2, B3-1A1 and C1--from a sheep, three calves and a dog, respectively) were classified as belonging to genetic group I of Andrews et al. (1989). DNA amplified from the VSP genes tsp11, tsa417 and vsp1267 of these isolates was indistinguishable in size and restriction characteristics from that amplified from group-I Giardia isolated from humans in Australia. One human-derived Swiss isolate (H2-17A1), typed as belonging to genetic group II, yielded a vsp1267-specific PCR product that was indistinguishable by size or restriction sites from the equivalent 1.6-kb product amplified from human-derived Australian group-II organisms. This isolate also yielded 1.8-kb tsp11 and 0.52-kb tsa417/tsp11-like PCR products possessing RFLPs typical of group-II organisms. Three isolates (O2-4A1, O3 and H3-15K2--originating from two sheep and a human, respectively) represent a novel genotype that is closely related to genetic groups I and II. These three isolates exhibited identical RFLPs in their tsp11 PCR products and failed to yield a vsp1267 PCR product. An antiserum specific for the 90-kDa VSP of the sheep-derived clone O2-4A1 reacted strongly by immunofluorescence and on Western blots with surface proteins from the O2, O3 and H3 isolates only--consistent with the genetic classification determined above. The data provide no evidence for the occurrence of host-specific genotypes.

Molecular genetic analysis of Giardia intestinalis isolates at the glutamate dehydrogenase locus.

Samples of DNA from a panel of Giardia isolated from humans and animals in Europe and shown previously to consist of 2 major genotypes--'Polish' and 'Belgian'--have been compared with human-derived Australian isolates chosen to represent distinct genotypes (genetic groups I-IV) defined previously by allozymic analysis. Homologous 0.52 kilobase (kb) segments of 2 trophozoite surface protein genes (tsa417 and tsp11, both present in isolates belonging to genetic groups I and II) and a 1.2 kb segment of the glutamate dehydrogenase (gdh) gene were amplified by the polymerase chain reaction (PCR) and examined for restriction fragment length polymorphisms (RFLPs). Of 21 'Polish' isolates that were tested, all yielded tsa417-like and tsp11-like PCR products that are characteristic of genetic groups I or II (15 and 6 isolates respectively) in a distinct assemblage of G. intestinalis from Australia (Assemblage A). Conversely, most of the 19 'Belgian' isolates resembled a second assemblage of genotypes defined in Australia (Assemblage B) which contains genetic groups III and IV. RFLP analysis of gdh amplification products showed also that 'Polish' isolates were equivalent to Australian Assemblage A isolates (this analysis does not distinguish between genetic groups I and II) and that 'Belgian' isolates were equivalent to Australian Assemblage B isolates. Comparison of nucleotide sequences determined for a 690 base-pair portion of the gdh PCR products revealed > or = 99.0% identity between group I and group II (Assemblage A/'Polish') genotypes, 88.3-89.7% identity between Assemblage A and Assemblage B genotypes, and > or = 98.4% identity between various Assemblage B/'Belgian' genotypes. The results confirm that the G. duodenalis isolates examined in this study (inclusive of G. intestinalis from humans) can be divided into 2 major genetic clusters: Assemblage A (= 'Polish' genotype) containing allozymically defined groups I and II, and Assemblage B (= 'Belgian' genotype) containing allozymically defined groups III and IV and other related genotypes.

Division of Giardia isolates from humans into two genetically distinct assemblages by electrophoretic analysis of enzymes encoded at 27 loci and comparison with Giardia muris.

Giardia that infect humans are known to be heterogeneous but they are assigned currently to a single species, Giardia intestinalis (syn. G. lamblia). The genetic differences that exist within G. intestinalis have not yet been assessed quantitatively and neither have they been compared in magnitude with those that exist between G. intestinalis and species that are morphologically similar (G. duodenalis) or morphologically distinct (e.g. G. muris). In this study, 60 Australian isolates of G. intestinalis were analysed electrophoretically at 27 enzyme loci and compared with G. muris and a feline isolate of G. duodenalis. Isolates of G. intestinalis were distinct genetically from both G. muris (approximately 80% fixed allelic differences) and the feline G. duodenalis isolate (approximately 75% fixed allelic differences). The G. intestinalis isolates were extremely heterogeneous but they fell into 2 major genetic assemblages, separated by fixed allelic differences at approximately 60% of loci examined. The magnitude of the genetic differences between the G. intestinalis assemblages approached the level that distinguished the G. duodenalis isolate from the morphologically distinct G. muris. This raises important questions about the evolutionary relationships of the assemblages with Homo sapiens, the possibility of ancient or contemporary transmission from animal hosts to humans and the biogeographical origins of the two clusters.

Giardia intestinalis: detection of major genotypes by restriction analysis of gene amplification products.

The polymerase chain reaction (PCR) has been used to amplify a 0.52 kb segment of Giardia intestinalis DNA, using primers specific for nucleotide sequences conserved within two genes (tsp11 and tsa417) that encode homologous, cysteine-rich trophozoite surface proteins. Using products amplified from axenic isolates belonging to genetic groups I and II (defined on the basis of allozyme electrophoresis data), restriction endonuclease analysis revealed both tsp11-like and tsa417-like fragments within all samples. The study also identified among the amplification products of group II organisms an additional fragment, containing a novel PstI site, that is not detected in the reaction products of group I isolates. The recovery of three distinct PCR products from each group II isolate was verified by cloning the fragments into the plasmid vector pGEM-7. Fragments containing the new PstI site possess the ClaI site common to both tsp11 and tsa417-like fragments, but they lack the HindIII site which characterizes tsp11-like fragments and also lack the PstI and KpnI sites which characterize tsa417-like fragments. Spot-blot analyses using cloned fragments of all three types as probes showed strong homologous hybridization but weak heterologous hybridization, indicating that each type differs substantially in nucleotide sequence from the others. Because the samples of Giardia DNA used in the PCR were purified from cultures that had been established from single trophozoites, the data indicate that individual trophozoites belonging to genetic group II possess three homologous genes defined by these related fragments. The presence of a PstI site in the amplified segment of the newly-discovered third gene of group II organisms provides a simple diagnostic means of differentiating group I and II isolates.

Two genes encoding homologous 70-kDa surface proteins are present within individual trophozoites of the binucleate protozoan parasite Giardia intestinalis.

A 0.52-kb DNA sequence encoding part of a major surface antigen of Giardia intestinalis trophozoites has been amplified by the polymerase chain reaction (PCR) using primers specific for nucleotide sequences that are conserved between two apparently related genes, tsp11 (cloned previously from the Australian G. intestinalis isolate, Ad-1) and tsa417 (cloned from the Afghanistani isolate, WB). Restriction analysis revealed that the DNA amplified from each of seven axenic isolates of G. intestinalis was not homogeneous, even though the template DNA had been purified from cultures that had been established from single trophozoites. Every isolate yielded two PCR products, whose respective cleavage fragments corresponded to tsp11 (HindIII+, PstI-, KpnI-) and to tsa417 (HindIII-, PstI+, KpnI+). This was confirmed by cloning individual amplification products into the plasmid vector, pGEM-7Zf(+). The sequence of one cloned fragment (1-P4), derived from the Ad-1 isolate, but possessing restriction sites characteristic of tsa417, exhibited 98.6% identity over 425 bp with tsa417 and only 63.8% identity with the corresponding region of tsp11. The data indicate that individual trophozoites of all the isolates examined contain a copy of each of these homologous genes.

Differentiation of major genotypes of Giardia intestinalis by polymerase chain reaction analysis of a gene encoding a trophozoite surface antigen.

The polymerase chain reaction (PCR) has been used to amplify in vitro a semi-conserved region of a gene encoding an M(r) 68-72,000 surface antigen of Giardia intestinalis trophozoites. Using primers specific for conserved nucleotide sequences identified within the promoter-distal portion of two homologous genes (tsp11 and tsa417) cloned previously from the G. intestinalis isolates Ad-1 (from Australia) and WB (from Afghanistan), a single PCR-amplified DNA fragment of the expected size (0.52 kilobases) was obtained in high yield from either purified DNA or whole trophozoites of the Ad-1 isolate and from every 1 to 9 other axenic G. intestinalis isolates belonging to genetic groups I and II (defined previously on the basis of allozyme electrophoresis data--Andrews et al. 1989). Discernible product was recovered from as few as 2-4 trophozoites. In contrast, 6 G. intestinalis isolates that were assigned by allozymic analysis to genetic groups III/IV yielded small amounts of a 0.37-kilobase (kb) amplification product (with evidence in some samples of an additional 0.4 or 0.18 kb fragment) but no 0.52 kb product. Two animal-derived isolates of G. duodenalis (one from an Australian native rodent, Notomys alexis, the other from a domestic cat) also yielded a single 0.37 kb PCR-amplified fragment, whereas an isolate from another cat produced a 0.34 kb fragment. No product was recovered from G. muris, a morphologically distinct species of Giardia. The results demonstrate that different genotypes of G. duodenalis can be distinguished using this assay and that it is diagnostic for isolates belonging to two major clusters (groups I/II and III/IV) of G. intestinalis. The amplified DNA segment appears to be relatively conserved among group I and group II isolates of G. intestinalis. A related but clearly distinct sequence seems to be conserved among group III/IV isolates of G. intestinalis and some isolates of G. duodenalis.

A gene encoding a 69-kilodalton major surface protein of Giardia intestinalis trophozoites.

A gene encoding a 68.5-kDa trophozoite surface protein (TSP11) of the Australian Giardia intestinalis (syn. G. lamblia) isolate, Ad-1, has been cloned from a genomic expression library screened with an antiserum specific for 3 major surface antigens. Sequence analysis of two overlapping genomic fragments identified a single open reading frame that contained no introns and predicted a cysteine-rich, 667-residue polypeptide with features common to other trophozoite surface proteins. These include the presence of 27 copies of the 4-amino acid Cys-X-X-Cys motif, an N-terminal signal sequence and a highly conserved, hydrophobic C-terminal segment. Transcripts from the tsp11 gene were detected as a single band on Northern blots using total RNA extracted from Ad-1 trophozoites. Primer extension analysis indicated that the mRNA has a 5' untranslated region of only 5 nt, similar to the very short (1-6 nt) leader sequences reported for other Giardia mRNAs. A large portion of the promoter distal segment of tsp11 has homology with tsa417, a gene encoding a 72.5-kDa trophozoite surface antigen of the Afghanistan-derived G. intestinalis isolate, WB [13].

Distinct genetic groups of Giardia intestinalis distinguished by restriction fragment length polymorphisms.

The taxonomic status of the parasitic protozoal species Giardia intestinalis depends on the morphological similarity of all Giardia isolated from humans and the presumption that Giardia are host-specific. On the basis of electrophoretic data derived from examination of 26 enzyme loci in Australian isolates, it has been proposed that G. intestinalis is a species complex comprising three or four genetically distinct (but morphologically cryptic) species. These received the tentative designations of genetic groups I-IV (R. H. Andrews, M. Adams, P. F. L. Boreham, G. Mayrhofer & B. P. Meloni. International Journal for Parasitology 19, 183-190, 1989). In the present study, two unrelated DNA probes (one specific for a gene encoding a trophozoite surface protein, the other detecting a non-coding repetitive sequence within the G. intestinalis genome) were used in Southern hybridization analyses to examine 10 axenic isolates of G. intestinalis, established from diverse geographical regions in Australia, together with the Portland-1 isolate from the USA. Both probes identified every isolate unambiguously as belonging to one or other of two genetic clusters. Electrophoretic analysis of the same samples indicated that these clusters correspond to the previously defined genetic groups I and II. No heterogeneity was apparent within the seven group I isolates using either probe. However, when probed with the repetitive sequence, the four isolates belonging to group II exhibited small differences in banding patterns, suggesting that this group may be less homogeneous than group I.(ABSTRACT TRUNCATED AT 250 WORDS)