Genotyping isolates and clones of Giardia duodenalis by polymerase chain reaction: implications for the detection of genetic variation among protozoan parasite species.

Detection of genetic variation among microorganisms can be done by DNA fingerprinting using the polymerase chain reaction (PCR). Application of primers directed to polymorphic DNA leads to the amplification of DNA fragments which differ in length when different species or isolates of a single species are compared. It has been demonstrated that PCR primers resembling eukaryotic repeat motifs enable the straightforward genetic differentiation of Giardia duodenalis isolates. Depending on the repeat motif, genetic variation between cloned G. duodenalis lines could also be detected. DNA polymorphisms could also be detected by random amplification of polymorphic DNA (RAPD) analyses. When the results obtained for G. duodenalis are compared to those found for another protozoan parasite, Naegleria fowleri, clear differences are encountered. In contrast to the findings for G. duodenalis, the repeat motif primers did not allow the discrimination of 'N. fowleri isolates. Apparently, as determined by this PCR-mediated genotyping, genetic variation occurs in G. duodenalis with increased frequency at the isolate level as compared to N. fowleri. The possible implications of this observation for clonality or the definition of a species in protozoan parasites will be discussed.

Detection of Echinococcus multilocularis in foxes in The Netherlands.

Echinococcus multilocularis was demonstrated in 5 out of 272 foxes in The Netherlands close to the border with Germany and Belgium. Besides microscopic examination of mucosal scrapings, two different PCR assays were used based on the detection of E. multilocularis DNA in colon content. Two distinct areas in The Netherlands were positive for E. multilocularis. Two positive foxes were found in the northern province of Groningen and three positive foxes were found in the southern province of Limburg. Both PCR assays detected more positive foxes compared to microscopic examination of the intestinal content. This is the first report of E. multilocularis in foxes occurring in The Netherlands.

Hepatitis in farmed hatchling Nile crocodiles (Crocodylus niloticus) due to chlamydial infection.

An investigation into the cause of acute mortality in farmed hatchling crocodiles Crocodylus niloticus led to the isolation of chlamydia from the livers of affected animals. Prominent pathological finds were acute hepatitis with intracellular chlamydial colonies and generalized oedema. A chlamydia presumed to be C. psittaci was isolated from livers of affected hatchlings. Mortality subsided after treatment with oxytetracycline. This disease is now recognized as being a major problem on crocodile farms in Zimbabwe.

Comparison of the internal transcribed spacer, ITS 1, from Toxoplasma gondii isolates and Neospora caninum.

The internal transcribed spacer (ITS1) region and the 5' part of the 5.8S ribosomal RNA gene of the ribosomal DNA repeat from 20 Toxoplasma gondil isolates was sequenced and found to be identical in all isolates, independent of host origin or virulence to mice. The ITS1 region from the closely related coccidian parasite Neospora caninum differed in 22% of its nucleotides. Hence, the ITS1 region provides a good marker for the distinction of T. gondii and N. caninum but is not useful for epidemiology studies of T. gondii.

Characterization of Giardia duodenalis by polymerase-chain-reaction fingerprinting.

The development of a polymerase chain reaction (PCR) based fingerprinting method for the characterization of Giardia duodenalis isolates is described. The method uses three different PCRs; one is specific for the A ("Polish") major group of G. duodenalis isolates, another is specific for the B ("Belgian") group of isolates; and one amplifies a fragment of the glutamate dehydrogenase gene present in all G. duodenalis isolates. The PCRs perform highly sensitively on DNA from cultured trophozoites. Isolates were further characterized by restriction-fragment-length polymorphism (RFLP) analysis of the PCR products. In this way, representative isolates from the A and B groups could be grouped together into a number of subgroups. The stability of the genotypes with time and the reproducibility of the two methods were tested on cloned and subcloned lines from a number of isolates and proved to be highly satisfactory. The PCR/RFLP method was evaluated on cysts derived from a number of human patients. It is concluded that the PCR fingerprinting method described in this paper provides a reliable characterization method for Giardia isolates and has the potential to be used as a direct method of typing G. duodenalis cysts from feces.

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.

Comparison of Giardia isolates from different laboratories by isoenzyme analysis and recombinant DNA probes.

A total of 13 new Giardia isolates were established in axenic culture. All of the new isolates were obtained by excystation of Giardia cysts from the feces of patients in Dutch hospitals. These isolates were subjected to isoenzyme and DNA analysis together with isolates from Poland, Belgium, and various other parts of the world. Isoenzyme analysis revealed that nearly all of the newly established isolates exhibited unique zymodemes. Isolates obtained from individuals from Belgium and Poland, on the other hand, displayed single zymodemes. Genomic DNA libraries were constructed from isolates belonging to the latter two zymodemes; specific and common recombinant DNA clones were selected from these libraries. Differential screening revealed that the two isolates had only 80% of the clones in common. Restriction-fragment-length polymorphism analysis using three different probes together with two synthetic probes that are complementary to Giardia structural protein genes led to the separation of all isolates into two major groups; within these groups, a further division could be made by application of other techniques or probes. The results of DNA analysis and zymodeme classification were in general agreement; in the present report they are compared with the data in the literature and discussed.