A new quantitative in vitro microculture method for Giardia duodenalis trophozoites

A reliable, rapid and low-cost method for drug sensitivity determination of Giardia duodenalis trophozoites (WB-strain) was developed in a 96-well plate. Using a standard inoculum of 5 x 10(4) trophozoites per well (300 microl), good growth was obtained after sealing the plate with an air-tight adhesive tape and incubation at 37 degrees C for 72 h in modified TYI-S-33 medium. Viable burdens were quantified using the formazan dyes MTT (100 microg/well) and XTT (20 microg/well) and the fluorescent substrate resazurin (2.5 microg/well). Prior removal of the culture medium is required since it causes spontaneous reduction of the substrate. Resazurin proved to be far superior to MTT and XTT with a level of sensitivity of about 3 x 10(4) trophozoites. Inhibitory concentrations (IC(50)) of several anti-giardial reference drugs were within the range of published values: metronidazole 2.25 microM, tinidazole 1.75 microM, albendazole 0.10 microM, furazolidone 2.00 microM and quinacrine 0.32 microM. The broad-spectrum antibiotics chloramphenicol, rifampicin, penicillin G+streptomycin and gentamycin were devoid of any inhibitory activity and are considered suitable for decontamination during excystation experiments.     

Improvement in cyst recovery and molecular detection of Giardia duodenalis from stool samples

Molecular Biology Reports - Molecular detection of Giardia duodenalis by polymerase chain reaction (PCR) is difficult in faecal samples due to inhibitors that contaminate DNA preparations, or due...     

A simple method for cloning leishmanial promastigotes

A method of cloning leishmanial promastigotes is described in which mid-exponential phase cultures are diluted to contain approximately 3 X 10(3) promastigotes per ml. Hanging-drop preparations made from 0.2-0.4 microliter volumes of the diluted culture seen to contain a single promastigote are picked-up in glass capillary tubes. Additional culture medium is taken into the capillaries which are then heat-sealed and incubated at 22 degrees C. Growth of leishmanial promastigotes inside the sealed capillary tubes is followed by direct microscopic observation through the walls of the tubes. When active promastigotes are seen the contents of the tubes are inoculated into small volumes of culture medium. The method is extremely easy to use, requires no specialised apparatus, and has been successfully used with different strains and species of Leishmania, with up to 100 percent of the cloned organisms growing.     

A simple and efficient method for obtaining urine samples from rats

A method for collection of urine from rats was developed that is simple, reliable, and efficient. A 5 ml. polystyrene beaker is placed over the urethra and the base of the tail is stimulated with the fingers of one hand. Depending on the quality and quantity of urine needed the perineal area may be shaved and the beaker may be held by hand or attached with tape.     

A simple method for obtaining blood samples from mature frogs

A technique was developed for obtaining periodic blood samples from mature frogs. Up to 150 microliter of blood was collected at weekly intervals. Anesthetization was not required. The data compiled with this technique indicated that activity-induced hyperglycemia was minimal.     

Virulent Avian Giardia duodenalis Pathogenic for Mice

Early in 1995, a sulphur-crested cockatoo captured in the wild died along with several other cage mates, apparently of an overwhelming, acute infection of Giardia. Trophozoites isolated from the dead bird and established in traditional Giardia axenic medium were infective to mice and established chronic infections associated with weight gain impairment. Genetically and morphologically, the Giardia isolated from the bird belonged to the duodenalis group. Here, Jacqui Upcroft, Ann McDonnell and Peter Upcroft present data on pathogenic avian Giardia with he potential to contaminate watersheds and discuss the implications.     

Multilocus genotyping of Giardia duodenalis from pigs in Korea

Giardia duodenalis (syn. G. intestinalis, G. lamblia) is an important zoonotic parasite infecting livestock (including pigs) through ingesting cysts in contaminated food or water. This parasite has been classified into eight different genetic assemblages, A to H. Here, we examined the individual-level prevalence of G. duodenalis in domestic pig farms and confirmed host specificity by genotype comparisons. Samples were collected from southern and central Korea, between May 2017 and January 2019. DNA directly extracted from 745 pig fecal specimens were tested by PCR for G. duodenalis small subunit ribosomal RNA (ssu rRNA), glutamate dehydrogenase (gdh), and β-giardin gene sequences. Based on ssu rRNA PCR, 110 (14.8%) were positive for G. duodenalis. Infection risk was the highest in the fattener group (31/139, 22.3%) and during the autumn season (52/245, 21.2%: p < .001). No statistically significant differences in risk for infection were observed between fecal types (normal versus diarrheal). Fifty ssu rRNA samples, three gdh samples, and five β-giardin samples were successfully sequenced and genotyped. Ssu rRNA assemblage sequence analysis identified E (40.0%, 20/50), D (34.0%, 17/50), C (24.0%, 12/50), and A (2.0%, 1/50). The gdh locus identified three samples as assemblage E, and the β-giardin locus identified four samples as assemblage E and one as assemblage C. Assemblage A sequences obtained (ssu rRNA; MK430919) had 100% identity with Giardia sequences isolated from a Korean individual (AJ293301), indicating the potential of zoonotic transmission. Continuous management and monitoring for prevention of transmission and protection of animal and human health are essential.     

A relatively simple and rapid multi-component method for analysis of steroid profiles in blood, fecal and liver samples

Various methods of steroid analysis were assessed using radiolabeled steroids and thin layer chromatography. Three reversed phase chromatography systems were evaluated for separation and recovery of steroids extracted from blood, liver tissue and feces. The use of different numbers of Sep-Pak C(18) cartridges for the purification of steroid extracts was examined and steroid recoveries were measured and compared. The results indicated that recoveries were best when 4-6 cartridges were used. Rapid and slow procedures of enzymatic hydrolysis and acidic solvolysis of steroid conjugates were compared. A new and relatively rapid method for analysis of steroid profiles in liver, blood and fecal samples was developed. Assessment of this method showed that steroid recoveries were improved compared to existing methods with percentage recoveries of 64.1-82.5 for liver samples, 55.2-75 for blood samples, and 65.1-76.3 for fecal samples.     

A novel Giardia duodenalis assemblage A subtype in fallow deer

The molecular identification of species and genotypes of Giardia spp. infecting wild mammals represents the most reliable tool to understand the role played by these animals as reservoirs of cysts infectious for human and other animals. Of 139 fecal samples collected from fallow deer (Dama dama L.) hunted in a Natural Reserve of northern Italy, the prevalence of Giardia sp. was 11.5% (16 of 139 animals), and it was higher in fawns than in older animals. Fragments of the betagiardin and triose phosphate isomerase (tpi) genes were successfully polymerase chain reaction amplified and sequenced from 8 isolates. No sequence variation was observed between isolates at the 2 genetic loci. Sequence and phylogenetic analyses identified a Giardia duodenalis subtype that clusters with assemblage A isolates and that shows homologies of 98 and 97% at the beta-giardin and tpi loci, respectively, compared with the A1 subtype. Because the G. duodenalis subtype found in fecal samples of fallow deer has never been detected previously, its role as a pathogen for humans and domestic animals is unknown, but, considering its genetic distinctiveness, it is likely to be low.     

A simple method for freeze-fracture of monolayer cultures

A simple method is described for the freeze-fracture in situ of monolayer cultures grown on gold carriers coated with a thin layer of silicon monoxide. Preliminary observations on 3T3 mouse embryo fibroblasts indicate that this technique exposes large areas of cell membrane, making it possible to determine how areas of membrane specialization are related to the cell as a whole and to regions of cellular interaction. 3T3 cells cultured on silicon monoxide show no modification of growth properties compared to cells growing on Falcon plastic, and other cell lines also appear to grow well on this substrate.     

Mixed Giardia duodenalis assemblage A and E infections in calves

A molecular epidemiological study was conducted on 100 dairy (499 calves) and 50 beef (333 calves) farms in Belgium to estimate the prevalence of different Giardia duodenalis assemblages in calves younger than 10 weeks of age. Positive samples from the epidemiological study and from a previous clinical study were selected and genotyped based on the amplification of the beta-giardin gene. To investigate the occurence of mixed assemblage A and E infections in calves, a novel assemblage-specific PCR was developed based on the triose-phosphate isomerase gene. The prevalence was 22% (95% Probability Interval (PI): 12-34%) in dairy calves and 45% (95% PI: 30-64%) in beef calves. In total, 120 Giardia-positive samples from dairy and beef calves collected in the epidemiological study and from clinically affected calves were identified based on the amplification of the beta-giardin gene. Overall G. duodenalis assemblage E was more prevalent (in 64% of the samples), although the majority (59%) of the dairy calves were infected with G. duodenalis assemblage A. Furthermore, mixed G. duodenalis assemblage A and E infections were identified in 31% of the calf samples (n=101) using the assemblage-specific PCR. We believe this is the first report of mixed infections in calves, and the results of the present study indicate that calves, although mainly infected with the host-specific G. duodenalis assemblage E, are frequently infected with the zoonotic assemblage A, either as a mixed or mono-infection, suggesting that calves might be underestimated as a potential zoonotic reservoir for human infections.     

A simple and rapid method for cloning insect vitellogenin cDNAs

We describe a simple and rapid method for cloning insect vitellogenin (Vg) cDNAs. The method relies on the facts that insect Vg amino acid sequences can be aligned confidently along their entire lengths and that a short, highly conserved GL/ICG motif and up to nine cysteine residues that follow at conserved locations are present near the C-termini. An adaptor-ligated double-strand cDNA library is constructed from poly(A)+ RNA prepared from vitellogenic female fat body tissues using a commercial kit, and subjected to PCR with each of the degenerate nucleotide sequences for the GL/ICG motif and the adaptor sequence as primers. The PCR products (0.7-0.9 kb, representing the 3' portion) are cloned, the nucleotide sequences are determined, and the deduced amino acid sequences are aligned with the known insect Vg sequences starting from the GL/ICG motif. Gene-specific primers corresponding to the sequences near the 5'-termini of the initial clones and the adaptor sequence are employed to obtain the remaining 5' portion of the Vg cDNAs. The method was successfully applied to the bean bug Plautia stali (Heteroptera), revealing three Vg genes.     

Detection of Giardia duodenalis antigen in human fecal eluates by enzyme-linked immunosorbent assay using polyclonal antibodies

The present study developed and standardized an enzime-linked immunosorbent assay (ELISA) to detect Giardia antigen in feces using rabbit polyclonal antibodies. Giardia cysts were purified from human fecal samples by sucrose and percoll gradients. Gerbils (Meriones unguiculatus) were infected to obtain trophozoites. Rabbits were inoculated with either cyst or trophozoite antigens of 14 Colombian Giardia isolates to develop antibodies against the respective stages. The IgG anti-Giardia were purified by sequential caprylic acid and ammonium sulfate precipitation. A portion of these polyclonal antibodies was linked to alkaline phosphatase (conjugate). One hundred and ninety six samples of human feces, from different patients, were tested by parasitologic diagnosis: 69 were positive for Giardia cysts, 56 had no Giardia parasites, and 71 revealed parasites other than Giardia. The optimal concentration of polyclonal antibodies for antigen capture was 40 g/ml and the optimal conjugate dilution was 1:100. The absorbance cut-off value was 0.24. The parameters of the ELISA test for Giardia antigen detection were: sensitivity, 100% (95% CI: 93.4-100%); specificity, 95% (95% CI: 88.6-97.6%); positive predictive value, 91% (95% CI: 81.4-95.9%); and negative predictive value, 100% (95% CI: 96.1-100%). This ELISA will improve the diagnosis of Giardia infections in Colombia and will be useful in following patients after treatment.     

Prevalence and genotyping of human isolates of Giardia duodenalis from Albania

Microscopical and PCR-based techniques were performed in order to investigate the prevalence of infection and the genotypes of Giardia duodenalis from 125 stool samples collected from children living in the urban and the rural areas of Tirana (Albania) and hospitalized with acute gastroenteritis. 7 out of 125 samples resulted positive for Giardia at the microscopic examination (5.6%). In 50 selected samples including the 7 samples positive for Giardia by microscopy, 3 and 15 additional positive samples were detected by immunofluorescence and PCR, respectively. Seasonality appeared as an important parameter to be evaluated in order to better understand the prevalence of infection. Sequence analysis revealed both human Assemblage A and B. This result represents the first data on G. duodenalis genotypes in Albania.     

A simple method to isolate salt-tolerant myxobacteria from marine samples

This paper describes a simple method for the isolation of salt-tolerant myxobacteria from marine conditions. As the results show in this paper, salt-tolerant myxobacteria are found to be able to grow, but unable to form fruiting bodies at high salt concentrations. The fruiting body structures of the salt-tolerant strains were all formed in conditions with lower seawater content, i.e. lower than 60% seawater (about 2.0% salt content) or distilled water supplemented with MgCl(2). The method picked up the fruiting bodies for isolation.     

Immunoblot patterns of Giardia duodenalis isolates from different hosts and geographical locations

Eighteen isolates of Giardia duodenalis from animal and human sources were studied for protein differences by polyacrylamide gel electrophoresis and for antigenic differences by immunoblot analysis. The polyacrylamide gels showed that whilst the isolates were for the most part homogeneous in their protein banding patterns, some isolates did show some differences. The immunoblot analysis yielded many bands, including prominent bands of 32 and 66 kilodaltons. Five of the six isolates that showed differences in protein banding pattern also showed differences in antigenic reactivity. Our findings suggest that differences can be seen with the use of immunoblotting and that this technique is a tool that may be useful for isolate differentiation when used in conjunction with other techniques.     

Antibody-magnetite method for selective concentration of Giardia lamblia cysts from water samples.

An antibody-magnetite method was developed in order to selectively concentrate Giardia cysts from water samples. The indirect technique employed a mouse immunoglobulin G anti-Giardia antibody as a primary antibody and an anti-mouse immunoglobulin G antibody-coated magnetite particle as a secondary labeling reagent. The magnetically labeled cysts were then concentrated by high-gradient magnetic separation. Ninety percent of the seeded cysts were recovered from buffer when this method was employed. An average of 82% of the seeded cysts were recovered from water samples with various turbidities. Significantly higher cyst recoveries were obtained from water samples with turbidities below 600 nephelometric turbidity units.