Excystation and culturing of human and animal Giardia spp. by using gerbils and TYI-S-33 medium.

Mongolian gerbils were used as an animal model to excyst and host Giardia spp. isolated from meadow voles, dogs, beavers, and humans. Both cysts and trophozoites were used to establish infections. Gerbils were infected with Giardia duodenalis from beaver, dog, and human sources, and the trophozoites were extracted and cultured in Diamond TYI-S-33 medium. The use of gentamicin and ampicillin in the medium, coupled with treatment of gerbils with gentamicin before they were sacrificed, permitted the elimination of trophozoite purification techniques before culturing. An extract of whole bovine calf blood, CLEX, was substituted for fetal bovine serum in TYI-S-33 medium and was found to be both adequate and less expensive.     

Serial subcultivation of Giardia lamblia in Keister's modified TYI-S-33 medium containing ultroser G.

The ability of Giardia strains of the duodenalis type to grow in Keister's modified TYI-S-33 medium varies with serum lot. Recently, strains of Giardia including MR4, WB, and Human-1-Portland, have been cultivated in Keister's modified TYI-S-33 medium containing the serum substitute Ultroser G and have been cultured serially as least 40 times. An optimal concentration of 8% Ultroser G promotes maximal growth in Keister's modified TYI-S-33 medium for all three strains. This concentration of Ultroser G will produce a two-log increase in the number of trophozoites in approximately three days post-inoculation. Generation times for the trophozoites ranging from 6 to 11 h have been achieved in Keister's modified TYI-S-33 containing 10% adult bovine serum and from 8 to 13 h in Keister's modified TYI-S-33 with 8% Ultroser G. Despite the excellent growth of Giardia strains in medium containing Ultroser G, the maximum trophozoite density is only about half of that achieved in Keister's modified TYI-S-33 medium supplemented with 10% adult bovine serum. Comparisons of trophozoites grown with serum or the serum substitute reveal no discernable differences in morphology and motility. Additionally, these strains have been successfully cryopreserved and revived in Keister's modified TYI-S-33 medium supplemented with Ultroser G. Because Ultroser G is a characterized mixture of six main groups of ingredients (growth factors, adhesion factors, mineral trace elements, hormones, binding proteins, and vitamins), the variability in cell proliferation that may occur when changing serum lots should be minimized when using this product.     

Excystation of Giardia muris induced by a phosphate-bicarbonate medium: localization of acid phosphatase

Giardia muris cysts were incubated briefly in an aqueous induction medium of 0.1 M potassium phosphate with 0.1, 0.2, or 0.3 M sodium bicarbonate. High rates of excystation (91.1-96.7%) were recorded within 5 min after the cysts were placed in trypticase-yeast extract-iron-serum (TYI-S) medium. Substitution of phosphate-buffered saline for TYI-S as the excystation medium resulted in high rates (95.9%) of excystation but required an incubation of 15 min. Excystation was inhibited by the presence of 4-4'-diisothiocyanatostilbene-2-2'-disulfonic acid (DIDS), a specific inhibitor of vacuolar and lysosomal acidification. Microscopic observation showed the loss of the peritrophic space and a change in the refractile nature of the cyst wall prior to excystation. Histochemical studies demonstrated a reaction product of acid phosphatase activity in the lysosomelike peripheral vacuoles in induced cysts and in the peritrophic space of cysts placed in excystation medium. Staining with acridine orange suggested that the peripheral vacuoles become acidified during induction. This staining was inhibited also by DIDS. These studies show that in vitro excystation can be produced at high rates by easily prepared media without exogenous enzymes, low pH, reducing agents, or complex components. The data also suggest that excystation may be stimulated by the bicarbonate-phosphate medium accompanied by acidification of the peripheral vacuoles and the release of their contents into the peritrophic space.     

Serial Subcultivation of Giardia lamblia in Keister's Modified TYI-S-33 Medium Containing Ultroser G

ABSTRACT. The ability of Giardia strains of the duodenalis type to grow in Keister's modified TYI-S-33 medium varies with serum lot. Recently, strains of Giardia including MR4, WB, and Human-1-Portland, have been cultivated in Keister's modified TYI-S-33 medium containing the serum substitute Ultroser G and have been cultured serially at least 40 times. An optimal concentration of 8% Ultroser G promotes maximal growth in Keister's modified TYI-S-33 medium for all three strains. This concentration of Ultroser G will produce a two-log increase in the number of trophozoites in approximately three days post-inoculation. Generation times for the trophozoites ranging from 6 to 11 h have been achieved in Keister's modified TYI-S-33 containing 10% adult bovine serum and from 8 to 13 h in Keister's modified TYI-S-33 with 8% Ultroser G. Despite the excellent growth of Giardia strains in medium containing Ultroser G, the maximum trophozoite density is only about half of that achieved in Keister's modified TYI-S-33 medium supplemented with 10% adult bovine serum. Comparisons of trophozoites grown with serum or the serum substitute reveal no discernable differences in morphology and motility. Additionally, these strains have been successfully cryopreserved and revived in Keister's modified TYI-S-33 medium supplemented with Ultroser G. Because Ultroser G is a characterized mixture of six main groups of ingredients (growth factors, adhesion factors, mineral trace elements, hormones, binding proteins, and vitamins), the variability in cell proliferation that may occur when changing serum lots should be minimized when using this product.     

Sub-typing of animal and human Campylobacter spp. using RAPD

R.H. MADDEN, L. MORAN AND P. SCATES. 1996. Based on a 10-mer primer (5'- CCTGTTAGCC-3'), a random amplified polymorphic DNA (RAPD) method for typing Campylobacter coli isolated from pigs was developed. The method proved effective with a high discrimination and good reproducibility. In contrast with serotyping no untypable strains were found out of a total of 269 isolates (veterinary, food and clinical) examined. The method was also successfully applied to typing Campylobacter jejuni from a similar range of sources.     

Comparative studies on the pattern of infection with Giardia spp. in mongolian gerbils

Mongolian gerbils (Meriones unguiculatus) were inoculated with known numbers of Giardia cysts isolated from humans, beavers and mice. The pattern of cyst release in the feces was studied for a period of 35 days. After a latent period of 5 days, animals infected with G. muris release cysts in their feces every day until day 14. Gerbils infected with human or beaver isolates released cysts in their feces intermittently for 30 days. These results indicated that the mode of cyst release in these animals was characteristic of the parasite, and was independent of the host. Mongolian gerbils acquire complete resistance upon homologous species challenge but demonstrate only partial protection when challenged with a different species of Giardia. We concluded that the Mongolian gerbil model could be useful in epidemiological studies for two reasons: it can be used for determination of cyst viability, and for the identification of the etiological agent.     

Advantages of using swine serum in culturing animal and human cell lines

Using the culture of lymphoid human cells, mice myeloma cells and hybridoma (the latter was obtained by fusion of myeloma cells with those of mice spleen immunized with phage lambda), the fetal serum was shown to be surpassed in growth activity by that from adult swines. This fact is especially pronounced at small serum concentrations. When hybridoma cells were cultured there were no differences in titre of monoclonal antibodies specific to phage lambda with the use of both sera. The possibility of substitution of swine serum for human one was demonstrated using the culture of lymphocytes from human peripheral blood.     

The biology of Giardia spp.

Gardia spp. are flagellated protozoans that parasitize the small intestines of mammals, birds, reptiles, and amphibians. The infectious cysts begin excysting in the acidic environment of the stomach and become trophozoites (the vegetative form). The trophozoites attach to the intestinal mucosa through the suction generated by a ventral disk and cause diarrhea and malabsorption by mechanisms that are not well understood. Giardia spp. have a number of unique features, including a predominantly anaerobic metabolism, complete dependence on salvage of exogenous nucleotides, a limited ability to synthesize and degrade carbohydrates and lipids, and two nuclei that are equal by all criteria that have been tested. The small size and unique sequence of G. lamblia rRNA molecules have led to the proposal that Giardia is the most primitive eukaryotic organism. Three Giardia spp. have been identified by light lamblia, G. muris, and G. agilis, but electron microscopy has allowed further species to be described within the G. lamblia group, some of which have been substantiated by differences in the rDNA. Animal models and human infections have led to the conclusion that intestinal infection is controlled primarily through the humoral immune system (T-cell dependent in the mouse model). A major immunogenic cysteine-rich surface antigen is able to vary in vitro and in vivo in the course of an infection and may provide a means of evading the host immune response or perhaps a means of adapting to different intestinal environments.     

The biology of Giardia spp.

Gardia spp. are flagellated protozoans that parasitize the small intestines of mammals, birds, reptiles, and amphibians. The infectious cysts begin excysting in the acidic environment of the stomach and become trophozoites (the vegetative form). The trophozoites attach to the intestinal mucosa through the suction generated by a ventral disk and cause diarrhea and malabsorption by mechanisms that are not well understood. Giardia spp. have a number of unique features, including a predominantly anaerobic metabolism, complete dependence on salvage of exogenous nucleotides, a limited ability to synthesize and degrade carbohydrates and lipids, and two nuclei that are equal by all criteria that have been tested. The small size and unique sequence of G. lamblia rRNA molecules have led to the proposal that Giardia is the most primitive eukaryotic organism. Three Giardia spp. have been identified by light lamblia, G. muris, and G. agilis, but electron microscopy has allowed further species to be described within the G. lamblia group, some of which have been substantiated by differences in the rDNA. Animal models and human infections have led to the conclusion that intestinal infection is controlled primarily through the humoral immune system (T-cell dependent in the mouse model). A major immunogenic cysteine-rich surface antigen is able to vary in vitro and in vivo in the course of an infection and may provide a means of evading the host immune response or perhaps a means of adapting to different intestinal environments.     

Studies of Giardia spp. and Cryptosporidium spp. in two adjacent watersheds.

Two adjacent British Columbia, Canada, watersheds with similar topographical features were studied. Both the Black Mountain Irrigation District (BMID) and the Vernon Irrigation District (VID) serve rural agricultural communities which are active in cattle ranching. The present study was carried out in five phases, during which a total of 249 surface water samples were tested in the study watersheds. The aims of these phases were to determine levels of parasite contamination in raw water samples collected from the intakes as well as from other sites in each watershed and to investigate cattle in the watersheds as potential sources of parasite contamination of surface drinking water supplies. Giardia cysts were not detected in the raw water samples collected from lake sources at the headwaters of both watersheds but were found in 100% (70 or 70) of water samples collected at the BMID intake and 97% (68 of 70) of water samples collected at the VID intake. Significantly higher levels (P < 0.05) of Giardia cysts were found at the BMID intake (phase 1, 7 to 2,215 cysts per 100 liters; phase 3, 4.6 to 1,880 cysts per 100 liters) when compared with that of the VID intake (2 to 114 cysts per 100 liters). The BMID watershed has a more complex system of surface water sources than the VID watershed. Cattle have access to creeks in the BMID watershed, whereas access is restricted in the VID watershed. Collection of raw water samples from a creek upstream and downstream of a cattle ranch in the BMID watershed showed that the downstream location had significantly higher (P < 0.05) levels (0.6 to 42.9 cysts per 100 liters and 1.4 to 300.0 oocysts per 100 liters) of both Giardia cysts and Cryptosporidium oocysts than those of the upstream location (0.5 to 34.4 cysts per 100 liters and 0.5 to 34.4 oocysts per 100 liters). Peak concentrations of both parasites coincided with calving activity. Fecal samples, collected from cattle in both watersheds, showed 10% (3 of 30) in the BMID and 50% (5 of 10) in the VID watersheds to be Giardia positive. No Cryptosporidium-positive fecal samples were found. Giardia cysts isolated from the BMID watershed were repeatedly infective to gerbils in contrast to those from the VID watershed. The 10 BMID drinking water Giardia isolates retrieved into culture and biotyped showed zymodeme and karyotype heterogeneity. The differences in patterns of parasite contamination and cattle management practices contribute to the unique watershed characteristics observed between two areas which are topographically similar and geographically adjacent.     

Variation between human and animal isolates of Giardia as demonstrated by DNA fingerprinting

Five Giardia stocks collected from animals and man in Alberta, Canada, were compared by DNA fingerprinting. Although many DNA bands were common to all stocks, differences in the DNA banding patterns were seen. These same stocks had previously been shown to be identical by restriction enzyme cleavage of genomic DNA.     

Enumeration of Klebsiella spp. in cold water by using MacConkey-inositol-potassium tellurite medium.

MacConkey-inositol-potassium tellurite agar was field tested for its ability to selectively enumerate Klebsiella species from the waters of the Saint John River Basin, which include fresh and marine waters. Water temperature varied from 1 to 6 degrees C during the survey period. Results of the study indicated that 77% of the typical colonies on MacConkey-inositol-potassium tellurite medium were Klebsiella species, but the total Klebsiella population enumerated was greatly underestimated.     

Similarities of Giardia antigens derived from human and animal sources

A total of 37 Giardia stocks isolated from humans and 14 stocks derived from animal sources have been analysed for antigenic differences. Separation of the proteins of the stocks by polyacrylamide gel electrophoresis showed no major differences among the stocks. Immunoblotting of these antigens demonstrated some minor differences which were not correlated with geographic location, allozyme type, virulence or any other distinguishing characteristic of the stocks. Immunofluorescence tests using monoclonal antibodies revealed some differences between stocks but the monoclonal antibodies did not significantly inhibit growth in inhibition assays.     

Prevalence of Cryptosporidium spp. and Giardia spp. in five marine mammal species

Cryptosporidium spp. and Giardia spp. are protozoan parasites that are often associated with severe diarrheal disease in a variety of mammals. Although these parasites have been extensively studied in terrestrial ecosystems, little is known about either parasite in the marine environment. Therefore, the objective of this study was to determine the prevalence of both Cryptosporidium spp. and Giardia spp. in 5 marine mammal species. Fecal samples were collected from 39 bowhead whales (Balaena mysticetus), 49 North Atlantic right whales (Eubalaena glacialis), 31 ringed seals (Phoca hispida), 22 bearded seals (Erignathus barbatus), and 18 beluga whales (Delphinapterus leucas) between 1998 and 2003. Using an immunofluorescent assay, parasites were detected in the feces of bowhead whales, right whales, and ringed seals, while neither parasite was detected in samples from bearded seals or beluga whales. Overall, prevalences were highest in ringed seals (Cryptosporidium spp., 22.6%; Giardia spp., 64.5%) and right whales (Cryptosporidium spp., 24.5%; Giardia spp., 71.4%) and lowest in bowhead whales (Cryptosporidium spp., 5.1%; Giardia spp., 33.3%). To our knowledge, this is the first report of Cryptosporidium spp. and Giardia spp. in either whale species and of Cryptosporidium spp. in the ringed seal.     

Enumeration of Klebsiella spp. in cold water by using MacConkey-inositol-potassium tellurite medium

MacConkey-inositol-potassium tellurite agar was field tested for its ability to selectively enumerate Klebsiella species from the waters of the Saint John River Basin, which include fresh and marine waters. Water temperature varied from 1 to 6 degrees C during the survey period. Results of the study indicated that 77% of the typical colonies on MacConkey-inositol-potassium tellurite medium were Klebsiella species, but the total Klebsiella population enumerated was greatly underestimated.     

Reservoirs of Giardia spp. in southwestern Alberta

A survey of potential hosts of Giardia spp. was carried out during 1982 and 1983 in the Kananaskis Valley and Banff National Park, Alberta, Canada. Diagnosis was based mainly on fecal analysis but a few animals were examined at necropsy and scrapings from the small intestine analyzed. A total of 304 specimens was examined from humans (Homo sapiens L.) and a variety of animal species. Cysts and/or trophozoites of Giardia were found in 10.5% of the specimens examined. Positive samples were found from 20 of 21 red-backed voles (Clethrionomys gapperi Vigors), two of six meadow voles (Microtus pennsylvanicus Ord), one of three long-tailed voles (Microtus longicaudus Allen), five of 50 deer mice (Peromyscus maniculatus Mearns), and two of 58 beavers (Castor canadensis Kuhl). Cysts obtained from a beaver were successfully introduced to gerbils (Meriones unguiculatus Milne-Edwards) and the trophozoites obtained were cultured in vitro.     

Improved Method for High-Yield Excystation and Purification of Infective Sporozoites of Eimeria spp.1

This report describes a new, gentle procedure for rapid and efficient excystation of large numbers of infective sporozoites of Eimeria vermiformis and Eimeria stiedai. Excysted sporozoites are purified using modifications of a previously described ion-exchange chromatography method. The procedure avoids physical breakage of oocysts and results in greater than 70% recovery of the sporozoites present as sporulated oocysts (i.e. 5–6 sporozoites per sporulated oocyst). The recovered sporozoites are greater than 95% pure and are infective in vivo. We routinely isolate greater than 2 times 10⁸ sporozoites without the use of specialized or expensive equipment.     

Giardia and Cryptosporidium spp. in filtered drinking water supplies.

Giardia and Cryptosporidium levels were determined by using a combined immunofluorescence test for filtered drinking water samples collected from 66 surface water treatment plants in 14 states and 1 Canadian province. Giardia cysts were detected in 17% of the 83 filtered water effluents. Cryptosporidium oocysts, were observed in 27% of the drinking water samples. Overall, cysts or oocysts were found in 39% of the treated effluent samples. Despite the frequent detection of parasites in drinking water, microscopic observations of the cysts and oocysts suggested that most of the organisms were nonviable. Compliance with the filtration criteria outlined by the Surface Water Treatment Rule of the U.S. Environmental Protection Agency did not ensure that treated water was free of cysts and oocysts. The average plant effluent turbidity for sites which were parasite positive was 0.19 nephelometric turbidity units. Of sites that were positive for Giardia or Cryptosporidium spp., 78% would have been able to meet the turbidity regulations of the Surface Water Temperature Rule. Evaluation of the data by using a risk assessment model developed for Giardia spp. showed that 24% of the utilities examined would not meet a 1/10,000 annual risk of Giardia infection. For cold water conditions (0.5 degree C), 46% of the plants would not achieve the 1/10,000 risk level.