William J. Bemrick

Axenic culture and characterization of Giardia ardeae from the great blue heron (Ardea herodias)

Trophozoites of Giardia ardeae were obtained from the great blue heron (Ardea herodias) and established in axenic culture using the TYI-S-33 medium. The generation time in culture for G. ardeae was 22-25 hr, which was 3-fold longer than for Giardia duodenalis (WB strain). A morphological comparison of trophozoites in the original intestinal isolate to those grown in culture revealed that they were identical for the following characteristics: a pyriform-shaped body, a ventral adhesive disc with a deep notch in the posterior border, teardrop-shaped nuclei, pleomorphism in median body structure ranging from a round-oval appearance (Giardia muris type) to that of a clawhammer (G. duodenalis type), and a single caudal flagellum on the right side (as viewed dorsally) with the left one being rudimentary. Analysis of the chromosomal migration patterns was performed by orthogonal-field-alternation gel electrophoresis and demonstrated that the pattern for G. ardeae was distinctly different from that for G. duodenalis (Portland 1-CCW strain). Bacterial symbionts were seen attached to trophozoites in the original isolate but could not be detected in cultured trophozoites using scanning electron microscopy, fluorescence light microscopy using the Hoechst 33258 dye for DNA localization, or by standard microbiological techniques using nonselective media for growing aerobic or anaerobic bacteria. This study demonstrated that avian-derived Giardia could be grown in axenic culture; based on morphological criteria and chromosomal migration patterns, that G. ardeae should be considered a distinct species; and that rationale for determining Giardia spp., based on median body structure alone, should no longer be considered adequate for classification at the species level.

SEM EVIDENCE FOR A NEW SPECIES, GIARDIA PSITTACI

The genus Giardia has been subdivided by Filice (1952) into 3 species, G. agilis, G. muris, and G. duodenalis, based on the morphology of the median body and subtle variations in the dimensions of trophozoites. Giardia trophozoites were isolated from the small intestine of budgerigars (parakeets) and examined morphologically with light and scanning electron microscopy. These trophozoites, like other Giardia spp., possessed a flattened dorso-ventral shape, 8 flagella, and an adhesive disc on the ventral surface. The presence of a claw hammer-shaped median body suggested classification of these trophozoites as G. duodenalis. However, unlike any known members of G. duodenalis, the Giardia trophozoites from budgerigars were morphologically distinct in that they lacked the ventrolateral flange and therefore did not have a marginal groove bordering the anterior and lateral border of the adhesive disc. This distinct morphology clearly indicated that trophozoites from budgerigars should be considered as a separate species, G. psittaci. Our evidence has demonstrated that median body shape cannot serve as a sole criterion for speciation of Giardia. In addition, if other avian species of Giardia also resemble G. psittaci, then this would suggest that evolutionary divergence has occurred in the genus Giardia.

Production of viable Giardia cysts in vitro: Determination by fluorogenic dye staining, excystation, and animal infectivity in the mouse and Mongolian gerbil

The purpose of this research was to document the formation of viable Giardia cysts in vitro. Viability staining, using fluorogenic dyes that required metabolic conversion for detection, and immunocytochemistry at the light microscopic level provided information on viability and for the identification of formed in vitro. Analysis of cysts formed in vivo and in vitro showed similar morphologic appearances by both light and electron microscopy. Cysts formed in vitro were capable of establishing infections in both mouse and gerbil models for giardiasis. Trophozoites obtained from mice experimentally infected with in vitro-formed cysts could be maintained in culture and induced a second time to form cysts in vitro. This model for the production of viable Giardia cysts in vitro should facilitate research on controlling the complete life cycle of Giardia outside an animal host.

High-resolution immunogold localization of Giardia cyst wall antigens using field emission SEM with secondary and backscatter electron imaging.

We describe here the ultrastructural localization of Giardia cyst antigens in the filaments associated with the outer portion of intact cysts and on developing cyst wall filaments in encysting trophozoites. Post-embedding immunogold labeling of thin sections of intact Giardia cysts with polyclonal and monoclonal antibodies specific for cyst wall antigens (major protein bands of approximately 29, 75, 88, and 102 KD on Western blots) showed strong labeling of the filamentous cyst wall, whereas no labeling was seen on the membranous portion. High-resolution field emission scanning electron microscopy (FESEM) of Giardia cysts revealed that the cyst wall-specific polyclonal rabbit antisera and monoclonal mouse antibody produced gold labeling of 20-nm filaments in the cyst wall as detected with secondary electron imaging (SEI) and backscatter electron imaging (BEI) at 10 kV, despite coating of the cells with platinum by ion sputtering. FESEM studies of encysting Giardia trophozoites demonstrated that immunostaining with antibodies to cyst wall antigens produced colloidal gold labeling of developing cyst wall filaments on the cell surface; however, the intervening membrane domains were unlabeled. Substitution of normal serum for cyst wall-specific antibodies, or preabsorption of specific antibodies with Giardia cysts, eliminated immunolabeling of the filaments.

High-resolution electron microscopic evidence for the filamentous structure of the cyst wall in Giardia muris and Giardia duodenalis.

High-resolution morphological studies of the cyst wall of Giardia spp. were performed using low-voltage scanning electron microscopy (LVSEM) and transmission electron microscopy (TEM). The cyst wall was composed of membranous and filamentous layers. The membranous layer consisted of an inner and an outer cyst membrane separated by a thin layer of cytoplasm. The filamentous layer contained individual filaments that ranged from 7 to 20 nm in diameter when measured by LVSEM, formed a dense meshwork with branches or interconnections, and were occasionally arranged on the surface in whorled patterns. Cysts of Giardia muris from mice, Giardia duodenalis from dogs, pigs, voles, beavers, muskrats, and humans, and Giardia psittaci from a bird (parakeet), possessed an essentially identical wall composed of filaments. Inducement of excystation in viable Giardia cysts produced a dramatic increase in the interfilament spacing over an entire cyst, but none was observed in heat-killed or chemically fixed control cysts. These results demonstrated that the cyst wall of Giardia spp. was composed of a complex arrangement of filaments, presumably formed during the process of encystment.

A comparison of Giardia microti and Spironucleus muris cysts in the vole: an immunocytochemical, light, and electron microscopic study.

We have shown that cysts of the genus Spironucleus share many common morphological features with Giardia cysts including: 2-4 nuclei, flagellar axonemes, a distinct cyst wall, and they even display the same immunostaining as Giardia cysts when labeled with antibodies specific for Giardia cyst wall. A direct comparison of Spironucleus muris and Giardia microti cysts have revealed that cysts of S. muris are significantly smaller than cysts of G. miroti. At the ultrastructural level, the cyst walls are similar in fibrillar appearance, but the width of the S. muris cyst wall is significantly less than that of G. microti. The cysts of S. muris also differ from G. microti in that they contain a striated rootlet fiber, flagellar sheath, and numerous glycogen rosettes. Characteristic features of Giardia include the adhesive disc and median body. Although the cysts of Spironucleus and Giardia are similar in appearance, these unique morphological features can be used to distinguish between the 2 protozoa and should be employed in the detection of Giardia cysts in water samples.

Some Perspectives on the Transmission of Giardiasis

A number of aspects of the genus Giardia have been a source of great controversy among parasitologists for decades. Much of the difficulty centers around the taxonomy of this flagellate. This has never been completely resolved; it will be discussed further later in this chapter. Giardia spp. investigations proceeded, at a fairly constant level, until the mid-1960s, when research publications pertaining to this parasite diminished considerably. The work with Giardia was continued, over a period of about a decade, by a small group of dedicated workers, without the benefit of much financial support for their research. One reason for this lack of support has been the belief that this organism is of no public health significance in our society. It was considered, and with considerable justification, to be a rather unusual flagellate of doubtful pathologic significance. Many considered it to be more of an exotic parasite, which on occasion produced a diarrheic syndrome in travelers. The research, of course, did progress, and a considerable amount of significant information was developed in such areas as in vitro cultivation (Meyer, 1976; Fortess and Meyer, 1976) and cellular morphology, using both transmission and scanning electron microscopy (Friend, 1966; Erlandsen, 1974; Erlandsen and Chase, 1974; Sheffield and Bjorvatn, 1977) and immunology, by Roberts-Thomson and his co-workers (1976, 1981), Stevens et al. (1978), Visvesvara and Healy (1980), Visvesvara et al. (1980), and many others.

The detection of Giardia muris and Giardia lamblia cysts by immunofluorescence in animal tissues and fecal samples subjected to cycles of freezing and thawing

The effects of freezing and thawing on the detection of selected Giardia spp. cysts were investigated using immunofluorescence, bright field microscopy, and low voltage scanning electron microscopy (SEM). Giardia muris cysts were obtained from either animal carcasses, fecal pellets, or isolated cyst preparations, whereas Giardia lamblia cysts were isolated from fecal samples. These samples were stained using an immunofluorescence technique after 1-3 freezing (-16 C) and thawing (20 C) cycles. Cysts were detected successfully by immunofluorescence in all samples. However, in those samples subjected to freeze-thawing, the cyst walls often became distorted and then were not detectable by bright field microscopy. Low voltage SEM demonstrated that the filaments in the distorted cyst wall underwent rearrangements of interfilament spacing. Quantitation of cyst recovery after freezing and thawing demonstrated that a substantial loss occurred after 1 cycle of alternating temperature when low concentrations of cysts were used, but not with high concentrations of cysts. Cyst recovery, after 3 freezing and thawing cycles, was dramatically lowered irrespective of the initial cyst concentration. These results demonstrated that immunofluorescence was an effective technique for the detection of Giardia spp. cysts in frozen samples and would suggest that freezing and thawing of fecal samples could prevent the detection of cysts when only bright field microscopy was employed.

ENZYME-LINKED IMMUNOSORBENT ASSAY AND INDIRECT HAEMAGGLUTINATION TECHNIQUES FOR MEASUREMENT OF ANTIBODY RESPONSES TO EIMERIA TENELLA IN EXPERIMENTALLY INFECTED CHICKENS

An indirect haemagglutination test (IHA) and ELISA technique were developed to detect antibodies against Eimeria tenella. The ELISA technique was relatively easy to perform, more sensitive than the IHA test, and needed only a fraction of the antigen required for IHA. The highest titers using ELISA were 1:16,384 compared to the IHA titers of 1:64 for the same sera. The ELISA titers depended upon the age of the birds when they were infected, the number of oocysts inoculated and the number of inoculations. Immunodeficient birds (cyclophosphamide-treated), when inoculated with several doses of oocysts of E. tenella (350, 3,000, 30,000) showed no IHA or ELISA antibody titers. The immuno-competent chickens of the same age, which received identical doses of oocysts responded with readily detectable antibody levels. Chickens inoculated with E. maxima or E. necatrix had sera titers of 1:50 or 1:400, respectively, when reacted with E. tenella antigen. The E. tenella inoculated birds had titers as high as 1:3,200 with the same antigen.

Cross‐species transmission of avian and mammalian Giardia spp: Inoculation of chicks, ducklings, budgerigars, mongolian gerbils and neonatal mice with Giardia ardeae, Giardia duodenalis (lamblia), Giardia psittaci and Giardia muris

Birds infected with Giardia may constitute a potential avenue for cross‐species transmission of giardiasis, especially during seasonal migration of infected birds from wetland habitats. We have investigated the potential for cross‐species transmission of avian Giardia species (Giardia ardeae from the great blue heron; Giardia psittaci from budgerigars) into unrelated avian hosts (chicks and ducklings) as well as established mammalian models for giardiasis, the Mongolian jird (gerbil) and the neonatal mouse. Avian Giardia species could not be transmitted to either young chicks, ducklings, or mammalian hosts; however, the transmission of G.psittaci to its natural host, the budgerigar, was successfully performed with either trophozoites or cysts. Attempts at transmission of mammalian Giardia spp. into avian hosts were consistently unsuccessful, although mammalian hosts were successfully infected with mammalian Giardia spp.. Experiments on the short term survival of cultured Giardia trophozoites inoculated di...