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.

Experimental bovine coccidiosis: control with monensin.

Young Holstein-Friesian bull calves were used in a controlled experiment to evaluate the efficacy of monensin against coccidiosis. The calves were given oocysts of Eimeria bovis and/or E. zurnii. Medication was started 3 days prior to inoculation and continued during the 30-day experimental period. Oocyst shedding was quantified prior to and throughout the experiment and demonstrated that monensin at the rate of 20 or 30 g ton-1 of feed significantly reduced oocyst shedding and clinical coccidiosis. Clinical infection with E. zurnii was very difficult to establish, even when calves were treated with 20 mg dexamethasone IM on Days 12, 15, and 16 post-inoculation.

Scanning electron microscopy of damage to the cecal mucosae of chickens infected with Eimeria tenella.

A group of chickens were inoculated with 100,000 Eimeria tenella oocysts each. The birds were sacrificed on days 4-14 postinoculation. Tissue samples from 4 different areas of each cecum examined were obtained and processed by the osmium-thiocarbohydrazide-osmium technique. Examination of the prepared tissues with the scanning electron microscope revealed lesions ranging from localized tissue swelling to complete disruption of the mucosal epithelium due to the rupture of large numbers of epithelial cells. The amount of damage to the tissue varied greatly, tending to be most obvious in the 2 more distal regions of the ceca examined. Observed in addition to the pathological changes demonstrated were some specific stages of the parasite, including schizonts, merozoites, and oocysts.

Scanning electron microscopy of damage to the cecal mucosa of turkeys infected with Eimeria adenoides.

White Wrolstad turkeys were each inoculated with 100,000 Eimeria adenoides oocysts and killed on days 4-14 postinoculation. Tissue samples, obtained from 4 areas of the ceca comparable to areas examined in chickens infected with E. tenella in previous studies, were processed by a modification of the osmium-thiocarbo-hydrazide-osmium technique and examined with a scanning electron microscope. The pathologic situation found in turkeys was slightly different from that in the ceca of chickens infected with E. tenella. The mucosal lesions are most severe at the proximal end of an infected cecum. Surface disruption was far less severe than with cecal coccidiosis in chickens of the same age exposed to an equal number of infective oocysts. Rupture of the epithelial cell often caused the mucosal surface to present a honeycomb appearance. Some specific stages of the life cycle were identified, including schizonts and oocysts.

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.

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.

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.

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.

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.

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.

Natural transmission of Dirofilaria immitis by Aedes vexans.

Aedes vexans mosquitoes were reared in the laboratory from field-collected fourth instar larvae. Mosquitoes were allowed to feed on dogs which were infected with Dirofilaria immitis and then the mosquitoes were housed in an environmental chamber with an ambient air temperature of approximately 26 C and a relative humidity of approximately 80%. Incandescent lighting was used to simulate daylight. On the 17th, 19th, 21st, and 28th day after their infected blood meal, the mosquitoes were allowed to feed on five microfilaria-negative dogs which had been reared in mosquito-free environments. Blood from four of the five exposed dogs became positive for microfilariae of D immitis in an average of 272 days after the feeding. The local strain of A vexans was established as a vector of canine heartworm disease in the Minneapolis-St. Paul metropolitan area.

Cytotoxic effects of Pasteurella haemolytica on bovine neutrophils.

The interaction of logarithmic- and stationary-phase organisms of Pasteurella haemolytica with bovine neutrophils was evaluated by an opsonocytophagic assay. Only 5% to 8% of the logarithmic-phase P haemolytica 12296 organisms opsonized with normal bovine serum or antiserum were phagocytized. Results from cytotoxicity assays, using the 51Cr-release technique and the trypan blue exclusion test, indicated that the logarithmic-phase organisms liberated a soluble material that was cytotoxic to neutrophils and destroyed their phagocytic capabilities. This hypothesis was verified by transmission electron microscopy studies. Opsonized stationary-phase organisms were completely phagocytized and degraded when exposed to neutrophils at a bacteria/neutrophil ratio of 10:1. However, at a high bacteria/neutrophil ratio of 100:1, only 31% of the bacteria were phagocytized. Prolonged incubation of this mixture resulted in cytotoxic changes in the neutrophils. Seemingly, excess unphagocytized bacteria liberated a soluble substance that was toxic to neutrophils. These findings were confirmed by cytotoxicity assays and transmission electron microscopy studies.

Interaction of Pasteurella haemolytica with bovine neutrophils: identification and partial characterization of a cytotoxin.

Timed cultures of Pasteurella haemolytica 12296 strain in RPMI 1640 medium (with L-glutamine, pH 7.4) were used to determine the correlation between cytotoxin production and the age of the culture. Cytotoxic activity was measured by a 51Cr-release assay and trypan blue exclusion test with bovine neutrophils as target cells. Results demonstrated that optimal cytotoxin production occurred during the logarithmic phase (peaked at 6 hours) and decreased during the stationary phase of bacterial growth. The cytotoxin was concentrated by sequential ultrafiltration on Diaflo XM 50, XM 100, and XM 300 membranes. The cytotoxin was retained on an XM 300 membrane. These studies indicated that the molecular weight of cytotoxic substance was 300,000 or more. The cytotoxin was heat labile, oxygen stable, and susceptible to extremes of pH and killed bovine neutrophils and mononuclear leukocytes. It was not hemolytic to bovine or ovine RBC. The cytotoxic activity was inactivated by trypsin and did not contain any detectable endotoxin. Bovine fetal serum and serum collected before immunization from neonatal calves did not neutralize the cytotoxic effects of toxin on neutrophils. However, adult bovine serum from 6 cows and an antiserum (against the cytotoxin) neutralized the cytotoxin, as revealed by both the 51Cr-release assay and the trypan blue exclusion test. This was confirmed by transmission electron microscopy. These results indicated that the cytotoxin may be antigenic in cattle. The significance and implications of these findings to bovine pasteurellosis are discussed.