Developmentally regulated expression of a Giardia lamblia cyst wall protein gene.

The protozoan Giardia lamblia is an obligate parasite of the mammalian small intestine. We studied the expression of a gene that encodes a protein component of the cyst wall, a complex structure assembled during the differentiation of trophozoites to cysts and which is critical to survival of the parasite outside its mammalian host. Transcripts from the cyst wall protein gene increase more than 100-fold during encystation, reaching a maximum between 5 and 24 hours after induction. Cyst wall protein expression also increases dramatically during encystation, and, prior to its incorporation into the nascent cyst wall, the protein is contained within the encystation-specific vesicles of encysting trophozoites. The sequence of the cloned gene predicts an acidic, leucine-rich polypeptide of M(r) 26,000 that contains 5.3 tandemly arranged copies of a degenerate 24-amino-acid repeat. A hydrophobic amino-terminal peptide probably serves as the initial signal that targets this protein to a secretory pathway involving vesicular localization during encystation and, ultimately, secretion to form the cyst wall.

Chemical characterization of vasoactive intestinal polypeptide-like immunoreactivity in ovine hypothalamus and intestine.

Two forms of pituitary adenylate cyclase activating polypeptides with 38 (PACAP38) and 27 residues (PACAP27) respectively were recently isolated from ovine hypothalamic tissues. The N-terminal 28 amino acids sequence of PACAP was found to have 68% homology with porcine vasoactive intestinal peptide (VIP). In order to determine whether the primary structure of VIP of ovine hypothalamus is identical with porcine VIP or similar to PACAP, VIP immunoreactivity as determined by radioimmunoassay for porcine VIP was isolated in a pure form from ovine hypothalamic extracts. VIP was also isolated from ovine intestine. Amino acid analysis as well as amino acid sequence analysis showed that ovine hypothalamic and intestinal VIP were identical to porcine VIP, but different from PACAP.

Identification of Giardia lamblia-specific antigens in infected human and gerbil feces by western immunoblotting.

Western immunoblot analysis of aqueous extracts of feces obtained from five giardiasis patients and from experimentally infected gerbils (Meriones unguiculatus) with rabbit antiserum to Giardia lamblia cysts has revealed antigens of three molecular weight groups. A stepladderlike, evenly-spaced set of strongly reactive antigens (darkest at a molecular weight [m.w.] of 55,000 to 70,000) appeared in the gerbil feces from day 4 (first experiment) or day 2 (second experiment) and lasted to about day 7 but disappeared completely by day 8 and did not reappear later. These antigenic bands were seen in gerbils infected with two isolates of G. lamblia. These bands were not revealed when antiserum to trophozoites was used as the probe, nor were they evident in specimens from the patients or in a preparation of sonicated cysts. A second group of antigens, represented by two to three low-m.w. bands of approximately 15,000 to 20,000, was evident in both the blots of gerbil feces after approximately day 8 and the specimens from the giardiasis patients. The third group of antigens revealed by blotting experiments was a high-m.w. band (approximately 110,000) which appeared on a number of days (beginning of day 8 of gerbil infection), but this band was not seen in the human specimens. A clear band corresponding to the previously reported GSA-65 antigen was not seen in either the gerbil or the human samples. Some low- and high-m.w. bands were also detected by antitrophozoite serum in the gerbil samples, but these were weak and unimpressive compared with those visualized using anticyst serum. A monoclonal antibody-based antigen capture enzyme-linked immunosorbent assay revealed that Giardia spp.-specific stool antigen rose suddenly at day 3 of gerbil infection, at the time when fecal cyst numbers began to rise rapidly.

A novel peptide which stimulates adenylate cyclase: molecular cloning and characterization of the ovine and human cDNAs.

A novel neuropeptide which remarkably stimulates adenylate cyclase in rat anterior pituitary cell cultures has been recently isolated from ovine hypothalami by A. Arimura and his collaborators (Biochem.Biophys.Res.Commun.164, 567-574(1989)). This peptide was designated as PACAP38(Pituitary Adenylate Cyclase Activating Polypeptide with 38 residues). In an attempt to investigate physiological implications of PACAP38, we have succeeded in cloning the cDNAs encoding the precursor of PACAP38 from ovine hypothalamus and human testis. An ovine cDNA encodes a protein of 176 amino acids in which PACAP38 is proceeded by a putative signal peptide and a "pro"-region (107 amino acids), and followed by a Gly-Arg-Arg sequence for proteolytic processing and amidation. Deduced amino-acid sequence of human PACAP38 was completely identical to that of the ovine isolated peptide. Cloning of PACAP38 cDNAs confirms the expression of the corresponding mRNAs and the presence of this neuropeptide in ovine hypothalamus and also in human testis.

Back-country water treatment to prevent giardiasis.

This study was conducted to provide current information on the effectiveness of water treatment chemicals and filters for control of Giardia cysts in areas where treated water is not available. Four filters and seven chemical treatments were evaluated for both clear and turbid water at 10 degrees C. Three contact disinfection devices were also tested for cyst inactivation. Filters were tested with 1-liter volumes of water seeded with 3 x 10(4) cysts of G. lamblia produced in gerbils inoculated with in vitro cultured trophozoites; the entire volume of filtrate was examined for cyst passage. Chemical treatments were evaluated at concentrations specified by the manufacturer and for contact times that might be expected of hikers (30 minutes) and campers (eight hours, i.e., overnight). Two of the four filter devices tested were 100 percent effective for Giardia cyst removal. Of the other two filters, one was 90 percent effective and the other considerably less effective. Among the seven disinfection treatments, the iodine-based chemicals were all significantly more effective than the chlorine-based chemicals. None of the chemical treatments achieved 99.9 percent cyst inactivation with only 30-minute contact. After an eight-hour contact each of the iodine but none of the chlorine preparations achieved at least 99.9 percent cyst inactivation. None of the contact disinfection devices provided appreciable cyst inactivation. Heating water to at least 70 degrees C for 10 minutes was an acceptable alternative treatment.

Monoclonal antibody-based enzyme immunoassay for Giardia lamblia antigen in human stool.

A visually readable monoclonal antibody-based antigen-capture enzyme immunoassay for the detection of Giardia lamblia antigen in human stool specimens was developed and found to be 97% (30 of 31 stool specimens) sensitive for formalinized stools and 82% (49 of 60 stool specimens) sensitive for unfixed stool specimens by visual reading. The storage of specimens in 10% Formalin resulted in increased absorbance in 20 of 26 G. lamblia-positive specimens tested as both formalinized and unfixed specimens; the increase averaged 1,336%. The assay was specific for antigens of this organism and for antigens derived from the cyst, as opposed to the trophozoite, stage. The assay could detect the antigens of five cysts per well, but could not detect antigen in in vitro-cultured trophozoites. A mouse monoclonal antibody of the immunoglobulin G1 (IgG1) subclass, which was prepared against cysts of G. lamblia, was used as the solid-phase capture antibody. The antibody was reactive with the cyst wall, as determined by immunofluorescence. Polyclonal rabbit anti-cyst IgG was used as the secondary antibody, and peroxidase-labeled goat anti-rabbit IgG was used as the tertiary antibody in the assay format. Maximal capture of antigen from stool specimens occurred by 30 min. Optimal dilution of specimens was in the range of 1:60 to 1:600. Preliminary characterization of affinity-purified antigen recognized by the monoclonal antibody showed that it is heat stable (100 degrees C, 12 min) and resistant to sodium periodate treatment and that it may exist in multiple molecular weights from 45,000 to 110,000.

Antigenic conservation and variation in Giardia cysts from various vertebrate hosts.

Monoclonal antibodies produced against Giardia muris cysts reacted in indirect immunofluorescence with homologous cysts and cysts from a Giardia-infected wild Norway rat but did not cross-react with Giardia lamblia cysts of human, dog, or beaver sources. Another monoclonal antibody raised against Giardia simoni cysts from the Norway rat reacted with homologous cysts (rat) and cross-reacted with cysts from a cow. The demonstration of antigenic differences at the cyst surfaces of Giardia organisms of animal and human origin suggests that it is possible to identify the animal source of Giardia cysts according to their pattern of reactivity with monoclonal antibodies. Such identification would have a useful application in affirming the possible zoonotic transmission of animal source Giardia species to humans.

Prevalence of Cryptosporidium infection in dairy calves in western Washington.

The prevalence and intensity of Cryptosporidium infection were examined in 445 Holstein calves at 10 dairy farms in western Washington, near Seattle. Fifty-one percent (176) of calves in the 7- to 21-day-old age group (n = 342) were positive for oocysts in the feces by carbolfuchsin staining. Prevalence and intensity of infection were highest in calves 8 to 14 days old; prevalence was 60% in this group, and 48% of the Cryptosporidium-positive calves had oocyst shedding at a 4+ level. A seasonal pattern in prevalence was not evident.

Enzyme immunoassay for detection of Giardia lamblia cyst antigens in formalin-fixed and unfixed human stool.

An antigen-capture enzyme-linked immunosorbent assay employing rabbit and mouse antisera to Giardia lamblia cyst antigens was developed for the diagnosis of Giardia infection through detection of G. lamblia-specific stool antigens in cell-free aqueous eluates of human stool. This is the first report of the use of anti-cyst antibodies in an enzyme immunoassay for G. lamblia. The assay gave a positive result with 54 of 59 stools from patients with symptomatic, clinically diagnosed giardiasis, giving the test a sensitivity of 91.5%. A negative reading was obtained with all of 25 stools from G. lamblia-negative control patients. The assay could detect as few as 20 sonicated cysts added to control stool eluate. The assay was more sensitive to cyst-derived antigens than to trophozoite-derived antigens. With two exceptions, the assay gave a negative result with stools from patients infected with Entamoeba histolytica (seven), Cryptosporidium sp. (four), or Blastocystis hominis (seven) and thus appears to be specific for G. lamblia antigens. Storage of stool eluates for more than 6 months at 4 degrees C as unpreserved aqueous eluates or as formalinized eluates did not affect the ability of the assay to detect the giardial antigens. The enzyme-linked immunosorbent assay proved useful for monitoring the levels of G. lamblia-specific stool antigens in the stool of patients undergoing antigiardial chemotherapy.

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.

Neurochemical changes in experimental African trypanosomiasis in voles and mice.

Dopamine, norepinephrine, serotonin and related metabolites were measured in the brain of meadow voles and mice after infection with Trypanosoma brucei gambiense with the goal of understanding the neurochemical changes accompanying infection with this parasite. Serotonin and 5-HIAA levels both fell by 26%, and HVA levels rose by 56%, in vole brains during infection. Dopamine, DHPG and norepinephrine levels, however, remained unchanged. In mice, serotonin levels dropped by 17 and 23%, respectively, in the pons-medulla and midbrain regions after infection; 5-HIAA and dopamine levels remained unaffected by infection in mice. The possible causes and the significance of these changes are discussed.

Identification of Cryptosporidium oocysts in river water.

Water samples were collected from four rivers in Washington State and two rivers in California and examined for the presence of Cryptosporidium oocysts. Oocyst-sized particles were concentrated from 20-liter samples of water by membrane filtration, centrifugation, and differential sedimentation. The particle concentrate was then deposited on a 25-mm-diameter membrane filter for oocyst identification by indirect immunofluorescence assay. The identification procedure had a limit of detection of about five oocysts per liter. Cryptosporidium oocysts were found in each of 11 river water samples examined. Concentrations ranged from 2 to 112 oocysts per liter. The finding of Cryptosporidium oocysts in all samples examined from six western rivers is noteworthy in light of recent reports indicating that Cryptosporidium sp. is a significant agent of human and animal disease. This finding suggests that waterborne oocysts of this parasite are more important than was previously recognized. More detailed studies are needed to define geographical and temporal distribution, to assess the viability of waterborne oocysts, and to determine the importance of water as a means of transmission.

Physical and chemical characterization of a Giardia lamblia-specific antigen useful in the coprodiagnosis of giardiasis.

We recently reported the isolation and identification of a Giardia lamblia-specific antigen (GSA 65) that is shed in the stool of giardiasis patients. In the present study, this antigen was affinity purified from sonic extracts of axenically cultured G. lamblia trophozoites and characterized to better understand its biological function and its potential usefulness in the design of coprodiagnostic assays for giardiasis. GSA 65 was resistant to proteolytic digestion with trypsin, chymotrypsin, and protease but was sensitive to treatment with NaIO4 as assessed by Western blotting. This antigen was also stable during prolonged storage at 4 and -20 degrees C in 10% Formalin or distilled H2O as assessed by counterimmunoelectrophoresis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing gel banding patterns, in conjunction with protein and carbohydrate assays and lectin binding studies, confirmed that this antigen is a highly glycosylated glycoprotein. The resistance of GSA 65 to proteolytic degradation, together with previous immunofluorescence data that indicate the antigen is an integral part of the G. lamblia cyst wall, suggests that this molecule may play a role in maintaining the integrity of the cyst in vivo. The ability of GSA 65 to maintain its antigenic structure under a wide variety of conditions makes it an ideal antigen around which to design sensitive immunodiagnostic assays for giardiasis.

Immunofluorescence detection of Cryptosporidium oocysts in fecal smears.

An indirect fluorescent antibody (IFA) procedure was developed for the detection of Cryptosporidium sp. oocysts in human, nonhuman primate, and bovine fecal smears. The procedure, which takes about 90 min to perform, involves the use of a rabbit antiserum against Cryptosporidium oocysts isolated from dairy cattle. Cross-specificity testing of the IFA method revealed no reactivity with yeasts, various amoebae, Giardia lamblia, Chilomastix sp., or Blastocystis sp. and only very weak cross-reactivity with coccidian oocysts of other genera. IFA detection of oocysts in human and nonhuman primate fecal smears was far more sensitive than was dimethyl sulfoxide-carbolfuchsin staining. Moreover, IFA detection was comparable in sensitivity to auramine O staining with samples of high oocyst concentration and somewhat more sensitive than auramine O with samples containing relatively few oocysts. The IFA procedure may be useful in the clinical diagnosis of human and animal cryptosporidiosis and also in the detection of oocysts in environmental samples.

Isolation and identification of a Giardia lamblia-specific stool antigen (GSA 65) useful in coprodiagnosis of giardiasis.

A Giardia lamblia-specific antigen (GSA 65) was isolated from stools of G. lamblia-positive patients by crossed- and line-immunoelectrophoresis and counterimmunoelectrophoresis (CIE) in agarose by using rabbit antiserum prepared against G. lamblia cysts. CIE with rabbit anti-GSA 65 monospecific antiserum revealed that GSA 65 was present in aqueous stool eluates of giardiasis patients and in cysts and trophozoites of the parasite. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of immunoaffinity-purified antigen followed by Western blotting showed that the molecular weight of this molecule was about 65,000. GSA 65 was detectable by CIE in stool eluates of 36 of 40 giardiasis patients but not in eluates of 10 G. lamblia-negative asymptomatic controls. GSA 65 was detected in stool eluates of 2 of 18 individuals with chronic diarrhea who were negative for parasites by microscopic examination. Cross-specificity studies with other genera of parasitic protozoa performed by using CIE and immunofluorescence indicated that GSA 65 was present only in strains of G. lamblia. Based on these findings, GSA 65 may prove to have an important application in the design of sensitive diagnostic tests for giardiasis.

Identification of a surface antigen of Trichomonas vaginalis.

A major surface antigen of Trichomonas vaginalis was purified by using three independently derived monoclonal antibodies (two immunoglobulin M and one immunoglobulin G1) prepared against T. vaginalis PHS-2J. A 115,000-molecular-weight antigen and one or more components with a molecular weight of 58,000 to 64,000 were recovered when any of the three antibodies was used as an immunoadsorbent. The purified antigen reacted with all three monoclonal antibodies in an enzyme-linked immunosorbent assay, indicating that the antibodies recognized the same antigen but not necessarily the same determinant. The purified antigen was sensitive to both pronase digestion and periodate oxidation. The antigen was shown to be on the external surface of some but not all T. vaginalis isolates by agglutination of live organisms with the monoclonal antibodies.

Changes in brain concentrations of catecholamines and indoleamines in Toxoplasma gondii infected mice.

Brain concentrations of dopamine, homovanillic acid, norepinephrine, serotonin and 5-hydroxyindoleacetic acid were measured in mice with acute and chronic, adult-acquired toxoplasmosis. Mice with acute infections showed a 40% rise in homovanillic acid levels as compared with controls; dopamine levels, however, remained unchanged. Norepinephrine levels in this group were 28% lower than in controls. Dopamine levels were 14% higher in the mice with chronic infections than controls. Serotonin and 5-HIAA levels were not altered in infected mice. These neurochemical changes may be factors contributing to mental and motor abnormalities that accompany or follow toxoplasmosis in rodents and possibly in man.

Effects of African trypanosomiasis on brain levels of dopamine, serotonin, 5-hydroxyindoleacetic acid, and homovanillic acid in the rabbit.

Serotonin (5-HT) levels fell by 21% in the mid-brain-thalamus-hypothalamus (MTH) region of the rabbit brain after chronic infection with the protozoan Trypanosoma brucei gambiense. 5-HT did not decrease significantly in the caudate/putamen (CP) or the pons/medulla (PM) region. 5-Hydroxyindoleacetic acid (5-HIAA) levels were unchanged in the MTH and caudate/putamen (CP) but increased by 17% in the pons/medulla (PM) after infection. Dopamine (DA) levels rose by 19% and homovanillic acid (HVA) by 33% in the PM during infection. DA and HVA tended to be lower in the CP of infected rabbits, but the apparent decreases were not statistically significant. DA and HVA levels in the MTH were also unchanged by infection. These neurochemical changes may be involved in the behavioral symptoms that frequently accompany this disease in man and cattle.

Identification and characterization of a major surface antigen of Giardia lamblia.

The surface antigens of Giardia lamblia trophozoites were characterized by crossed immunoelectrophoresis, radioiodination, and immunoprecipitation. Crossed immunoelectrophoretic analysis of trophozoites with hyperimmune rabbit anti-trophozoite antiserum revealed a prominent precipitin peak that disappeared upon adsorption of the antiserum with live or formaldehyde-fixed trophozoites. This peak was intensely labeled when the antigen was derived from surface-radioiodinated trophozoites. An antiserum monospecific for the antigen contained in this precipitin peak was prepared. The precipitin peak was shown to contain an antigen with an apparent molecular weight of 82,000 by Western blotting. The antiserum also detected this 82,000-molecular-weight antigen on nitrocellulose blots of trophozoites analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On radioiodination of live trophozoites, an iodinated molecule of 82,000 apparent molecular weight was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was immunoprecipitated by the monospecific antiserum. Preliminary characterization of this antigen with the monospecific antiserum in crossed immunoelectrophoresis revealed that the surface antigen is hydrophobic and thus may be anchored in the plasma membrane, and that it is heat sensitive, but only partially sensitive to pronase or periodate. This antigen was shared by the four G. lamblia strains examined.