In vitro phagocytosis of Giardia duodenalis cysts by hemocytes of the Asian freshwater clam Corbicula fluminea

Hemocytes of the Asian freshwater clam Corbicula fluminea, phagocytosed in vitro infectious Giardia duodenalis cysts. After 15, 30, 60, 90, and 120 min of incubation an average of 22%, 32%, 43%, 54%, and 72% of the cysts were phagocytosed by 22%, 55%, 63%, 81%, and 86% of the hemocytes, respectively. The number of hemocytes showing phagocytosis and the mean number of cysts ingested per hemocyte increased␣significantly over time (P < 0.01); the numbers of nonphagocytosed cysts significantly decreased (P < 0.02). Extrapolation reveals that C. fluminea can retain by phagocytosis an average of 1.6 × 10⁶ G. duodenalis cysts/ml hemolymph. The phagocytic capacity of C. fluminea hemocytes indicates the applicability of this freshwater benthic bivalve for bioindication of contamination of waste waters and agricultural drainage with Giardia cysts. Received: 28 March 1997 / Accepted: 6 June 1997     

Scanning electron microscopy of Blastocystis hominis cysts

Scanning electron microscopy of Blastocystishominis cysts reveals that some cysts have an outer coat, whereas others are naked. If intact, the outer coat forms a fan-like structure around the cyst and its surface is granular. The fragmented outer coat adheres to other cysts and bacteria, forming irregular clumps. Received: 15 September 1997 / Accepted: 2 December 1997     

Host cells of Toxoplasma gondii encystation in infected primary culture from mouse brain

In order to identify brain cell types that serve as host cells of Toxoplasma gondii encystation primary cultures from murine brain were infected and stained for neural and parasite stage-specific markers. In mixed culture inoculated with T. gondii tachyzoites, MAP2⁺ neurons, GFAP⁺ astrocytes, F4/80⁺ microglia, and O1⁺ oligodendrocytes proved to be infected as detected by parallel labeling of SAG1. At 4 days following infection with bradyzoites, cysts developed in neuronal, astroglial, and microglial host cells as clarified using bradyzoite-specific antibody 4F8. Additional staining of SAG1 revealed that astrocytes in bradyzoite-infected brain cell culture can also harbor tachyzoite-containing vacuoles. Stage conversion was observed shortly after inoculation and was accompanied by an increase in parasite proliferation. However, tachyzoites became rare in prolonged culture. By contrast, the numbers of cysts and of the bradyzoites isolated multiplied during long-term culture. These findings demonstrate that both glial and neuronal host cells allow T. gondii encystation in the absence of T cell-derived cytokines and imply that a brain-internal spreading of bradyzoites may sustain chronic infection. Received: 25 March 1997 / Accepted: 16 April 1997     

Echinococcus granulosus: membrane permeability of secondary hydatid cysts to albendazole sulfoxide

The objectives of the present study were, first, to establish a methodology for evaluation of the permeability in vitro of hydatid cysts to different drugs and, second, to compare the permeability to albendazole sulfoxide of cysts from untreated animals, cysts from animals treated with 50 mg/kg netobimin for 5 days, and cysts from animals treated with 50 mg/kg netobimin plus 1.1 mg/kg fenbendazole for 5 days. The drug flow follows the Fick law, i.e., the uptake occurs by simple diffusion. We calculated the permeability constant of the cyst membrane by taking into account the disappearance velocity constant, the cyst area, and the incubation solution volume. The permeability value obtained for albendazole sulfoxide was 8.06 ± 2.30 × 10⁻⁶ cm s⁻¹ in cysts from untreated animals, 5.56 ± 2.53 × 10⁻⁶ cm s⁻¹ in cysts from animals treated with netobimin, and 7.05 ± 3.04 × 10⁻⁶ cm s⁻¹ in cysts from animals treated with netobimin + fenbendazole. These permeability values show significant differences (P < 0.05). Received: 20 September 1997 / Accepted: 15 October 1997     

First isolation and characterization in humans of Entamoeba histolytica (laboratory-made) zymodeme XX

Isoenzyme analysis by starch-gel electrophoresis has proved to be a useful method for the biochemical differentiation of pathogenic Entamoeba histolytica and non-pathogenic E. dispar isolates. Of the 24 known zymodemes, 3 are laboratory-made and have not previously been identified in humans. Parasitology screening was carried out in a psychiatric institution. Two amebic stocks were isolated and characterized that had never previously been found in humans and that have protein patterns identical to that of the laboratory-made zymodeme XX. Received: 15 February 1997 / Accepted: 10 March 1997     

Cyst formation by Toxoplasma gondii in vivo and in brain-cell culture: a comparative morphology and immunocytochemistry study

Formation of Toxoplasma gondii cysts was examined in cultured murine brain cells and was compared with the development of cysts in mouse-brain tissue. Cultures of mixed glial cells from neonatal mouse brain were infected with bradyzoites of the avirulent T. gondii strain DX. The development and maturation of Toxoplasma cysts was monitored for up to 63 days after inoculation. Transmission electron microscopy indicated that in-vitro-derived cysts were morphologically similar to tissue cysts and were located intracellularly, even for up to 63 days postinfection. For immunohistological and immunocytochemical examination of both in-vivo- and in-vitro-infected material, monoclonal antibody (mAb) CC2 was used. MAb CC2 was shown to detect specifically the underlying granular material of the cyst wall without binding to the limiting membrane of the parasitophorous vacuole. This reactivity of mAb CC2 allows the distinction of bradyzoite-containing cysts from parasitophorous vacuoles harboring tachyzoites both in vitro and in vivo. Received: 15 February 1997 / Accepted: 18 March 1997     

Force and myosin content variation in isolated intact single muscle fibres from Rana temporaria

 We studied the relation between force normalized by dry mass per unit length and the myosin fraction of muscle dry mass. The two tibialis anterior muscles were dissected from 12 frogs (Rana temporaria). Then, from one muscle, two single fast-twitch fibres were isolated. Each fibre was mounted isometrically in Ringer’s solution, and electrically stimulated using a standardized protocol. Peak force production, normalized by the fibre’s dry mass per unit length, varied by a factor of 1.4. Little variation in normalized force was measured between fibres from the same animal, whereas between animals a significant difference was found (P<0.05). The contralateral muscle was used to determine the myosin fraction of the dry mass. The relationship between the fraction myosin of the dry mass and force normalized by dry mass per unit length showed a high correlation (r = 0.81; n = 12). From this we conclude that variation in normalized tetanic force is determined greatly (65%) by variations in myosin content. Received: 23 January 1997 / Received after revision: 14 March 1997 / Accepted: 26 March 1997     

Improved immunofluorescence assay for detection of Giardia and Cryptosporidium from asymptomatic adult cervine animals

We tested an improved immunofluorescence assay (IFA) in detecting Giardia and Cryptosporidium from feces of asymptomatic adult cervine animals. Samples were concentrated by sucrose flotation before being stained by fluorescent monoclonal antibody and examined microscopically. The detection limit was determined as 500 G. intestinalis cysts or 200 C. parvum oocysts/g of sample. Among the 82 samples collected from adult fallow deer, Columbian black-tailed deer, and Tule elk in northern California, 3 (3.7%) contained G. intestinalis cysts, which were confirmed by a species-specific polymerase chain reaction (PCR) following immunomagnetic capture (IC) of cysts. C. parvum oocysts were detected in a total of 13 (15.9%) samples, and oocysts from 2 such samples were smaller than oocysts from the other 11 samples. C. parvum identification was also confirmed by specific IC-PCR and sequencing of the PCR product. In addition, a C. muris-like organism was detected in 2 (2.4%) samples. Findings obtained with the improved IFA confirmed that cysts/oocysts may pass unnoticed in adult cervine animals and that subclinically infected individuals could serve as potential carriers of infection for humans and other animals via contaminated feces or water. Received: 19 January 1999 / Accepted: 29 March 1999     

Irregular shedding of Blastocystis hominis

The shedding pattern of the protozoan parasite, Blastocystis hominis, is investigated in man and in experimental animal infections. The shedding pattern of the vacuolar and cystic forms of Blastocystis hominis in infected individuals have been shown in the present study to be irregular. The study shows that there is marked fluctuation in the shedding of the parasite from day to day, varying from as high as 17 to 0 per ×40 microscopic field. The cystic stages when estimated in 8 Blastocystis-infected individuals ranged from as high as 7.4 × 10⁵ cysts per gram of stool to 0. The shedding of cystic and vacuolar forms observed over a period of 20 days in experimentally-infected Wistar rats were not only shown to be irregular but the amount varied from host to host. The study has important diagnostic implications in that the stool samples must be collected more than once from patients showing clinical signs and symptoms to eliminate the cause of it to Blastocystis. The study also shows that there are asymptomatic individuals who pass a large amount of cysts as such individuals should be treated to prevent transmission to others. Received: 13 March 1998 / Accepted: 5 April 1998     

cGMP facilitates calcium current via cGMP-dependent protein kinase in isolated rabbit ventricular myocytes

 The effect of guanosine 3′,5′-cyclic monophosphate (cGMP) on L-type Ca current (I _Ca) was investigated in a study of rabbit ventricular myocytes using the whole-cell patch-clamp technique. Intracellular application of cGMP (100 μM) increased I _Ca in the absence of isoprenaline or forskolin. 8-Bromo-cGMP (100 μM) and 8-(4-chlorophenylthio)-cGMP (8-pCPT-cGMP, 400 μM), relatively specific stimulators of cGMP-dependent protein kinase (cGMP-PK), also increased I _Ca. The stimulatory effect of 8-pCPT-cGMP was suppressed by Rp-8-chlorophenylthio-cGMP (400 μM), a phosphodiesterase-resistant cGMP-PK inhibitor. When I _Ca was increased by bath application of the non-specific phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX, 100 μM), 8-pCPT-cGMP (400 μM) resulted in additional stimulation of I _Ca. In the presence of 8-pCPT-cGMP, additional applications of isoprenaline (1 μM) or forskolin (1 μM) induced a further increase in I _Ca. From these results, it could be concluded that the activation of cGMP-dependent protein kinase is involved in the facilitation of I _Ca by cGMP in rabbit ventricular myocytes. Received: 17 March 1997 / Received after revision: 28 August 1997 / Accepted: 16 September 1997     

The parasitism of fish from Lake Constance – a comparison of present and earlier data

Commercial fish (perch, roach, bream, dace, and burbot) were caught below Langenargen in 1988–1990. Their infestation with cestodes and digenean trematodes was studied. The results were compared with earlier data. The infestation of perch with Diplostomum spathaceum and Ichthyocotylurus variegatus noted in 1988–1990 had increased as compared with that observed in 1974–1976. The infestation of perch with Triaenophorus nodulosus seen in 1990 had increased as opposed to that noted in 1988. The infestation of bream with D. spathaceum and of perch and roach with Tylodelphys clavata observed in 1988–1990 had remained the same as that seen in 1974–1976. The infestation of perch with D. spathaceum noted in 1988–1990 had decreased as opposed to that reported for 1974–1976. The infestation of bream, dace, and burbot with T. clavata noted in 1988–1990 had decreased as compared with that observed in 1974–1976. The infestation of perch with T. nodulosus seen in 1988/1989 had decreased as opposed to that noted in 1974–1976. All three cyprinids studied as well as the burbot had been less intensively infested in 1988–1990 than in 1974–1976. Only perch had been more intensively infested in 1988–1990 than in 1974–1976, especially with D. spathaceum and I. variegatus. Received: 7 March 1997 / Accepted: 15 May 1997     

Evidence for the existence of genetically distinct strains of Enterocytozoon bieneusi

Enterocytozoon bieneusi is the most frequently found microsporidium in human infections. In all, 3 distinct genotypes were detected in 12 stool samples from 8 patients with acquired immunodeficiency syndrome (AIDS). A total of 9 polymorphic sites were found in the 243-bp-long internal transcribed spacer (ITS) of the rDNA gene, whereas none was found in 241 bp of adjacent rRNA coding regions. The genotype was stable in samples taken during 11 weeks of infection from one of the patients. The existence of and the ability to discriminate among strains of E. bieneusi are important prerequisites for elucidation of the hitherto unknown reservoirs of this pathogen and the mode of its transmission and may explain its pathogenicity. Received: 18 March 1997 / Accepted: 10 April 1997     

Decline in isokinetic force with age: muscle cross-sectional area and specific force

 Humans produce less muscle force (F) as they age. However, the relationship between decreased force and muscle cross-sectional area (CSA) in older humans is not well documented. We examined changes in F and CSA to determine the relative contributions of muscle atrophy and specific force (F/CSA) to declining force production in aging humans. The proportions of myosin heavy chain (MHC) isoforms were characterized to assess whether this was related to changes in specific force with age. We measured the peak force of isokinetic knee extension in 57 males and females aged 23–80 years, and used magnetic resonance imaging to determine the contractile area of the quadriceps muscle. Analysis of MHC isoforms taken from biopsies of the vastus lateralis muscle showed no relation to specific force. F, CSA, and F/CSA decreased with age. Smaller CSA accounted for only about half of the 39% drop in force that occurred between ages 65–80 years. Specific force dropped about 1.5% per year in this age range, for a total decrease of 21%. Thus, quantitative changes in muscle (atrophy) are not sufficient to explain the strength loss associated with aging. Received: 12 November 1996 / Received after revision: 10 March 1997 / Accepted: 19 March 1997     

Seasonal differences in infestation of the perch Perca fluviatilis L. from Lake Constance with digenean trematodes

Perch (Perca fluviatilis L.) caught in Lake Constance every 2 months over a period of 3 years below six towns or villages (Langenargen, Nonnenhorn, Rorschach, Romanshorn, Bottighofen, and the Lake of überlingen) were examined for parasites. In contrast to Diplostomum spathaceum (Rudolphi, 1819) and Tylodelphysclavata (Nordmann, 1832), Bunoderaluciopercae (Müller, 1776) and Ichthyocotylurusvariegatus (Creplin, 1825) showed marked seasonal differences. These differences were influenced by various factors: the different numbers of the first intermediate hosts (snails, copepods), the water temperature, the physiological state of the fish, and its way of life. Received: 7 March 1997 / Accepted: 15 May 1997     

Role of actin microfilament in osmotic stretch-induced increase of voltage-operated calcium channel current in guinea-pig gastric myocytes

 Using the whole-cell patch clamp technique, the role of actin microfilament in hyposmotic increase of voltage-operated calcium channel current (I _Ba) was studied in guinea-pig gastric myocytes. Hyposmotic superfusate (212 mOsm) increased peak I _Ba amplitude by 32.7 ± 6.5%; when cytochalasin-D (Cyt-D, 20 μM), an actin cytoskeleton disruptor, was used, an increase of only 9.7 ± 3.1% was seen. I _Baresponse to osmotic stress was potentiated (45.1 ± 4.1% increase) by 20 μM phalloidin, an actin microfilament stabilizer. However, colchicine (100 μM), an microtubule cytoskeleton disruptor, had no effect on either I _Ba or its response to hyposmotic solution. Phalloidin also induced a rightward shift of the I/V relationship of I _Ba, while Cyt-D itself had no effect. These results suggest that actin cytoskeleton may mediate hyposmotic stretch-induced I _Ba increase in gastric smooth muscle. Received: 26 March 1997 / Received after revision: 28 May 1997 / Accepted: 3 June 1997     

In vitro encystation of Blastocystis hominis : a kinetics and cytochemistry study

The kinetics of in vitro encystation of Blastocystis hominis was studied over 9 days. The differentiation between trophic (TF) and cyst forms (CF) was determined by differential counts before and after treatment with distilled water. A cytochemistry study using acridine orange and Calcofluor white wet-mount preparations of CF was carried out. The growth curves of TF and CF were related because the decrease in TF was followed by an increase in CF, and vice versa. The maximum of CF counts was obtained on the 6th or 7th day. Using the differential acridine orange stain, two subpopulations of CF, yellow-orange fluorescent cells or precysts and green fluorescent cells or cysts, were detected and their curves were also related. CF was stained by Calcofluor white, which suggested the existence of β-(1-4)-glycosyl residues in the wall cysts of B. hominis. Received: 27 May 1997 / Accepted: 15 July 1997     

Observations on the ultrastructure and viability of the cystic stage of Blastocystis hominis from human feces

 This report describes the ultrastructure and viability of cysts of Blastocystis hominis from feces of infected patients. The cysts were round to ovoid, measured 2–5 μm in size, and contained a condensed cytoplasm that had vacuoles of varying sizes, four nuclei, and as many as six cristate mitochondria. The cell wall was rather electron-lucent. Surprisingly, chromatoid-like structures were found in the cytoplasm and nucleus of some of the cysts. These have not previously been reported in Blastocystis. The cysts can survive in water for up to 19 days at normal temperatures but are fragile at extreme temperatures and in common disinfectants. Received: 7 September 1995 / Accepted: 18 January 1996     

Cholinergic agonists decrease quantal output at the frog neuromuscular junction by targeting a calcium channel blocked by ω-conotoxin

 Nicotinic cholinergic agonists are known to decrease synchronous evoked quantal output at the frog neuromuscular junction [Van der Kloot 1993, J Physiol (Lond) 468:567–589]. Here we also show that carbachol decreases the frequency of miniature endplate potentials (F _MEPP) in solutions containing elevated levels of K⁺ and Ca²⁺. Carbachol did not decrease F _MEPP in hypertonic solutions or in solutions containing the Ca²⁺ ionophore ionomycin and Ca²⁺. We conclude that the nicotinic agonists decrease Ca²⁺ influx through voltage-gated Ca²⁺ channels. Carbachol did not alter two-pulse facilitation. A blocker of N-type Ca²⁺ channels, ω-conotoxin GVIA, antagonized the nicotinic agonist-induced decrease in evoked quantal output. The effect of carbachol was not altered by ω-conotoxin MVIIC, a blocker of P-type and certain other Ca²⁺channels. The Ca²⁺ channel targeted by the nicotinic agonists appears to be of the N-type. Received: 6 March 1997 / Received after revision: 6 May 1997 / Accepted: 4 June 1997     

Characterization and partial purification of the cytoplasmic factor that maintains cardiac Ca2+ channel activity

 Using the patch clamp method we attempted to characterize the cytoplasmic factor in guinea-pig cardiac myocytes which restores L-type Ca²⁺ channel activity after run-down. The factor was eluted from a diethylaminoethyl (DEAE) sepharose column by KCl at 100–360 mM. On gel filtration the factor had an apparent molecular mass (M _r) of 250–300 kDa. Two-dimensional electrophoresis of the partially purified factor showed at least nine spots, of which the major spot had a M _r of about 100 kDa and an isoelectric point of 4.8, suggesting that the physicochemical properties of the factor resemble those of calpastatin, an endogenous inhibitor of Ca²⁺-activated protease, calpain. Calpastatin activity was increased in the partially purified cytoplasm and an antibody raised against calpastatin recognized the major band. Reduction of calpastatin in the cytoplasm decreased the potency of Ca²⁺ channel activation. These results suggest that calpastatin might interact with the Ca²⁺ channel and maintain channel activity. Received: 26 March 1997 / Received after revision: 8 August 1997 / Accepted: 5 September 1997     

Evaluation of the recovery of waterborne Giardia cysts by freshwater clams and cyst detection in clam tissue

The Asian freshwater clam, Corbicula fluminea, inhabits environments recognized to be contaminated with waterborne Giardia cysts. Sixty-four tissue samples of Giardia-free clams were spiked with various numbers of Giardia duodenalis cysts within the range of 50–700 cysts. Regression analysis showed that paired numbers of spiked (x) versus recovered (y) cysts regressed significantly (P < 0.01) according to the equation y= 42.57 + 1.81x (±64.3). The cyst detection threshold was 43 cysts/clam, the coefficient of determination was 77%, and the overall sensitivity of cyst detection was 42.9%. All 20 values of cyst numbers in clam tissue samples that were processed blind were located within the 95% prediction limits of the linear regression equation. The cyst retention rate of 160 clams kept in an aquarium with 38 l of water spiked with 1.00 × 10⁵ G. duodenalis cysts was approximately 1.3 × 10³ cysts/clam. No waterborne cysts were detected by the membrane filtration method 90 min after spiking the aquarium water. G. duodenalis cysts were detected in clam tissue up to 3 weeks post-exposure. Filtration of water by clams substantially depleted the aquarium water of its particulate matter. The sampling program demonstrated that the population of 160 clams examined during the study could be accurately assessed for exposure to waterborne Giardia cysts by random sampling of 86 (54%) clams. The results indicate that C. fluminea clams can be used␣for biological monitoring of contamination with Giardia. Received: 27 May 1998 / Accepted: 7 July 1998