Morphological changes and viability of Cryptosporidium parvum sporozoites after excystation in cell-free culture media.

Cryptosporidium parvum, belonging to the phylum Apicomplexa, is a major cause of waterborne gastroenteritis throughout the world. The sporozoites are thought to invade host enterocytes using an active process termed gliding motility. However, the biological and morphological changes within the sporozoites during this process are not fully understood. In the present study, excysted sporozoites of C. parvum were analysed ultrastructurally in vitro and their viability was evaluated using fluorescent dyes. The sporozoites excysted from oocysts changed morphologically from banana-shaped to rod-shaped and finally to a rounded shape, in culture media in 3 h. Transmission microscopy revealed that the distance between the apical end and the nucleus was markedly reduced, dense granules were present close to the rhoptry in the apical region, amylopectin granules were absent, and membranes of round sporozoites were less clear. A fluorescent assay showed that the rate of survival decreased from 89% to 56% at 0-3 h (84.3% for banana-shaped and 49.2% for rod-shaped sporozoites). Therefore, post-excysted sporozoites in vitro underwent morphological changes and a rapid loss of viability. This staining method is useful, inexpensive and provides an alternative to more costly and intensive flow cytometric assays or infectivity assays with host cells in vitro.

Electron microscopic observation of cytoskeletal frame structures and detection of tubulin on the apical region of Cryptosporidium parvum sporozoites.

Cryptosporidium parvum is an intracellular protozoan parasite belonging to the phylum Apicomplexa, and a major cause of waterborne gastroenteritis throughout the world. Invasive zoites of apicomplexan parasites, including C. parvum, are thought to have characteristic organelles on the apical apex; however, compared with other parasites, the cytoskeletal ultrastructure of C. parvum zoites is poorly understood. Thus, in the present study, we ultrastructurally examined C. parvum sporozoites using electron microscopy to clarify the framework of invasive stages. Consequently, at the apical end of sporozoites, 3 apical rings and an electron-dense collar were seen. Two thick central microtubules were seen further inside sporozoites and extended to the posterior region. Using anti-alpha and -beta tubulin antibodies generated from sea urchin and rat brain, both antibodies cross-reacted at the apical region of sporozoites in immunofluorescent morphology. The molecular mass of C. parvum alpha tubulin antigen was 50 kDa by Western blotting and the observed apical cytoskeletal structures were shown to be composed of alpha tubulin by immunoelectron microscopy. These results suggested that C. parvum sporozoites were clearly different in their cytoskeletal structure from those of other apicomplexan parasites.

Inactivation of Cryptosporidium parvum by ultrasonic irradiation.

The inactivation of Cryptosporidium parvum was investigated by the use of three different sonicators utilizing the squeeze-film effect, which may occur when ultrasound is irradiated into an extremely thin space and generate intensified pressure in the sample suspension. To expand from the small-scale horn-type sonicator to large-scale cylindrical or cleaning bath sonicators, the inactivation effectwas improved. In the case of the cylindrical sonicator (26.6 kHz, 30 W), 97% of the initial concentration of 2260 oocysts mL(-1) was inactivated at33 mL min(-1) (residence time of approximately 5.2 min). Hundreds of cubic meters of water can be treated per day at several kW using this sonicator. In addition, the simultaneous use of sonication and chlorination showed a beneficial effect on inactivation for C. parvum based on the evaluation of infectivity testing and morphological observation.

Outbreak of cryptosporidiosis after contamination of the public water supply in Saitama Prefecture, Japan, in 1996.

An outbreak of cryptosporidiosis occurred in Ogose Town, Saitama Prefecture. Japan, in June 1996. Of 12,345 respondents to a questionnaire sent to households in the town (population; 13,809), 8,812 (71.4%) reported an acute gastrointestinal illness some time between May and July. In addition, 274 traceable visitors at local inns, golf courses, and the like during this period and 54 employees from out of town were infected. Cases of cryptosporidiosis were estimated to 9,140. Of these, 2,856 subjects were treated at outpatient clinics and 24 subjects were hospitalized (some subjects counted twice). No deaths were attributed to the outbreak. Among the visitors to Ogose who were traced, 7 persons who stayed only one day during the outbreak and drank half a glass to 2 glasses (100 to 360 ml) of tap water had cryptosporidiosis confirmed by laboratory tests. The median incubation period for the 14 persons for whom this calculation was possible was 6.4 days (range, 5 to 8 days). Of 469 pupils reporting details of their fever and diarrhea, abdominal cramps, or these combined signs and symptoms, the median maximum body temperature was 37.8 degrees C (range, 36.7 to 40.3 degrees C). The duration of illness, reported by 608 of the pupils, was 5.2 days (range, 1 to 15 days), and that reported by 187 employees was 4.8 days (range, 1 to 18 days). The longest known time for discharge of oocysts after onset was 44 days. Blood was not found in the 609 stool specimens examined. The outbreak was caused by contamination of the town's potable water by Cryptosporidium parvum oocysts. The town's water treatment plant treated river water by coagulation, sedimentation, sand filtration, and chlorination. Contamination arose because of various natural and artificial factors: one was that the monthly precipitation in May was much lower than average, causing the river water level to drop. Another factor was heavy rainfall one night in May that increased water turbidity. The amounts of the coagulant added seemed to be insufficient. There are two inns, three public lavatories, and two small-scale wastewater treatment plants upstream 400 m and 1,200 m of the intake point of the town's water treatment plant. However, there are no farms with livestock in the area. We suggest that the location of the water treatment facilities was inappropriate, and that oocysts had circulated from the potable water to humans to sewage to the river and back to the potable water.

Specific PCR primers for Cryptosporidium parvum with extra high sensitivity.

One pair of high-sensitive polymerase chain reaction (PCR) primers for Cryptosporidium parvum was constructed based on the sequence of random amplified polymorphic DNA. PCR with this primer pair amplified only the DNA of C. parvum, not the control DNA including Cryptosporidium muris. This primer pair had most advantageous in its sensitivity over the six pairs of primers reported elsewhere. The minimum amount of template DNA required to produce visible bands after gel electrophoresis and ethidium bromide staining was 0.156 pg of C. parvum or just a single oocyst in the PCR tube.

Heterogeneous distribution of membrane cholesterol at the attachment site of Cryptosporidium muris to host cells.

Distribution of membrane cholesterol at the attachment site of Cryptosporidium muris was investigated by freeze-fracture cytochemistry using a polyene antibiotic filipin. Since the host plasma membrane enveloped C. muris, the inner and outer membranes were continuous with the parasite plasma membrane at the annular ring and with host membrane at the dense band, respectively. Although many filipin-cholesterol complexes were observed on the plasma membrane of host cells and parasites, a line showing no complexes was evident at the above two membrane junctures. These observations indicate that parasitic infection of C. muris altered the organization of membrane cholesterol.

[An outbreak of waterborne Cryptosporidiosis in Kanagawa, Japan].

An outbreak of diarrhea due to infection with Cryptosporidium occurred among the staff members and customers who visited one of the 10 public houses or a dancing school in a building in Hiratsuka, Kanagawa Prefecture, at the end of summer in 1994. The epidemiological surveys by a questionnaire revealed that 461 out of 736 persons investigated complained of cholera-like or flu-like illness. The clinical manifestations included mucous and/or watery diarrhea (96.7%), abdominal pain (61.6%), fever (54.2%: lower than 39 degrees C = 84.1%, higher than 39 degrees C = 15.9%), malaise (37.1%), nausea (32.8%) and headache (29.3%). The polluted drinking water was strongly suspected to be the immediate cause of infection. Although several species of pathogenic bacteria were isolated both from stool and water samples, they were not supposed to be linked to the outbreak. No known enteropathogenic virus was found in either of the samples. Oocysts of Cryptosporidium parvum were identified in 12 (48.0%) of the 25 stool samples. The oocysts were also found in tap water and other water samples from a receiving tank which was directly connected with the public waterworks, and an elevated tank on the roof, a wastewater pits, a soil pit and artesianspring water tank. These tanks and pits except for the elevated tank were built adjucent to each other on an underground floor of the building. These tanks and pits were connected with openings in the upperpart of the tank walls. These openings might have functioned to discharge excess of drinking water in the receiving tank to the wastewater pit. The water level of the wastewater pit is kept down below the openings by pumping out the sanitary sewage to the public drain. According to the declaration of the owner of the building, however, the wastewater pump was broken at the time of outbreak. Accidental malfunction of the drainage system caused contamination of drinking water with sanitary sewage through the connecting pipes.

Geographic distribution of Gongylonema pulchrum and Gongylonema macrogubernaculum from Macaca fuscata in Japan.

After a first report on the gullet nematode, Gongylonema pulchrum Molin, 1857, being found in the Japanese macaque, Macaca fuscata, in Kyushu, Japan, the geographic distribution of the parasite, a causative agent of gongylonemiasis in cattle and man, was examined in 181 monkeys transferred to the Japan. Monkey Centre from 23 sites in Japan, including Yaku-shima (Island). Yaku-shima is included in the World Natural Heritage List of the United Nations for its subtropical forests, which have an unusual variety of plant and animal species. G. pulchrum was found in M. fuscata yakui monkeys inhabiting Yaku-shima and M. fuscata fuscata monkeys inhabiting Honshu and an is and near Honshu. G. macrogubernaculum was found in M. fuscata yakui monkeys. Comparison of the two kinds of parasite specimens obtained from the variety M. fuscata yakui confirmed that G. macrogubernaculum Lubimov, 1931 is a valid species. Thus, the finding of G. macrogubernaculum constitutes a record of a newly identified host. M. fuscata yakui, and shows that Yaku-shima, Japan, is a natural locality of G. macrogubernaculum.

[Cryptosporidium diarrhea developing in two Japanese adults--one in AIDS and the other in a normal host. Research Group for Infectious Enteric Diseases, Japan].

Case I: A middle-age homosexual male developed AIDS with Pneumocystis carinii pneumonia (PCP) and esophageal candidiasis in 1986 during his stay in an European country about five months prior to transfer to Tokyo Metropolitan Komagome Hospital, Tokyo, in 1987. He was also diagnosed as having cryptosporidiosis presenting with mild diarrhea a month following the diagnosis of PCP. Diarrhea was successfully treated with spiramycin. On transfer to Tokyo Metropolitan Komagome Hospital, he was febrile but had no diarrhea. Serum HIV and TPHA were positive and his blood lymphocyte subset T4a was markedly decreased. On the 13th day after transfer to the hospital, watery diarrhea appeared. Cryptosporidium oocysts were detected from the feces taken on the 17th hospital day. The patient died of Escherichia coli septicemia on the 38th hospital day. Autopsy finding yielded Cryptosporidium infection widely spread over the stomach, ileum, bile and pancreatic ducts. Case II: A 31-year-old previously healthy female presented with abrupt onset of mucous stool five times daily. Mucous passage continued on the subsequent days despite administration of loperamide, and the passage increased to 20 times daily with mucous to watery diarrhea associated with mild abdominal cramps and nausea on the 4th day after onset of illness. On the 6th day of illness, she visited Tokyo Metropolitan Komagome Hospital. She denied close contact with pet animals or contact with any person presenting diarrhea. She had no recent history of travelling anywhere outside Tokyo. On examination she was an apparently healthy woman except for a slightly distended abdomen with localized tenderness in the right upper quadrant.(ABSTRACT TRUNCATED AT 250 WORDS)

Chemotherapeutic effect of azithromycin and lasalocid on Cryptosporidium infection in mice.

Prednisolone-immunosuppressed mice (ICR, 7-wk-old female) were each inoculated with 1 x 10(5) oocysts of Cryptosporidium parvum. Medication with azithromycin (400 mg/kg/day) or lasalocid (64, or 128 mg/kg/day) was started 13 h after inoculation and continued for 3 days. The number of oocysts discharged by each mouse was calculated on days 4-12 post-inoculation. Compared with non-medicated controls, oocyst production by the medicated mice was markedly reduced; some mice did not discharge oocysts and the remaining mice discharged less than 1/100 the number of oocysts of the control mice. These results indicate that both azithromycin and lasalocid have prophylactic or therapeutic activity against Cryptosporidium.

Ultrastructural changes associated with reversal of chloroquine resistance by verapamil in Plasmodium chabaudi.

Reversal of chloroquine (CQ) resistance by verapamil, a Ca2+ antagonist, has been shown in CQ-resistant human and rodent malaria parasites. Here, we report ultrastructural changes associated with this phenomenon in CQ-resistant Plasmodium chabaudi (AS strain) after infected mice were administered CQ and verapamil. At parasitaemias of 5-7%, CQ at 6 mg/kg caused little morphological effect on CQ-resistant parasites. In contrast, co-administration of CQ and verapamil at 50 mg/kg induced swelling of food vacuoles with clumped pigment at 2.5 h. Morphological changes other than food vacuole enlargement occurred at 21 and 45 h: disappearance of endoplasmic reticulum, formation of myelin structures, focal cytoplasmic vacuolization and coarse clumping of electron-dense material in nuclei. These structural changes appeared to be very similar to those observed in CQ-sensitive P. chabaudi in mice injected with CQ alone or CQ plus verapamil. On the other hand, verapamil at 50 mg/kg alone did not induce any effect on both CQ-sensitive and CQ-resistant P. chabaudi. These results suggest that swelling of the food vacuoles is an initial event associated with reversal of CQ-resistance by verapamil.

Secretion by Toxoplasma gondii of an antigen that appears to become associated with the parasitophorous vacuole membrane upon invasion of the host cell.

Monoclonal antibodies against Toxoplasma gondii were prepared to characterize antigens of the parasite. Immunoperoxidase staining of parasites fixed with paraformaldehyde and glutaraldehyde (PFAGA) followed by Triton X-100 treatment showed that the antibody of clone I-63 recognized an antigen located in the anterior part of the parasite. When analysed by SDS-PAGE and immunoblotting, the antigen migrated in a 66 x 10(3) Mr region. The parasite antigen diminished greatly in parasites after invasion of host cells, but reappeared around a time when intracellular T. gondii multiplied. Immunodetection on PFAGA-fixed T. gondii-infected cells, whose membranes were permeabilized by freeze-thawing in the presence of 5% glycerol, demonstrated that, immediately after parasite invasion, the I-63 antibody-reactive antigen appeared to become associated with the parasitophorous vacuole (PV) membrane, that had been formed mainly by invagination of the host-cell plasma membrane so as to surround an invading parasite. The antigen remained associated with the PV membrane for some time, but disappeared later when the PV increased in size after the parasites had multiplied several times. These results were strengthened by immunoelectron microscopic observations: the antigen that had been localized at the anterior part of the parasite before invasion appeared in an area of the host cell cytoplasm around the tips of penetrating parasites and, thereafter, extended throughout the surface of the PV membrane when parasites completed invasion. Thus, it appears that the I-63-reactive antigen is secreted by T. gondii upon invasion of the host cell and becomes associated with the PV membrane shortly after invasion.

Multiplication of Toxoplasma gondii in maturing erythroid cells.

Multiplication of Toxoplasma gondii was examined in vitro in murine erythroid cells at different stages of development. Toxoplasma gondii multiplied in nucleated erythroblasts and enucleated reticulocytes from the foetal liver, and in immature reticulocytes and yolk-sac derived erythrocytes from the foetal circulation. However, the rate of multiplication was lowered as erythroblasts matured, and multiplication was not detected in foetal erythrocytes. A decrease in the multiplication rate with maturation was also observed with yolk-sac erythrocytes.

Penetration of chick embryo erythrocytes by toxoplasma gondii tachyzoites in simplified incubation media.

The ability of Toxoplasma gondii tachyzoites to penetrate chick embryo erythrocytes (CEE) and the extent of penetration were examined in various simplified, incubation media. Toxoplasma gondii penetrated CEE well in serum-free Eagle's minimum essential medium or in Hanks' balanced salt solution, but much less in phosphate buffered saline (PBS). However, the addition of glucose alone to PBS substantially increased the penetration. A similar effect was seen with other monosaccharides, except galactose. A further, marked increase in penetration was noticed if magnesium ion (but not calcium ion) was added to PBS containing glucose. The present results indicate that T. gondii can penetrate CEE well in simplified incubation solutions such as PBS only when magnesium ion and a monosaccharide are added. Their possible role is discussed in relation to cellular adhesiveness, because the adherence of the parasite to the host cell membrane may be the first step in the penetration process.

Penetration of maturating red blood cells by Toxoplasma gondii.

Penetration of Toxoplasma gondii tachyzoites was studied in vitro using murine erythroid cells at different stages of development. Toxoplasma gondii penetrated nucleated erythroblasts and macroreticulocytes from foetal mouse liver and the circulating erythrocytes of foetal, neonatal or severely anaemic adult mice. Immature reticulocytes were more susceptible to penetration than mature ones, indicating that some change in their membrane properties occurred during maturation. The present results confirmed our previous finding that the major erythrocyte membrane-specific proteins do not prevent erythrocyte penetration since these proteins are known to be present in the reticulocyte membrane.