Development of the rapid and simple elisa (whole blood-ELISA) using coconut water-tween as a wash solution for whole blood sample from Schistosoma japonicum-infected rabbit and human.

PBS-Tween as a wash solution, prepared with distilled water, is used in ELISA. In areas where schistosomiasis is endemic, however, distilled water is hard to come by. We have modified a WHOLE BLOOD-ELISA test to use coconut water-Tween as a wash solution, because coconut water is easy to come by and cheap in the tropics. We applied the test to whole blood samples from rabbits and humans infected with Schistosoma japonicum. This modified WHOLE BLOOD-ELISA was confirmed to be a rapid, simple, and cost-effective method.

Comparative studies on the pathological findings and mortality in Schistosoma mansoni infected mice after treatment with artesunate and the current antischistosomal drugs.

The effect of artesunate (ART) on the pathology and mortality rate of in Schistosoma mansoni infected mice was comparatively studied with the current drugs of choice for the treatment of schistosomiasis mansoni: praziquantel (PZQ) and oxamniquine (OX). S. mansoni experimentally infected mice were treated at 9th week of infection with ART, PZQ or OX at an oral dosage of 300 mg kg(-1), 600 mg kg(-1) and 100 mg kg(-1), respectively. Untreated, infected mice and non-infected mice were added as controls. Samples of mice were sacrificed and examined for the pathological findings at 1 week, 1 month, and 3 months after treatment. At 1 week after treatment, both gross and microscopic lesions were observed. No significant differences were noted among the infected groups. Differences were observed at 1 month after treatment. The lesions decreased more rapidly in groups treated with PZQ and OX. At 3 months after treatment, there were significant differences in the pathological findings among groups. In the groups treated with PZQ and OX, the lesions were markedly reduced and rarely found, but they were clearly observed in the group treated with ART and in the untreated, infected group. High mortality was also recorded in the group treated with ART and in the untreated, infected group. Therefore, the treatment of S. mansoni infected mice at 9 weeks of infection with ART did not reduce the pathological findings or the mortality rate compared to treatment with the current recommended schistosomicides, PZQ and OX.

Therapeutic effect of subcurative dose praziquantel on Schistosoma mansoni infected mice and resistance to challenge infection after treatment.

The therapeutic effect of a subcurative dosage of praziquantel (PZQ) on Schistosoma mansoni infected mice and resistance to challenged worm infection after treatment were assessed and compared with conventional treatment using a curative dosage of PZQ. S. mansoni infected mice were treated with PZQ at a curative dosage (600 mg kg(-1)) or a subcurative dosage (300 mg kg(-1)) at 9 weeks after infection. Untreated mice and non-infected mice were added as controls. The therapeutic effect of the drug was evaluated in terms of the mortality of mice after treatment, and the parasitological and pathological findings in mice sacrificed at 1 week, 1 month, or 3 months after treatment. Another sample of mice was not killed but challenged with S. mansoni cercariae at 1 week, 1 month, or 3 months after treatment. Resistance to re-infection was evaluated by the extent of challenged worm reduction. In conclusion, there was no significant difference in mortality, or parasitological and pathological findings between mice treated with PZQ at the two dosages. However, resistance to challenged worm infection was more sustained in the group treated with subcurative dose PZQ, especially at 3 months after treatment.

A rapid and simplified ELISA using whole blood samples of Schistosoma japonicum-infected rabbits.

A rapid and simplified ELISA using whole blood samples of Schistosoma japonicum-infected rabbits was compared with a conventional ELISA. This whole-blood ELISA has advantages. The volume of crude egg antigens, whole blood samples, and conjugates was only 0.05 ml. The incubation time was shortened to 5 minutes. Wells were washed three to five times with PBS-Tween after each procedure. Optical density values were measured in 10 minutes after transfer of 0.1 ml of substrate. Constant temperature was not necessary. The entire procedure took only 20-30 minutes.

Development of the rapid and simplified ELISA (WHOLE BLOOD-ELISA) using samples of Schistosoma japonicum-infected human whole blood.

An ELISA technique was developed using samples of Schistosoma japonicum-infected human whole blood based on the conventional ELISA. In this study, the following were demonstrated. 1) Whole blood samples could be used. 2) The volume of whole blood and conjugate could be reduced to 0.05 ml. 3) The incubation time was shortened to 5 minutes. 4) The optical density could be measured at 10 minutes after transferring the substrate and the volume was reduced to 0.1 ml. 5) It did not require a fixed temperature setting. 6) The operation time was as short as 20 to 30 minutes. 7) The optical density values were almost the same as the conventional ELISA and were not influenced by other common intestinal helminthic infections. 8) The observed variations from day to day including effects of sampling in stool examination were negated by the results of this ELISA technique. 9) Based on correlation with stool examination results, criteria can be formulated in which optical density values of 0.3 and above as positive, 0.1 to less than 0.3 as doubtful, and less than 0.1 as negative. Whenever an immunological field survey is necessary, before and after a selective or a mass treatment control program, this WHOLE BLOOD-ELISA, which was shown to be rapid and simple, is recommended.

A comparative study on mouse MHC class I sequences detected in Schistosoma japonicum recovered from BALB/c (H-2d) and C57BL/6 (H-2b) mice.

The mouse major histocompatibility complex (MHC) class I sequence was detected in all the 8-week-old Schistosoma japonicum recovered from BALB/c (H-2d) and C57BL/6 (H-2b) mice by in situ polymerase chain reaction (in situ PCR). The signals of the mouse class I MHC sequence were observed in the nuclei of the mesenchymal and reproductive cells of 8-week-old S. japonicum. Furthermore, the class I MHC sequence was detected in each DNA extracted from S. japonicum cercariae maintained in BALB/c and C57BL/6 mice by nested PCR. To prove both horizontal and vertical transmission of this sequence in schistosomes, we have used cercariae obtained from parasites maintained in BALB/c mice to infect C57BL/6 and BALB/c mice, and vice versa. The MHC sequences from adult worms were compared to the cercarial MHC and host MHC sequences. Nucleotide sequence comparisons between adult worm DNA, host (H-2d and H-2b mice) DNA and cercarial DNA used for the infection suggested that the sequence of mouse class I MHC was incorporated into schistosome adults and inherited throughout their life-cycle.

Immunoblot analysis of Schistosoma japonicum egg antigens with sera from patients with acute and chronic schistosomiasis japonica.

Humoral immune responses of IgG, IgM, IgA, IgE and IgG subclass antibodies to Schistosoma japonicum egg antigens were determined by immunoblotting with serum samples from individuals in China with acute (n=24) or chronic (n=35) schistosomiasis. In general, IgM, IgA, and IgE in sera from acute patients exhibited strong binding to antigens but binding was much weaker in chronic cases. Reaction of IgG4 of chronic cases was stronger than that of IgG4 of acute cases. The recognition profile of each antibody isotype in sera was analyzed for 11 major antigen molecules (antigens with apparent molecular weights of 82, 76, 61, 57, 53, 46, 40, 32, 27, 10 and less than 6.5 kDa). Except for the 10 kDa molecule, they were well-recognized by IgA and IgE in sera of acute cases. In other combinations of antibody class and clinical phase, recognition patterns against these molecules differed among individuals. Notably, the 10 kDa molecule was specifically recognized by total IgG and IgG4 in sera from most of the chronic patients, but in sera from only one acute case. This result suggests that the 10 kDa molecule is one of the major target antigens of IgG4 and may be useful as a marker antigen to characterize the clinical phases of S. japonicum infection.

The first reported cases of canine schistosomiasis mekongi in Cambodia.

We have been conducting surveys of schistosomiasis mekongi along the Mekong river in Cambodia since 1997. We attempted to detect canine schistosome infection during the survey in 2000 because dogs were reported to be natural reservoirs of the Mekong schistosome in Lao PDR. A total of 28 canine fecal samples were collected in Kbal Chuor village, Kratie Province and examined for schistosome eggs. One specimen had schistosome eggs (positive rate = 3.6%; egg density = 100/gram stool), which showed characteristics of Schistosoma mekongi. During the 2001 survey, one out of 310 canine stool samples was positive for schistosome eggs (positive rate = 0.32%; egg density = 3,456/gram stool). These are the first confirmed cases of canine schistosomiasis mekongi in Cambodia, which suggests that dogs are animal reservoirs of S. mekongi in the survey area. We further tried to detect S. mekongi in cows, water buffalos, pigs,horses, and field rats in five villages in Kratie Province; no schistosome egg was found in the stools of these animals.