Description and classification of abnormal cercariae of Schistosoma mattheei and a method of concentration and collection

Over a period of about 12 years, 30 abnormal Schistosoma mattheei cercariae were found among a total of approximately 2.8 million examined. Initially seven were recovered from about 1.02 million (0.0007%), which were examined individually while being counted with the aid of a stereoscopic microscope. Subsequently, on the strength of relatively high percentages of abnormal individuals recovered when counting cercariae that failed to penetrate into oxen, it appeared that the morphologically abnormal cercariae were unable to swim and would mostly sediment out of a suspension while most of the normal cercariae would remain swimming. This surmise is supported by recovery of 23 morphologically abnormal cercariae (0.001%) from about 1.8 million, by examining the sediment after the cercarial suspension had been left standing undisturbed in glass measuring cylinders. The abnormalities ranged from aberrant tails only (e.g. an underdeveloped tail, or different degrees of schism) or aberrant heads only, to abnormalities of both the heads and tails. A suggested schematic classification of abnormal cercariae is presented. A young, adult hamster was exposed to eight S. mattheei cercariae with complete schism of the shaft of the tail, by pipetting the cercariae onto the shaved abdominal skin of the anaesthetised animal. Two underdeveloped females were subsequently encountered in squash preparations of the liver when the hamster was killed for worm recovery 10 weeks after infection, thus showing that some of the abnormal cercariae were viable. A method is also described for killing and fixing cercariae while retaining some of the shining brilliance of live cercariae, without them becoming shrivelled, granular and semi-opaque, as occurs when cercariae die spontaneously or are killed with heat. This is apparently the first report of abnormal cercariae of S. mattheei. In addition, a method of concentrating abnormal cercariae after emergence from a snail, a schematic classification of abnormal cercariae and a method for killing and fixing cercariae while retaining much of the shiny brilliance of live cercariae are also reported for the first time as far as is known.     

Application of an immunomagnetic bead ELISA based on IgY for detection of circulating antigen in urine of mice infected with Schistosoma japonicum

Schistosomiasis is an important zoonosis and some livestock especially bovine and swine play a crucial role on the disease transmission in endemic areas. The gold standard for animal Schistosoma japonicum infection is fecal examination although indirect agglutination assay (IHA) is so far mostly used in field survey and laboratory examination. Lack of sensitivity, poor practicality and high false positivity limit the use of those methods for routine veterinary detection as well as human diagnosis. A novel immunomagnetic bead ELISA based on IgY (egg yolk immunoglobulin) was developed for detection of circulating schistosomal antigen (CSA) in sera of hosts infected with S. japonicum. To assess the application of this method for diagnosis of domestic animal schistosomiasis with urine sample, the immunomagnetic bead ELISA based on IgY (IgY-IMB-ELISA) was employed in the present study to detect CSA in urine of murine schistosomiasis with either light (10 S. japonicum cercariae infection per mouse) or heavy infection (30 S. japonicum cercariae infection per mouse). The results showed that the CSA levels in urine of heavily and lightly infected mice reached a peak in 8 and 10 weeks after infection, respectively, remaining at a constant plateau in both groups by the end of the experiment (14 weeks after infection). The CSA level in urine of heavily infected mice was much higher than that of lightly infected mice from 8 to 14 weeks after infection. The effect of praziquantel treatment on the CSA level in urine of heavily infected mice was also investigated. It was found that the CSA level in urine of heavily infected mice with treatment was much lower than that of untreated mice at 4 weeks post-treatment, although still higher than that of control mice, and then gradually descended to the background level by 8 weeks after treatment. Our findings suggested that the IgY-IMB-ELISA may be an efficient and practical tool in non-invasive diagnosis of schistosome infection based on antigen detection, and evaluation of the efficacy of chemotherapy as well.     

A fatty acid binding protein from Fasciola hepatica induced protection in C57/BL mice from challenge infection with Schistosoma bovis.

Three strains of mice (NMRI, C57/BL, BALB/c) were each immunized with a 12 kDa purified, native Fasciola hepatica fatty acid binding protein (Fh12) and challenged percutaneously with Schistosoma bovis cercariae. C57/BL mice immunized with Fh12 had significant reductions in S. bovis worm burden recoveries (96 and 87% reductions over controls in two separate experiments). When using NMRI or BALB/c mice, Fh12 alone or in Freund's adjuvant failed to induce significant protection against S. bovis. In C57/BL mice vaccinated against Fh 12, antibodies to the IgG2a isotype, but not to the IgG1 isotype, increased by 2 weeks after the second immunization and remained high through 8 weeks of S. bovis infection. Antibodies to S. bovis increased after 4 weeks of infection. Regarding cytokine production by spleen mononuclear cells, C57/BL mice vaccinated with Fh12 in adjuvant, and having the highest protective response against challenge infection with S. bovis, had an increase of IFN-gamma production with Concanavalin A but no increase of IL-4 in similarly stimulated cells. These results suggest that the protection obtained in this group of mice is mediated by a Th1 immune response.     

东方田鼠感染日本血吸虫前后血常规和血清生化指标的动态变化

东方田鼠具有天然抗日本血吸虫病特征,为了探索东方田鼠抗日本血吸虫机制,本研究分析了东方田鼠和小鼠感染日本血吸虫尾蚴前后血液细胞以及生化指标的动态变化。东方田鼠和BALB/c鼠定量感染日本血吸虫尾蚴,于感染后第0、1、3、5、8、12、16、21、28、35、42 d分别采集抗凝血和血清,进行血常规和血清生化分析。血常规检测结果表明,东方田鼠感染后第3~16 d,WBC数量显著升高(Neu数量显著升高),而小鼠血常规检测结果正常;血清生化结果显示,东方田鼠感染日本血吸虫后ALB、HDL-C显著升高,小鼠各指标变化不明显。本研究结果显示东方田鼠天然免疫细胞Neu和血清ALB可能与其抗日本血吸虫特性相关,对补体杀伤日本血吸虫机制研究具有重要的指导意义。     

Comparison of the vaccine efficacy of gamma-irradiated Schistosoma japonicum cercariae with the defined antigen Sj62(IrV-5) in pigs

Development of a vaccine against Schistosoma japonicum which can protect both man and the domestic animal zoonotic reservoirs of infection would be an invaluable tool in attempts to control this infection in those areas in which conventional control methods have failed to break transmission. The pig is a natural host of S. japonicum and because of its anatomical and immunological similarities to humans, it is a potentially valuable host for studies on S. japonicum in particular and schistosomes in general. Radiation-attenuated cercariae are highly effective in inducing immunity in experimental schistosomosis and there are promising reports of partial protection against schistosomes with recombinant-derived individual antigens. In the present study we have set out to establish a protocol for inducing protection with gamma-irradiated cercariae in pigs and to assess the protective capacity of recombinant and naked DNA formulations of Sj62, a 62kDa region of S. japonicum myosin. The corresponding S. mansoni version or Sj62, recombinant IrV-5, has previously been implicated in irradiated vaccine immunity in S. mansoni infections and has been shown to induce high levels of immunity in a variety of hosts.Groups of pigs were immunised three times at 2-week intervals with 2000 cercariae irradiated at 20krad, with Sj62 as a recombinant (rSj62) incorporated in Freund's adjuvant, a micellar preparation, or as a naked DNA construct. Vaccination with irradiated cercariae did not induce significant anti-Sj62 antibody but following intramuscular challenge with 2000 cercariae, the vaccinated pigs showed >95% resistance as assessed by reduced faecal egg output, worm tissue egg burdens and also reduced septal fibrosis. Immunisation with each of the Sj62 formulations induced significant anti-Sj62 antibody responses, the highest titre (>12,800) being with the Freund's preparation but none of the Sj62-immunised groups showed significant resistance to challenge. The data suggest that Sj62 shows little promise as a vaccine candidate for schistosomosis.     

Schistosoma mattheei in the ox: clinical pathological observations

Twenty-eight Friesian calves were infected between seven and 11 months of age with 5000 to 45,000 cercariae of Schistosoma mattheei. They developed anaemia, lymphopaenia and hypoalbuminaemia during the period of acute clinical illness after the infection became patent, and lymphocyte counts remained depressed after clinical recovery. Neutrophil counts rose and later fell before returning to normal. Eosinophilia and hypergammaglobulinaemia were marked during the period of recovery. The changes in haemoglobin, neutrophils and serum proteins were proportional to the level of infection. The eosinophil response was reduced in animals subjected to nutritional stress. The aetiology of the changes is discussed.     

Experimental infection of rhesus monkeys with Schistosoma incognitum and Orientobilharzia dattai

Rhesus monkeys inoculated subcutaneously with 800-2500 cercariae of Schistosoma incognitum did not yield adult worms. Percutaneous exposures, however, resulted in the presence of adult flukes 21-35 days post infection. The infection became very mild (0-4 flukes) 45-100 days after exposure and no eggs were excreted. Monkeys percutaneously exposed to cercariae of Orientobilharzia dattai and treated with prednisolone yielded a very small number of flukes 22 days after exposure while non treated controls yielded no adult worms.     

东方田鼠和小鼠感染日本血吸虫后组织与血清蛋白组分差异比较研究

探讨东方田鼠和小鼠感染日本血吸虫前后不同时点组织与血清中蛋白质电泳谱的变化。分别提取东方田鼠感染日本血吸虫(0、7、12、25 d) 和小鼠感染日本血吸虫(0、7、12、45 d)后的肝脏、脾脏、肺脏蛋白及血清进行聚丙烯酰胺凝胶电泳,对结果比较分析并进行组织病理形态学观察。结果显示,小鼠感染日本血吸虫第7和12天后肝脏150 ku附近蛋白质条带较东方田鼠表达量明显增高;东方田鼠在感染日本血吸虫后40~50 ku附近蛋白质条带较正常东方田鼠表达明显上调;感染第7天后肺脏40 ku附近蛋白质条带较其他时间点变化明显;小鼠感染日本血吸虫第7、12和45天后血清中IgG类蛋白质表达量较东方田鼠显著增高。东方田鼠和小鼠在日本血吸虫感染后不同时点间的组织及血清蛋白组分存在差异,东方田鼠肝脏和血清中150 ku附近蛋白条带较其他动物特异,蛋白质表达丰度高,该蛋白可能与东方田鼠抗日本血吸虫相关。     

Histopathology of Experimental Schistosoma bovis Infection in Goats

The inflammatory host response to Schistosoma bovis in young goats was studied at necropsy by light microscopy 34 weeks after primary exposure to 3,000 cercariae (group B, n=6), 34 weeks after primary exposure to 3,000 cercariae followed by challenge with 2,500 cercariae at week 17 (group C, n=5), and 17 weeks after primary exposure to 2,500 cercariae, given on week 17 of the experiment (group D, n=6). Three goats served as uninfected controls. The faecal egg output had been minimal for 17 weeks prior to necropsy in groups B and C and only for the last 2 weeks in group D.Histological studies were carried out on the small intestine, liver, lung and spleen, and tissue egg counts were performed. In sections of the small intestine and liver, a panel of histopathological variables were quantitated to characterize the host response and differences between groups of animals were evaluated with one way analysis of variance. The mean tissue egg count in the small intestine was slightly but not significantly higher in group C than group B and about twice as high in group D (D vs B or C p<0.01). Group means of numbers of inflammatory foci per section of gut wall corresponded well with those of tissue egg counts, suggesting that the rate of inflammatory destruction of eggs did not differ markedly between the groups. Egg material was less commonly seen in granulomas of the small intestine in group B than in group D (p<0.01), suggesting lower passage of eggs through the gut wall during the later than during the earlier phase of patent primary infection. The frequency of eosinophil-rich hepatic inflammatory foci was much higher in group D than in the other groups (D vs B p<0.05, D vs C p< 0.01), and coincided with a high degree of blood eosinophilia in this group at the time of sacrifice. Challenged goats showed a significantly higher frequency of markedly fibrotic inflammatory foci in the liver and of liver granulomas with a marked giant cell component than goats of the other groups. Hepatic portal fibrosis was least prominent in animals with 17- week- old primary infections, implying a possible relation between this change and duration of infection.     

IgG1型单克隆抗体和吡喹酮协同预防日本血吸虫病的研究

6组昆明系小鼠人工感染日本血吸虫尾蚴前2小时,组1和组2胃饲低剂量吡喹酮(20mg/kg),并分别腹腔注入鼠抗日本血吸虫IgG_1型单克隆抗体ISj51和ISj55作被动免疫,4天后重复1次,4周后剖杀集虫。结果表明,单抗合并吡喹酮的组1和组2的减雌率分别为45.32％和42.95％,均明显高于(p<0.01)仅作腹腔被动免疫转移ISj 51和ISj 55单抗的组3和组4(分别为15.11％和19.44％),亦高于(p>0.05)仅用单剂吡喹酮的组5(37.41％)。2株单抗在巨噬细胞和嗜酸性粒细胞的介导下,杀伤童虫率接近1:2稀释的8周阳性鼠血清(分别为37.50％,46.88％和48.00％,41.18％)。提示:鼠抗日本血吸虫IgG_1型单抗协同低剂量吡喹酮,具有预防日本血吸虫感染,提高灭雌率,降低吡喹酮毒副作用的优点,对控制和预防日本血吸虫病的传播具有一定的意义。     

Vaccination of mice against schistosoma bovis with a recombinant fatty acid binding protein from Fasciola hepatica

Two strains of mice (NMRI and C57/BL) were each immunized with a 15kDa recombinant Fasciola hepatica fatty acid binding protein (FABP) (Fh15) and challenged percutaneously with Schistosoma bovis cercariae. C57/BL mice immunized with Fh15 had significant reductions in S. bovis worm burden recoveries (72% reductions over controls). When using NMRI mice, Fh15 in Freund's adjuvant failed to induce significant protection against S. bovis. In C57/BL mice, only antibodies to the IgG2a isotype increased after the second immunization and remained high through 8 weeks of S. bovis infection. This is the first time that a heterologous recombinant molecule from F. hepatica has been used in vaccination against S. bovis, obtaining a significant reduction in the number of worms in C57/BL mice.     

Parasitological survey of the first intermediate host of Paragonimus westermani in Iga area of Mie Prefecture, Japan

During the period from September 1985 to March 1988, the freshwater snails, Semisulcospira libertina, were collected from 4 mountain rivers in Ayama County of Mie Prefecture, which is known as a heavily infected locality with Paragonimus westermani (Kerbert, 1878) Braun, 1899, and were examined for cercariae and rediae of this lung fluke. Of 3,000 snails studied, 80 (2.67%) harbored Paragonimus larvae. The infected snails were found at 3 sites in the Nishitani (A), Nenobi(B), and Higashitani (C) rivers. The infection rate of Paragonimus cercariae at these sites varied from 0.13 to 6.08%. The highest incidence (6.08%) of cercarial infection occurred at site A (Nishitani river), where the prevalence and intensity of infection with P. westermani metacercariae were considerably greater in the 2nd intermediate host crabs. No Paragonimus was observed in 776 snails collected at site D (Taki river), where the incidence and degree of metacercarial infection in crabs were quite small. There was a positive correlation between the infection incidence of P. westermani and the size of S. libertina; the percentage of infected snails increased in larger size classes, reaching 24.14% at a shell length of over 40 mm. In the 80 positive snails there were 51 mixed infections with P. westermani and 1 or 2 other species of trematode larvae. Of these, 43 were double infections and 8 triple. The morphological features of P. westermani cercaria and redia are described.     

Immunologic and hematologic responses in ponies with experimentally induced Strongylus vulgaris infection

Immunologic and hematologic responses were examined in 4 ponies with experimentally induced Strongylus vulgaris infection and in 5 helminth-free ponies. Two ponies were inoculated with 200 larvae and 2 were inoculated with 700 larvae of S vulgaris and then were reinoculated with the same numbers of larvae 34 weeks later. Initial response of the ponies inoculated with S vulgaris was S vulgaris antigen-induced lymphocyte response that developed 1.5 to 3 weeks after inoculation and did not persist. Development of antigen-reactive lymphocytes was followed sequentially by a biphasic complement-fixing antibody response, then biphasic eosinophilia. Antibody titer to S vulgaris antigen was higher in ponies inoculated with 700 larvae, compared with that in ponies given 200 larvae of S vulgaris. Also, the second peak in antibody titer and in absolute number of eosinophils was observed earlier in ponies inoculated with 700 larvae, compared with ponies inoculated with 200 S vulgaris larvae, and subsided before or from about 24 weeks after inoculation. The prepatent period for S vulgaris infection was 24 to 25 weeks. After reinoculation with S vulgaris, a degree of increased lymphocyte responsiveness was apparent but, by 17 weeks after reinoculation, only the primary peak in the absolute number of eosinophils indicated an anamnestic response. Essentially, antibody was not detectable after reinoculation.     

Peroral infection of pigs with Schistosoma japonicum cercariae.

Infections with the zoonotic trematode, Schistosoma japonicum in pigs serves as a valuable model for studying natural definitive host/parasite relationships and a model for human schistosomosis japonica. In the present study the efficiency of a peroral infection route was compared with that of an intramuscular route of infection. Eleven specific pathogen-free Danish Landrace/Yorkshire/Duroc crossbred male and female pigs were divided into two groups of five and six pigs, respectively. Each pig was given 1000 cercariae, either placed in droplets on the mucosa in the buccal cavity, or as medium-suspended cercariae injected into musculus biceps femoris of one of the hindlegs. Ten weeks post infection, all pigs were killed with pentobarbital and the venous system perfused. Worm burdens and liver egg counts were determined and worm fecundity was calculated. S. japonicum infections were established in all individuals in both groups of pigs. When comparing the two groups, the peroral group had significantly higher number of immature worms, whereas the intramuscularly infected group had significantly more worm nodules. However, no difference was seen in total number of worms. No statistical significant differences were found in neither tissue egg counts nor worm fecundity when comparing the two groups. The results from the present study showed a delay in maturation of infection following a peroral infection as compared with an intramuscular infection, but comparability was seen between overall worm establishment and egg production.     

Factors affecting the bluetongue virus neutralizing antibody response and the reaction between virus and antibody.

A study was made of different aspects of the bluetongue virus neutralizing antibody response and the reaction between the virus and antibody. Optimum neutralization was obtained in a 2mM Tris-HCl buffer, pH 9,0, at a temperature of 4 degrees C. The reaction of virus and antibody could be demonstrated by electron microscopy in the formation of clumps which were shown to be serotype specific. It was found that both IgM and IgG antibodies can neutralize the virus, but that IgM reached its maximum level sooner after infection than IgG.     

Comparative study of sheep and buffalo isolates of Fasciola gigantica in the intermediate host Lymnaea auricularia

A comparison was made of the biological behaviour of two isolates of Fasciola gigantica, obtained from sheep and buffalo, in the aquatic snail Lymnaea auricularia. The sheep isolate exhibited a lower rate of infection, slower larval development and the production of fewer cercariae. Larger eggs, miracidia and metacercariae were obtained from the buffalo isolate while the rediae and cercariae were smaller. Metacercariae of sheep origin were dark brown with a mortality after 80 days of storage at 4 degrees C of 30.6 per cent while those of the buffalo isolate were straw coloured and had a mortality of 10.2 per cent.     

The epidemiology of fasciolosis in Papua New Guinea

Fasciola hepatica metacercariae were present on pasture throughout the year at the trial site. The highest infection rate in trial sheep occurred in the first year after the beginning of the wet season, in December/January, when contamination of the pasture was maintained by other sheep. Variation in rates of infection in consecutive years was linked to differences in grazing pressure and to a change from sheep to cattle as accompanying grazing stock. A minimum precipitation of 125 mm over 4 consecutive weeks appears to be necessary for infected snails to move from their normal habitats and to contaminate wet pasture with cercariae. Infection of sheep can occur during a dry season if pasture has permanent seepage sites or swampy areas where infected snails can remain active and continue to liberate cercariae.     

Transmission of Ehrlichia risticii, the agent of Potomac horse fever, using naturally infected aquatic insects and helminth vectors: preliminary report

Ehrlichia risticii, the agent of Potomac horse fever (PHF), has been recently detected in trematode stages found in snail secretions and in aquatic insects. Based on these findings, horses could conceivably be exposed to E. risticii by skin penetration with infected cercariae, by ingestion of infected cercariae in water or via metacercariae in a second intermediate host, such as an aquatic insect. In order to test this hypothesis, horses were challenged with infectious snail secretions and aquatic insects collected from a PHF endemic region in northern California. Two horses stood with their front feet in water harbouring E. risticii-infected cercariae, 2 horses drank water harbouring E. risticii-infected cercariae, and 6 horses were fed pools of different aquatic insects harbouring E. risticii-infected metacercariae. In this preliminary study, only the one horse infected orally with mature caddisflies (Dicosmoecus gilvipes) developed the clinical and haematological disease syndrome of PHF. The agent was isolated from the blood of the infected horse in a continuous cell line and identified as E. risticii by characterisation of the 16S rRNA gene. Therefore, E. risticii is maintained in nature in a complex aquatic ecosystem and transmission to horses can occur through accidental ingestion of insects such as caddisflies containing infected metacercariae. At present, the small number of horses used in this study does not exclude other insects and free trematode stages as potential sources of infection.     

Clinical pathology of experimental Schistosoma curassoni infections in sheep and goats

The clinical pathology of Schistosoma curassoni infection in sheep and goats was studied for 22 weeks following experimental infection with 7000 and 4000 cercariae, respectively. Excretion of eggs began at week 7 after infection: in goats the numbers increased to 30 to 50 eggs per gram faeces (epg) at weeks 8 to 18, followed by a reduction. In a pregnant goat, epg values increased markedly before and after parturition. The mean faecal egg counts in sheep were lower than in goats, increasing to a maximum level of 30 epg at weeks 16 and 17 after infection. Infected sheep maintained growth rates roughly comparable with controls, whereas infected goats failed to gain as much weight as the controls. Infected goats and sheep produced eosinophil counts of about 3 x 10(3) mm-3, five and eight weeks after infection, respectively. Sheep developed a progressive anaemia from week 11 after infection, in goats blood values remained within normal limits. Differences in serum protein concentration were observed between infected and uninfected goats about nine weeks after infection, but not in sheep. Increased total protein values, hyperglobulinaemia and lowered albumin to globulin ratios were features of infected goats. Serum glutamate oxaloacetate transaminase, glutamate pyruvate transaminase, gamma-glutamyl transferase, total lactate dehydrogenase and bilirubin were not significantly changed. The mean recovery in sheep was 608 worms, in goats 428 worms, but the total tissue egg counts were higher in the latter. Of the total eggs deposited in the goats 92 per cent were found in the liver with 51.5 per cent in the ovine liver. The histopathological changes were studied.     

Evaluation of immunological, parasitological and molecular methods for the diagnosis of Schistosoma mansoni infection before and after chemotherapy treatment with praziquantel in experimentally infected Nectomys squamipes

In low endemicity areas of schistosomiasis, the recommended diagnostic method of coprological examination results in an underestimation of infection cases. Alternative diagnostic methods have been developed, such as immunodiagnostic and molecular techniques. In this study we evaluated three methods used in the diagnosis of Schistosoma mansoni infection: parasitological (Kato-Katz), immunological (ELISA) and molecular (real time PCR), and also investigated the sensitivity of each technique in the cure determination after treatment with praziquantel using the water rat Nectomys squamipes, a natural reservoir for S. mansoni, as an experimental model. Two infection laboratory experiments were carried out. The first experiment aimed to observe the evolution of the immunological response in the first moments after infection and in the first months after treatment. The second experiment aimed to compare the efficacy of the three diagnostic techniques after infection and after treatment over a more extended time period. In the first experiment, 44% of the infected animals showed IgG reactivity after two weeks of infection, and 94% were positive based on serology 30 days after infection. The serological IgG titers increased just after infection but decreased gradually after treatment. In the second experiment, 89% of the animals showed positive IgG titers 22 days after infection. Only 68% of the animals showed positive results on the coproscopic diagnostic analysis and 79% did so by qPCR, 50 days after infection. Treated animals showed negative results on coproscopy one month after treatment but remained positive by serology even 12 months after treatment, although showing a decline in immunologic reaction after treatment. By qPCR analysis, all animals showed negative results three months after treatment, except for one animal. The parasitosis can be detected by coproscopy only six weeks after infection, and by serology 14 days after infection. The qPCR was a better diagnostic method for confirming the infection cure of S. mansoni. In early infection, this method was less efficient than serology but was slightly more efficient than the Kato-Katz method. We suggest that the methods should be used in low endemic areas as follows: serology should be used in the initial diagnosis in a population with potential positive cases; subsequently, coproscopy should be used in IgG positive cases to confirm the current infection; and qPCR should be used to evaluate the infection cure after treatment and is also a very valuable tool when there are cases showing positive IgG and negative coproscopy.