Fasciola hepatica: an antigen fraction derived from newly excysted juveniles, containing an immunoreactive 32-kDa protein, induces strong protective immunity in rats

Crude antigens of adult Fasciola hepatica and of newly excysted juveniles (NEJ) and a low-molecular-weight fraction of antigen from NEJs were tested for inducing protective immunity in rats. Two routes of vaccination were applied. The results showed that intraperitoneal vaccination induced significantly better protection (P <0.05) than intramuscular vaccination. Intraperitoneal vaccination with antigens from NEJs induced more effective protection: after challenge infection, rats that were so vaccinated had 92.6% (+/-2.5% SEM) fewer parasites in their liver and 57.3% (+/-13.3% SEM) fewer parasites penetrating the gut wall than control rats. Rats that were vaccinated with a low-molecular-weight fraction of antigen from NEJs were also highly protected against a challenge. F. hepatica antigens that are immunoreactive were identified on immunoblots, using sera collected from highly protected rats that had been vaccinated with NEJ antigens and also sera from cattle and rats that were experimentally infected with F. hepatica. The low-molecular-weight fraction of antigen from NEJs contained an immunodominant 32-kDa protein that was recognized by serum antibodies of vaccinated rats and immune cattle. This 32-kDa protein was not detected in partially purified antigens from adult flukes. We conclude that antigens of NEJs of F. hepatica, when injected intraperitoneally in rats, are highly protective. In particular, the 32-kDa protein contained in these antigens may be highly valuable for the development of an effective vaccine against F. hepatica.     

Fasciola hepatica: attempts to induce protection against infection in rats and mice by injection of excretory/secretory products of immature worms

In vitro excretory/secretory products of 4-week (immature) and 8-week-old (mature) Fasciola hepatica parasites, derived from rats, were injected together with adjuvant into naive rats and mice. Resistance to infection was assessed in rats by counting adults in the bile ducts at 9 weeks, or in mice by recording deaths after oral challenge with a high dose of viable metacercariae. Exposure of rats to excretory/secretory products of immature F. hepatica conferred a significant degree of resistance which was comparable to the level of resistance induced following oral administration of a low number of metacercariae. No protection against infection was seen in rats injected with excretory/secretory products from mature, bile duct-derived worms. In mice, no obvious mouse strain variation in susceptibility to first infection existed and hypothymic nude mice were as susceptible to infection as intact mice. As determined by protection against death, vaccination with excretory/secretory products derived from immature F. hepatica was without effect in mice. It is concluded that "host protective antigens", at least for rats, were present in the excretory/secretory products of immature F. hepatica larvae.     

肝片形吸虫对肝纤维化的影响及治疗

作为一种可同时感染家畜和人类的寄生虫,肝片形吸虫对人类和家畜的身体机能造成了不可修复的损伤,在世界范围内造成了巨大的经济损失,尤其是感染早期对肝脏造成的肝纤维化症状不明显,不易被发现。本文主要综述了肝片形吸虫、肝纤维化、三氯苯哒唑的相互关系,并从Th1、Th2类细胞因子、T细胞、血红素氧合酶-1(heme oxygenase 1, HO-1)、巨噬细胞等几个方面探讨了肝片形吸虫对大鼠肝纤维化的影响,包括细胞间的相互作用以及它们所诱导的细胞因子(如:IL-4、IL-10、IFN-γ、TNF-α等)对肝纤维化的促进或抑制作用。最后,还综述了当前唯一可以治疗人类和家畜寄生虫感染的药物三氯苯哒唑对肝片形吸虫所致肝纤维化的治疗作用。     

Preliminary survey of Capillaria hepatica (Bancroft, 1893) in Malaysia.

The prevalence of Capillaria hepatica (Bancroft, 1893) infection in a total of 2324 rats trapped from 25 localities in West Malaysia was 15.5%. Infection rates in males (16.0%) and females (15.1%) are similar. A significantly higher percentage of adults (18.1%) than young (7.7%) was infected. Capillaria hepatica infection rates among urban (0.7%) and jungle (0.0%) rats was very low as compared to field rats (17.7%) trapped from agricultural areas such as oil palm estates and rice growing areas. Prevalence of C. hepatica infection in rats is not evenly distributed throughout West Malaysia. There seem to be localised foci of infection. In some areas as many as 77.8% of the adult rats are found to be infected while in other areas the same species of rats are found free of infection.     

Fasciola hepatica: Surface tegumental responses to in vitro and in vivo treatment with the experimental fasciolicide OZ78

The tegumental alterations in adult Fasciola hepatica induced by the experimental fasciolide OZ78 were investigated utilizing scanning electron microscopy (SEM). Twelve weeks post-infection with F. hepatica, rats were treated with a single 100mg/kg oral dose of OZ78 and flukes were recovered from the bile ducts after 24-72 h. In vitro F. hepatica were incubated with OZ78 for 48 h at a concentration of 10 microg/ml in the absence or presence of haemin. Twenty-four and 48 h post-treatment of rats disruption of the tegument of F. hepatica as blebbing, swelling and furrowing was evident. The recovery of flukes 72 h post-treatment was low. Flukes examined at this time point showed an increasing severity of tegumental damage as sloughing and absence of spines, in particular in the tail region. SEM analysis of F. hepatica incubated in the presence of OZ78 without haemin showed only minor and localized damage of the tegument. In the presence of haemin extensive tegumental damage, including sloughing or blebbing, in particular in the anterior part, was observed. In conclusion, our experiments confirm the interesting fasciocidal properties of OZ78. The tegument of adult F. hepatica might play a role in the action of this drug.     

Immunological tolerance to pig-serum partially inhibits the formation of septal fibrosis of the liver in Capillaria hepatica-infected rats

Systhematized septal fibrosis of the liver can be induced in rats either by repeated intraperitoneal injections of pig-serum or by Capillaria hepatica infection. The relationship between these two etiological factors, as far as hepatic fibrosis is concerned, is not known, and present investigation attempts to investigate it. C. hepatica-induced septal fibrosis of the liver was considerably inhibited in rats previously rendered tolerant to pig-serum. Pig-serum-tolerant rats developed antibodies against pig-serum when infected with C. hepatica, but this did not happen when the infection occurred in normal rats. On the other hand, anti-C. hepatica antibodies failed to recognize any epitope in pig-serum, by Western blot. However, no evidence of an immunological cross reactivity was found, at least at the humoral level. Alternatively, cell-mediated mechanisms may be involved, and further investigations are warranted.     

Attempts to immunise rats and mice against infection with fasciola hepatica using antigens prepared from taenia hydatigena.

Attempts were made to immunise rats and mice against infection with F. hepatica by oral dosing with T. hydatigena eggs, or by vaccination with various T. hydatigena antigen preparations. These antigens included extracts from T. hydatigena cysticerci and cyst fluid, and antigens collected during short-term (48 h) and long-term (14 days) in vitro cultivation of larvae. Immunity was assessed by the numbers of F. hepatica recovered from the challenge infection in rats, and the mortality rates of infected mice. None of the immunisation regimes with T. hydatigena antigens induced consistent, significant immunity. This was in contrast to the high level of immunity shown by rats dosed orally with F. hepatica metacercariae four weeks prior to challenge infection.     

First report of Lymnaea columella Say, 1817 (Pulmonata: Lymnaeidae) naturally infected with Fasciola hepatica (Linnaeus,1758) (Trematoda: Digenea) in Argentina

We report the first evidence of natural infection of Lymnaea columella with Fasciola hepatica in Argentina. A sample of 601 snails was collected in May 2003 in northeastern Corrientes, a province bounded on the north by Paraguay, on the east by Brazil and on the southeast by Uruguay. Among 500 examined snails, 44 (8.8%) were exclusively infected with F. hepatica. Parasite identification was based on morphological features of cercariae from snails, and of eggs and adult flukes from Wistar rats. We discuss the events suggesting that an enzootic transmission cycle of F. hepatica has been recently established in northeastern Corrientes.     

Glutathione S-transferases in Fasciola hepatica

Glutathione S-transferases (GST's) are widespread in the tissues of the liver fluke, Fasciola hepatica, and consist of multiple isozymes. Following purification to apparent homogeneity by affinity chromatography on glutathione agarose, fluke GST's were shown to comprise 2 components with molecular weights of about 25,000. Fluke GST's were immunogenic to rats, but when used as a vaccine conferred no protection on the animals against a challenge infection with F. hepatica metacercariae.     

A contribution to the diagnosis of Capillaria hepatica infection by indirect immunofluorescence test

A highly specific pattern of immunofluorescence was noted when sera from Capillaria hepatica-infected rats were tested against the homologous worms and eggs present either in paraffin or cryostat sections from mouse liver. The pattern was represented by a combined apple green fluorescence of the internal contents of worms and eggs, which persisted in serum-dilutions of 1:400 up to 1:1600. Unequivocal fluorescent pattern was observed from 15 days up to 3 months following inoculation of rats with embryonated C. hepatica eggs and such result was confirmed by the ELISA. After the 4th month of infection, the indirect immunofluorescence test turned negative, probably revealing the extinction of parasitism, however the ELISA was contradictory, disclosing high levels of antibodies in this period. The IIF was also negative when control normal rat sera and sera from rats administered by gavage with immature C. hepatica eggs (spurious infection), or for reactions made against Schistosoma mansoni eggs, although a weakly positive pattern occurred with Fasciola hepatica eggs. The indirect immunofluorescence test may be recommended for use with human sera to detect early C. hepatica infection in special clinical instances and in epidemiological surveys, since it is a simple, inexpensive, and reliable test, presenting excellent sensitivity and specificity. Although the diagnosis is positive only during early infection, this is the period when the symptoms are usually more severe and the need for differential diagnosis is greater.     

Ecology of Capillaria hepatica (Bancroft 1893) (Nematoda). II. Egg-releasing mechanisms and transmission

The egg-releasing mechanism and transmission ecology of Capillaria hepatica among Norway rat populations of the Baltimore Zoo were studied from 1972 to 1974. Nearly all adult rats were infected, while 65% of juveniles had infections. The mean egg count per liver was calculated to be 457,783 (N = 39 livers) and ranged from 11,270 to 1,400,000 eggs per liver. Data from the present study suggest that cannibalism serves as a primary egg-releasing mechanism and is a source of infection within the burrows. Increased infection rates among juveniles in spring support the hypothesis of maintenance of C. hepatica infections within the burrow system through cannibalism. Predation was responsible for scattered foci of infection throughout the study area and considered as a secondary source of infection. Decomposition was an important egg-releasing mechanism in secondary foci and in the warmer season when insects were active. However, of 849 carrion-associated insects and soil invertebrates collected from around decomposing rats, eggs of C. hepatica were found in only two species of beetles. This suggests a minor role for insects and soil invertebrates as egg disseminators.     

Capillaria hepatica (Nematoda) infections in human-habituated mountain gorillas (Gorilla gorilla beringei) of the Parc National de Volcans, Rwanda

Habituation to humans of free-ranging populations of endangered mountain gorillas (Gorilla gorilla beringei) raised concern of anthropozoonotic transmission of parasitic helminths and protozoans. Examinations of liver tissue of 19 gorillas found dead in the Parc National de Volcans, Rwanda, revealed 10 cases of hepatic nematodiasis due to Capillaria hepatica. Identifiable C. hepatica eggs were present in the liver of 4 gorillas (3 juveniles, 1 adult), and nematode cross-sections were found in 1 juvenile gorilla. Six other adult gorillas had areas of periportal and subcapsular fibrosis with calcified eggs. Histologically, the lesions surrounded by the areas of mild inflammatory reaction were characterized by subcapsular, periportal foci of fibrosis in which were embedded numerous C. hepatica eggs. Control of hepatic capillariasis in the remaining populations of mountain gorillas should be focused on eradication or control of populations of rodent pests (i.e., mice and rats) that sustain the reservoir of C. hepatica in habitats shared by gorillas and humans.     

Disruption of spermatogenesis in Fasciola hepatica by some anthelmintics.

Eight-weeks old F. hepatica were recovered from rabbits and rats that had been treated with known fasciolicides and anthelmintics between 4 and 40 h previously. Examination of the surviving flukes showed that spermatogenesis was significantly disrupted by most of the fasciolicides but by none of the other anthelmintics. It is suggested that disruption of spermatogenesis is particularly associated with compounds showing activity against mature F. hepatica.     

口服卡介菌治疗实验性自身免疫性脑脊髓炎的研究

目的:观察口服卡介菌对实验性变态反应性脑脊髓炎(EAE)的治疗效果。方法:制备EAE大鼠模型,随机分为BCG高、中、低剂量组和PBS对照组,每组各15只,对大鼠治疗后的临床症状及病理组织学进行评估,提取脾脏淋巴细胞,流式细胞术检测T淋巴细胞亚群,3H-TdR掺入法检测淋巴细胞增殖能力。结果:BCG组EAE大鼠与对照组相比,临床症状减轻,发病时间延迟,炎性细胞浸润数减少;急性期,口服BCG各组CD4+、CD8+T细胞的数量随剂量增加而增加,缓解期CD4+、CD8+T细胞数量减少;口服BCG可促进EAE大鼠T淋巴细胞增殖能力;高、中剂量组上述变化均较其它分组明显。结论:口服BCG可很好的诱导免疫耐受,延迟EAE发病,减轻炎症反应,改善临床症状。     

Antigenicity of a proteolytic enzyme of Fasciola hepatica

The possibility was examined of using a haemoglobinase released during in vitro incubation of adult Fasciola hepatica for immunodiagnosis of fascioliasis. By SDS gel electrophoresis the enzyme appeared as two closely migrating bands with a molecular weight of approximately 27,000 daltons. After Western blotting the bands reacted with serum from rats infected with F. hepatica and mice infected with Schistosoma mansoni. The enzyme was therefore not a good antigen for immunodiagnosis.     

Patterns in size and shedding of Fasciola hepatica eggs by naturally and experimentally infected murid rodents

Using samples collected on the island of Corsica, a comparative study was done of the morphometry of Fasciola hepatica eggs shed by cattle and by naturally and experimentally infected murid rodents (wild Mus musculus and Rattus rattus and Rattus norvegicus Wistar laboratory strain). Eggs shed by murids are smaller in size than those shed by naturally infected cattle. A second study analyzed the number of F. hepatica eggs shed in murid feces at different time intervals, i.e., months, days, and 6-hr periods, by the Kato-Katz technique. Both experimentally and naturally infected black rats (R. rattus) were used, and Wistar rats were experimentally infected and included for comparison. The present studies prove that black rats R. rattus are able to shed eggs independently from the liver fluke isolate and that egg shedding occurs throughout the life of this host species, uninterrupted during all the months analyzed in a 2-yr period. Moreover, the results suggest that this shedding is continuous, with eggs appearing in the feces daily. The results on egg shedding by wild black rats R. rattus reach their maximum shedding in spring and autumn and a maximum during twilight hr. These chronobiological patterns appear to favor parasite transmission, both seasonally and daily.     

Composition and metabolism of phospholipids of Fasciola hepatics, the common liver fluke.

1. The phospholipid composition of Fasciola hepatica, the common liver fluke, was compared to that of the liver of the host animals (rats and cattle). Considerable differences were found:monoacyl-sn-glycero-3-phosphorylcholine, hardly detectable in the liver, was found in significant amounts in the parasite. On the other hand, sphingomyelin, a normal constituent in the liver, appears to be absent in the liver fluke. Fasciola hepatica isolated from rat and cow liver had a strikingly similar phospholipid composition. 2. Qualitative and quantitative differences were also found between the fatty acyl constituents of the phospholipids of the parasite and the liver. The major difference was the presence of eicosaenoic and eicosadienoic acids in the parasite, whereas these acids were not detected in the liver. 3. In vitro incubations of Fasciola hepatica in the presence of (32P)phosphate and (2-3H)glycerol resulted in the labelling of all phospholipids of the parasite, except that the 3H label did not incorporate into ethanolamine plasmalogen. This is in agreement with the concept that in animals, glycerol is introduced into plasmalogens via dihydroxyacetonephosphate. 4. Homogenates of liver flukes were found to catalyze the synthesis of phosphatidylcholine from 1,2-diacyl-sn-glycerols and CDPcholine. 5. These results strongly suggest that Fasciola hepatica is capable of synthesizing at least part of its fatty acids and phospholipids.     

Fasciola hepatica in rats: transfer of immunity by serum and cells from infected to F. hepatica naive animals

Immune, hyperimmune, and nonimmune serum samples were collected from inbred rats following 10 to 15 weeks of one [5 metacercariae (mc)/rat], two (5 mc followed by 30 mc/rat) or no (uninfected) exposure to Fasciola hepatica. Lymphoid cells also were collected from these donors. Inbred, naive rats in groups receiving immune serum, hyperimmune serum, nonimmune serum (serum control), immune cells, hyperimmune cells, and nonimmune cells (cell control) received intraperitoneally either a total of 20 ml of serum or a total of 3 x 10(8) viable lymphoid cells. A challenge infection of 30 mc/rat was administered orally at about the time of serum or cell transfer. The transfer of immunity was evaluated by examining recipient rats for parasites 4 and 8 weeks after challenge. Some hematological parameters and the precipitating antibody response of the recipients were monitored also. Hyperimmune serum, unlike immune serum, consistently provided a significant degree of protection in recipient rats. The precipitating antibody titre of this serum was higher than that obtained from the immune donor group. The importance of a second sensitization to obtain sufficiently potent serum was demonstrated. Lymphoid cells from infected donors did not consistently confer protection on recipients. Thus, the expression of protective immunity against F. hepatica seemed to be more dependent on the presence of antibodies than on cells. The hematological parameters of the recipients, in general, supported this observation. The precipitating-antibody response of protected rats was lower than that of unprotected animals following challenge, presumably because the development of fewer worms in the former provided less antigenic stimulation.     

Fasciola hepatica: collagen deposition and other histopathology in the rat host's bile duct caused by the parasite and by proline infusion

Bile ducts were examined histochemically to compare the effects of proline infusion with Fasciola hepatica implantation in rats. After 3 weeks of infusion or implantation, both proline and F. hepatica produced increases in the luminal perimeter and collagen content of the bile duct. However, the effect of the parasite was significantly greater than that of proline, and the parasite produced significant increases in the bile duct wall. These results corroborate earlier biochemical and histological studies indicating the important role of proline in the enlargement of the bile duct in fascioliasis.     

口服卡介菌诱导免疫耐受的CD4+CD25+调节性T 细胞机制研究

目的:研究口服卡介菌诱导免疫耐受对CD4+CD25+调节性T细胞的影响。方法:采用口服MPB制备EAE大鼠模型,随机分为BCG组(0.5mg/kg)和EAE模型组(PBS),每组各15只,连续经口灌服给药14d,同时选取15只健康大鼠作为对照组。分别于免疫后15d、27d流式细胞术检测外周血、胸腺及脾脏中CD4+CD25+T淋巴细胞百分率,ELISA检测血清IL-6、TGF-β、IgE、IgG含量。结果:与EAE模型组相比,免疫后BCG组大鼠外周血、胸腺及脾脏中CD4+CD25+T淋巴细胞百分率增加,血清IL-6、TGF-β含量上升,血清IgE、IgG抗体水平下降。结论:口服BCG通过上调淋巴器官中CD4+CD25+T淋巴细胞比例,抑制效应性T细胞活性,发挥免疫耐受作用。