两种片形吸虫感染家兔的试验比较

用大片形吸虫和肝片形吸虫感染家兔以便选择大片形吸虫对动物的最佳感染量,及明确肝片形吸虫和大片形吸虫的生物学和对动物宿主的致病力的差别。结果显示肝片形吸虫虫体在兔体内发育成熟的时间早于大片形吸虫,感染成活率更高,对动物的病理损害明显比感染大片形吸虫兔的病变要轻。本试验证实这两种片形吸虫除了形态学的差异外,在对动物致病力、病理损害等方面确实存在差别。     

大片形吸虫幼虫的培育及其感染小鼠的研究

本试验旨在培育大片形吸虫囊蚴,着重研究大片形吸虫在中间宿主体内的发育过程和临床病理变化.人工培养和孵化大片形吸虫虫卵,用孵化出来的大片形吸虫毛蚴感染中间宿主小土蜗螺,收集大片形吸虫囊蚴,再用囊蚴感染试验小鼠.结果显示,囊蚴经口感染试验小鼠后1周即可在肝脏找到虫体,幼虫可在小鼠体内发育生存7～8周,随着时间的推移和感染囊蚴数量不同,可给小鼠肝脏造成不同程度的病变,甚至造成死亡.剖检小鼠可见肝脏质地脆,颜色发黄,脾脏肿大等严重病理变化.因此,可利用小鼠作为大片形吸虫幼虫感染的试验动物,进行片形吸虫病的早期诊断、免疫预防和治疗等方面的研究.     

Serum antibody isotype responses of Fasciola-infected sheep and cattle to excretory and secretory products of Fasciola species

This study investigated the immunoglobulin isotype responses of sheep and cattle chronically infected with Fasciola hepatica and Fasciola gigantica to adult F. hepatica excretory/secretory products (Fh-ES) or F. gigantica excretory/secretory products (Fg-ES), respectively. An antibody enzyme-linked immunosorbent assay (Ab-ELISA) was used to determine serum antibody (total Ig, IgG(1), IgM, IgG(2) and IgA) responses. At necropsy, the mean number of flukes recovered was lower in cattle than in sheep. All F. hepatica and F. gigantica infected sheep and cattle showed an increased total Ig levels from 3 to 4 weeks post-infection (wpi). Among isotypes IgG(1) was most dominant while IgM was the earliest (2 wpi) to be detected in both sheep and cattle infected with both F. hepatica and F. gigantica animals. IgG(2) response was early (2 wpi) in sheep infected by F. hepatica but there was no response in sheep infected with F. gigantica. There was a late and strong IgG(2) response in cattle infected with both flukes. The IgA isotype showed an early and a clear biphasic response in sheep with F. hepatica but was less pronounced in F. gigantica infected sheep. While IgA response to Fh-ES was noticed 5 wpi in F. hepatica infected cattle, it appeared much later (21 wpi) in those infected with F. gigantica. The dominance of IgG(1) isotype in infected sheep and cattle suggest an associated Th2 response. This early response to adult Fasciola spp. ES antigen suggests an early exposure to the antigen presumably through the cross-reacting ES products of juvenile flukes. There is clearly difference in IgG(2) isotype response in cattle (resistant) compared to sheep (susceptible). The late IgG(2) response in cattle may suggest late Th1 involvement in bovine cellular responses to adult Fh-ES/Fg-ES.     

分子分类结合形态学观察鉴定重庆地区片形吸虫种类

目的明确重庆地区片形吸虫的种类,并为重庆地区片形吸虫的分类研究提供科学的参考依据。方法在对重庆地区黄牛、水牛肝脏上寄生的片形吸虫形态结构进行观察后,根据片形吸虫第一内转录间隔区(ITS-1)和第二内转录间隔区(ITS-2)基因设计特异性引物,运用多聚酶链式反应(PCR)技术,以法国肝片吸虫gDNA为对照,对所采样品gDNA用大片吸虫和肝片吸虫的ITS-1和ITS-2特异性引物进行扩增。结果形态学鉴定均为“不规则”片形吸虫,电泳结果显示均分别扩增出特异性ITS-1和ITS-2条带。结论综合形态学和PCR鉴定结果,初步认为重庆地区存在着大片吸虫和肝片吸虫的“中间型”。     

Host responses during experimental infection with Fasciola gigantica or Fasciola hepatica in Merino sheep I. Comparative immunological and plasma biochemical changes during early infection

This study reports the early biochemical changes in plasma, comparative host-immune responses and parasite recovery data in Merino sheep during the first 10 weeks of infection with Fasciola gigantica and Fasciola hepatica. One group of sheep were uninfected, four groups of sheep received incremental challenge doses of F. gigantica metacercariae (50, 125, 225 and 400, respectively) and the sixth group was challenged with 250 F. hepatica metacercariae. At 10 weeks post infection (wpi), sheep challenged with F. hepatica showed the greatest fluke recovery (mean 119, range 84-166); a significantly higher biomass of parasites recovered (2.5-fold greater than the highest dose of F. gigantica); and a greater mean % parasite recovery (39.3%, range 27-55%) than any group challenged with F. gigantica. Within the groups dosed with F. gigantica a strong dose-dependent response was observed in both fluke recovery and fluke biomass with increasing dose of metacercariae. The mean % parasite recovery of F. gigantica infected groups 1-5 were 26, 23, 26 and 25%, respectively, suggesting a uniform viability of parasite establishment independent of infection dose. At 6 wpi, elevated levels of plasma GLDH were observed in the F. gigantica infected groups compared to the uninfected sheep (p<0.005) whereas the F. hepatica challenged group had four-fold higher levels of GLDH compared to the F. gigantica infected group (p<0.001). Elevated levels of GGT as an indicator of epithelial damage in the bile duct was only seen in the group challenged with F. hepatica at 10 wpi when it rose from below 100 IU/l to approximately 250 IU/l (p<0.0001) whereas no detectable increase in GGT was observed in any of the groups challenged with F. gigantica. The white blood cell response to F. hepatica infection was biphasic with the initial peak at 4 wpi and a second peak at 9 wpi, corresponding to the period of migration of juvenile fluke in the liver and the time when adult flukes are migrating into the bile duct, respectively. This biphasic response was also evident in the changes in the eosinophil counts and serum haemoglobin levels. There was a trend toward higher parasite-specific IgG2 titres in sheep infected with lower worm burdens, suggesting that higher F. gigantica or F. hepatica burdens suppress IgG2 responses. The findings of this study suggest that, in early infection in a permissive host, F. hepatica appears to be more pathogenic than F. gigantica because of its rapid increase in size and the speed of its progression through the migratory phases of its life cycle.     

Innate and adaptive resistance of Indonesian Thin Tail sheep to liver fluke: a comparative analysis of Fasciola gigantica and Fasciola hepatica infection

In the current study, three independent trials directly compared Fasciola gigantica and Fasciola hepatica infection of ITT sheep. In all trials, F. hepatica infection resulted in higher worm burden recoveries and greater physiological damage to ITT sheep. Developmental differences of the two Fasciola species were also observed during the first twelve weeks of a primary infection, where the migration and growth of F. hepatica was more rapid than F. gigantica. Various immunological blood parameters were measured and indicated similar kinetics in the humoral and cellular responses during the time course of infection with each Fasciola species. In contrast to F. hepatica infection, we demonstrate an innate and adaptive comparative ability of ITT sheep to resist the early stages of infection with F. gigantica infection. Unraveling the mechanisms leading to this differential resistance may potentially lead to new methods for the control of fasciolosis and other human liver flukes.     

Fasciola in horses in the Republic of South Africa: a single natural case of Fasciola hepatica and the failure to infest ten horses either with F. hepatica or Fasciola gigantica

The faeces of 11 horses were examined for Fasciola spp. eggs. One of them was positive for Fasciola hepatica, a finding which was confirmed post-mortem. The 10 negative horses were subsequently infested with either F. hepatica or Fasciola gigantica, each animal receiving orally from 500-9,500 metacercariae. No clinical signs were observed and no fluke eggs were detected in the faeces, and neither immature nor adult Fasciola worms were recovered from the horses slaughtered 16-26 weeks post-infestation with F. hepatica and 28-34 weeks post-infestation with F. gigantica, respectively. The results indicate that horses have a high level of resistance to both South African Fasciola spp. Factors which can probably explain the conflicting reports for the prevalences of fascioliasis in horses throughout the world and the varying results obtained in experimental infestations are briefly considered.     

Studies on the prevalence and laboratory transmission of fascioliasis in animals in the Kashmir Valley

In Kashmir, 85.1% of cattle, 51.3% of sheep and 14.8% of goats were found infected with Fasciola spp. The prevalence rate varied from 66.6 to 100.0%, 25.0 to 100% and nil to 66.0% in cattle, sheep and goats respectively in different months of the year. Fasciola gigantica was the predominant species in all animal species but sheep harboured both F. gigantica and F. hepatica. The prevalence of F. hepatica infection in sheep happens to be the first report from India. Lymnaea auricularia sensu stricto supported the development of F. gigantica under laboratory conditions. The incubation temperature affected the shedding of the cercariae. Snails maintained at 25-27 degrees C started cercarial shedding as early as day 20 post-infection (PI), whereas those maintained at 10-12 degrees C commenced it from day 64 PI. One out of three experimentally infected guinea pigs aged 1 month revealed adult flukes in the liver at necropsy on day 52 PI.     

Identification of the Egyptian species of Fasciola.

Reports on the species of Fasciola present in the Nile Delta, Egypt, appear controversial. Some authors reported the presence of both Fasciola gigantica and Fasciola hepatica, others reported F. gigantica only and mentioned that F. hepatica was found only in imported animals.This study was an attempt to identify the species of Fasciola flukes collected from locally bred animals. Morphologic, morphoanatomic, morphometric, and chemotaxonomic criteria of the fluke isolates were studied. Speciation based on morphologic and morphometric data was not decisive due to overlap in the values of most measurements. Morphoanatomic data proved the presence of both the species, and isoelectric focusing (IEF) of fluke soluble protein confirmed the presence of both F. gigantica and F. hepatica in Egypt.     

The influence of different food sources on cercarial production in Lymnaea truncatula experimentally infected with Digenea

Experimental infections of Lymnaea truncatula with a digenean species (Fasciola gigantica, F. hepatica, or Paramphistomum daubneyi) were performed under laboratory conditions to study the effect of four sources of food (microalgae, romaine lettuce, wheat germs, or modified Boray diet) on cercarial production. The mean number of F. hepatica, F. gigantica, or P. daubneyi cercariae was significantly greater when snail food was microalgae or Boray diet. The lowest cost prices for 100 metacercariae of F. hepatica (9.7-10.1 euros) were noted when algae of Boray diet were used as food for snails. In contrast, the highest prices (18.0-18.5 euros) were found in lettuce-reared snails. The choice of a source of food for breeding the intermediate hosts of F. hepatica and collecting metacercariae must take into account the importance of the commercial demand for metacercariae and will depend on the strategy the producer wishes to develop.     

Characterisation of Fasciola species from Mainland China by ITS-2 ribosomal DNA sequence

Isolates of Fasciola (Platyhelminthes: Trematoda: Digenea) from different host species and geographical locations in Mainland China were characterised genetically. The second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA (rDNA) was amplified from individual trematodes by polymerase chain reaction (PCR), and the representative amplicons were cloned and sequenced. The length of the ITS-2 sequences was 361-362bp for all Chinese Fasciola specimens sequenced. While there was no variation in length or composition of the ITS-2 sequences among multiple specimens from France, Sichuan and Guangxi, sequence difference of 1.7% (6/362) was detected between specimens from France and Sichuan, and those from Guangxi. Based on ITS-2 sequence data, it was concluded that the Fasciola from Sichuan represented Fasciola hepatica, the one from Guangxi represented Fasciola gigantica and the one from sheep from Heilongjiang may represent an "intermediate genotype", as its ITS-2 sequences were unique in that two different ITS-2 sequences exist in the rDNA array within a single Fasciola worm. One of the sequences is identical to that of F. hepatica, and the other is almost identical to that of F. gigantica in that nucleotides at five of the six polymorphic positions represent F. gigantica. This microheterogeneity is possibly due to sequence polymorphism among copies of the ITS-2 array within the same worm. Based on the sequence differences, a PCR-linked restriction fragment length polymorphism (PCR-RFLP) assay was established for the unequivocal delineation of the Fasciola spp. from Mainland China using restriction endonuclease Hsp92II or RcaI. This assay should provide a valuable tool for the molecular identification and for studying the ecology and population genetic structures of Fasciola spp. from Mainland China and elsewhere.     

The serum glucose and beta-hydroxybutyrate levels in sheep with experimental Fasciola hepatica and Fasciola gigantica infection

The influence of Fasciola hepatica and Fasciola gigantica infection on serum glucose and beta-hydroxybutyrate (beta-HOB) in sheep was evaluated. This was done by setting up two groups of sheep. The first group (n=13) was split in two sub-groups, one experimentally infected with F. hepatica (n=9) and the other (n=4) as uninfected control. A second group consisting of a sub-group experimentally infected with F. gigantica (n=9) the other sub-group (n=6) left as uninfected control was also set up. The results of weight gain, parasitological and serum liver enzymes activity (glutamate dehydrogenase [GLDH] and gamma glutamyltransferase [gamma-GT]) used in monitoring the infection showed that all infected animals developed fasciolosis. It was observed that a reduction in serum glucose levels was significantly lower (p<0.05) in F. hepatica infected sheep than in uninfected control sheep starting from 5 weeks post-infection (wpi) to the end of the experiment. Similar reduction was recorded in F. gigantica infected sheep between 8 and 19 wpi. In contrast, serum beta-HOB levels were elevated in F. hepatica infected sheep between 6 and 16 wpi and in F. gigantica infected sheep between 7 and 15 wpi. It would appear from these serum glucose and beta-HOB levels that fasciolosis does lead to energy deficiency (low glucose) and ketosis (increased beta-HOB). The decrease in serum glucose and increase in serum beta-HOB levels in infected sheep may help in understanding the interaction between fasciolosis and nutritional status of infected ruminants especially in young growing animals.     

Rapid identification and differentiation of Fasciola hepatica and Fasciola gigantica by a loop-mediated isothermal amplification (LAMP) assay

The present study developed and validated a species-specific loop-mediated isothermal amplification (LAMP) assay for the rapid detection and discrimination of Fasciola hepatica and Fasciola gigantica. The LAMP assay is inexpensive, easy to perform and shows rapid reaction, wherein the amplification can be obtained in 45 min under isothermal conditions of 61 °C or 62 °C by employing a set of four species-specific primer mixtures and results can be checked through naked-eye visualization. The optimal assay conditions with no cross-reaction with other closely related trematodes (Clonorchis sinensis, Opisthorchis viverrini, Orientobilharzia turkestanicum and Schistosoma japonicum) as well as within the two Fasciola species were established. The assay was validated by examining F. gigantica DNA in the intermediate host snails and in faecal samples. The results indicated that the LAMP assay is approximately 10(4) times more sensitive than the conventional specific PCR assays. These findings indicate that this Fasciola species-specific LAMP assay may have a potential clinical application for detection and differentiation of Fasciola species, especially in endemic countries.     

Comparative study of the reproductive organs of Fasciola groups by optical microscope

Reproductive organs of stained and mounted whole specimens of different types of Fasciola (F. hepatica, F. gigantica, and parthenogenetic diploid and triploid flukes) were observed to clarify the structure of their reproductive organs. The results are as follows; 1. Basic structure differences could not be identified. 2. The flukes without sperm, or those with an extremely small quantity in the seminal vesicle. are parthenogenetic Fasciola sp. 3. It was newly discovered that the surface of the cirrus is surrounded by many shallow gutters, and that spines form a line in the gutters. 4. The structure of the reproductive organ on the genus Fasciola are shown in detail in the figures.     

片形吸虫DNA随机扩增多态性分析

为区别从南京市江宁县采集的片形吸虫非典型形态虫体，应用随机扩增多态性DNA（RAPD）技术，对6株片形吸虫总DNA进行了扩增。结果，10条引物中有8条能产生扩增图谱，电泳图谱经聚类分析，与传统的分类结果一致，并表明来自江宁的片形吸虫既有形态典型的肝片形吸虫，也有形态不典型的大片形吸虫。     

A TaqMan real-time PCR-based assay for the identification of Fasciola spp

Real time quantitative PCR (qPCR) is one of the key technologies of the post-genome era, with clear advantages compared to normal end-point PCR. In this paper, we report the first qPCR-based assay for the identification of Fasciola spp. Based on sequences of the second internal transcribed spacers (ITS-2) of the ribosomal rRNA gene, we used a set of genus-specific primers for Fasciola ITS-2 amplification, and we designed species-specific internal TaqMan probes to identify F. hepatica and F. gigantica, as well as the hybrid 'intermediate'Fasciola. These primers and probes were used for the highly specific, sensitive, and simple identification of Fasciola species collected from different animal host from China, Spain, Niger and Egypt. The novel qPCR-based technique for the identification of Fasciola spp. may provide a useful tool for the epidemiological investigation of Fasciola infection, including their intermediate snail hosts.     

湖南省猬迭宫绦虫的线粒体cox1和nad1基因的序列测定及种系发育分析

本研究旨在阐明猬迭宫绦虫湖南分离株线粒体细胞色素c氧化酶第I亚基(cox1)基因部分序列(pcox1)和烟酰胺腺嘌呤二核苷酸(NADH)脱氢酶亚单位l基因(nad1)部分序列(pnad1)的遗传变异情况,并用pcox1和pnad1序列重构猬迭宫绦虫与其它绦虫的种群遗传关系。利用聚合酶链反应(PCR)扩增猬迭宫绦虫的pcox1和pnad1,应用ClustalX1.81程序对序列进行比对,再用Phylip3.67程序MP法和Mage4.0程序NJ法绘制种系发育树,并用Puzzle5.2程序构建最大似然树,同时利用DNAStar5.0中的Megalign程序进行同源性分析。结果显示所获得各样品株的pcox1和pnad1序列长度一致,分别为396和566bp,湖南分离株与已知猬迭宫绦虫位于同一分枝。由于猬迭宫绦虫pcox1和pnad1序列种内相对保守,种间差异较大,故均可作为种间遗传变异研究的标记,从而为猬迭宫绦虫的种群体遗传学研究和其相关疾病的诊断奠定基础。     

Molecular characterization of parthenogenic Fasciola sp. in Korea on the basis of DNA sequences of ribosomal ITS1 and mitochondrial NDI gene

Nucleotide sequences of ribosomal internal transcribed spacer (ITS1) and mitochondrial NADH dehydrogenase I (NDI) gene were analyzed to genetically characterize aspermic Fasciola forms in Korea. From the difference in ITS1 sequences, Korean flukes were divided into 3 haplotypes represented by Kor1, Kor2 and Kor1/2, which had nucleotides identical to F. hepatica, F. gigantica and those overlapped between the two species, respectively. NDI sequences also showed that Korean flukes could be classified into 3 distinct haplotypes (Kor1: F. hepatica-type, Kor2a and Kor2b: F. gigantica-type). The sequences of Kor1 and Kor2a were 100% identical to those of the haplotypes Fsp1and Fsp2, respectively, which are major Fasciola forms in Japan. These findings strongly suggest that aspermic Fasciola forms in Korea and Japan originated from same ancestors and have recently spread throughout both countries.     

鸡蛔虫线粒体cox1和nad4基因的遗传变异分析

为探究鸡蛔虫的种内遗传变异和系统发育关系,对分离自四川省西昌市的15个鸡蛔虫分离株的线粒体细胞色素c氧化酶第Ⅰ亚基（cox1）基因部分序列和烟酰胺腺嘌呤二核苷酸脱氢酶亚单位Ⅳ（nad4）基因全序列进行PCR扩增、序列测定及分析,并用cox1和nad4基因部分序列构建鸡蛔虫与其他蛔虫的NJ树和贝叶斯树。测序结果显示,所获得的鸡蛔虫cox1和nad4基因全序列长度分别为1 152和1 236 bp,分别有33和40个变异位点,碱基变异率分别为0~2.1%和0~2.3%;分别检测到8和11个单倍型,总的单倍型多样性分别为0.733±0.124和0.933±0.054,核苷酸多样性分别为0.00433±0.00153和0.00541±0.00157,平均遗传距离分别为0.004和0.005。构建的种系发育树均显示15个鸡蛔虫西昌分离株与其他国家/地区的鸡蛔虫分离株聚类形成一个分支,与鸽蛔虫的亲缘关系最近,与其他蛔虫所属的分支相隔较远。研究结果表明鸡蛔虫西昌分离株的遗传变异程度低,且nad4基因比cox1基因更适合作为研究鸡蛔虫分离株遗传变异的分子标记。     

The prevalence and economic importance of bovine fasciolosis in Kenya--an analysis of abattoir data

A retrospective study covering a period of 10 years (1990-1999) was carried out using post mortem meat inspection records at the Veterinary Department Headquarters at Kabete to determine the prevalence and economic importance of bovine fasciolosis in Kenya. Meat inspection records from abattoirs in 38 districts distributed over seven out of the eight provinces of Kenya were examined. Prevalence of fasciolosis was calculated as the number of cattle found to be infected with Fasciola, expressed as a percentage of the total number of cattle slaughtered. Using the average weight and market price of a bovine liver, the monetary loss occasioned by condemnation of Fasciola infected livers was calculated. A survey was also carried out at Dagoretti slaughterhouse complex in Nairobi to determine the relative occurrence of F. gigantica and F. hepatica in slaughtered cattle. Cattle slaughtered at Dagoretti slaughterhouse originate from all parts of the country. A total of 5,421,188 cattle were slaughtered in the seven provinces of Kenya during the 10-year period and 427,931 (8%) of these cattle were infected with Fasciola. The region with the highest prevalence of fasciolosis was Western Province (16%) followed, in descending order, by Eastem Province (11%), Nyanza Province (9%), Rift Valley Province (8%), Central Province (6%), Nairobi Province (4%) and Coast Province (3.5%). The total economic loss incurred by the country during the 10-year period as a result of condemnation of the infected livers was approximately US$2.6 million. The total annual economic losses during this period ranged from approximately US$0.2-0.3 million. The highest total economic losses for the 10-year period were recorded in Western Province (US$0.8 million) and Central Province (US$0.7 million). A total of 1584 cattle originating from five provinces of Kenya were slaughtered at Dagoretti slaughterhouse over a period of two months of which 147 (9.3%) were infected with liver flukes. All the liver flukes obtained from the infected livers were identified as F. gigantica. It is concluded that fasciolosis is prevalent in cattle in all provinces of Kenya, that it causes great economic losses as a result of condemnation of infected livers, and that F. gigantica is the main species of liver flukes affecting cattle in Kenya. Local climatic factors, cattle trade, rustling and population numbers, and the presence of the snail intermediate hosts are probably the main factors influencing the incidence of the disease in the various regions of the country.