Detection of Fasciola gigantica infection in snails by polymerase chain reaction

Polymerase chain reaction (PCR) was used to detect Fasciola gigantica infection in the snail intermediate host. Fasciola specific primers amplified a 124 bp fragment in PCR when the genomic DNA isolated from F. gigantica infected Lymnaea auricularia snails was used as template. In addition to the 124 bp amplicon, a ladder of DNA fragments representing amplification of the 124 bp repetitive sequences was observed. Genomic DNA of the parasite was used as a positive control, which also gave an amplification of the 124 bp fragment. DNA isolated from non-infected snails was used as a negative control and no amplification of this sequence was observed. This technique is highly specific and sensitive and possesses fairly good prospects of its utility as an epidemiological tool for ascertaining the infectivity status in ubiquitous snail populations.     

Epidemiological studies of Fasciola gigantica infections in cattle in the highveld and lowveld communal grazing areas of Zimbabwe

During the period between January 1999 and December 2000, the distribution and seasonal patterns of Fasciola gigantica infections in cattle in the highveld and lowveld communal grazing areas of Zimbabwe were determined through monthly coprological examination. Cattle faecal samples were collected from 12 and nine dipping sites in the highveld and lowveld communal grazing areas respectively. Patterns of distribution and seasonal fluctuations of the intermediate host-snail populations and the climatic factors influencing the distribution were also determined by sampling at monthly intervals for a period of 24 months (November 1998 to October 2000) in six dams and six streams in the highveld and in nine dams in the lowveld communal grazing areas. Each site was sampled for relative snail density and the vegetation cover and type, physical and chemical properties of water, and mean monthly rainfall and temperature were recorded. Aquatic vegetation and grass samples 0-1 m from the edges of the snail habitats were collected monthly to determine the presence or absence of F. gigantica metacercariae. Snails collected at the same time were individually checked for the emergence of larval stages of F. gigantica. A total of 16264 (calves 5418; weaners 5461 and adults 5385) faecal samples were collected during the entire period of the study and 2500 (15.4%) of the samples were positive for F. gigantica eggs. Significantly higher prevalences were found in the highveld compared to the lowveld (P < 0.001), for adult cattle than calves (P < 0.01) and in the wet season over the dry season (P < 0.01). Faecal egg output peaked from August/September to March/April for both years of the study. Lymnaea natalensis, the snail intermediate host of F. gigantica was recorded from the study sites with the highveld having a significantly higher abundance of the snail species than the lowveld (P < 0.01). The snail population was low between December and March and started to increase in April reaching a peak in September/October. The number of juvenile snails peaked between April and August. The mean number of snails collected was negatively correlated with rainfall and positively correlated with temperature. Mean number of snails collected was also positively correlated with Potamogeton plant species and negatively correlated with Cyperus plant species. However, none of the L. natalensis collected from the habitats were found shedding Fasciola cercariae. Metacercariae were found on herbage from the fringes of the snail habitats between February and August for both years, with most of the metacercariae concentrated on herbage 0-1 m from the banks of the habitats. Based on the findings of this study, anthelmintic treatment should be administered in December/January to control chronic and mature fasciolosis. A second treatment should be given in April/May to reduce pasture contamination and subsequently snail infection, as this is the time the snail population starts to build up. To control acute fasciolosis due to the immature liver flukes a third treatment should be given in August. The first application of molluscicides to control the snail intermediate hosts can be done in June the time when the snail is harbouring the parasite and a second application in September in order to kill new generations of infected snails     

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.     

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.     

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。     

Epidemiology and control of trematode infections in cattle in Zimbabwe: a review

In this paper the main epidemiological aspects of the major domestic ruminant trematode infections in Zimbabwe are reviewed and discussed with regard to the available options for control. Seasonal occurrence of amphistomes, Fasciola gigantica and Schistosoma mattheei are considered both in the definitive and intermediate hosts. The regional distribution of the trematodes is reviewed in relation to the distribution patterns of their snail intermediate hosts. Based on the epidemiological features of the trematodes, practical control measures are suggested.     

Bovine fascioliasis in the Manawatu: epidemiology and farmer awareness

The prevalence of Fasciola hepatica was found to be 5.1% in slaughtered cattle originating from the Palmerston North Veterinary District. Approximately 4% of beef breeding, 7% of dairy and 21% of drystock herds showed evidence of infection. Of 34 dairy herd and 14 beef breeding herd owners, 0 and 21% respectively were aware of the presence of fluke on their properties. Distribution of affected dairy farms conformed with the ecological requirements of the intermediate host Lymnaea columella. The high prevalence of infection in drystock herds is consistent with the extensive movements known to occur into these herds from liver fluke-affected parts of the country. In view of the expanding geographical range of Fasciola hepatica and its intermediate host Lymnaea columella, farmers should be alerted to the potential risks that result from the introduction of infected cattle to properties which contain suitable habitats for the snail.     

Epidemiological survey of Neospora caninum infection in dogs from Romania

Native cathepsin-L cysteine proteinase (28 kDa) was purified from the excretory secretory products of Fasciola gigantica and was used for sero-diagnosis of F. gigantica infection in buffaloes by Dot-enzyme-linked immunosorbent assay (Dot-ELISA). The test detected F. gigantica field infection in these animals with a sensitivity of ～ 90%. No specific IgG antibody binding was displayed by sera obtained from 76 buffaloes considered to be Fasciola and other parasite-free by microscopic examination of faeces and necropsy examination of liver, rumen and intestine. Additionally, sera from 156 Fasciola-free buffaloes, yet infected with Gigantocotyle explanatum, Paramphistomum epiclitum, Gastrothylax spp., Strongyloides papillosus and hydatid cyst were all negative, indicating that F. gigantica cathepsin-L cysteine proteinase does not cross-react with these helminth parasites in natural infection of the host. The data indicated that cathepsin-L cysteine proteinase based Dot-ELISA reached ～ 90% sensitivity and 100% specificity with relation to above parasites in the detection of bubaline fasciolosis. The present Dot-ELISA diagnostic assay is relevant to the field diagnosis of F. gigantica infection in buffaloes.     

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.     

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.     

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.     

Ecological basis of parasite control: Trematodes with special reference to fascioliasis

In this paper the main ecological components of selected animal trematode infection cycles are considered with regard to the possibilities of control. The geographical distributions of Fasciola hepatica, F. gigantica, Paramphistomum microbothrium, Schistosoma bovis and S. mattheei are compared in relation to the distribution patterns of their snail intermediate hosts. In order to constitute an effective lifecycle host, intermediate host and the eggs and freeliving stages of the parasite overlap in the effective biotope. Comparing the biotopes of the different trematodiases it becomes evident that they have much in common particularly as the snail intermediate hosts occupy identical niches in a food-chain in which the pollution by the host species results in abundant food for the snail. Moreover, the physical activaties of herbivores, e.g. trampling down the turf, create favourable habitats for the intermediate host.The dynamic nature of the freshwater snail habitats characteristic for trematodiasis, regularly results in a complete disappearance of the effective biotopes in the field. With regard to the parasite these effects are overcome by a long life-expectancy of egg producing parasites in the host population. Small scale changes in availability of appropriate hosts for successful cercarial establishment are compensated by the extended period of cercarial shedding from the snail population.These features in combination with the survival of eggs and the grazing and drinking behaviour characteristics of the hosts tend to stabilize the potential oscillations in transmission rate. Because of the complicated nature of interdependency within the host-trematode-intermediate host complex, the ecological base with regard to practical measures is fundamentally wide. However, the pressure upon the situation by social and economic conditions limits operative possibilities considerably.     

Dynamics of Fasciola hepatica infection in two species of snails in a rural locality of Mexico

The aim of the present study was to identify the species that are natural intermediate hosts of Fasciola hepatica in a Research Centre in Chapa de Mota, State of Mexico, Mexico, where temperate and humid conditions prevail. Also, the magnitude and dynamics of the natural infection in four biotopes were analysed. The study was performed between October 2000 and November 2001. The molluscs were collected, identified and studied for trematode infection. Soil humidity and temperature were determined monthly. A total of 4042 snails were collected 3372 (83%) were Fossaria humilis and 670 (17%) F. bulimoides. F. hepatica was present in 2537 (75.2%) and 515 (76.9%) of the two snail species, respectively. Temperature, soil humidity and microenvironment affected the dynamics of the host population and the parasite infestation, with a significant increase from August to November 2001. The highest prevalence was observed in the warmer and more humid biotopes.     

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.     

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”。     

Infection of Lymnaea truncatula and Lymnaea glabra by Fasciola hepatica and Paramphistomum daubneyi in farms of central France

Natural infection of Lymnaea glabra and Lymnaea truncatula by Fasciola hepatica and/or Paramphistomum daubneyi was studied at two periods of risk (June-July and September-October) in 11 French farms known for their high prevalences of F. hepatica infection in ruminants. A total of 1,778 L. truncatula and 2,396 L. glabra measuring 6 mm or more in height were collected to determine the prevalence of natural infection with F. hepatica and P. daubneyi. The role of four factors, i.e. season, snail co-infection (F. hepatica-P. daubneyi), ruminant host and the existence of single or mixed intermediate host communities, was investigated. There were no differences in prevalences between the two risk periods. Co-infections in L. glabra were more frequent than expected. The prevalences of infection with F. hepatica or P. daubneyi in L. glabra were higher in farms rearing sheep than in farms rearing cattle. A similar finding was also noted for L. truncatula infected with P. daubneyi only. L. glabra was a much better intermediate host for F. hepatica and P. daubneyi when it was the only available snail, possibly indicating an adaptation of parasites to their less usual host in local conditions. The two trematodes preferably developed in L. truncatula rather than in L. glabra when both host species lived in the same places.     

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.     

Cathepsin L cysteine proteinase in the diagnosis of bovine Fasciola gigantica infection

Cathepsin L cysteine proteinase from Fasciola gigantica was evaluated for its potential in the early prepatent detection of this helminth infection in bovine calves. Five cross-bred bovine calves were experimentally infected with 400 metacercariae/calf and evaluated for anti-cathepsin L antibody response. F. gigantica infection in these calves could be detected 4 weeks post-infection using an ELISA, dipstick ELISA and Western blotting with 100% sensitivity. The antigen was also used to detect F. gigantica field infection in cattle, by screening 256 sera of these animals by an ELISA, which demonstrated an overall infection rate of 26.95%. Preliminary studies showed that F. gigantica cathepsin L cysteine proteinase does not cross-react with Paramphistomum epiclitum, Gigantocotyle explanatum and hydatid cyst antigens. However, extensive studies on the cross-reactivity of this antigen with related helminth parasites of cattle and buffaloes are required, before this antigen can be considered suitable for immuno-diagnosis of fasciolosis in these ruminants.     

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.     

Protection against Fasciola gigantica infection in rats administered metacercarial antigens

Immature worms were recovered from the liver parenchyma of rats which received extracts of metacercariae or adult worms of Fasciola gigantica, after challenge infection. When resistance was assessed by counting the number of immature worms, exposure of the animals to extracts of these two stages of the flukes conferred a significant degree of protection against challenge with F gigantica metacercariae. Rats given the extract of metacercariae were more resistant to infection than rats immunised with the extract of adult worms.