PCR diagnosis of Fasciola hepatica in field-collected Lymnaea columella and Lymnaea viatrix snails

Fasciolosis, caused by the trematode Fasciola hepatica, is a zoonosis of economic importance in livestock that is emerging as a chronic disease in humans. The intermediate hosts are lymnaeid snails, in which diagnosis of infection is traditionally based on cercarial shedding, tissue sectioning and crushing. We developed a PCR assay for the sensitive and specific detection of F. hepatica in field-collected Lymnaea sp. snails. A primer pair was designed to amplify a 405 bp fragment of the cytochrome c oxidase subunit 1 gene of F. hepatica. The PCR assay showed a limit of detection of 10 pg of genomic F. hepatica DNA. No cross-reactions were observed with samples from other related trematode species or from the snail hosts Lymnaea columella and Lymnaea viatrix. DNA sequencing of the amplicon showed 100% homology with F. hepatica, and 75-89% homology with other trematodes on regions that did not include the entire set of primers. Two samples from Argentina were analysed. For snails in sample 1 (n = 240), identified as L. columella, the infection rate was 17.5 and 51.3% by direct examination and PCR, respectively. For snails in sample 2 (n = 34), identified as L. viatrix, the infection rate was 2.9 and 61.8% by direct examination and PCR, respectively. Differences in infection rates between these diagnosis methods were significant for both samples. Our PCR technique showed to be effective for detecting specific F. hepatica infections of low intensity in the intermediate host, and hence it could be used to study the epidemiological situation in a given area, as well as to assess host suitability for the parasite.     

The distribution of Fasciola hepatica in Queensland, Australia, and the potential impact of introduced snail intermediate hosts

A survey was conducted to establish the distribution of the liver fluke, Fasciola hepatica, in the state of Queensland, Australia, and to evaluate the impact of the introduced snail intermediate hosts, Pseudosuccinia columella and Austropeplea viridis. Serum samples from a total of 5103 homebred cattle in 142 beef herds distributed throughout the state and 523 pooled milk samples from dairy herds from the state's major dairying regions were tested for antibodies to F. hepatica by ELISA. Snails were collected on infected properties around the limits of the F. hepatica distribution. F. hepatica infection was detected in 44 dairy herds and two beef herds. The distribution of infected herds indicates that F. hepatica is established only in southeast Queensland. The distribution there was patchy but the parasite was more widespread than suggested by an earlier survey. The predominant intermediate host species found along the northern limit of the distribution was P. columella. We conclude that the introduction of P. columella and A. viridis has not yet had a major impact on the distribution of F. hepatica in Queensland. However, the presence of P. columella, which is much more adaptable to tropical habitats than the native intermediate host, Austropeplea tomentosa, at the northern limit of the F. hepatica distribution suggests that there is potential for the parasite to expand its range.     

The susceptibility of Lymnaeid snails to Paramphistomum cervi infections in Mexico

The occurrence of Paramphistomum cervi has been reported in Mexico, but its intermediate hosts have not been identified. Five species of snails of the genus Lymnaea, L. bulimoides, L. columella, L. cubensis, L. humilis and L. palustris, were collected in Mexico. Only three of the exposed species, L. palustris, L. cubensis and L. humilis, became infected with P. cervi. The first of these snail species was highly susceptible to infection and a higher number of the exposed snails shed cercariae. It is suggested that L, palustris may act as an important vector of P. cervi in Mexico.     

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.     

Geographical distribution and habitat preferences of the invader freshwater snail species Lymnaea columella (Mollusca: Gastropoda) in South Africa

The present geographical distribution of Lymnaea columella, as recorded in the National Freshwater Snail Collection, is described and discussed. It appears that L. columella is the most successful colonist of all the freshwater snail species in South Africa, and, together with Bulinus tropicus and Lymnaea natalensis, it forms the most widely distributed freshwater snail species in the region. Data regarding the 2,341 discovery sites of L. columella recorded by collectors during surveys are analysed. L. columella cluster mainly in rivers or streams with perennial, slow flowing, or stagnant freshwater with many plants and a mud substrate. L. columella is regarded as a more effective intermediate host for the transmission of fascioliasis than the indigenous snail species. The economical implications of this are discussed briefly in the light of its success as an invader species.     

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.     

Prevalence of Fasciola hepatica in the intermediate host Lymnaea truncatula detected by real time TaqMan PCR in populations from 70 Swiss farms with cattle husbandry

Bovine fasciolosis is an economically important parasitic disease. Quantitative real time PCR was utilized to determine the prevalence of Fasciola hepatica in the snail intermediate host Lymnaea truncatula from 70 selected, infected Swiss cattle farms, and to gain information on the infection risk to the definitive host. Snails from 130 habitats (36 streams, 21 wells, 24 drainage ditches, 33 spring swamps, 14 reeds, 1 drainage shaft and 1 pond) originating from 71 dairy cow pastures, 39 pastures for young stock, 14 hay fields and 6 dry cow pastures were collected. Of these, 51 populations were found to be infected with F. hepatica. A total of 4733 snails were examined of which 331 were infected (7.0%). The numbers of snails collected from different sites ranged from 1 to 159 snails. Clustering of infection in snails was found on the farm of origin with a mixed logistic model with random effects. The risk of infection of L. truncatula with F. hepatica was significantly higher in populations originating from spring swamps, wells and reeds compared to populations from streams. In addition the risk of snail infection was significantly lower in populations collected in young stock and dry cow pastures compared to dairy cow pastures. The greater the population size collected from a habitat also increased the risk of an individual snail being infected.     

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 of Fasciola hepatica infection in the Paraíba River Valley, S?o Paulo, Brasil

The study revealed that although Lymnaea columella and Stenophysa marmorata were found in the same habitat, only L. columella harbored intramolluscan stages of F. hepatica. The population density of L. columella cyclically decreased between September and February, and increased from March to September. The drought that occurred between June and November 1981 dramatically reduced the number of snails collected. Larger snails were collected in March-July and November-December, while the smaller snails were collected in January-February and August-October, suggesting that there may be two generations of L. columella per year. The highest prevalence levels of infection in L. columella were: first year, June 8.82%, September 9.09% and October 10.52%; second year, March 6.25% and April 6.89%; third year, July 7.69%, August 10.25%, April 17.91% and May 13.91%. Results obtained with tracer sheep showed that the largest numbers of metacercariae were on pasture between June and October, and between March and April, but the infection was present during most of the year.     

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.     

THE OCCURRENCE OF LYMNAEA (PSEUDOSUCCINEA) COLUMELLA, AN INTERMEDIATE HOST OF FASCIOLA HEPATICA, IN AUSTRALIA

An established population of the eastern North American freshwater snail Lymnaea (Pseudosuccinea) columella Say is reported from Middle Creek and Centennial Park, Sydney. This species is an important intermediate host of the liver fluke (Fasciola hepatica Linné) and its introduction and spread in New Zealand has resulted in an increase in the liver fluke problem in New Zealand.     

A survey of fascioliasis in beef cattle killed at abattoirs in southern Queensland

The overall prevalence of fascioliasis in beef cattle found in an abattoir survey completed in May 1983 in southern Queensland, was 1.1%. Prevalence was 36% in the Stanthorpe Shire, varied from greater than 1 to 10% in 12 shires and was 1% or less in 24 shires. Fascioliasis was not recorded from the other 46 shires in the area of the survey. Prevalence was less than 1% in female cattle of all ages. In males, prevalence increased with age from 0.6% in 17-month-old cattle to 4.7% in 4-year-old cattle. The differences in sex-specific prevalences may be due to differences in susceptibility to the parasite between sexes, differences in susceptibility to the parasite between sexes, differences in management between sexes, or artefacts associated with sampling for a disease of very low prevalence. The presence of the exotic snail species, Lymnaea columella and L. viridis was confirmed in southern Queensland in 1977. These snails are suitable intermediate hosts for Fasciola hepatica and may widen the endemic area for liver fluke because of their more aquatic character and greater tolerance to high temperatures than L. tomentosa, the traditional snail host in Queensland. We suggest that introduced snails have not yet had a substantial impact on the importance of fascioliasis to the beef industry of southern Queensland. The survey provides baseline data for the future monitoring of this potentially important parasitic disease.     

肝片形吸虫囊蚴培育及其中间宿主的研究

本文报告了广西肝片形吸虫中间宿主的种类为小土蜗螺和斯氏萝卜螺。对不同剂量毛蚴感染中间宿主椎实螺的存活率，椎实螺的繁殖及其对干旱的抵抗力；尾蚴的逸出时间、数量；囊蚴对终宿主山羊羔的感染情况及其特点进行了观察。     

An interactive map to assess the potential spread of Lymnaea truncatula and the free-living stages of Fasciola hepatica in Switzerland

The intermediate host of Fasciola hepatica is Lymnaea truncatula in Switzerland. The snail and the free-living stages of the parasite require a moderate climate and moisture for survival, reproduction, and transmission. In Switzerland, these conditions are present in many regions, resulting in a mean prevalence of bovine fasciolosis from 8.4 to 21.4%. An interactive map was created in order to demonstrate the relative risk of transmission by modelling the environmental conditions that promote the survival and reproduction of the larval stages of the parasite and the parasite's intermediate host. The map is based on temperature and rainfall data, soil conditions including ground water and forest cover in Switzerland. Extensive information on the free-living stages of F. hepatica and the intermediate host L. truncatula and how the development of these are affected by these environmental factors was used to create the interactive risk map.     

The occurrence of the snail Lymnaea columella on grazing areas in New South Wales and studies on its susceptibility to Fasciola hepatica

Field surveys were carried out in the Lismore and Casino area for the presence of fresh water snails potentially responsible for the transmission of trematodes in ruminants. Although the North American snail, Lymnaea columella has previously only been reported from metropolitan areas, large populations of the snail were found east of Lismore. Natural infection with F. hepatica was detected in some of the snails and laboratory studies showed that this snail species was highly susceptible to infection with miracidia of F. hepatica and produced viable metacercariae. Three species of planorbid snails, Helicorbis australiensis, Pygmanisus pelorius and Gyraulus gilberti were also found. The first 2 planorbid species were infected with paramphistomid cercariae.     

Evidence to support horses as natural intermediate hosts for Sarcocystis neurona

Opossums (Didelphis spp.) are the definitive host for the protozoan parasite Sarcocystis neurona, the causative agent of equine protozoal myeloencephalitis (EPM). Opossums shed sporocysts in feces that can be ingested by true intermediate hosts (cats, raccoons, skunks, armadillos and sea otters). Horses acquire the parasite by ingestion of feed or water contaminated by opossum feces. However, horses have been classified as aberrant intermediate hosts because the terminal asexual sarcocyst stage that is required for transmission to the definitive host has not been found in their tissues despite extensive efforts to search for them [Dubey, J.P., Lindsay, D.S., Saville, W.J., Reed, S.M., Granstrom, D.E., Speer, C.A., 2001b. A review of Sarcocystis neurona and equine protozoal myeloencephalitis (EPM). Vet. Parasitol. 95, 89-131]. In a 4-month-old filly with neurological disease consistent with EPM, we demonstrate schizonts in the brain and spinal cord and mature sarcocysts in the tongue and skeletal muscle, both with genetic and morphological characteristics of S. neurona. The histological and electron microscopic morphology of the schizonts and sarcocysts were identical to published features of S. neurona [Stanek, J.F., Dubey, J.P., Oglesbee, M.J., Reed, S.M., Lindsay, D.S., Capitini, L.A., Njoku, C.J., Vittitow, K.L., Saville, W.J., 2002. Life cycle of Sarcocystis neurona in its natural intermediate host, the raccoon, Procyon lotor. J. Parasitol. 88, 1151-1158]. DNA from schizonts and sarcocysts from this horse produced Sarcocystis specific 334bp PCR products [Tanhauser, S.M., Yowell, C.A., Cutler, T.J., Greiner, E.C., MacKay, R.J., Dame, J.B., 1999. Multiple DNA markers differentiate Sarcocystis neurona and Sarcocystis falcatula. J. Parasitol. 85, 221-228]. Restriction fragment length polymorphism (RFLP) analysis of these PCR products showed banding patterns characteristic of S. neurona. Sequencing, alignment and comparison of both schizont and sarcocyst DNA amplicons showed 100% identity. Although Koch's postulates have not been demonstrated in this case study, the finding of mature, intact S. neurona schizonts and sarcocysts in the tissues of this single horse strongly suggests that horses have the potential to act as intermediate hosts. Further studies are needed to demonstrate Koch's postulates with repeated transfer of S. neurona between opossums and horses.     

Genetic diversity and relatedness of Fasciola spp. isolates from different hosts and geographic regions revealed by analysis of mitochondrial DNA sequences

The present study examined sequence variability in a portion of the mitochondrial cytochrome c oxidase subunit 1 (pcox1) and NADH dehydrogenase subunits 4 and 5 (pnad4 and pnad5) among 39 isolates of Fasciola spp., from different hosts from China, Niger, France, the United States of America, and Spain; and their phylogenetic relationships were re-constructed. Intra-species sequence variations were 0.0-1.1% for pcox1, 0.0-2.7% for pnad4, and 0.0-3.3% for pnad5 for Fasciola hepatica; 0.0-1.8% for pcox1, 0.0-2.5% for pnad4, and 0.0-4.2% for pnad5 for Fasciola gigantica, and 0.0-0.9% for pcox1, 0.0-0.2% for pnad4, and 0.0-1.1% for pnad5 for the intermediate Fasciola form. Whereas, nucleotide differences were 2.1-2.7% for pcox1, 3.1-3.3% for pnad4, and 4.2-4.8% for pnad5 between F. hepatica and F. gigantica; were 1.3-1.5% for pcox1, 2.1-2.9% for pnad4, 3.1-3.4% for pnad5 between F. hepatica and the intermediate form; and were 0.9-1.1% for pcox1, 1.4-1.8% for pnad4, 2.2-2.4% for pnad5 between F. gigantica and the intermediate form. Phylogenetic analysis based on the combined sequences of pcox1, pnad4 and pnad5 revealed distinct groupings of isolates of F. hepatica, F. gigantica, or the intermediate Fasciola form irrespective of their origin, demonstrating the usefulness of the mtDNA sequences for the delineation of Fasciola species, and reinforcing the genetic evidence for the existence of the intermediate Fasciola form.     

Effect of parasite burden on the detection of Fasciola hepatica antigens in sera and feces of experimentally infected sheep

The effect of Fasciola hepatica parasite burden on the detection of excretory/secretory (E/S) antigens in sera and feces of experimentally infected sheep was evaluated using a double antibody-based capture enzyme-linked immunosorbent assay (ELISA). Four groups of five sheep each were used. The first three groups were infected with 50, 100 and 200 metacercariae of F. hepatica, and the fourth group remained as non-infected control. On the day of infection and weekly thereafter, serum and fecal samples were taken. ELISA detected F. hepatica E/S antigen levels in serum from the first week post-infection (wpi) and in fecal supernatant from the fourth wpi, which were significantly (p<0.05) higher than controls. F. hepatica eggs were not detected until after the eighth wpi. The correlation between absorbance of E/S antigens in serum with the fluke burden was 0.77 (p<0.0001) and in feces 0.76 (p<0.0001) at 12th wpi. The sensitivity of the assay to detect E/S antigens in serum was 86.6% and in feces 93.3%. It is concluded that the ELISA technique used in this study offers a diagnostic alternative for detecting early infections of F. hepatica in sheep.     

Uptake of albendazole and albendazole sulphoxide by Haemonchus contortus and Fasciola hepatica in sheep

The pattern of in vivo uptake of albendazole (ABZ) and its major metabolite, ABZ-sulphoxide (ABZSO), by Haemonchus contortus and Fasciola hepatica recovered from ABZ-treated sheep, was investigated. Concentration profiles of both compounds were simultaneously measured in target tissues/fluids from the same infected sheep. In addition, the proportion of the (+) and (-) ABZSO enantiomers was determined in plasma, bile and F. hepatica recovered from treated sheep. Sheep naturally infected with H. contortus were intraruminally (i.r.) treated with ABZ (micronized suspension, 7. 5mg/kg) and the plasma concentrations of ABZSO and ABZ-sulphone (ABZSO(2)) determined in addition to the concentration of ABZ and ABZSO in H. contortus, abomasal mucosa and fluid content samples. In addition, F. hepatica artificially infected sheep were treated i.r. with the same ABZ suspension (7.5mg/kg), and samples of blood, bile, liver tissue and adult flukes were collected and analysed by HPLC to determine the concentrations of ABZ and both enantiomers of ABZSO. ABZSO and ABZSO(2) were the analytes recovered in plasma with ABZ and ABZSO present in H. contortus. ABZ was the analyte recovered at the highest concentration in H. contortus and abomasal mucosa, whereas higher concentrations of ABZSO were measured in abomasal fluid content. Only low concentrations of ABZ were detected in F. hepatica and bile, but markedly higher concentrations of ABZ were measured in liver tissue. ABZSO was the main molecule recovered in F. hepatica, plasma and bile samples collected from ABZ-treated sheep. The (+) enantiomer of ABZSO was recovered at a higher proportion in plasma (75%), bile (78%) and F. hepatica (74%) after ABZ administration to infected sheep.     

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.