The laboratory maintenance of field-collected Lymnaea tomentosa for the production of Fasciola hepatica metacercariae

A culture method is described for the production of Fasciola hepatica metacercariae in Lymnaea tomentosa collected from the field habitat. Two-litre glass jars, filled with artificial spring (A.S.) water and having a substratum of soil-bentonite (S.B.) agar to provide habitat and food, maintain groups of 40 to 100 snails graded according to size. Small quantities of washed lettuce are fed as a supplement. Compressed air, pre-warmed to 25°C, is piped to each jar to maintain thermoregulation, aeration, filtration and circulation of the water. Infection is achieved by reducing the A.S. water level to 3–4 mm and exposing the snails to miracidia for 6 h. During the aquatic developmental phase of 5–6 weeks, twice-weekly changes of A.S. water and the filter minimise proliferation of protozoans and Chaetogaster spp. Accumulation of mature cercariae is achieved by sustaining the infected snails on moist S.B. agar while humidification within the jar is maintained. Alternatively, groups of five to ten snails may be held in disposable petri dishes lined with S.B. agar. Maturation of cercariae may be delayed by refrigeration at 4–5°C. Upon maximum accumulation, the cercariae are harvested by dissection of the snails and metacercariae are stored in plastic containers at 4°C.     

The potential impact of exotic Lymnaea spp. on fascioliasis in Australasia

The presence of the following exotic Lymnaea spp. has been recently confirmed in Australia: L. columella (North and South America, South Africa, New Zealand,) L. viridis (China, Japan, Philippines, New Guinea, Pacific Area), L. auricularia rubiginosa (Indochina, Malaysia, Indonesia) and L. peregra (Europe).In the present studies the relative susceptibility of the exotic snails noted above to Fasciola hepatica was investigated. Two of the species, L. columella and L. viridis, were found to be highly susceptible to the Australian strain of F. hepatica and a substantial number of viable metacercariae were produced. However a strong disparity in the host— parasite relationship was evident when the parthenogenetic development of the fluke in the two exotic snails was compared with that in the native L. tomentosa. It was concluded that L. tomentosa is a more suitable intermediate host for F. hepatica than any of the introduced exotic species, but it has to be considered that the host—parasite relationships between new combinations of Fasciola and Lymnaea spp. may improve by adaptation. In view of the results of the present studies the potential danger of fascioliasis spreading to areas currently not endemic in Australasia through introduced exotic snails is discussed with particular reference to Queensland, Western Australia and the Northern Territory.There is strong circumstantial evidence that the exotic snails were introduced with aquatic plants through the relatively free overseas and Australian trade. Similarly, through the introduction of infected snails, dangerous parasites such as F. gigantica of cattle and sheep or Schistosoma ssp. which cause serious diseases of man and animals could enter the subtropical or tropical regions of Australia. Appropriate State and Federal quarantine regulations are essential to prevent further introduction and spread of the snails, and monitoring of the distribution of established exotic snails in Australasia must be continuous.     

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.     

Fascioliasis in New Zealand: A review

Intermediate hosts of Fasciola hepatica in New Zealand are the native species Lymnaea tomentosa, and introduced species, L. columella and L. truncatula. The latter is of little significance in New Zealand. Fascioliasis has become more widespread since 1950 following colonisation of much of the North Island by L. columella. L. tomentosa can produce eggs at 5°C and L. columella at 2°C. L. columella produces about the three times as many eggs as L. tomentosa and the eggs of the former develop more rapidly and over a wider range of temperatures. Both species inhabit ponds and marshes, although L. columella is better suited to pond habitats. Neither species will survive in marsh habitats that dry out in summer. Both species tolerate wide ranges of Ca and Mg concentrations and pH. Marsh habitats of the two species are indistinguishable though there is evidence of subtle differences in habitat preferences. Snail populations are minimal in winter and fluctuate irregularly throughout the rest of the year. In general, L. tomentosa populations contain proportionally more mature snails than those of L. columella. The epidemiology of fascioliasis in New Zealand is discussed. The absence of outbreaks of acute disease, the multiplicity of snail hosts and variations in climate pose problems for investigators. Overwintering infections of snails have been observed in L. tomentosa, but not in L. columella. With the latter host, transmission of Fasciola occurred over 6–7 months from mid-summer to mid-winter.The rate of infection of tracer sheep over 5 years varied but was not related to temperature or increased summer rainfall. Infection rates tended to increase when summers were dry and animals forced to graze the snails habitats. Rates in infection were not related to snail densities but to the numbers of mature snails on habitats. The infection rate of L. columella was extremely low ($\text{1}\text{790}$ snails examined). Sheep appear to be more important than cattle in maintaining Fasciolainfections from year to year. The control of fascioliasis is discussed briefly. It is argued that prevention of infection is preferable to removing infections already acquired, and low this might be achieved by a strategic anthelmintic treatment when L. columella is involved is described. Further studies on the prevalence and epidemiology of fascioliasis in New Zealand are required.     

Maintenance in vitro of adult Fasciola hepatica in a continuous-flow system

A continuous-flow apparatus is described for the maintenance of groups of adult Fasciola hepatica. The ammonia produced by the flukes was used as a measure of their metabolic activity and it was shown that this is best maintained in culture fluids containing a large proportion of bovine serum. More ammonia per fluke was consistently produced when the flow rate of the medium was increased but including small pieces of clotted sheep blood or increasing the number of flukes per culture had no significant effects.     

Experimental Infection of Lymnaea palustris snails with Fascioloides magna

This report describes experimental infection of Lymnaea (Galba) palustris. (Müller, 1774) snails with Fascioloides magna (Bassi, 1875). Of 725 L. palustris, 400 survived the experimental infection and in 172 (43%) the developmental cycle of the fluke was completed. Free emergence of cercariae was obtained was obtained 68 days after infection. The infectivity of metacercariae thus obtained was demonstrated in guinea-pigs, rabbits and a Cameroon goat. In sheep, the infection produced only changes in the organs, but no flukes could be recovered. Considering that L. palustris are found frequently in natural habitats in Czechoslovakia, they should be considered as possible intermediate hosts for F. magna.     

The role of the gut in acquired resistance to Fasciola hepatica in the rat

The role of the gut in acquired resistance to Fasciola hepatica in the rat was assessed by comparison of the number of flukes recovered 4 and 10 weeks after oral or intraperitoneal challenge infection of male outbred Wistar rats previously infected by the oral or intraperitoneal routes.Previous infection given by either route generated signigicant protection against both oral and intraperitoneal challenge. The rats were resistant to Fasciola challenge in the presence of the primary infection or after its removal by anthelmintic treatment.It was concluded that passage of juvenile flukes through the gut was not essential for either the acquisition or the expression of acquired resistance to F. hepatica in the rat.     

A three-year study on seasonal transmission and control of Fasciola hepatica of cattle in Louisiana

Seasonal transmission data on sentinel calves, herd prevalence and snail population dynamics over a 3-year period indicate that major transmission of Fasciola hepatica to cattle occurs between February and July in Louisiana. Soil temperature and water budget were used as indicators of microhabitat suitability for development of snail populations and extra-mammalian stages of F. hepatica and were related to wide annual variation in fascioliasis risk.     

Fasciola hepatica: Attempts to immunise rats using fluke eggs and in vitro culture products

Rats were sensitised on days 0 and 12 by subcutaneous injection of Fasciola hepatica ova and/or the excretion/ secretion products obtained from in vitro maintained adult flukes and then challenged orally with 30 F. hepatica metacercariae on day 14. Worm burdens were determined 3 and 8 weeks after infection. No resistance to challenge infection was detected, but those fluke recovered from rats sensitised with excretion/secretion products were smaller than those from control groups.     

Precipitating antibodies against excretory/secretory antigens of Fasciola hepatica in sheep serum

Gel diffusion techniques were used to study antigen-antibody reactions in precipitates forming around juvenile, semi-mature and adult Fasciola hepatica cultured in serum from infected sheep. The number of reactions was analysed in both primary and challenge infections. The number of antigens shared by the various developmental stages of the fluke was also examined.At least 2 antigens were involved in precipitate formation around juvenile flukes. These antigens were also produced by the later stages of development. Two additional antigens were produced by semi-mature flukes and these, together with two others, were produced by adult flukes.Challenge infections had little effect on the number of antigens or antibodies in the precipitate.     

Observations on mass production of Calicophoron microbothrium metacercariae from experimentally and naturally infected Bulinus tropicus

In an attempt to establish an ideal method for mass production of Calicophoron microbothrium metacercariae, a study was carried out to compare the shedding capacities of Bulinus tropicus naturally and experimentally infected with C. microbothrium. A total of 906 F1 B. tropicus between 4 and 5 weeks old were each experimentally infected with two C. microbothrium miracidia and monitored for 12 weeks. The infected snails were fed on dried lettuce and fish flakes and were kept in 1 l plastic aquaria housed in a snail room where temperature, light and humidity were controlled. Seventy-four percent of the experimentally infected snails died during the prepatent period and of the remaining, only 13.2% developed patent infection, while 12.5% were refractory. Snail growth rate was poor and the average shedding rate was 20 cercariae per snail per day. Compared to the experimentally infected snails, 2200 adult B. tropicus, collected from the field and naturally infected with C. microbothrium, yielded high numbers of metacercariae. Eighty-four percent of the snails died within 7 weeks of the study with peak mortality occurring from the 2nd to the 4th week of infection and coinciding with an overall decrease in the number of cercariae shed.     

Characteristics of snail farming in Edo South Agricultural Zone of Edo State, Nigeria

The study was carried out to determine the characteristics of snail farming in Edo South Agricultural Zone of Edo State Nigeria. The interview schedule was used to collect data from 60 snail farmers randomly selected from six cells in the study area. Information on the socioeconomic status of the farmers, production system, management practices and production constraints in the snail farms were elicited. The constraints were determined using a four-point Likert-type scale; a mean score of ≥2.5 was considered as a production constraint. Majority (85.0 %) of the respondents were part-time snail farmers. The major species of snails reared were Achatina achatina and Archachatina marginata, reared by 43.3 and 26.7 % of the farmers, respectively. Semi-intensive system of production was practised by 40.0 % of the farmers. Majority (78.0 %) of the respondents used car tyres to house their snails. About 56 % of the respondents kept their snails for 1–2 years before sale. Up to 51.7 % of the respondents separated their snails into different pens according to their size/age. The most commonly used feeds were vegetables (71.2 %), plant leaves (67.8 %) and kitchen waste (59.3 %). Records of snail production activities were kept by 75.0 % of respondents. The major constraints identified were lack of capital (3.31), inability to get good laying stock (3.00), lack of formulated feed to buy (2.98) and slow growth rate of snails (2.52). The potentials of snail farming in the study area have not been fully exploited as farmers produced at subsistence level.     

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.     

Myocastor coypus as a reservoir host of Fasciola hepatica in France

To clarify the role of the nutria Myocastor coypus in the epidemiology of domestic fasciolosis in Loire-Atlantique (department of western France), 438 nutrias were trapped in 9 humid areas of the department and 304 nutrias were trapped in 3 farms where Fasciola hepatica was present; all animals were necropsied. Liver flukes were found in 160 nutrias: 38 nutrias randomly taken in the department (8.7%) and 122 trapped in fasciolosis areas (40.1%). The average parasitic burden was 5.7 flukes per nutria. Sixty-five percent of the liver flukes measured more than 18 mm (size of sexual maturity). The coproscopic examinations carried out on 144 infected nutrias showed that 90% of the infected nutrias shed fluke eggs. The hatching rate was 39.6%. Two groups of 100 Lymnaea truncatula snails, originating from 2 different populations, were exposed to F. hepatica miracidiae hatched from eggs collected from infected nutrias. The prevalence of the infection was 74% and 58.6% in the 2 groups of snails. The average redial burden was 6.2 rediae per snail. The total number of metacercariae was 72.4 metacercariae per snail producing cercariae. Two groups of 5 sheep were orally infected by 150 metacercariae of nutria or sheep origin, respectively. The installation rates of F. hepatica in sheep were respectively 31.6% and 29.6% for the two groups. Specific antibody kinetics of sheep were similar whether the metacercariae were of nutria or sheep origin. M. coypus allows the complete development of F. hepatica and releases parasitic elements that are infective for domestic ruminants. Because of its eco-ethologic characteristics, the nutria could be a potential wild reservoir of F. hepatica in France.     

MM3-ELISA evaluation of coproantigen release and serum antibody production in sheep experimentally infected with Fasciola hepatica and F. gigantica

During an experimental infection of sheep with Fasciola hepatica or F. gigantica, MM3-SERO and MM3-COPRO ELISA tests were applied to compare the kinetics of antibody production and coproantigen release between the 2nd and 32nd week post-infection (wpi). The Kato-Katz technique was used to measure the kinetics of egg shedding by both Fasciola species (eggs per gram of feces, epg). The kinetics of IgG antibodies for all sheep infected with F. hepatica and F. gigantica followed a similar pattern. Optical density (OD) increased rapidly between the 4th until the 12th wpi, when the highest values were reached and then decreased slowly until the 32nd wpi. Coproantigen levels increased above the cut-off value between 6 and 9 wpi in the F. hepatica group, and between 9 and 11 wpi in the F. gigantica group. The comparison between coproantigen levels and epg indicated that F. hepatica-infected sheep had detectable amounts of coproantigens 4–7 weeks before patency (egg shedding), while F. gigantica-infected sheep had detectable amounts of coproantigens 3–6 weeks before patency. When comparing the kinetics of coproantigen release vs the kinetics of epg, a similar pattern emerged, but with a two-week time-lag in epg, for both F. hepatica and F. gigantica infections. The amount of coproantigen release by each adult was not burden dependent for F. hepatica infection (burden of 33–66 adults), while it was for F. gigantica infection (burden of 17–69 adults). The results demonstrate the usefulness of the MM3-SERO and MM3-COPRO ELISAs as tools for the diagnosis of early as well as long-term fascioliasis infections, and suggest that they can potentially be applied to human fascioliasis even in countries where F. hepatica and F. gigantica co-exist. These tests can be employed not only in the diagnosis, but also in studies on epidemiology as well as pathogenesis and treatment in animals and humans since they allow post-treatment infection monitoring.     

The effect of age of host in resistance expressed at the gut level in rats infected with Fasciola hepatica

The development of oral and intraperitoneal infections with Fasciola hepatica in young and old rats showed that the gut was involved in the expression of age resistance. The role of the gut was assessed by comparison of the number of flukes recovered 4 and 10 weeks after oral or intraperitoneal infection of 2-, 6-, 15-, and 35-week-old male Wistar rats and 6- and 15-week-old male DA rats with encysted metacarcariae of F. hepatica.Rats of both strains behaved similarly in their response to F. hepatica infection. For both routes of infection the number of flukes recovered decreased as host age increased. In 22- and 6-week-old rats equal numbers of flukes were recovered at 4 and 10 weeks after oral or intraperitoneal infection. In 15-week-old rats, fluke burdens 4 weeks after infection were significantly greater following intraperitoneal infection than oral infection. A significant loss of flukes from the intraperitoneal infection of 15-week-old Wistar rats occured between 4 and 10 weeks after infection. In 35-week-old Wistar rats there was no significant effect for route or age of infection.As intraperitoneal infection (to by-pass rejection at the gut level) only partially eliminates the age response, additional age related mechanism(s), able to reject flukes at some time after they have entered the peritoneal cavity, must be operative in the peritoneal cavity and/or the liver.     

Failure to induce homologous immunity to Fasciola hepatica in sheep vaccinated with irradiated metacercariae

Vaccination of sheep with either 100 or 1000 γ-irradiated (2.5 krad) metacercariae of Fasciola hepatica, on two occasions six weeks apart, did not generate significant protection against intraruminal challenge with F. hepatica six weeks after the second vaccinating dose as measured by recovery of flukes from liver and bile ducts, twenty weeks after challenge. There was, however, a significant increase in the proportion of flukes retarded in the parenchyma of both vaccinated groups. The percentage of retarded flukes was positively correlated with the degree of liver damage and increased weight of the hepatic lymph nodes. It was not possible to determine if the retarded flukes were derived from the vaccine or challenge infections or both.Challenge infection of both vaccinated and unvaccinated sheep significantly increased the numbers of eosinophils and globule leucocytes in the parenchymal bile duct and the numbers of mast cells and globule leucocytes in the abdominal bile duct. In addition the numbers of eosinophils and globule leucocytes in the parenchymal bile duct were significantly correlated with the percentage of retarded flukes in both vaccinated groups. In the abdominal bile duct, only the numbers of eosinophils in the low level vaccination group were significantly correlated with fluke retardation.Vaccination did not protect against the pathogenic effects of challenge infection as measured by reduced packed cell volumes and weight gain.     

Pathophysiology of ovine fascioliasis: The influence of dietary protein and iron on the erythrokinetics of sheep experimentally infected with Fasciola hepatica

Worm-free sheep were fed diets with a high or low protein and iron content and infected with 1,000 F. hepatica metacercariae. The adult fluke burdens of the sheep on each diet were similar, but animals on the poorer plane of nutrition developed an earlier and ultimately more severe anaemia and as a result had to be necropsied earlier. By measuring the attendant changes in blood volume and erythrokinetics, it was found that the underlying cause of the anaemia in all animals was a combination of haemodilution, intra-hepatic and biliary haemorrhage, and its earlier appearance and faster development in the poorer-fed group was a refelection of the earlier onset of these changes in association with a faster rate of fluke migration. The ultimate degree of anaemia was not related to the severity of biliary haemorrhage, but rather to the animals' erythropoietic capacity. This was substantially lower in the sheep maintained on the poorer diet, which unlike their better-fed counterparts, became iron deficient by virtue of their inability to replace from the diet iron lost in the faeces as a result of haemorrhage. It was concluded that this difference was primarily a reflection of the better appetite and hence iron intake of the supplemented sheep, but differences in the efficiency of dietary iron utilization, possibly related to protein intake, may also have been involved.In a further experiment involving sheep infected with 600 metacercariae, there was no evidence of haemodilution. When provided with a diet of high protein and iron content, these animals were able to maintain constant, albeit reduced PCV values in the face of substantial biliary haemorrhage. However, when switched to an inferior diet, the same degree of haemorrhage produced a sharp fall in PCV, suggesting that erythropoiesis was adversely affected by reduced protein and iron intake.     

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     

Early Pathological Changes Associated with Fasciola hepatica Infection in White-tailed Deer

Three white-tailed deer (Odocoileus virginianus) were inoculated with 1000 metacercariae of Fasciola hepatica and examined on days 7, 14, and 28 postinoculation to determine the early response of a resistant host to this infection. It was concluded that only small numbers of the metacercariae penetrated the intestinal wall into the peritoneal cavity. Flukes that migrated to the liver penetrated through Glisson's capsule, primarily on the parietal surface. Marked fibroplasia and cellular infiltration of the capsule were induced and flukes were killed and destroyed in granulomas immediately beneath the capsule. Migration in hepatic parenchyma was minimal and immature flukes or migratory tracks were not found. There were infiltrations of eosinophils and mononuclear cells, bile duct hyperplasia and fibroplasia in portal areas. A few flukes penetrated through the diaphragm within 14 days postinoculation and on day 28 granulomas were observed on the dorsal surface of the lung where F. hepatica had penetrated this organ. The early reaction of Glisson's capsule to F. hepatica infection in white-tailed deer has not been described in cattle, sheep or swine infected with this fluke.