Detection of Lymnaea columella infection by Fasciola hepatica through Multiplex-PCR

From complete mitochondrial DNA sequence of Fasciola hepatica available in Genbank, specific primers were designed for a conserved and repetitive region of this trematode. A pair of primers was used for diagnosis of infected Lymnaea columella by F. hepatica during the pre-patent period simultaneously with another pair of primers which amplified the internal transcribed spacer (ITS) region of rDNA from L. columella in a single Multiplex-PCR. The amplification generated a ladder band profile specific for F. hepatica. This profile was observed in positive molluscs at different times of infection, including adult worms from the trematode. The Multiplex-PCR technique showed to be a fast and safe tool for fascioliasis diagnosis, enabling the detection of F. hepatica miracidia in L. columella during the pre-patent period and identification of transmission areas.     

Larval development of Fasciola hepatica in experimental infections: variations with populations of Lymnaea truncatula

A retrospective study was undertaken on 70 French populations of Lymnaea truncatula experimentally infected with Fasciola hepatica to determine whether or not susceptibility of snails to infection influenced redial and cercarial production. Results were compared with those obtained from two control populations, known for prevalences higher than 60% when experimentally infected with F. hepatica. In the 70 other populations examined, the prevalences ranged from 2 to 75%. In 55 of these populations, where the prevalence was more than 20%, a high proportion (50.1-56.8%) of snails died after cercarial shedding, whereas in the other groups (non-shedding snails with the most differentiated larvae being free cercariae, rediae containing cercariae, immature rediae, or sporocysts, respectively), snail death was significantly less. In 11 populations, where the prevalence values were 5-19%, only 14% of snails died after cercarial shedding, whereas snails with free cercariae, rediae with cercariae, or immature rediae showed significant increases in snail mortality. In the remaining four snail populations, with prevalences of less than 5%, the most differentiated larval forms were only immature rediae and/or sporocysts. Overall, the number of rediae containing cercariae significantly decreased with decreasing prevalence values. The low prevalence of experimental infection in several populations of snails might be explained by the occurrence of natural infections with miracidia originating from a mammalian host other than cattle, and/or by genetic variability in the susceptibility of snails to infection.     

Comparison of humoral response in sheep to Fasciola hepatica and Fasciola gigantica experimental infection

Humoral response of sheep to F. gigantica was compared with the well known humoral response to F. hepatica, in order to explain the difference of susceptibility of sheep to these two parasites. In this work, a lesser susceptibility of sheep to F. gigantica than to F. hepatica infection was confirmed. Humoral response to F. hepatica infection is similar to that previously described by several authors. IgG level of F. gigantica infected sheep increased from week 2 post-infection (2WPI) and displayed a peak at 13WPI. F. gigantica excretory-secretory products (FgESP) analyzed by SDS-PAGE showed at least 31 bands from 12.0 to 127.6 kDa in FgESP. Western blot indicated that F. gigantica infected sheep sera recognized, in FgESP, at least 30 antigens from 7.8 to 119.2 kDa of which 12 major bands recognized after OWPI. In FhESP and FgESP, F. hepatica infected sheep serum reacted only with the lower molecular mass antigens, while F. gigantica infected sheep serum reacted with the lower and the higher molecular mass antigens. These differences of antigenic recognition might be associated with the difference of susceptibility of sheep. Further investigation must be done to study the mechanism of resistance between the sheep infected with F. hepatica or F. gigantica.     

Fasciola hepatica and Fasciola gigantica: comparison of cellular response to experimental infection in sheep

Cellular responses to Fasciola gigantica and to Fasciola hepatica infection in sheep were compared. Eosinophil numbers increased more quickly and strongly in F. gigantica-infected sheep than in F. hepatica-infected sheep. In both groups, peripheral blood mononuclear cell (PBMC) proliferation in response to the parasitic excretory-secretory products (ESP) showed similar kinetics. Interferon-gamma (IFN-gamma) production by ESP-stimulated PBMC was early and showed similar kinetics in both groups. Interleukin-10 (IL-10) production by FhESP-stimulated PBMC was very high throughout infection even at 0 weeks post-infection (WPI) in F. hepatica-infected sheep, while in F. gigantica-infected sheep, IL-10 production by FgESP-stimulated PBMC increased between 1 and 4 WPI. IL-10 production in F. gigantica-infected sheep was significantly lower than in F. hepatica-infected sheep during infection. The lower susceptibility to F. gigantica infection in sheep could be explained by the more intense cellular response induced by the parasite and the weaker capacity of F. gigantica to evade the immune response.     

The stress of Lymnaea truncatula just before miracidial exposure with Fasciola hepatica increased the prevalence of infection.

Single-miracidium infections of Lymnaea truncatula with Fasciola hepatica were carried out under laboratory conditions to determine whether the stress of snails just before miracidial exposure had any influence on the prevalence of Fasciola infection, redial burden, and cercarial shedding. Three methods, i.e., the fasting of L. truncatula for 3 days in water filtered through a Millipore membrane, the effect of 6-8 degrees C water for 15 min, or the immersion of L. truncatula in a detergent solution at low concentration for 15 min, were used to stress snails. Enhanced susceptibility of snails to F. hepatica infection was noted in stressed groups (93-96% vs 48-50% in controls). The number of free rediae did not show any variation in controls as well as in stressed groups, except for fasted snails in which free rediae were significantly fewer. No differences in cercarial production between controls and the cold group were noted. Fasting, cold shock, or detergent exposure prior to exposure to F. hepatica miracidia might have weakened the snails so that they were not as efficient in avoiding miracidial penetration, thus leading to higher infection rates.     

Resistance to experimental infection with Fasciola hepatica in exotic and domestic breeds of sheep

Significant breed differences were detected in fecal egg counts and fluke counts following experimental infection of five breeds of sheep with metacercariae of F. hepatica. Two experiments compared the responses of Barbados Blackbelly, St. Croix, Florida Native, Finn X Rambouillet and Targhee sheep to primary and challenge infections (Expt. 1) and to multiple infections (Expt. 2). Barbados Blackbelly sheep were more susceptible to infection than the other breeds in both experiments. St. Croix and Florida Native sheep were the most resistant breeds in Expt. 1, while Targhee and Florida Native were the most resistant in Expt. 2. There was no evidence of acquired immunity in any breed in either experiment.     

Fasciola hepatica: characteristics of infection in Lymnaea truncatula in relation to the number of miracidia at exposure.

Experimental infections of Lymnaea truncatula by Fasciola hepatica were carried out in three snail populations to determine whether the number of miracidia used for each snail at exposure (1, 2, 5, 10, or 20 per snail) had any influence on the characteristics of Fasciola infection and metacercarial production. The number of miracidia had a significant influence on snail survival at day 30 postexposure and the frequency of infected L. truncatula that died without shedding (NCS snails). The frequency of NCS snails, the growth of cercaria-shedding snails throughout the experiment, the time between exposure and the first cercarial shedding, the duration of shedding, and the number of metacercariae were independent of the number of miracidia used for each snail. The highest metacercaria productivity for each miracidium was found in single-miracidium infections. Single-miracidium infections were the most effective, as the mean number of cercariae was the same as in other groups, whereas their survival rate was much higher.     

Effect of concurrent infection with Muellerius capillaris on the development of redial generations of Fasciola hepatica in Lymnaea truncatula.

The rediae of Fasciola hepatica were counted according to generation in adult and juvenile Lymnaea truncatula following single infection with Fasciola hepatica, double infection with F. hepatica and then Muellerius capillaris, or double infection with M. capillaris and F. hepatica. The rediae found in double infections were essentially first generation and an early cohort from the second generation. The following differences were observed in adult snails which underwent double infection when compared to corresponding single infections: i) dependent rediae were almost completely absent; ii) degenerating independent rediae were found in identical or decreased numbers; iii) living independent rediae of the first generation were decreased in number, while those of the second generation had variable decreased numbers. The results were similar in juvenile snails with double infections, except that the numbers of degenerating independent rediae were higher than those found in corresponding single infections and the numbers of rediae of the second generation were increased. The order of exposure in double infections had no influence on the number and maturation of fasciolid rediae.     

The presence of predators modifies the larval development of Fasciola hepatica in surviving Lymnaea truncatula

Experimental infections of Lymnaea truncatula with Fasciola hepatica were performed to study the consequences of the presence of predators (sciomyzid larvae or zonitid snails) on the characteristics of larval F. hepatica development in surviving snails. Controls consisted of infected snails that were not subjected to predators. Compared to controls, the survival rate at day 30 post-exposure, the duration of cercarial shedding, and the number of cercariae shed by surviving snails were significantly lower when predators were present in snail breeding boxes, whatever the type of predator used. In contrast, the prevalences of Fasciola infections in snails, and the length of time between exposure and the onset of cercarial shedding showed no significant variation. The progressive development of a stress reaction in surviving snails against predators during the first 30 days of experimental exposure to F. hepatica would influence snail survival during the cercarial shedding period and, consequently, the number of cercariae shed by the snails.     

Effect of BCG on the resistance of rats to infection with Fasciola hepatica

The effects of three different doses of BCG, given at various periods before infection, on the subsequent establishment of Fasciola hepatica metacercariae were studied. Although evidence was found to suggest that rats which received BCG had mounted a cell mediated immune response, there were no significant differences in worm recovery between BCG-treated rats and controls. The significance of these results in the light of current knowledge on immunity to F. hepatica in rats is discussed.     

Epidemiological observations on iridovirosis of Lymnaea truncatula, host mollusca of Fasciola hepatica]

Epidemiological studies were carried out in 11 populations of Lymnaea truncatula in 1983-1984 and 1987-1989 to determine the prevalence of snail infection by an iridovirus. The virus was found in the different populations and samples, with a frequency ranging from 1.6 to 87%. The virosis would be endemic.     

Hepatic fibrosis and Fasciola hepatica infection in cattle

This study focuses on the development of fibrosis of the liver of cattle with Fasciola hepatica infection, correlating with the intensity of infection. Animals with an established diagnosis of chronic F. hepatica infection were identified in a slaughterhouse in Lima, Peru. The study included 24 fresh cattle livers from infected animals and two uninfected controls. Tissues were stored at 4 degrees C for approximately 8 h after which they were brought to a necropsy room and examined. Between 9 and 12 biopsies were randomly obtained from each liver. Histological staining of formalin-fixed liver sections with haematoxylin and eosin (H & E) and Masson's trichrome were performed. Liver samples were examined using a pathology protocol that included 30 items. Histopathologically, 16 out of 30 liver specimens (67.6%) showed diffuse fibrotic lesions (cirrhosis) with a mean number of Fasciola of 116 +/- 30 (range 4-435). Pathological data were matched to number of adult parasites and presence of cirrhosis after being reviewed by two independent pathologists. There was concordance between the two pathologists (K = 0.72). The group with cirrhosis showed an average of 116 +/- 30 adult parasites whereas the group not showing cirrhosis contained 56 +/- 28 flukes (P = 0.2). To measure how number of flukes and diagnosis of cirrhosis are related we used Kendall's tau-b coefficient; the correlation was +0.296 (P = 0.04). Receiver Operating Characteristic (ROC) curve results showed that the best point was 38 parasite adults, which had 93.8% sensitivity and 75% specificity. We conclude that as the number of F. hepatica adult forms increases, the likelihood of developing liver fibrosis will also increase in cattle.     

Influence of shell size of Lymnaea columella on infectivity and development of Fasciola hepatica

Experimental infections of Lymnaea columella with Fasciola hepatica were carried out to determine the influence of shell size on the infection rate and on the outcome of rediae and cercariae. Snails were divided into seven groups according to shell size: 2-4 mm, 5-6 mm, 7-8 mm, 9-10 mm, 11-12 mm, 13-14 mm and 15 mm or more. One hundred snails in each group were infected by using four miracidia for each snail. Snails with larger shell size showed a lower infection rate, the groups presenting the highest (79%) and lowest (2%) proportions of positives being those of 5-6 mm and 15 mm or more, respectively. Cercariae were present in 21% of them at 31 days post-infection, and cercarial shedding was observed 61 days post-infection. It was concluded that there is a non-linear negative association between shell size and infection rate.     

Development of Fasciola hepatica in Lymnaea columella infected with miracidia derived from cattle and marmoset infections

The development of Fasciola hepatica from two species of definitive hosts, i.e. cattle (Bos taurus) and a marmoset (Callithrix penicillata) in the snail Lymnaea columella was determined based on the production of rediae and cercariae and snail survival rate. More rediae and cercariae at 60-74 days post-infection were produced by snails infected by cattle-derived miracidia (cattle group) than by those infected by marmoset-derived miracidia (marmoset group). Among the L. columella parasitized by the marmoset group, the survival rate and the percentage of positive snails were higher than among those parasitized by the cattle group. Eggs of F. hepatica released in cattle faeces were significantly bigger than those released in marmoset faeces. Miracidia originating from parasites that completed their development in cattle were more efficient in infecting the intermediate host. These results suggest that vertebrate-host origin influences the eggs produced by the parasite and the infection rates in the snail host L. columella.     

Population dynamics of Lymnaea columella and its natural infection by Fasciola hepatica in the State of Minas Gerais,Brazil

Studies on the population dynamics and natural infection of Lymnaea columella by Fasciola hepatica were carried out from September 1999 to December 2000 in a low-lying area near Itajubá in the Brazilian State of Minas Gerais. A total of 626 snails were collected monthly at nine different sites, counted, and dissected to search for larvae of F. hepatica. The highest populations of L. columella were reached in October of 1999 and August of 2000, and the highest natural infection rates of snails by F. hepatica were reached in September 1999 (5.2%) and July 2000 (3.9%). The removal by farmers of aquatic plants from the drainage furrows caused a drastic reduction in this snail population.