Adam Novobilský

SEQUENCE ANALYSIS OF RIBOSOMAL AND MITOCHONDRIAL GENES OF THE GIANT LIVER FLUKE FASCIOLOIDES MAGNA (TREMATODA: FASCIOLIDAE): INTRASPECIFIC VARIATION AND DIFFERENTIATION FROM FASCIOLA HEPATICA

Complete sequences of ribosomal and mitochondrial genes of the giant liver fluke Fascioloides magna are presented. In particular, small subunit (18S) and internal transcribed spacers (ITS1 and ITS2) of the ribosomal gene (rDNA), as well as cytochrome c oxidase subunit I (cox1) and nicotinamide dehydrogenase subunit I (nad1) of the mitochondrial DNA (mtDNA), were analyzed. The 18S and ITS sequences were compared with previously published sequences of the liver fluke Fasciola hepatica. Fixed interspecific genetic differences were determined that allow molecular differentiation of F. magna and F. hepatica using either the PCR-RFLP method or PCR amplification of species-specific DNA regions. Additionally, intraspecific sequence polymorphism of the complete cox1 and nad1 mitochondrial genes in geographically distinct F. magna populations was determined. Based on the sequence divergences, short (<500 bp) variable regions suitable for broader biogeographical studies of giant liver fluke were designed.

The giant liver fluke Fascioloides magna (Bassi 1875) in cervids in the Czech Republic and potential of its spreading to Germany

The giant liver fluke Fascioloides magna is an important parasite of cervids in Europe. From September 2003 to December 2005, faecal samples and livers of red deer (Cervus elaphus) and fallow deer (Dama dama) were investigated to determine the current distribution of the fluke in the Czech Republic. Faecal samples were collected from 20 different areas, and livers of hunted deer were dissected from each locality to confirm F. magna infection. The prevalence of F. magna in examined areas determined by coprological examination varied from 4% to 95%. Moreover, new foci of F. magna infection were discovered in all localities in the Šumava mountains where F. magna was observed; this has epizootiological importance due to the possibility of the spread of F. magna into the German territory (Bavaria).

Is Radix peregra a new intermediate host of Fascioloides magna (Trematoda) in Europe? Field and experimental evidence

Rediae and immature cercariae of the giant liver fluke, Fascioloides magna, a pathogenic and important parasite of game animals, were found in 6 out of 7,277 Radix peregra (Gastropoda, Lymnaeidae) from two localities in Western and Central Bohemia, Czech Republic. In one of these localities a relatively high infection of Galba truncatula (26 out of 76) with mature cercariae of F. magna was detected. R. peregra, which is dominant over Galba truncatula, has never been reported as the natural intermediate host of F. magna. Its potential susceptibility to F. magna infection was confirmed experimentally with 67 snails of R. peregra (out of 371) infected. The present data indicate the capability of F. magna to expand the spectrum of its intermediate hosts and demonstrate the potential epizootiological importance of lymnaeid snails, other than G. truncatula, for transmission of the parasite in Europe.

The field evaluation of albendazole and triclabendazole efficacy against Fasciola hepatica by coproantigen ELISA in naturally infected sheep

Albendazole (ABZ) has been used for control of ovine fasciolosis in Sweden for several decades. However, increasing prevalence of Fasciola hepatica in Sweden requires attention to diagnostic tools and current control strategies. The coproantigen reduction test (CRT) using commercial Bio-X K201 Fasciola coproantigen ELISA (Bio-X Diagnostics, Jemelle, Belgium) was recently suggested as a novel method for diagnosis of patent F. hepatica infection. The aim of this study was to compare the efficacy of albendazole and triclabendazole (TCBZ) treatment against F. hepatica in naturally infected sheep in south-western Sweden by CRT, and also to evaluate the usefulness of this test as a diagnostic marker for anthelmintic efficacy following treatment with ABZ. Three weeks after housing, 24 serologically positive ewes on a commercial farm in Sweden were randomly allocated into three groups, which were either dewormed with 5mg/kg ABZ, 10mg/ml TCBZ or left untreated. Twenty-six days after initial administration of these anthelmintics, all of the sheep in the ABZ group and untreated control groups were treated with 10mg/kg TCBZ. While TCBZ caused elimination of coproantigen and eggs in faeces 7 days after application, ABZ treatment failed completely. Neither anthelmintic resistance, underdosing, nor lack of efficacy due to the presence of immature flukes can be out ruled as possible causes. Despite some deviations in conformity between coproantigen levels and presence of fluke eggs, the CRT was a useful tool for measuring treatment efficacies.

First report of closantel treatment failure against Fasciola hepatica in cattle.

Control of Fasciola hepatica infection in livestock is based on annual treatment using flukicides such as triclabendazole, albendazole and closantel. However, triclabendazole resistant F. hepatica populations are emerging worldwide and resistance is emerging to albendazole, whereas it has until now never been described for closantel. In Sweden, a topical formulation containing a combination of closantel and ivermectin (Closamectin Pour On) has been registered for use in cattle only since 2011. This study evaluated the efficacy of closantel against F. hepatica in naturally infected beef cattle using both coproantigen and faecal egg count reduction tests. Faecal egg counts (FEC) and coproantigen ELISA examinations were conducted in February 2014 in three beef cattle herds (A, B, C) in south-western Sweden. On each farm, 10 F. hepatica coproantigen-positive and F. hepatica egg-positive animals were allocated after 12–16 weeks of housing into groups and treated topically with a minimum of 20 mg closantel per kg body weight. Faecal samples were collected from selected animals on 0, 7 and 21 day post-treatment (PT). Based on FEC, closantel efficacy 21 days PT was 72% (95% CI: 65–77%) and 97% (95% CI: 95–98%) on farms A and B, respectively. No FEC reduction at all was observed on farm C. In total, 4, 1 and 6 animals remained coproantigen-positive at 21 days PT on farms A, B and C, respectively. Closantel treatment failure was confirmed on two of the farms. As the animals were housed 12–16 weeks before treatment and thereafter during the entire study, failure due to the presence of juvenile flukes was excluded. Although the cause of closantel failure currently remains unclear, development of resistance or/and absorption failure of topical administration should be considered. To our knowledge, this is the first report of closantel treatment failure against F. hepatica in cattle.

Lymnaea neotropica and Lymnaea viatrix, potential intermediate hosts for Fascioloides magna

Experimental infections of two South American lymnaeid populations with Fascioloides magna were carried out to determine whether these snails may sustain larval development of this digenean and, if so, to quantify their potential for cercarial production. The reference group was a French population of Galba truncatula infected and raised according to the same protocol. According to the internal transcribed sequence (ITS)-1 segment of their genomic rDNA, these South American populations were identified as Lymnaea neotropica (origin, Argentina) and Lymnaea viatrix var. ventricosa (origin, Uruguay). In the snail groups followed for cercarial shedding, longer prepatent periods and lower numbers of shed cercariae were noted in South American lymnaeids. In other snails dissected at day 65 post-exposure, the redial and cercarial burdens of F. magna found in the bodies of L. neotropica and L. v. ventricosa were significantly lower than those noted in G. truncatula. Compared to the total cercarial production noted in the dissected snails, the percentage of cercariae that exited from snails was 51.3% for G. truncatula, 32.2% for L. neotropica and 46.8% for L. v. ventricosa. The two South American species of snails can thus be considered as potential intermediate hosts of F. magna.

Distribution of Fasciola hepatica in Swedish dairy cattle and associations with pasture management factors.

The geographic distribution of Fasciola hepatica infection in relation to management routines was studied in Swedish dairy herds by testing for F. hepatica antibodies with the enzyme-linked immunosorbent assay (ELISA). In addition, all farmers were sent a questionnaire asking for information about type of production, management routines and historical record of F. hepatica at slaughter. A total of 176 farmers (41%) responded to the questionnaire. A total of 426 bulk tank milk (BTM) samples were randomly selected from the period September to October 2012 representing approximately 10% of all herds in Sweden. The overall seroprevalence was 25% (n = 107; 95% confidence interval = 21-29%) with a concentration of herds located in south-western Sweden. Among the seropositive herds, 31 (29%) had antibody levels indicating production loss. There were no significant differences in seropositivity between organic and conventional herds or due to pasture management routines. The length of grazing period, which increased the risk for heifers, was found to be the most influential factor. A discrepancy was noted between reported F. hepatica presence at meat inspection and herds that were seropositive based on BTM-ELISA results. Although the largest proportion of seropositive BTM samples (80%) came from herds where liver fluke presence had been observed at meat inspection after slaughter, seropositive BTM samples were also diagnosed in five herds (17%) with no remarks at slaughter. In conclusion, F. hepatica is a common parasite in Swedish dairy herds and the month of heifer turn-out and the grazing period length were the most influential factors observed.

Prevalence and intensity of infections in the lymnaeid snail Omphiscola glabra experimentally infected with Fasciola hepatica , Fascioloides magna and Paramphistomum daubneyi

Single and double infections of juvenile Omphiscola glabra (Gastropoda: Lymnaeidae) with Paramphistomum daubneyi and/or Fasciola hepatica were carried out to determine the redial burden and cercarial production in snails dissected at day 60 or at day 75 post-exposure (p.e.) in the laboratory at 20 degrees C. The results were compared with those obtained with single-miracidium infections by Fascioloides magna. Compared to F. hepatica, low values were noted at day 75 p.e. for the prevalence of snail infections with P. daubneyi (4.6-8.3% instead of 23.6-25.9%), the total number of free rediae (10.7-17.9 per snail instead of 26.3-34.7), and that of free cercariae (112.8-136.9 per snail instead of 177.8-248.5). Despite a greater number of free rediae at day 75 p.e. (36.2-45.6 per snail), the prevalences of snail infections with F. magna and cercarial production were similar to those noted for F. hepatica. The results concerning F. hepatica and P. daubneyi might partly be explained by a progressive adaptation of O. glabra to sustain the larval development of these digeneans over the years, as this snail is a natural intermediate host of F. hepatica and P. daubneyi in central France since 1995. Compared with the high number of fully-grown rediae of F. magna in O. glabra, cercarial production seemed limited and this might be explained by the presence of high numbers of rediae which reduced the avaibility of nutrients for cercarial differentiation within the snail.

Comparison of molecular and conventional methods for the diagnosis of Fasciola hepatica infection in the field

The liver fluke, Fasciola hepatica, is one of the major parasite threats to livestock industries world-wide. In sheep and cattle, F. hepatica infection is commonly diagnosed using a range of methods. Aside from conventional coprological and serological diagnostic methods, there are also several molecular methods available based on the detection of liver fluke DNA in faeces. In this study, the outcomes of faecal egg count (FEC), serology and coproantigen ELISA (cELISA) were compared with the performance of polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) in diagnosis of F. hepatica from naturally infected cattle and sheep. A total of 64 individual faecal and serum samples were collected from four sheep and beef cattle herds with previous histories of F. hepatica infection. FEC and coproantigen levels were measured in faecal samples and anti-F.hepatica antibody levels were measured in serum samples. DNA samples isolated from faeces were examined both by PCR and LAMP, targeting the internal transcribed spacer 2 (ITS2) region of the F. hepatica genome. Results showed that F. hepatica eggs were present in 28 animals, while coproantigen and specific anti-F. hepatica antibodies were detected in 36 and 53 animals, respectively. Only 3 and 6 samples were positive by PCR and LAMP, respectively. To calculate method specificity and sensitivity, a combination of FEC and cELISA was selected as the composite reference standard (CRS). When compared to the CRS, PCR had a sensitivity of 10.7% and specificity of 100%, whereas LAMP had a sensitivity and specificity of 17.9% and 97.2%, respectively. PCR and LAMP in this field study were highly specific, but both had poor sensitivity compared with FEC and cELISA. Potential reasons for PCR and LAMP failure were inadequate amounts of amplifiable F. hepatica DNA, possibly due to the choice of DNA extraction procedure, amount of faecal material processed, as well as different faeces consistency and composition between different animal species.

Lymnaea fuscus (Pfeiffer, 1821) as a potential intermediate host of Fascioloides magna in Europe

Experimental infections of two different populations of Lymnaea fuscus in France and Sweden, with a Czech isolate of Fascioloides magna were carried out to determine if this lymnaeid species enables parasite larval development. Species identification of both snail populations was performed using the morphology of the copulatory organ, and also confirmed by sequencing of the internal transcribed spacer 2 (ITS2) region of the snail genomic rDNA. Only juvenile snails measuring less than 3mm (1-3 weeks of age) were successfully infected (the viable cercariae were recorded) and infection prevalence decreased with age, as documented by increased shell height. In both French and Swedish L. fuscus populations, prevalence ranged between 1.1% and 58.8%. The mean number of metacercariae obtained from cercariae-shedding snails was 13.7 (±11.4), while the total cercarial production noted in snails dissected at day 85 post-exposure was 147.5 (±56.6). Compared to uninfected control snails, we observed reduced growth of infected snails. Despite age-related resistance of snail to the parasite, and limited cercarial production in these experimentally infected snails, F. magna was still able to complete larval development in L. fuscus.