Occurrence of the giant liver fluke, Fascioloides magna, in sympatric wild ungulates in one area in the Upper Palatinate Forest (northeastern Bavaria, Germany).

Associated with the spreading in (north)western direction of Fascioloides magna from its historic endemic area in Bohemia with its cervid hosts, unusual noticeable hepatic lesions (black-colored tissue, hemorrhage) were observed in deer harvested in hunting grounds and one deer farm located in the Upper Palatinate Forest close to the border to the Czech Republic, initially in the years of 2007 and 2009, respectively. Confirmation of the suspected diagnosis of F. magna infection in October 2011 prompted investigations on the occurrence of "fascioloidosis" among wild ungulates in that locality. From October 2011 to January 2014, livers from 89 cervids and two wild boars were examined for flukes. Thirty-seven livers (40.6%) harbored F. magna: 17 of 21 red deer, nine of 24 sika deer, six of eight fallow deer, four of 36 roe deer, one of two wild boars. Fluke burdens ranged from 2 up to 151 in red deer, from 2 up to 37 in fallow deer, and from 1 up to 7 in sika deer and in roe deer; one fluke was recovered from the liver of one wild boar. No other parasites were recovered from the livers. The rate of recovery of F. magna differed significantly (p < 0.001) among the species of deer (red deer, 81.0%; sika deer, 37.5%; fallow deer, 75.0%; roe deer, 11.1%) and between the age groups (< 1 year: 22.2%, 1 to 2 years: 26.0%, and > 2 years: 70.0%, respectively). There was no association (p > 0.1) between the rate of recovery of F. magna and the sex of the combined 80 deer of ≥ 1 year of age (male: 41.8% and female: 31.4%). The occurrence of F. magna in the wild ungulates in the Upper Palatinate Forest area in northeastern Bavaria is of epidemiological importance for the further spreading of the parasite into Germany with migrating deer.

An Autochthonous Human Case of Fasciolopsiasis in Nepal.

Fasciolopsiasis is rarely known as the parasitic disease in Nepal. Herein, we report a case of fasciolopsiasis in a 22-year-old man who was admitted in the hospital with abdominal pain, distension and loss of appetite for a month. He had previously diagnosed with acute viral hepatitis but, his abdominal pain was not resolving despite improvement in his liver function and general condition. During endoscopy an adult digenean worm was seen in the first part of the duodenum. After isolation, the worm was identified morphologically as Fasciolopsis buski. Microscogic examination of the patient's stool revealed eggs with a morphology consistent with F. buski. Eggs were yellow-brown, ellipsoidal, unembmbryonated, operculated, filled with yolk cells, with thin shell and ranging 118-130 µm in length and 60-69 µm in width. The abdominal pain of the patient was resolved after treatment with praziquantel. By the present study, it was confirmed for the first time that fasciolopsiasis is indigenously transmitted in Nepal. Accordingly, the epidemiological studies in humans and reservoir host animals should be performed intensively in near future.

Establishment of Fascioloides magna in a new region of Hungary: case report.

During the monitoring of red deer (N = 124) and fallow deer (N = 13) populations in four neighbouring areas, the presence of Fascioloides magna was confirmed in southwestern Hungary. The prevalence and the mean intensity of the infection within the host populations ranged between 0 and 100% and 0-36.3, respectively. The determined prevalences are similar to that observed earlier in other European natural foci. The authors hypothesise that the appearance of F. magna in this region should have been a partly natural- and partly human-influenced process.

Landscape influence on spatial patterns of meningeal worm and liver fluke infection in white-tailed deer.

Parasites that primarily infect white-tailed deer (Odocoileus virginianus), such as liver flukes (Fascioloides magna) and meningeal worm (Parelaphostrongylus tenuis), can cause morbidity and mortality when incidentally infecting moose (Alces alces). Ecological factors are expected to influence spatial variation in infection risk by affecting the survival of free-living life stages outside the host and the abundance of intermediate gastropod hosts. Here, we investigate how ecology influenced the fine-scale distribution of these parasites in deer in Voyageurs National Park, Minnesota. Deer pellet groups (N = 295) were sampled for the presence of P. tenuis larvae and F. magna eggs. We found that deer were significantly more likely to be infected with P. tenuis in habitats with less upland deciduous forest and more upland mixed conifer forest and shrub, a pattern that mirrored microhabitat differences in gastropod abundances. Deer were also more likely to be infected with F. magna in areas with more marshland, specifically rooted-floating aquatic marshes (RFAMs). The environment played a larger role than deer density in determining spatial patterns of infection for both parasites, highlighting the importance of considering ecological factors on all stages of a parasite's life cycle in order to understand its occurrence within the definitive host.

Molecular identification of Fascioloides magna (Bassi, 1875) from red deer from South-Western Poland (Lower Silesian wilderness) on the basis of internal transcribed spacer 2 (ITS-2).

The study was conducted in 2012-2013 on 75 fecal samples of red deer from the Lower Silesian Wilderness which were examined to determine the prevalence of Fascioloides magna in the game population. Finding liver fluke eggs in a single sample which were larger in size than Fasciola hepatica eggs indicated that further molecular analysis was necessarily. The partial sequence (116 bp long) of ITS-2 of the investigated eggs was identical to the sequences of F. magna from red deer (Cervus elaphus) (GenBank, EF534993; GenBank, EF534992) and from wapiti deer (Cervus elaphus canadensis) (GenBank, EF534994) from Slovakia, as well as from fallow deer (Dama dama) from the USA (GenBank, EF051080). This is the first molecular confirmation of the occurrence of F. magna in Poland.

The origin of the giant liver fluke, Fascioloides magna (Trematoda: Fasciolidae) from Croatia determined by high-resolution melting screening of mitochondrial cox1 haplotypes.

The high-resolution melting (HRM) method, recently optimized as a reliable technique for population study of the European Fascioloides magna populations, was applied to determine an origin of F. magna individuals from Croatia. The structure and frequency of mitochondrial cytochrome c oxidase subunit I (439 bp; cox1) haplotypes of 200 Croatian flukes coming from 19 red deer (Cervus elaphus elaphus) livers were screened and compared with recently determined reference samples of F. magna from all European foci-Italy, Czech Republic, and Danube floodplain forests. While the reference haplotypes Ha1 and Ha2 were specific for flukes from the first European focus of fascioloidosis, the Natural Park La Mandria in Italy, the remaining three haplotypes (Ha3, Ha4, and Ha5) represented parasites from the second focus, Czech Republic. Besides, Ha3 and Ha4 were found also in the third, latest, and still expanding European focus, the Danube floodplain forests. The HRM screening of cox1 haplotypes of Croatian F. magna individuals resulted in classification of samples into the two mitochondrial haplogroups characterized by well-distinguished melting curves. They corresponded to Ha3 and Ha4 reference haplotypes that confirmed the Danube origin of F. magna from Croatia. The results support the theory that the Danube floodplain forests population of F. magna represents uniform genetic pool of the parasite. The spread of F. magna alongside the Danube River down to Croatia was possible due to suitable ecological conditions for definitive and intermediate hosts present in this unique biotope.

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.

Morphological characterization of adult Fascioloides magna (Trematoda: Fasciolidae): first SEM report.

Five adult Fascioloides magna specimens were recovered from the livers of naturally infected calves from Texas, USA. Scanning electron microscopy was used to study the morphological characteristics of the trematodes. These mature flukes measured 35-100 mm in length by 15-25 mm in width and had an oval dorsoventrally flattened body, with no anterior cone. The tegument was armed with sharp spines. Around the oral and ventral suckers, some of the spines were small, with a sharp point, while others had serrated edges with 15-22 sharp points. The surface of the oral sucker was covered by an interesting pattern of tegument, small dome-shaped and ciliated papillae. The ventral sucker showed a smooth surface and two unknown spine-like structures. There were fewer spines at the base of the genital pore than on other parts of the anterior end of the worm. At the anterior end of the ventral side, well-developed spines were observed, while at the posterior end of the ventral side, the spines were small, mostly with one or three points and blunted edges. At the posterior end of the dorsal side, the spines became progressively fewer, smaller, and shorter. Around the excretory pore, the tegument was folded, with no spines, and small groups of dome-shaped and ciliated papillae were present. The cirrus organ showed a smooth surface, with small pores on the dorsal side and small groups of tiny spines between the folds. The eggs measured 168 × 101 µm and had a protoplasmic appendage at the pole opposite the operculum. At the posterior end of the dorsal side, and toward the right, a pore with a very thin rim was present, which could be the terminus of Laurer's canal.

Liver enzymes and blood metabolites in a population of free-ranging red deer (Cervus elaphus) naturally infected with Fascioloides magna.

We investigated the effects of Fascioloides magna infection on the serum biochemistry values of the naturally infected red deer population in eastern Croatia. The investigation was performed on 47 red deer with F. magna infection confirmed patho-anatomically in 27 animals (57.4%). Fibrous capsules and migratory lesions were found in 14 deer while only fibrous capsules without migratory lesions were found in 13 deer. In 13 deer both immature and mature flukes were found, in 5 deer only immature flukes were found and in 9 deer only mature flukes were found. Fascioloides magna infected deer with fibrous capsules and migratory lesions had significantly higher values for lactate dehydrogenase (LDH), glutamate dehydrogenase (GLDH) and globulin, and lower values for albumin/globulin ratio and glucose compared to uninfected deer. Fascioloides magna infected deer with fibrous capsules without the presence of migratory lesions had higher values for alanine aminotransferase (ALT) and globulin, and lower values for albumin/globulin ratio and glucose, than the uninfected deer. The number of immature flukes was positively correlated with values of aspartate aminotransferase (AST), γ-glutamyltransferase (GGT), LDH, GLDH, urea and triglycerides. The number of migratory lesions was positively correlated with GGT, GLDH, globulin and urea values. The creatinine value was positively correlated with the number of mature flukes. The trial showed that F. magna infection causes significant changes in serum biochemistry. Moreover, these changes do not completely resemble changes following F. hepatica infection. Further investigation of changes in liver enzymes and other serum metabolites in controlled, experimentally induced fascioloidosis in red deer is needed to better understand the pathogenesis of F. magna.

Development of high-resolution melting (HRM) analysis for population studies of Fascioloides magna (Trematoda: Fasciolidae), the giant liver fluke of ruminants.

The high-resolution melting (HRM) technique was successfully optimized as fast and effective method for population study of digenetic fluke, Fascioloides magna (Trematoda: Fasciolidae), originally North American liver parasite of free-living and domestic ruminants. Previously selected variable region (439 bp) of mitochondrial cytochrome c oxidase subunit I (cox1) of 249 fluke individuals from enzootic European and North American regions were sequenced and mutually compared. The sequence analysis of partial cox1 revealed presence of seven structurally different haplotypes. Based on the sequence structure and alignments of six of them (Ha1-Ha6), three internal probes were designed and applied in HRM-based haplotype determination of all F. magna specimens. HRM analysis, performed with three designed probes, resulted in classification of samples into the seven haplogroups, equally with their assortment according to the sequence analysis. The representative of the haplotype, which was not involved in probe design (Ha7), was characterized by a unique melting curve shape as well. This provided an evidence of optimally settled conditions in HRM assay and indicated a probability of successful discrimination of novel haplotypes in future population studies on F. magna. The successful optimization of HRM method stands for an opportunity of detection of genetically unknown North American variants of F. magna and promises its application as fast and cheap screening technique for phylogeography studies of the giant liver fluke on its original continent.

A comparative study of karyotypes and chromosomal location of rDNA genes in important liver flukes Fasciola hepatica and Fascioloides magna (Trematoda: Fasciolidae).

Chromosomal characteristics, i.e., number, size, morphology, and location of ribosomal DNA (rDNA) clusters were examined in two medically important liver flukes, Fasciola hepatica and Fascioloides magna (Fasciolidae), using conventional Giemsa staining and fluorescent in situ hybridization (FISH) with ribosomal 18S rDNA probe. A comparison of F. magna and F. hepatica karyotypes confirmed significant differences in all chromosomal features. Whilst the karyotype of F. hepatica comprised ten pairs of chromosomes (one metacentric and nine medium-sized subtelocentrics and submetacentrics; 2n = 20, n = 1 m + 5 sm + 4 st; TCL = 49.9 µm), the complement of F. magna was composed of 11 pairs of medium-sized subtelocentrics and submeta-metacentrics (2n = 22, n = 9 st + 1 sm + 1 sm-m; TCL = 35.2 µm). Noticeable differences were found mainly in length and morphology of first chromosome pair. It was metacentric and 9.0 µm long in F. hepatica while subtelocentric and 4.7 µm long in F. magna. Although FISH with rDNA probe revealed a single cluster of ribosomal genes in both species, conspicuous interspecific differences were displayed by chromosomal location of ribosomal loci (i.e., NORs). The signals were found on short arms of fifth homologous pair in F. hepatica; however, they were detected in pericentromeric regions of the long arms of tenth pair in F. magna. The observed cytogenetic differences were interpreted in terms of karyotype evolution of fasciolid flukes; F. hepatica may be regarded phylogenetically younger than F. magna. The present paper provides a pilot study on molecular cytogenetics within a group of hermaphroditic digenetic flukes.

Fasciolopsiasis presenting as intestinal perforation: a case report.

Infestation by the zoonotic trematode Fasciolopsis buski (fasciolopsiasis) is seen in several parts of South-East Asia. Abdominal pain, diarrhoea, mucosal ulceration, intestinal obstruction, anasarca, and even fatality are described following heavy infestation. We present here the case of a 10-year-old boy from the Barabanki district of Uttar Pradesh, India with heavy infestation by Fasciolopsis buski causing intestinal perforation. Fasciolopsiasis is by no means rare but its presentation as a case of intestinal perforation is extraordinary.

[Age structure and dynamics of abundance of the adult Trematode Opisthioglyphe ranae (Fasciolida, Plagiorchidae) hemipopulation in lake frog from the Saratov reservoir].

Age structure and dynamics of abundance of adult Opisthioglyphe ranae, a parasite of intestine of lake frog, are analyzed. In the period of activity of the host (May-October), the hemipopulation of O. ranae maritae is represented by two generations, which are situated in host lesser than one year and differ from each other in the rate of maturation.

[Improved staining method for permanent specimen of Fasciolopsis buski].

Fasciolopsis buski specimens were collected, fixed with neutral formalin fixative solution, stained with alum-carmine staining solution, discolored with 2% kalium alum. The fixed and stained specimen shows clear internal structure with bright color, and can be stored for long time.

Fasciolopsis buski (Digenea: Fasciolidae) from China and India may represent distinct taxa based on mitochondrial and nuclear ribosomal DNA sequences.

BACKGROUND: Fasciolopsis buski is a zoonotic intestinal fluke infecting humans and pigs, but it has been seriously neglected. It is yet to know whether there is any genetic diversity among F. buski from different geographical locations, particularly in sequences of nuclear ribosomal DNA (rDNA) and mitochondrial (mt) DNA. Therefore, we determined the sequences of partial 18S, the complete internal transcribed spacer (ITS) rDNA and the complete mt genome of F. buski from China, compared the rDNA and mtDNA sequences with those of isolates from India and Vietnam, and assessed the phylogenetic relationships of this fluke and related fasciolid trematodes based on the mtDNA dataset. RESULTS: The complete mt genome sequence of F. buski from China is 14,833 bp, with 36 genes, including 12 protein-coding genes (PCGs), 22 tRNA genes, and two rRNA genes (rrnL and rrnS). The AT content of F. buski from China is 65.12%. The gene content and arrangement of the F. buski mt genome is similar to that of Fascioloides magna. Genetic distances between isolates of F. buski from China and India were high (28.2% in mtDNA, 13.2% in ITS-1 and 9.8% in ITS-2) and distinctly higher than the interspecific differences between Fasciola hepatica and Fasciola gigantica. The rDNA and mtDNA datasets for F. buski from China (isolate from pigs) and Vietnam (isolates from humans) were identical. The intergeneric differences in amino acid and nucleotide sequences among the genera Fasciolopsis, Fascioloides and Fasciola ranged between 24.64-25.56% and 26.35-28.46%, respectively. CONCLUSIONS: Our results indicate that F. buski from China and India may represent distinct taxa, while F. buski in Vietnam and China represent the same species. These findings might have implications for the implementation of appropriate control strategies in different regions. Further studies are needed to decode mtDNA and rDNA sequences of F. buski from various geographical isolates for the better understanding of the species complex of F. buski.

Fasciolopsiasis in children: Clinical, sociodemographic profile and outcome.

PURPOSE: To describe the clinical and sociodemographic profile of fasciolopsiasis in children. MATERIALS AND METHODS: A chart review of 56 children presenting with the passage of adult Fasciolopsis buski per stool from February 2015 to January 2016 was done for their clinical profile and risk factors for acquiring fasciolopsiasis in the Paediatric Unit of a medical college of Northern India. RESULTS: The mean age of presentation was 8.2 years (2-14 years age group). Persistent diarrhoea (85.71%) was the most common presentation, whereas anaemia (71.42%) was the most common sign. Protein-energy malnutrition (PEM) and tuberculosis were well-associated comorbid conditions in this study. Polyparasitism was an important finding, Hymenolepis nana being the most common associated parasite. Patients were treated either with praziquantel or nitazoxanide. CONCLUSION: All patients recovered well except one who died due to severe PEM and disseminated tuberculosis and two cases presented with relapse. Most of the cases of polyparasitism were associated with tuberculosis.

Better Alone or in Ill Company? The Effect of Migration and Inter-Species Comingling on Fascioloides magna Infection in Elk.

Migratory movements and alteration of host communities through livestock production are examples of ecological processes that may have consequences on wildlife pathogens. We studied the effect of co-grazing of cattle and wild elk, and of elk migratory behaviour on the occurrence of the giant liver fluke, Fascioloides magna, in elk. Migratory elk and elk herds with a higher proportion of migratory individuals were significantly less likely to be infected with F. magna. This may indicate a decreased risk of infection for migratory individuals, known as the "migratory escape" hypothesis. Elk herds overlapping with higher cattle densities also had a lower prevalence of this parasite, even after adjustment for landscape and climate variables known to influence its life cycle. Serological evidence suggests that even in low-prevalence areas, F. magna is circulating in both elk and cattle. Cattle are "dead-end" hosts for F. magna, and this may, therefore, indicate a dilution effect where cattle and elk are co-grazing. Migratory behaviour and host community composition have significant effects on the dynamics of this wildlife parasite; emphasizing the potential impacts of decisions regarding the management of migratory corridors and livestock-wildlife interface.

Pulmonary embolization of immature Fascioloides magna causing fatal hemothorax confirmed by molecular technique in a heifer in the United States.

The current report describes the use of a molecular technique to identify immature Fascioloides magna An 18-month-old Brangus heifer was found dead in the field without any prior clinical signs. The cause of death was exsanguination into the thoracic cavity associated with pulmonary embolization and infection by immature Fascioloides magna resulting in 2 large foci of pulmonary necrosis and focal arteriolar and lung rupture. The liver had a few random migratory tracts with typical iron and porphyrin fluke exhaust, but no identified fluke larvae. A single immature fluke was found in the lungs, and species level identification as F. magna was confirmed by DNA sequence analysis of the ribosomal internal transcribed spacer regions (ITS1 region, 5.8S rRNA gene, and ITS2) and of partial 28S rRNA gene sequence. This is one of only a few pulmonary fascioloidiasis cases associated with hemothorax in the veterinary literature.