The stimulation of resistance in sheep to Fasciola hepatica by infection with Cysticercus tenuicollis

Infection of sheep with Cysticerus tenuicollis for 12 weeks generated a high level of protection (> 95%) against intra-ruminal challenge with metacercariae of Fasciola hepatica as measured by recovery of flukes from liver and bile ducts and counts of fluke eggs in faeces. The animals were resistant to Fasciola whether challenge was superimposed upon the cestode infection or after removal of the cestode with mebendazole.Previous infection with C. tenuicollis also protected against the pathogenic effects of challenge infection with F. hepatica. Liver fibrosis was much less extensive in resistant sheep than controls and PCV's were not affected although these were reduced during fluke infection in the control animals.     

Characterization of sheep antibodies involved in precipitate formation with surface antigens of Fasciola hepatica in vitro

Sandeman R. M. and Howell M. J. 1982. Characterization of sheep antibodies involved in precipitate formation with surface antigens of Fasciola hepatica in vitro. International Journal for Parasitology12: 467–471. The role of sheep antibodies which precipitate with surface antigens of Fasciola hepatica is unclear. In an attempt to clarify their function these antibodies were characterized as to their immunoglobulin class and ability to affect the survival of fluke in rats. The ability of fluke antigens complexed with sheep antibody to vaccinate rats against infection was also tested. IgM antibodies were involved in precipitate formation on the teguments of fluke 3 weeks after infection but IgG₁ predominated at later stages of infection. The decreased survival of fluke in rats after culture with increasing levels of sheep antibodies suggests that the antibodies exert some deleterious effect on the fluke in vitro. The fluke antigen-sheep antibody complex failed to immunize rats against infection. Since sheep antibodies to F. hepatica can impair the ability of fluke to resist further attack in rats but not sheep, it is suggested that some effector mechanism other than antibody is defective in the latter.     

Isolation of Fasciola hepatica genus-specific antigens

Santiago de Weil N., Hillyer G. V. and Pacheco E. 1984. Isolation of Fasciola hepatica genus-specific antigens. International Journal for Parasitology14: 197–206. The Fasciola hepatica antigens which induce antibody formation in acute fascioliasis were isolated by acid elution after reacting an F. hepatica tegument antigen extract with a CNBr-Sepharose 4B column coupled with IgG obtained from the serum of rabbits infected with fascioliasis for 6–10 weeks. These isolated antigens were further separated by gel filtration using a column packed with Sephacryl S-200. In this manner three major peaks were obtained. The best serologic antigens were found in peak 2 which had a mol. wt range of 14,000–43,000. This peak contains genus-specific F. hepatica antigens which are highly reactive with fascioliasis serum. These antigens do not cross-react with either Schistosoma mansoni or with bovine serum albumin by gel diffusion. Monitoring by ELISA and gel diffusion with heterologous and homologous antisera showed that as purification by antibody affinity chromatography proceeded, cross reactivity with S. mansoni was eliminated. The rabbit antiserum obtained against peak 2, when tested by immunoelectrophoresis with a crude F. hepatica extract shows one main band identical to the main band observed with serum from acutely infected rabbits. Up to two other minor bands can be detected using concentrated homologous antisera. Fractions obtained from preparative iso-electric focusing of the F. hepatica tegument extract were reacted with sera from rabbits with acute fascioliasis. Two main bands were observed in immunodiffusion with antigens eluting in a pH range of 7.4–8.7. When these fractions were monitored with anti peak 2 antisera, two precipitin bands appeared with antigens eluting in a pH range of 7.4–7.9. The F. hepatica genus-specific antigen pool was applied to ELISA to evaluate its ability to detect antibody in a primary F. hepatica infection in rabbits. A rise in absorbance values could be detected by 2 weeks of infection, reached high levels by 6 weeks and remained high through 28 weeks of infection.     

Age-associated responses in susceptible and resistant rats to infection with Fasciola hepatica

Rajasekariah G. R. and Howell M. J. 1981. Age-associated responses in susceptible and resistant rats to infection with Fasciola hepatica. International Journal for Parasitology11: 59–65. Groups of susceptible (5-week-old) and age resistant (25-week-old) outbred male Wistar rats were infected with metacercariae of Fasciola hepatica and the establishment of the parasite was assessed in terms of worm reocvery, and haematological, histopathological and immunological criteria, 2, 4, 6 and 8 weeks after infection. Apart from 2 weeks after infection, there was a significant difference between groups in the recovery of F. hepatica, with resistant rats infected with consistently fewer parasites than susceptible animals. The juvenile worms which invaded the livers of resistant rats elicited a number of host reactions, marked by an intensive cellular infiltration into migratory tracks of the parasite, heavy deposition of fibrous tissue in the liver parenchyma and a rapid antibody response. These responses were not as striking in susceptible animals even though more worms were present. The ability of resistant rats to mount an enhanced response seems related to the maturation of their haemopoietic system.     

Stimulation of resistance to Taenia taeniaeformis in the rat by infection with Fasciola hepatica

Homologous resistance to F. hepatica and T. taeniaeformis and cross resistance between these two parasites was investigated in the rat. Rats given a primary infection with F. hepatica were challenged with either F. hepatica or T. taeniaeformis. Conversely rats given a primary infection with T. taeniaeformis were challenged with either F. hepatica or T. taeniaeformis.Infection with F. hepatica generated significant resistance against challenge with F. hepatica given 9 weeks later. Similarly, infection with T. taeniaeformis protected against challenge with T. taeniaeformis given 6 weeks later. Infection with F. hepatica also generated significant resistance against challenge with T. taeniaeformis given 4, 8 or 9 weeks later. Primary infection with T. taeniaeformis did not protect against challenge with F. hepatica.     

Schistosoma mansoni and Fasciola hepatica: Cross-resistance in mice

Cross-resistance in Schistosoma mansoni and Fasciola hepatica infections were studied in mice. A primary infection of S. mansoni, 7 to 28 days old, did not stimulate a significant level of resistance to heterologous challenge with F. hepatica. In contrast, in older S. mansoni infections (54–65 days old) there was a significant level of resistance to a challenge with F. hepatica. The F. hepatica worm burden was reduced by 34.0 to 72.5% in separate experiments. Challenge infection with F. hepatica did not influence the number of S. mansoni in primary infections. No heterologous resistance to S. mansoni was found in mice with 7- and 23-day-old F. hepatica infections. However, primary infections with F. hepatica, 28, 32, 42, and 50 days old, conferred significant resistance to a heterologous challenge with S. mansoni. The established schistosome worm burden was reduced by 41.5 to 50.4%. In no case was the primary F. hepatica burden reciprocally influenced by challenge infection with S. mansoni.     

Fasciola hepatica: Comparative studies on fascioliasis in rats and mice

Chapman C. B. and Mitchell G. F. 1982. Fasciola hepatica: comparative studies on fascioliasis in rats and mice. International Journal for Parasitology12: 81–91. Certain characteristics of infection differ between rats and mice exposed to metacercariae of the trematode parasite, Fasciola hepatica. Rats develop a degree of age-related resistance (and infected older females contain fewer parasites than older males), resistance to reinfection in infected rats is demonstrated readily though is partial, and a comparable degree of resistance can be obtained in recipients of infected rat serum provided the serum is given at about the time of challenge. None of these features of F. hepatica infection is seen in mice. Rats also differ from mice in that they can be vaccinated against infection (although again, resistance is incomplete) using larval antigen mixtures in adjuvants. Mice do respond to infection by production of antilarval antibodies and a slight IgG₁ hypergammaglobulinaemia and larvae will sensitize mice for delayed hypersensitivity. The results of this study indicate that sera from infected rats versus infected mice will be useful in pinpointing antigens of F. hepatica larvae which are involved in expression of partial host protection.     

Fasciola hepatica: An immunofluorescent study of antigenic changes in the tegument during development in the rat and the sheep

Serum from sheep was collected throughout a 30-week period of infection with Fasciola hepatica and specificity for the tissues of flukes of various ages was tested by an indirect fluorescent antibody labeling technique, using as antigen JB4 plastic-embedded sections of flukes up to 30-weeks old grown in rats. Quantitative estimates of host antibody concentration and fluke tissue antigenicity were determined by titration using serially diluted serum. Serum from early infections (pre-7 weeks) gave strong labeling over the tegument of young flukes, but the reaction became progressively weaker with older fluke tissue. This was associated with a decline in the number of T1 bodies in the tegument as revealed by electron microscopy. T1 bodies contain glycocalyx precursor substances and during development they replace the antigenically similar T0 secretory bodies characteristic of early juvenile flukes. Glycocalyx turnover may help protect the pre-bile duct flukes against immunological attack. Serum from sheep with F. hepatica infections older than 7 weeks gave moderate reaction with T2 bodies which accumulated in the tegument during the early stages of infection but only expressed their antigens on the surface about the time of entry into the host's bile ducts. The antigenicity of the gut and excretory system of flukes seemed to remain unchanged throughout adult life. Levels of host antibody specific for juvenile tegument, gut, and excretory system peaked at 3–5 weeks postinfection, and declined once the flukes entered the bile ducts. Anti-T2 antibody appeared 6 weeks postinfection and began to decline 5–6 weeks later.     

Schistosoma mansoni and Fasciola hepatica: Cross-resistance in mice with single-sex schistosome infections

Primary Schistosoma mansoni single-sex infections in mice, i.e., either male only or female only, did not stimulate any detectable level of heterologous resistance to challenge with Fasciola hepatica after 22 to 76 days, while statistically significant resistance to a challenge with F. hepatica was demonstrated in the presence of patent mixed-sex S. mansoni infections. Simultaneous infections with S. mansoni and F. hepatica induced a statistically significant reduction in the number of schisto-some worms established, i.e., the burden being reduced by 40.1 and 43.9%, respectively. There was no reduction of the F. hepatica worm burden. Similar features could be observed with a time interval of 48 hr between the S. mansoni infection and the F. hepatica challenge, i.e., the schistosome burden being reduced by 34.2 and 45.6%, respectively. Furthermore, simultaneous infections with S. mansoni and F. hepatica induced a statistically significant reduction of the egg production capacity per paired female schistosome worm as compared with that of the S. mansoni control group. Tissue egg counts of the various intestinal sections were reduced by 92.8–99.6%.     

An immunofluorescence reaction for Fasciola hepatica using the Defined Antigen Substrate Spheres (DASS) system

The Defined Antigen Substrate Spheres (DASS) system, using Fasciola hepatica antigens, proved to be a promising immunofluorescent antibody test for fascioliasis. The antigen bound beads could be freeze-dried and reconstituted to a spherical form for measurement.Sera of individuals with F. hepatica infections were examined with the Indirect Fluorescent Antibody (IFA) technique on frozen sections of the adult parasite and with the DASS system.Sera of experimentally F. hepatica infected rabbits were examined with the IFA technique, the DASS system, and the Soluble Antigen Fluorescent Antibody (SAFA) technique. A few bovine sera with F. hepatica infection were examined with the DASS system.     

Molecular characterization of Fasciola flukes using mitochondrial 28S rRNA gene in Naimi Saudi sheep

Fasciolosis is a parasitic disease of medical and economic importance. This retrospective study was conducted on 110 Fasciola flukes collected from livers of 14 infected Naimi sheep slaughtered at Riyadh abattoir in Saudi Arabia during winter season of 2016. Collected specimens were analyzed for their species identification on the basis of partial sequences of mitochondrial 28S rRNA gene. Results have shown the presence of both Fasciola hepatica (F. hepatica) and Fasciola gigantica (F. gigantica) species. Where Fasciola hepatica was predominate (80%). Both intra-species and interspecies genetic distance was studied and results showed that the intraspecific variability among individuals of both species i.e., F. hepatica and F. gigantica, ranging between 0 and 1% while the interspecific diversity between F. hepatica and F. gigantica was only 1%. In conclusion, mitochondrial 28S rRNA gene is a proved as a good marker in identifying Fasciola of different species. Where, the F. hepatica and F. gigantica are present in sheep breed in Riyadh region, Saudi Arabia.     

A Case of Fasciola hepatica Infection Mimicking Cholangiocarcinoma and ITS-1 Sequencing of the Worm

Fascioliasis is a zoonotic infection caused by Fasciola hepatica or Fasciola gigantica. We report an 87-year-old Korean male patient with postprandial abdominal pain and discomfort due to F. hepatica infection who was diagnosed and managed by endoscopic retrograde cholangiopancreatography (ERCP) with extraction of 2 worms. At his first visit to the hospital, a gallbladder stone was suspected. CT and magnetic retrograde cholangiopancreatography (MRCP) showed an intraductal mass in the common bile duct (CBD) without proximal duct dilatation. Based on radiological findings, the presumed diagnosis was intraductal cholangiocarcinoma. However, in ERCP which was performed for biliary decompression and tissue diagnosis, movable materials were detected in the CBD. Using a basket, 2 living leaf-like parasites were removed. The worms were morphologically compatible with F. hepatica. To rule out the possibility of the worms to be another morphologically close species, in particular F. gigantica, 1 specimen was processed for genetic analysis of its ITS-1 region. The results showed that the present worms were genetically identical (100%) with F. hepatica but different from F. gigantica.     

Fasciola hepatica in Snails Collected from Water-Dropwort Fields using PCR

Fasciola hepatica is a trematode that causes zoonosis mainly in cattle and sheep and occasionally in humans. Fascioliasis has been reported in Korea; however, determining F. hepatica infection in snails has not been done recently. Thus, using PCR, we evaluated the prevalence of F. hepatica infection in snails at 4 large water-dropwort fields. Among 349 examined snails, F. hepatica-specific internal transcribed space 1 (ITS-1) and/or ITS-2 markers were detected in 12 snails and confirmed using sequence analysis. Morphologically, 213 of 349 collected snails were dextral shelled, which is the same aperture as the lymnaeid snail, the vectorial host for F. hepatica. Among the 12 F. hepatica-infected snails, 6 were known first intermediate hosts in Korea (Lymnaea viridis and L. ollula) and the remaining 6 (Lymnaea sp.) were potentially a new first intermediate host in Korea. It has been shown that the overall prevalence of the snails contaminated with F. hepatica in water-dropwort fields was 3.4%; however, the prevalence varied among the fields. This is the first study to estimate the prevalence of F. hepatica infection using the vectorial capacity of the snails in Korea.     

Fasciola hepatica: role of developmental stages in the rat's resistance to challenge

Rats were sensitized by subcutaneous implantation of either metacercariae, 4 week-old juveniles, adult worms, or eggs of Fasciola hepatica and then challenged with 30 metacercariae 2 weeks later. Worm burdens were determined 8 weeks after challenge. Apart from adult worms, all implanted stages conferred a significant degree of protection on the recipients. The effectiveness of adult worm implants was not improved by using worms from different sources (sheep and cattle rather than rats) nor by extending the period of sensitization prior to challenge.     

FhCaBP3: A Fasciola hepatica calcium binding protein with EF-hand and dynein light chain domains

A DNA sequence encoding a protein with predicted EF-hand and dynein light chain binding domains was identified in a Fasciola hepatica EST library. Sequence analysis of the encoded protein revealed that the most similar known protein was the Fasciola gigantica protein FgCaBP3 and so this newly identified protein was named FhCaBP3. Molecular modelling of FhCaBP3 predicted a highly flexible N-terminal region, followed by a domain containing two EF-hand motifs the second of which is likely to be a functioning divalent ion binding site. The C-terminal domain of the protein contains a dynein light chain like region. Interestingly, molecular modelling predicts that calcium ion binding to the N-terminal domain destabilises the β-sheet structure of the C-terminal domain. FhCaBP3 can be expressed in, and purified from, Escherichia coli. The recombinant protein dimerises and the absence of calcium ions appeared to promote dimerisation. Native gel shift assays demonstrated that the protein bound to calcium and manganese ions, but not to magnesium, barium, zinc, strontium, nickel, copper or cadmium ions. FhCaBP3 interacted with the calmodulin antagonists trifluoperazine, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide and chlorpromazine as well as the myosin regulatory light chain-binding drug praziquantel. Despite sequence and structural similarities to other members of the same protein family from F. hepatica, FhCaBP3 has different biochemical properties to the other well characterised family members, FH22 and FhCaBP4. This suggests that each member of this trematode calcium-binding family has discrete functional roles within the organism.     

Genotyping of Taenia hydatigena isolated from sheep and goats in KSA based on Cox1 gene

Sheep and goats are among other herbivorous animals that serve as intermediate hosts (containing the larval stage Cysticercus tenuicollis) of Taenia hydatigena tapeworm. This infection can lead to serious complications or cause death. The genetic diversity and epidemiological significance of cysticercosis due to T. hydatigena is poorly understood. We examined 11,651 goats and 23,542 sheep slaughtered at the municipal abattoir in Makkah, for C. tenuicollis infection. The resulted DNA sequences were compared with previously available sequences from different hosts. Phylogenetic analysis and Pairwise nucleotide variations of cox1 gene were performed. Sheep and goats revealed infection rates of (4.95%) and (4.75%) respectively. DNA sequence analysis of all isolates from both sheep and goats showed that the total haplotypes number was 7. T. hydatigena population with high haplotypes diversity values. The nucleotide diversity was low, while Tajima’s D and Fu’s tests were negative (with no statistical significance). The present work will give valuable information regarding the prevalence and implementation of control and prevention measures of C. tenuicollis.     

Identification and differentiation of Fasciola hepatica and Fasciola gigantica using a simple PCR-restriction enzyme method

Accurate morphological differentiation between the liver fluke species Fasciola hepatica and Fasciola gigantica is difficult. We evaluated PCR-restriction enzyme profiles of internal transcribed spacer 1 (ITS1) that could aid in their identification. Fifty F. hepatica and 30 F. gigantica specimens were collected from different hosts in three provinces of Iran. For DNA extraction, we crushed fragments of the worms between two glass slides as a new method to break down the cells. DNA from the crushed materials was then extracted with a conventional phenol-chloroform method and with the newly developed technique, commercial FTA cards. A primer pair was selected to amplify a 463-bp region of the ITS1 sequence. After sequencing 14 samples and in silico analysis, cutting sites of all known enzymes were predicted and TasI was selected as the enzyme that yielded the most informative profile. Crushing produced enough DNA for PCR amplification with both the phenol-chloroform and commercial FTA card method. The DNA extracted from all samples was successfully amplified and yielded a single sharp band of the expected size. Digestion of PCR products with TasI allowed us to distinguish the two species. In all samples, molecular identification was consistent with morphological identification. Our PCR-restriction enzyme profile is a simple, rapid and reliable method for differentiating F. hepatica and F. gigantica, and can be used for diagnostic and epidemiological purposes.     

Fasciola hepatica: influence of extrahepatic adult flukes on infections and immunity in rats

Surgical transfer of adult Fasciola hepatica from sheep, goats, and cattle to subcutis of rats 4 wk before infection with metacercariae resulted in a 50% decrease in infection rate as compared to nonoperated controls.Infection was established in 25 out of 77 rats with F. hepatica implants, while 54 out of 79 were infected in the control group. The protective effect of the fluke implantation is discussed. It is suggested that production of protective antibodies is stimulated by the undamaged living flukes, although the antigen itself has not been demonstrated.     

Molecular characterization of Fasciola hepatica from Sardinia based on sequence analysis of genomic and mitochondrial gene markers

The aim of the present study is to investigate for the first time the genetic diversity of samples identified morphologically as Fasciola hepatica (Platyhelminthes: Trematoda: Digenea) (n = 66) from sheep and cattle from two localities of Sardinia and to compare them with available data from other localities by partial sequences of the first (ITS-1), the 5.8S, and second (ITS-2) Internal Transcribed Spacers (ITS) of nuclear ribosomal DNA (rDNA) genes, the mitochondrial cytochrome c oxidase subunit I (COI), and nicotinamide adenine dinucleotide dehydrogenase subunit I (ND1) genes. Comparison of the sequences from Sardinia with sequences of Fasciola spp. from GenBank confirmed that all samples belong to the species F. hepatica. The nucleotide sequencing of ITS rDNA showed no nucleotide variation in the ITS-1, 5.8S and ITS-2 rDNA sequences among all Sardinian samples, comparing with two ITS-2 haplotypes in standard F. hepatica, showing a substitution C/T in 20 position 859, reported previously from Tunisia, Algeria, Australia, Uruguay and Spain. The present study shows that in Sardinian sheep and cattle there is the most frequent haplotype (FhITS-H1) of F. hepatica species from South Europe. Considering NDI sequences, the phylogenetic trees showed reliable grouping among the haplotypes of F. hepatica from Sardinia and the mitochondrial lineage I, including the main N1 haplotype, observed previously from Europe (Russia, Belarus, Ukraine and Bulgaria), Armenia, West Africa (Nigeria), America (Uruguay and USA), Asia (Turkey, Japan, and China), Georgia, Turkmenistan, Azerbaijan and Australia. Furthermore, common haplotypes FhCOI-H1 and FhCOI-H2 of F. hepatica from Sardinia also corresponded mostly to the first lineage including the main C1 haplotype reported previously from Eastern European and Western Asian populations, they belonged just to a phylogenically distinguishable clade, as F. hepatica from Australia, France, Turkey, Uruguay, Russia, Armenia, Ukraine, Belarus, Turkmenistan, USA, Tunisia and Algeria, indicating that this is the main haplotype involved in the spread of F. hepatica throughout all continents.     

Genotypic characterization and species identification of Fasciola spp. with implications regarding the isolates infecting goats in Vietnam

Ribosomal RNA sequences (361 or 362 bp) of the second internal transcribed spacer 2 (ITS-2) and a portion of mitochondrial cox1 (423 bp) for Fasciola spp. obtained from specimens collected in indigenous and hybrid goats and sheep in Vietnam were characterized for genotypic status and hybridization/introgression. Alignment of 48 ITS-2 sequences (also those from goats and sheep in this study) indicates that F. gigantica and F. hepatica differ typically from each other at seven sites whereas one of these is a distinguishing deletion (T) at the 327th position in F. gigantica relative to F. hepatica. The isolates from the mountainous goats in the North of Vietnam (Yen Bai province) showed the ITS-2 composition relatively identical to that of F. hepatica. The ITS-2 sequences from populations of Fasciola isolates in goats had probably experienced introgression/hybridization as reported previously in other ruminants and humans. All Vietnamese goat-of-origin specimens had high pairwise percentage of mitochondrial cox1 sequences to F. gigantica (97-100%), and very low identity to F. hepatica (91-93%), suggesting their maternal linkage to be traced to F. gigantica. The presence of hybrid and/or introgressed populations of liver flukes bearing genetic material from both F. hepatica and F. gigantica in the goats/sheep in Vietnam, regardless of indigenous or imported hosts, appears to be the first demonstration from a tropical country.