Development and application of a fecal antigen diagnostic sandwich ELISA for estimating prevalence of Fasciola gigantica in cattle in central Java, Indonesia

The purpose of this study was to compare the sensitivity and specificity of an ELISA test to detect Fasciola gigantica antigens (coproantigens) in bovine feces, with fecal egg counting and an ELISA for detecting anti-F. gigantica antibodies in serum. Monoclonal antibodies to cathepsin L were generated and used to capture this antigen in feces of infected cattle. Blood, feces, and livers were collected from 150 cattle at an abattoir in Jakarta, Indonesia, for anti-Fasciola antibodies, coproantigen detection, and F. gigantica egg and worm counts. Fluke recovery varied from 1 to 426 per host, with a mean of 32 flukes. The results showed that the sensitivity and specificity of coproantigen detecting ELISA (95 and 91%, respectively) was better than the anti-F. gigantica antibody ELISA (91 and 88%, respectively) and to fecal egg counting (87 and 100%, respectively). The coproantigen ELISA was able to detect 100% of the cattle with >15 flukes. A survey of 305 cattle in central Java over a 10-mo period validated this test in the field, demonstrating a high prevalence of fascioliasis and establishing the test as a useful diagnostic method to determine patent F. gigantica infections in cattle.     

Prevalence of amphistomiasis and its association with Fasciola gigantica infections in Zambian cattle from communal grazing areas

To estimate prevalence of amphistomiasis and its association with bovine Fasciola gigantica infections in Zambia, 709 faeces of cattle presented for slaughter from three major cattle rearing areas of Central, Southern and Western provinces were analysed. The prevalence rate of amphistomes was 51.6%. Egg counts per gram (EPG) of faeces ranged from 0 to 385 with a mean (+/- SEM) of 11.96 +/- 1.07. The origin of the cattle had a significant influence (P < 0.001) on the prevalence rate. Fasciola gigantica infections accounted for 46.7% of the cattle examined. The mean EPG count (+/- SEM) was 6.3 +/- 0.66 with a range of 0 to 223. A total of 34.6% were mixed infections while single Fasciola and amphistome infections represented 12.1% and 17.1%, respectively. Significantly more cattle (63.8%) were infected with either single or both trematode infections (P < 0.0001) than not. Mixed trematode infections were highest in the Southern province (80.0%) while the lowest were recorded in Central province (16.3%). A positive correlation (r(2) = 0.0428) was present in mixed infections. In order to minimize losses, epidemiology and cross-resistance of amphistomiasis and fascioliasis should be studied in different ecological regions of Zambia to formulate efficient control programmes.     

The relationship between haematological indices, serum gamma-glutamyl transferase and glutamate dehydrogenase, visual hepatic damage and worm burden in cattle infected with Fasciola gigantica

The association between visual hepatic damage, burden of Fasciola gigantica, serum levels of gamma glutamyl transferase (GGT) and glutamate dehydrogenase (GLDH) is described from an abattoir study of 70 cattle in the Philippines. In another abattoir study of 60 cattle, the relationship between burden of F. gigantica and haematological indices was investigated. The degree of visual hepatic damage and burden of F. gigantica were significantly positively related to levels of GGT and GLDH. Red blood cell counts and packed cell volume were significantly inversely related to worm burden, but animals compensated for reduced numbers of red blood cells by increasing red cell haemoglobin content.     

Studies on heterologous resistance between Schistosoma bovis and Fasciola gigantica in Sudanese cattle

Using the local strains of Schistosoma bovis and Fasciola gigantica, it was shown that Sudanese zebu calves with mature primary infections of F. gigantica were highly resistant to challenge with S. bovis cercariae, and vice versa. Liver enzyme tests showed that, in both cases, the primary infections had caused some liver damage. Primary infection with irradiated S. bovis cercariae, which did not cause significant liver damage, did not protect significantly against challenge with F. gigantica.     

Molecular identification of Fasciola spp. (Digenea: Platyhelminthes) in cattle from Vietnam

Fasciola spp. were collected from naturally infected cattle at a local abattoir of Khanh Hoa province, Vietnam, for morphological and genetic investigations. Microscopic examination detected no sperm cells in the seminal vesicles, suggesting a parthenogenetic reproduction of the flukes. Analyses of sequences from the first and second internal transcribed spacers (ITS1 and ITS2) of the ribosomal RNA revealed that 13 out of 16 isolates were of Fasciola gigantica type, whereas three isolates presented a hybrid sequence from F. gigantica and Fasciola hepatica. Interestingly, all the mitochondrial sequences (partial COI and NDI) were of F. gigantica type, suggesting that the maternal lineage of the hybrid form is from F. gigantica. No intra-sequence variation was detected.     

Characterization of Fasciola samples by ITS of rDNA sequences revealed the existence of Fasciola hepatica and Fasciola gigantica in Yunnan Province, China

On mainland China, liver flukes of Fasciola spp. (Digenea: Fasciolidae) can cause serious acute and chronic morbidity in numerous species of mammals such as sheep, goats, cattle, and humans. The objective of the present study was to examine the taxonomic identity of Fasciola species in Yunnan province by sequences of the first and second internal transcribed spacers (ITS-1 and ITS-2) of nuclear ribosomal DNA (rDNA). The ITS rDNA was amplified from 10 samples representing Fasciola species in cattle from 2 geographical locations in Yunnan Province, by polymerase chain reaction (PCR), and the products were sequenced directly. The lengths of the ITS-1 and ITS-2 sequences were 422 and 361-362 base pairs, respectively, for all samples sequenced. Using ITS sequences, 2 Fasciola species were revealed, namely Fasciola hepatica and Fasciola gigantica. This is the first demonstration of F. gigantica in cattle in Yunnan Province, China using a molecular approach; our findings have implications for studying the population genetic characterization of the Chinese Fasciola species and for the prevention and control of Fasciola spp. in this province.     

Fasciola gigantica fatty acid binding protein (FABP) as a prophylactic agent against Schistosoma mansoni infection in CD1 mice

Although schistosomicidal drugs and other control measures exist, the advent of an efficacious vaccine remains the most potentially powerful means for controlling this disease. In this study, native fatty acid binding protein (FABP) from Fasciola gigantica was purified from the adult worm's crude extract by saturation with ammonium sulphate followed by separation on DEAE-Sephadex A-50 anion exchange chromatography and gel filtration using Sephacryl HR-100, respectively. CD1 mice were immunized with the purified, native F. gigantica FABP in Freund's adjuvant and challenged subcutaneously with 120 Schistosoma mansoni cercariae. Immunization of CD1 mice with F. gigantica FABP has induced heterologous protection against S. mansoni, evidenced by the significant reduction in mean worm burden (72.3%), liver and intestinal egg counts (81.3% and 80.8%, respectively), and hepatic granuloma counts (42%). Also, it elicited mixed IgG(1)/IgG(2b) immune responses with predominant IgG1 isotype, suggesting that native F. gigantica FABP is mediated by a mixed Th1/Th2 response. However, it failed to induce any significant differences in the oogram pattern or in the mean granuloma diameter. This indicated that native F. gigantica FABP could be a promising vaccine candidate against S. mansoni infection.     

Trematode infections in freshwater snails and cattle from the Kafue wetlands of Zambia during a period of highest cattle-water contact

A total of 984 snails, comprising nine species, were collected from six areas in the Kafue wetlands between August and October 2003 to assess larval trematode infections. Of these, 135 (13.7%) were positive. Most trematode infections were recorded from Lymnaea natalensis (42.8%), which harboured four of the five morphologically different cercariae found. No trematodes were recovered from Bellamya capillata, Biomphalaria pfeifferi, Melanoides tuberculata, Physa acuta and Cleopatra nswendweensis. One snail (0.2%) of 416 Bulinus snails shed brevifurcate-apharyngeate distome cercariae while three (0.7%) shed amphistomes. Gymnocephalous and longifurcate-pharyngeate distome were the commonest types of cercariae recorded while xiphidiocercaria was the least common. The highest prevalence rates of F. gigantica (68.8%) and amphistomes (50.0%) in cattle (n = 101) were in Chiyasa while those in Kaleya had the lowest (9.1 and 18.2%, respectively). In most habitats, infections were recorded in both cattle and snails. Critical determinants of infection may have been the distance of settlements and/or cattle kraals, the number of animals in nearby homesteads and the presence of susceptible host snails. This study suggests that fascioliasis and amphistomiasis could be major constraints of cattle production in the Kafue wetlands because favourable factors were available to introduce and maintain the infections. It further provides a starting point for some comprehensive studies on snail-related aspects of transmission and snail host ecology in Zambia.     

Identification of Fasciola flukes in Thailand based on their spermatogenesis and nuclear ribosomal DNA, and their intraspecific relationships based on mitochondrial DNA

We analyzed 147 Fasciola flukes obtained from cattle in Thailand based on their spermatogenetic ability, and nuclear ribosomal internal transcribed spacer 1 (ITS1) and mitochondrial nicotiamide adenine dinucleotide dehydrogenase subunit 1 (ND1) genes as molecular markers. One hundred twenty-eight flukes, which had abundant sperm in their seminal vesicles (spermic) and showed the PCR-RFLP pattern of F. gigantica in the ITS1, were accurately identified as F. gigantica. The other 19 flukes that had no sperm in their seminal vesicles were aspermic Fasciola sp. with the RFLP patterns identical to that of F. gigantica. Twenty-nine ND1 haplotypes (Fg-ND1-Thai 2-30) were distinguished in the 128 F. gigantica flukes and were divided into haplotypes unique to Thailand and those common to other countries, suggesting the possibility that ancestral haplotypes were introduced into Thailand. Three haplotypes (Fg-ND1-Thai 7, 9 and 27) appeared to be the major haplotypes found in F. gigantica from Thailand. Only one haplotype (Fg-ND1-Thai 1) was found in the 19 aspermic Fasciola sp. flukes obtained from geographical regions, and the nucleotide sequence of Fg-ND1-Thai 1 was identical to that of the aspermic Fasciola sp. from Japan, Korea, China, Vietnam and Myanmar, suggesting that they were descendants with a common provenance and expanded to these countries in the relatively recent past.     

An evaluation of the efficacy of monensin against Fasciola hepatica in the albino rat

The in vivo efficacy of monensin against Fasciola hepatica was determined in the albino rat. The results were variable, with monensin generally showing greater activity against juvenile (two-week-old) than adult (12-week-old) flukes. Significant (p less than 0.005) reductions in worm burdens were obtained only following treatment of adult flukes with 2 X 10 mg/kg monensin (52.9% efficacy), and of juvenile flukes with 1 X 10 mg/kg (42.4% efficacy) and 2 X 10 mg/kg (56.23% efficacy). Monensin administered in the diet (200 ppm) had a negligible effect on juvenile and adult fluke burdens. Prophylactic treatment of rats with monensin (100 ppm) produced a 45.5% efficacy, but this was not statistically significant. At doses of 1 X 5 mg/kg and 2 X 2.5 mg/kg, monensin had little effect on egg output by F. hepatica. No clear relationship was established between egg output and worm burden, and so faecal egg counting was not a reliable indicator of fluke burdens in the rat.     

Protective role of purified cysteine proteinases against Fasciola gigantica infection in experimental animals

Fascioliasis is one of the public health problems in the world. Cysteine proteinases (CP) released by Fasciola gigantica play a key role in parasite feeding, migration through host tissues, and in immune evasion. There has been some evidence from several parasite systems that proteinases might have potential as protective antigens against parasitic infections. Cysteine proteinases were purified and tested in vaccine trials of sheep infected with the liver fluke. Multiple doses (2 mg of CP in Freund's adjuvant followed by 3 booster doses 1 mg each at 4 week intervals) were injected intramuscularly into sheep 1 week prior to infect orally with 300 F. gigantica metacercariae. All the sheep were humanely slaughtered 12 weeks after the first immunization. Changes in the worm burden, ova count, and humoral and cellular responses were evaluated. Significant reduction was observed in the worm burden (56.9%), bile egg count (70.7%), and fecel egg count (75.2%). Immunization with CP was also found to be associated with increases of total IgG, IgG(1), and IgG(2) (P<0.05). Data showed that the serum cytokine levels of pro-inflammatory cytokines, IL-12, IFN-γ, and TNF-α, revealed significant decreases (P<0.05). However, the anti-inflammatory cytokine levels, IL-10, TGF-β, and IL-6, showed significant increases (P<0.05). In conclusion, it has been found that CP released by F. gigantica are highly important candidates for a vaccine antigen because of their role in the fluke biology and host-parasite relationships.     

Mitochondrial DNA polymorphism of a triploid form of Fasciola in Japan

Mitochondrial DNA polymorphism was characterized in a triploid form of Fasciola found in Japan in comparison with F. hepatica, F. gigantica and Korean Fasciola worm. Seventy worms of Fasciola from Japan, three of F. hepatica from Uruguay and Australia, two of F. gigantica from Thailand and one of Fasciola from Korea were used in the study. Mitochondrial DNA polymorphism was detected by restriction fragment length polymorphism (RFLP) using eight restriction enzymes, BamH I, Bgl II, Dra I, EcoR I, EcoR V, Hind III, Mfl I and Sca I. Three different types (types 1, 2 and 3) were detected from 76 Fasciola worms used in the study. Eight of 70 Japanese worms were categorized in type 2 (F. gigantica type), and the remaining 62 were in type 3 (F. hepatica type).     

Human fascioliasis and the presence of hybrid/introgressed forms of Fasciola hepatica and Fasciola gigantica in Vietnam

The two species common of liver fluke, Fasciola hepatica and Fasciola gigantica, cause human fascioliasis. Hybrids between these species, and introgressed forms of Fasciola, are known from temperate and subtropical regions of eastern Asia. Here, we report the presence of hybrid and/or introgressed liver flukes in Vietnam where it has recently been recognised that human fascioliasis is an important zoonotic disease. Specimens examined came from domestic stock (cattle and buffalo) at slaughter and also from human patients. DNA sequences were obtained from the nuclear ribosomal second internal transcribed spacer (ITS-2) and from portions of two mitochondrial protein-coding genes. Mitochondrial sequences in every case were similar to those of Fasciola gigantica. Nuclear ITS-2 sequences belonged to one or other of the Fasciola species, or, sequences from both were found in the same individual worm. This study extends the known range of hybrids or introgressed forms of Fasciola into tropical regions of Asia.     

The protective effect of irradiated metacercariae of Fasciola gigantica against homologous challenge in sheep

Five lambs were each sensitized with 400 metacercariae of Fasciola gigantica irradiated at 3 kr gamma rays. Eight weeks later, they were each challenged together with 5 controls with 500 non-irradiated metacercariae. Eight weeks later, all of the animals were necropsied and the worm burdens determined. The lambs which received the sensitizing infections had 80% less worms than the controls. The sensitized lambs also showed less hepatic damage compared with the controls as indicated by lower levels of the serum enzyme glutamate dehydrogenase. The blood indices showed insignificant reductions in the sensitized lambs whereas marked reductions were found in the erythrocytes, packed cell volume and haemoglobin values of the challenge controls. Bilirubinaemia was also evident in this latter group.     

Studies on the viability of metacercariae of Fasciola gigantica.

The viability of metacercariae of Fasciola gigantica was tested by in vitro and in vivo methods. In vitro testing was based upon the motility of juvenile flukes within the inner cyst as examined under the light microscope. In vivo testing was undertaken through experimental infections of rabbits (two groups) and natural definitive hosts, lambs (one group). In the first group, out of six rabbits each given 25 metacercariae, worm establishment only took place in one rabbit with a single fluke recovery on 60 days post infection. In the second group of six rabbits each given 200 metacercariae, five were infected, with two or three flukes per host. All the lambs given 250 metacercariae became infected showing prevalences of 7.2-40% in comparison with rabbits in which low prevalences (0-4%) were recorded. The results indicated that even viable metacercariae which were already tested in vitro could not readily establish in rabbits. Such variability in worm establishment suggests that immunological and chemotherapeutic studies in rabbits infected with F. gigantica are likely to be unreliable.     

Opisthorchis viverrini: identification of a glycine-tyrosine rich eggshell protein and its potential as a diagnostic tool for human opisthorchiasis

A cDNA encoding a novel eggshell protein (OvESP) with high-glycine (49.2%) and -tyrosine (27.8%) content was cloned from the human liver fluke Opisthorchis viverrini. In the adult parasite, the RNA products of the OvESP gene are limited to the vitelline follicles. They have a size of 800 nucleotides and are already present in 2-week-old juveniles. Immune sera of hamsters, experimentally infected, and humans, naturally infected with O. viverrini, detect bacterially expressed recombinant OvESP (rOvESP). A rabbit anti-rOvESP antiserum only reacts with the shells of intrauterine eggs in tissue sections of the parasite. Comparison of rOvESP with the parasite's excretion/secretion products as diagnostic tools for human opisthorchiasis shows a higher sensitivity (0.82-0.48) and specificity (0.97-0.91) of the recombinant protein in the ELISA technique. But the observed weak cross-reactivity of immune sera from mice infected with Schistosoma mansoni, Schistosoma mekongi, and Fasciola gigantica in Western blots of rOvESP indicates that the diagnostic quality of this protein might be compromised if infections by other trematodes are present.     

Population dynamics of the parasitic stages of Oesophagostomum dentatum in pigs in single and trickle infections

A mathematical model was developed to describe the dynamics of the parasitic stages of Oesophagostomum dentatam in pigs. An immigration-death model with constant establishment, development and death rates was fitted to L3, L4 and adult worm burdens observed in a single-infection experiment. Female worm length was modelled by a function of worm age and total worm burden, while worm egg production (eggs per gram faeces per female worm) was modelled by a function of worm age and worm length. The model was then used to predict worm burdens observed in a trickle-infection experiment. The predicted worm burdens were much higher than those observed, suggesting that worm death rates were higher during the trickle infection. After increusing worm death rates to fit the observed worm burdens, female worm lengths and egg production in the trickle infection were predicted. At the medium- and high-dose rates, predicted worm lengths and, thus, egg preduction were lower than observed, while at the low-dose rate predicted egg production was too high. It appeared that in the trickle infections, total worm burden had less influence on observed female worm length and egg production than in the single infections. The results suggest that the demography of O. dentatum in pigs differs between single and trickle infections.     

Early immunodiagnosis of fasciolosis in ruminants using recombinant Fasciola hepatica cathepsin L-like protease

A diagnostic ELISA with recombinant Fasciola hepatica cathepsin L-like protease as antigen was developed to detect antibodies against F. hepatica in sheep and cattle. The recombinant cathepsin L-like protease was generated by functional expression of the cDNA from adult stage F. hepatica flukes in Saccharomyces cerevisiae. Specificity and sensitivity of the cathepsin L enzyme-linked immunosorbent assay (ELISA) was assessed using sera from sheep and calves experimentally or naturally mono-infected with F. hepatica and six-seven other parasites. The sensitivity of the cathepsin L ELISA for sheep and cattle sera was 99.1 and 100%, respectively. In the experimental setting with established mono-infections, the specificity of the cathepsin L ELISA was 98.5% for cattle sera and 96.5% for sheep sera. In experimentally infected cattle and sheep, the first detection of F. hepatica-specific antibodies appeared first between 5 and 7 weeks post-infection, but depended on the infectious dose of F. hepatica. In ELISA the detection preceded first detection of the infection based on egg counts and remained detectable till at least 23 weeks after a primary F. hepatica infection. Detection of Fasciola gigantica infections was similar to detection of F. hepatica. The first detection occurred at week 5 and signals persisted for at least 20 weeks. All sera from naturally F. hepatica infected sheep were seropositive in the cathepsin L-like ELISA. The relevance of this ELISA format was also evaluated using sera from naturally infected cattle in the Netherlands, Ecuador and Vietnam and compared with results from egg-counts. For the latter two endemic areas with mixed parasitic infections the 'apparent' sensitivity of the cathepsin L ELISA was calculated for all serum samples together to be 90.2%. The 'apparent' specificity under these conditions was calculated to be 75.3%. In cattle, the cathepsin L ELISA was superior to the concurrently evaluated peptide ELISA format using a single epitope as the antigen both in controlled natural infections as well as in infections in endemic areas. The present ELISA-format contributes a relatively sensitive and reliable tool for the early serodiagnosis of bovine and ovine fasciolosis.     

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%.