Newly excysted juveniles (NEJs) of Fasciola gigantica induce mice liver fibrosis and M2 macrophage-like phenotype in vivo.

Liver flukes of animals are parasitic flatworms of major socioeconomic importance in many countries. Particularly, Fasciola gigantica is a leading cause of production losses to the livestock (mainly sheep and cattle) and meat industries due to clinical disease, reduced weight gain and milk production, and deaths. Immune responses induced by helminth have been extensively studied, but there is limited information on this aspect by F. gigantica, especially on macrophages induced with this parasite. Studies have shown that host immune responses induced by parasitic infection is greatly correlated with the macrophage polarization axis. In the present study, we used the murine model of F. gigantica to explore the interaction of host and F. gigantica. We found F. gigantica NEJs promoted pathology and fibrosis of mice liver, and the enlargement of mice spleen. We also showed that macrophages were recruited to mice peritoneal cavity at 5 days post infection. By evaluating the expression of genetic markers of M2 macrophages such as Arg-1, Ym1 and RELMɑ, and genetic marker of M1 macrophages iNOS, we showed that M2 macrophages were induced by F. gigantica. M2 macrophages are central to the immune response during helminth infection, and our findings in this study provided insight into the immune interaction between F. gigantica and host.

Complex and dynamic transcriptional changes allow the helminth Fasciola gigantica to adjust to its intermediate snail and definitive mammalian hosts.

BACKGROUND: The tropical liver fluke, Fasciola gigantica causes fasciolosis, an important disease of humans and livestock. We characterized dynamic transcriptional changes associated with the development of the parasite in its two hosts, the snail intermediate host and the mammalian definitive host. RESULTS: Differential gene transcription analysis revealed 7445 unigenes transcribed by all F. gigantica lifecycle stages, while the majority (n = 50,977) exhibited stage-specific expression. Miracidia that hatch from eggs are highly transcriptionally active, expressing a myriad of genes involved in pheromone activity and metallopeptidase activity, consistent with snail host finding and invasion. Clonal expansion of rediae within the snail correlates with increased expression of genes associated with transcription, translation and repair. All intra-snail stages (miracidia, rediae and cercariae) require abundant cathepsin L peptidases for migration and feeding and, as indicated by their annotation, express genes putatively involved in the manipulation of snail innate immune responses. Cercariae emerge from the snail, settle on vegetation and become encysted metacercariae that are infectious to mammals; these remain metabolically active, transcribing genes involved in regulation of metabolism, synthesis of nucleotides, pH and endopeptidase activity to assure their longevity and survival on pasture. Dramatic growth and development following infection of the mammalian host are associated with high gene transcription of cell motility pathways, and transport and catabolism pathways. The intra-mammalian stages temporally regulate key families of genes including the cathepsin L and B proteases and their trans-activating peptidases, the legumains, during intense feeding and migration through the intestine, liver and bile ducts. While 70% of the F. gigantica transcripts share homology with genes expressed by the temperate liver fluke Fasciola hepatica, gene expression profiles of the most abundantly expressed transcripts within the comparable lifecycle stages implies significant species-specific gene regulation. CONCLUSIONS: Transcriptional profiling of the F. gigantica lifecycle identified key metabolic, growth and developmental processes the parasite undergoes as it encounters vastly different environments within two very different hosts. Comparative analysis with F. hepatica provides insight into the similarities and differences of these parasites that diverged > 20 million years ago, crucial for the future development of novel control strategies against both species.

Molecular detection of natural infection of Lymnaea (Pseudosuccinea) columella (Gastropoda: Lymnaeidae) with Fasciola gigantica (Digenea: Fasciolidae) from two provinces of South Africa.

The main intermediate host of Fasciola gigantica in sub-Saharan Africa is Lymnaea (Radix) natalensis. Lymnaea (Pseudosuccinea) columella is capable of transmitting both F. gigantica and F. hepatica and has been reported to be present in South Africa. To date, no natural infection with F. gigantica has been reported despite the wide distribution of the snail. The aim of this study was to confirm whether L. (P.) columella was transmitting F. gigantica and/or F. hepatica in selected locations of KwaZulu-Natal and Eastern Cape provinces of South Africa. Lymnaea (Pseudosuccinea) columella snails were collected from two locations in two provinces of South Africa and screened for cercariae shedding. This was followed by humanely sacrificing the screened snails, and whole tissue of each individual snail was homogenized and amplified using primers designed to amplify the ITS-1 region of Fasciola spp. No cercariae were shed from the screened snails and molecular analysis showed that snails from the two locations were infected with F. gigantica. This study confirms natural infection of L. (P.) columella with F. gigantica in South Africa, where F. gigantica and F. hepatica have already been reported to coexist. Although L. (P.) columella is able to transmit the two species, surprisingly no infection with F. hepatica was detected from the screened snails. The natural intermediate host of F. gigantica in southern Africa, including South Africa, is Lymnaea (Radix) natalensis and comparative studies are needed to determine the competence of the two snail species in the transmission of F. gigantica.

Characterization of a Fasciola gigantica protein carrying two DM9 domains reveals cellular relocalization property.

Even at the present age of whole-organism analysis, e.g., genomics, transcriptomics, and proteomics, the biological roles of many proteins remain unresolved. Classified among the proteins of unknown function is a family of proteins harboring repeats of the DM9 domain, a 60-75 amino acids motif first described in a small number of Drosophila melanogaster proteins. Proteins may carry two or more DM9 domains either in combination with other domains or as their sole constituent. Here we have characterized a 16.8 kDa Fasciola gigantica protein comprising two tandem repeated DM9 domains (FgDM9-1). The protein was located in the parenchyma of the immature and mature parasite and consequently it was not detected in the ES product of the parasite but only in the whole worm extract. Interestingly, extraction with SDS yielded a substantially higher amount of the protein suggesting association with insoluble cell components. In Sf9 insect cells a heterologously expressed EGFP-FgDM9-1 chimera showed cell-wide distribution but relocated to vesicle-like structures in the cytoplasm after stimulating cellular stress by bacteria, heat shock or chloroquine. These structures did not colocalize with the markers of endocytosis/phagocytosis ubiquitin, RAB7, GABARAP. The same behavior was noted for Aedes aegypti PRS1, a homologous mosquito DM9 protein as a positive control while EGFP did not exhibit such relocation in the insect cells. Cross-linking experiments on soluble recombinant FgDM9-1 indicated that the protein can undergo specific oligomerization. It is speculated that proteins carrying the DM9 domain have a role in vesicular transport in flatworms and insects.

Comparative toxicity of Paraquat herbicide and some plant extracts in Lymnaea natalensis snails.

Paraquat has been shown to be a highly toxic compound for humans and animals, and many cases of acute poisoning and death have been reported over the past few decades. The present study was undertaken to evaluate comprehensively herbicides (Paraquat) and some plant extracts to biochemical aspects of Lymnaea natalensis snails. It was found that the exposure of L. natalensis to Paraquat and plant extracts led to a significant reduction in the infectivity of Fasciola gigantica miracidia to the snail. The glucose level in hemolymph of exposed snails was elevated, while the glycogen showed a decrease in soft tissues when compared with the control group. In addition, the activity level of some enzymes representing glycolytic enzymes as hexokinase (HK), pyruvate kinase (PK), phosphofructokinase (PFK), lactate dehydrogenase (LDH), and glucose phosphate isomerase (GPI) in snail's tissues were reduced in response to the treatment. It was concluded that the pollution of the aquatic environment by herbicide would adversely affect the metabolism of the L. natalensis snails. Snails treated with Agave attenuate, Ammi visnaga, and Canna iridiflora plant had less toxic effect compared to snails treated with Paraquat.

Fasciola gigantica cathepsin B5 is an acidic endo- and exopeptidase of the immature and mature parasite.

Cysteine proteases of the liver fluke Fasciola have been described as essential molecules in the infection process of the mammalian host. Destinct cathepsin Bs, which are already expressed in the metacercarial stage and released by the newly excysted juvenile are major actors in this process. Following infection their expression is stopped and the proteins will not be detectable any longer after the first month of development. On the contrary, the novel cathepsin B5 of Fasciola gigantica (FgCB5) described in this work was also found expressed in later juvenile stages and the mature worm. Like all previously described Fasciola family members it was located in the cecal epithelium of the parasite. Western blot analysis of adult antigen preparations detected procathepsin B5 in crude worm extract and in small amounts in the ES product. In support of these data, the sera of infected rabbits and mice were reactive with recombinant FgCB5 in Western blot and ELISA. Biochemical analysis of yeast-expressed FgCB5 revealed that it has properties of a lysosomal hydrolase optimized for activity at acid pH and that it is able to efficiently digest a broad spectrum of host proteins. Unlike previously characterized Fasciola family members FgCB5 carries a histidine doublet in the occluding loop equivalent to residues His110 and His111 of human mature cathepsin B and consequently showed substantial carboxydipeptidyl activity which depends on these two residues.

Juvenile-specific cathepsin proteases in Fasciola spp.: their characteristics and vaccine efficacies.

Fasciolosis, caused by Fasciola hepatica and Fasciola gigantica, is one of the most neglected tropical zoonotic diseases. One sustainable control strategy against these infections is the employment of vaccines that target proteins essential for parasites' invasion and nutrition acquiring processes. Cathepsin proteases are the most abundantly expressed proteins in Fasciola spp. that have been tested successfully as vaccines against fasciolosis in experimental as well as large animals because of their important roles in digestion of nutrients, invasion, and migration. Specifically, juvenile-specific cathepsin proteases are the more effective vaccines because they could block the invasion and migration of juvenile parasites whose immune evasion mechanism has not yet been fully developed. Moreover, because of high sequence similarity and identity of cathepsins from juveniles with those of adults, the vaccines can attack both the juvenile and adult stages. In this article, the characteristics and vaccine potentials of juvenile-specific cathepsins, i.e., cathepsins L and B, of Fasciola spp. were reviewed.

Isolation and characterization of Cu/Zn-superoxide dismutase in Fasciola gigantica.

A full-length complementary DNA (cDNA) encoding Cu/Zn-superoxide dismutase was isolated from Fasciola gigantica that on nucleotide sequencing showed a close homology (98.9%) with Cu/Zn-superoxide dismutase (SOD) of the temperate liver fluke, F. hepatica. Expression of the gene was found in all the three developmental stages of the parasite viz. adult, newly excysted juvenile and metacercaria at transcriptional level by reverse transcription-polymerase chain reaction (RT-PCR) and at the protein level by Western blotting. F. gigantica Cu/Zn-SOD cDNA was cloned and expressed in Escherichia coli. Enzyme activity of the recombinant protein was determined by nitroblue tetrazolium (NBT)-polyacrylamide gel electrophoresis (PAGE) and this activity was inactivated by hydrogen peroxide but not by sodium azide, indicating that the recombinant protein is Cu/Zn-SOD. The enzyme activity was relatively stable at a broad pH range of pH 4.0-10.0. Native Cu/Zn-superoxide dismutase protein was detected in the somatic extract and excretory-secretory products of the adult F. gigantica by Western blotting. NBT-PAGE showed a single Cu/Zn-SOD present in the somatic extract while three SODs are released ex vivo by the adult parasite. The recombinant superoxide dismutase did not react with the serum from buffaloes infected with F. gigantica. The role of this enzyme in defense by the parasite against the host reactive oxygen species is discussed.

Protection against Fasciola gigantica infection in mice by vaccination with recombinant juvenile-specific cathepsin L.

Fasciola gigantica cathepsin L1H (FgCatL1H) is one of the major cathepsin L released by juveniles of F. gigantica to aid in the invasion of host's tissues. Due to its high sequence similarity with other cathepsin L (CatL) isoforms of late stage F. gigantica, it was considered to be a good vaccine candidate that can block all CatL-mediated protease activities and affect juveniles as well as adult parasites. In this study, recombinant proFgCatL1H protein expressed in yeast, Pichia pastoris, system was mixed with Freund's adjuvants and used to subcutaneously immunize mice that were later challenged with metacercariae of F. gigantica. The percentage of worm protection in the rproFgCatL1H-vaccinated mice compared to the non-immunized and adjuvant control mice were approximately 62.7% and 66.1%, respectively. Anti-rproFgCatL1H antisera collected from vaccinated mice reacted specifically with rproFgCatL1H and other cathepsin L isoforms of F. gigantica, but the antibodies did not cross react with antigens from other trematode and nematode parasites, including Eurytrema pancreaticum, Opisthorchis viverrini, Fischoederius cobboldi, Cotylophoron cotylophorum, Gigantocotyle explanatum, Paramphistomum cervi, and Setaria labiato-papillosa. The levels of IgG1 and IgG2a in mouse sera increased significantly at two weeks after immunization and were highest during the sixth to eighth weeks after immunization. The IgG1 level was higher than IgG2a at all periods of immunization, implicating the dominance of the Th2 response. The levels of IgG1 and IgG2a in the immune sera were shown to be strongly correlated with the numbers of worm recovery, and the correlation coefficient was higher for IgG1. The levels of serum aspartate aminotransferase and alanine transaminase were significantly lower in the sera of rproFgCatL1H-vaccinated mice than in the infected control mice indicating a lower degree of liver damage. This study demonstrated a high potential of FgCatL1H vaccine, and its efficacy is currently being studied in the larger economic animals.

Protection against Fasciola gigantica using paramyosin antigen as a candidate for vaccine production.

Yet no vaccine to protect ruminants against liver fluke infection has been commercialized. In an attempt to develop a suitable vaccine against Fasciola gigantica (F. gigantica) infection in rabbits, using 97 kDa Pmy antigen. It was found that, the mean worm burdens and bile egg count after challenge were reduced significantly by 58.40 and 61.40%, respectively. On the other hand, immunization of rabbits with Pmy induced a significant expression of humoral antibodies (IgM, total IgG, IgG1, IgG2 and IgG4) and different cytokines (IL-6, IL-10, L-12 and TNF-alpha). Among Ig isotypes, IgG2 and IgG4 were most dominant Post-infection (PI) while, recording a low IgG1 level. The dominance of IgG2 and IgG4 suggested late T helper1 (Th1) involvement in rabbit's cellular response. While, the low IgG1 level suggested Th2 response to adult F. gigantica worm Pmy. Among all cytokines, IL-10 was the highest in rabbits immunized with Pmy PI suggesting also the enhancement of Th2 response. It was clear that the native F. gigantica Pmy is considered as a relevant candidate for vaccination against fascioliasis. Also, these data suggested the immunoprophylactic effect of the native F. gigantica Pmy which is mediated by a mixed Th1/Th2 response.

Analysis of thioredoxin peroxidase as a promising antigen for diagnosis of Fasciola gigantica infection: a preliminary study.

Buffalo fasciolosis induced by Fasciola gigantica causes important economic losses in tropical areas of Asia. Detection of prepatent infection is essential to control this disease. Classical tools such as coprology, necroscopy or ELISA based on crude extracts from F. gigantica are poorly sensitive or specific. Purified antigens could be used to increase these parameters. Western blot analysis and mass spectrometry of a fraction of F. gigantica excretory-secretory products obtained by gel filtration showed that thioredoxin peroxidase could be a potential antigen for serodiagnosis: it was recognized from the 2nd week after infection, by all buffalo experimentally or naturally infected with F. gigantica but not by healthy animals.

Comparative analysis of two fatty acid binding proteins from Fasciola gigantica.

Fatty acid binding proteins are considered to be promising vaccine candidates against trematodiasis. In order to provide additional information about their function in Fasciola gigantica we performed a comparative analysis of FgFABP1 and FgFABP3, two isoforms with quite different isoelectric points of 4.9 and 9.9 and 67% sequence identity. Both are expressed in the juvenile and adult parasite but differ in their tissue-specific distribution. In addition, the sequence of FABP3 is identical in F. hepatica and F. gigantica indicating the protein's functional importance in this genus. Immune sera produced against soluble recombinant FgFABPs reacted with 14 kDa antigens in crude worm, soluble egg, cirrus sac extracts, and excretion/secretion product. Both FgFABPs were located in the parenchyma of the parasite but in addition, FgFABP1 was abundant in testes and spermatozoa while FgFABP3 was abundant in vitelline cells, eggs, and caecal epithelium. Mass spectrometry identified FgFABP1 and FgFABP3 in the ES product whereas only FgFABP3 was identified in egg extract. Serum samples of an experimentally infected rabbit reacted from week 6 post-infection with FgFABP3 and from week 12 with FgFABP1 while sera of infected sheep were not reactive. The results suggest differences in the biological functions of these 2 isoforms and differences in the host/parasite interaction that should be considered for their potential as vaccines against fascioliasis.

Hydrographic network structure and population genetic differentiation in a vector of fasciolosis, Galba truncatula.

We report a preliminary analysis on the relationships between drainage basin structure and genetic structure of populations of the European vector of fasciolosis, Galba truncatula. In the study area, 251 snails belonging to 12 populations were collected along different ditches of a same river network. Each snail was genotyped at six variable microsatellite loci. Our results show that all sample sites are characterized by a low level of polymorphism and a very high and significant heterozygote deficiency. Our data reveal a significant genetic differentiation, even at a small scale, and failed to delimit clear patterns of isolation by euclidian distance. Our study shows that genetic differentiation significantly increases with hydrographic distance along the streams (p<0.002), in consistence with the hypothesis that dispersion along the stream is dependent on the direction of water flow. This study shows that relationships can exist between the organization of the hydrological network and population biology of a disease vector, which has strong potential applications to drainage network management issues.

Fasciola gigantica: immunolocalization of 28.5 kDa antigen in the tegument of metacercaria and juvenile fluke.

Specific monoclonal antibody (MoAb) to 28.5 kDa tegumental antigen (TA) was used to localize this antigen in the tissues of metacercariae, newly excysted juvenile (NEJ), 1, 3, 5, and 7-week-old juveniles of Fasciola gigantica by using indirect immunofluorescence, immunoperoxidase and immunogold techniques. Both indirect immunofluorescence and immunoperoxidase detections showed that this antigen was concentrated in the tegument particularly in its outer rim, tegumental cells and their processes as well as epithelial linings of the oral sucker. Unlike adult F. gigantica, it was not detected in spermatogenic cells in the testes, cells of Mehlis'gland, oocytes within the ovary, and ovum within the egg of parasites. At the ultrastructural level, the immunogold labeling showed deposit of gold particles specifically in G2 tegumental granules and on the surface membrane. Thus, this 28.5 kDa antigen is expressed in the tegument and associated structures of juvenile parasites, and it could be a major component of the G2 granules which are shown to fuse with the surface membrane and contribute material to replace the casted-off membrane. This process is the replenishment and turnover of the surface membrane to prevent the attachment of the host immune effector cells.

Efficacy of temperature, and two commonly used molluscicides and fertilizers on Fasciola gigantica eggs.

This study evaluated the effect of temperature, molluscicides (Copper sulphate and Niclosamide), fertilizers (Superphosphate and Ammonium sulphate) on the hatchability of Fasciola gigantica eggs. The results showed that hatchability decreased to 4% when eggs were incubated in bile secretion at 37 degrees C for 5 days and to 1.4% for 10 days, but few eggs incubated in water at 37 degrees C hatched. Bile secretion at 37 degrees C was a poor medium for in-vitro egg preservation. But, hatching occurred only when eggs were transferred to water at 26 degrees C. Temperature fluctuation from 26-4 degrees C or from 32-4 degrees C had an inhibitory effect on embryos development (35.2% & 32.3%, respectively) as compared to controls (60% & 63.9%, respectively). The incubation period (19 & 17 days) was higher than controls (14 & 12 days, respectively). The LC50 & LC90 of Copper sulphate and Niclosamide against Biomphalaria alexandrina and Lymnaea natalenesis had no toxic effect on Fasciola eggs. The higher concentrations of Copper sulphate (30 ppm) and Niclosamide (1 ppm) slightly lower eggs hatchability rate than controls. The rate decreased by increasing the exposure time from 3 to 6 hours with both molluscicides. Ammonium sulphate had a lethal effect on eggs, but Superphosphate had some inhibitory effect on egg development, which increased by increasing Superphosphate concentration or with the prolongation of the exposure time.

Fasciola gigantica: evaluation of the effect of phenyl vinyl sulfone in vitro.

Phenyl vinyl sulfone is a synthetic inhibitor of cysteine protease and has antihelminthic and antiprotozoal properties. Phenyl vinyl sulfone was assayed in vitro for antifasciola activity against adult Fasciola gigantica worms using a well-established culture medium. Worms were treated with phenyl vinyl sulfone for incubation periods ranging from 0 to 12h and its activity was assessed in terms of viability, motility and death of worms. Phenyl vinyl sulfone exhibited a minimum effective concentration of 50 ppm after 12h. Three hundred parts per million concentrations were most potent causing immediate death of adult flukes in vitro. Histopathological studies showed that there was tegumental flattening, rupture of vesicles, and spine loss. Marked reduction in size and number of ova and sperms in the convoluted tubules of the reproductive organs was observed in comparison to the untreated control group. In conclusion, phenyl vinyl sulfone shows potent activity against F. gigantica in vitro, and the authors recommend carrying out more studies to detect its efficacy in vivo.

Efficacy of five chemicals on Fasciola gigantica encysted metacercariae infectivity.

The efficacy of treating encysted metacercariae (EMC) of Fasciola gigantica with different concentrations (conc.) of acetic acid, citric acid, cetrimide, potassium permanganate and sodium hydroxide, for 15 & 30 minutes was evaluated. The efficacy of these chemicals on the vitality and infectivity of the EMC was evaluated by the development of fascioliasis infection, and the histopathological changes in the livers of experimentally infected Albino rabbits. The results showed that 1% sodium hydroxide had a lethal effect on EMC, 10% to 40% potassium permanganate destroyed the infectivity power of EMC, and acetic acid gave an adverse effect on the EMC in conc. more than 2.5%. But, neither citric acid nor cetrimide affected the vitality or infectivity of EMC and all rabbits acquired fascioliasis.

Host responses during experimental infection with Fasciola gigantica or Fasciola hepatica in Merino sheep I. Comparative immunological and plasma biochemical changes during early infection.

This study reports the early biochemical changes in plasma, comparative host-immune responses and parasite recovery data in Merino sheep during the first 10 weeks of infection with Fasciola gigantica and Fasciola hepatica. One group of sheep were uninfected, four groups of sheep received incremental challenge doses of F. gigantica metacercariae (50, 125, 225 and 400, respectively) and the sixth group was challenged with 250 F. hepatica metacercariae. At 10 weeks post infection (wpi), sheep challenged with F. hepatica showed the greatest fluke recovery (mean 119, range 84-166); a significantly higher biomass of parasites recovered (2.5-fold greater than the highest dose of F. gigantica); and a greater mean % parasite recovery (39.3%, range 27-55%) than any group challenged with F. gigantica. Within the groups dosed with F. gigantica a strong dose-dependent response was observed in both fluke recovery and fluke biomass with increasing dose of metacercariae. The mean % parasite recovery of F. gigantica infected groups 1-5 were 26, 23, 26 and 25%, respectively, suggesting a uniform viability of parasite establishment independent of infection dose. At 6 wpi, elevated levels of plasma GLDH were observed in the F. gigantica infected groups compared to the uninfected sheep (p<0.005) whereas the F. hepatica challenged group had four-fold higher levels of GLDH compared to the F. gigantica infected group (p<0.001). Elevated levels of GGT as an indicator of epithelial damage in the bile duct was only seen in the group challenged with F. hepatica at 10 wpi when it rose from below 100 IU/l to approximately 250 IU/l (p<0.0001) whereas no detectable increase in GGT was observed in any of the groups challenged with F. gigantica. The white blood cell response to F. hepatica infection was biphasic with the initial peak at 4 wpi and a second peak at 9 wpi, corresponding to the period of migration of juvenile fluke in the liver and the time when adult flukes are migrating into the bile duct, respectively. This biphasic response was also evident in the changes in the eosinophil counts and serum haemoglobin levels. There was a trend toward higher parasite-specific IgG2 titres in sheep infected with lower worm burdens, suggesting that higher F. gigantica or F. hepatica burdens suppress IgG2 responses. The findings of this study suggest that, in early infection in a permissive host, F. hepatica appears to be more pathogenic than F. gigantica because of its rapid increase in size and the speed of its progression through the migratory phases of its life cycle.

Seasonal pattern of bovine amphistomosis in traditionally reared cattle in the Kafue and Zambezi catchment areas of Zambia.

Seasonality of bovine amphistomosis in the Southern province of Zambia was established after examining 268 faecal samples from cattle presented for slaughter at Turnpike slaughter slab, Mazabuka. Amphistomosis was found present throughout the year but the highest abundance rate was found during the post-rainy season (47.8%) and the lowest during the cold dry season (24.8%). In the rainy and post-rainy seasons, higher mean egg counts and cattle found positive were recorded than in any other season. The distribution of amphistome eggs was significantly different (p < 0.001) among the four seasons, with the rainy season having higher median egg counts than others. There were no significant differences in abundance rates between sexes or between ages of cattle. A similar seasonality to that of fasciolosis exists and may help in strategic management of Fasciola and amphistomes.

The effect of temperature and humidity on longevity of metacercariae of Fasciola gigantica.

Studies were undertaken on the viability of the metacercariae of Fasciola gigantica when stored in water at 13 degrees C for periods up to 23 weeks, exposed to the sunlight for up to 8 h or stored at a range of temperatures and humidities for up to 10 weeks. Excysted metacercariae were catergorized microscopically as viable (motile and undamaged), dubious (not motile and undamaged) or dead (visible necrosis). The infectivity of viable and dubious metacercariae and unselected reference metacercariae held in water at 7 degrees C for 20 days or longer was assessed by comparing numbers of flukes recovered from infected Merino sheep. Mean recovery rates were 54.6%, 7.2% and 37.2%, respectively, for viable, dubious and unselected metacercariae. Metacercariae immersed in water remained viable longer than those allowed to desiccate. Viability was promoted by decreasing temperature and increasing humidity. Exposure to direct sunlight killed metacercariae within 8 h. Results indicated that in lowland Indonesian irrigated rice paddies, metacercariae immersed in water are likely to survive for less than 5 weeks while those that become desiccated will survive less than 2 weeks. This information, together with the option of exposing fresh rice stalks to direct sunlight before feeding them to livestock, can assist farmers in reducing infection with F gigantica.