Exploring and Expanding the Fatty-Acid-Binding Protein Superfamily in Fasciola Species.

The liver flukes Fasciola hepatica and F. gigantica infect livestock worldwide and threaten food security with climate change and problematic control measures spreading disease. Fascioliasis is also a foodborne disease with up to 17 million humans infected. In the absence of vaccines, treatment depends on triclabendazole (TCBZ), and overuse has led to widespread resistance, compromising future TCBZ control. Reductionist biology from many laboratories has predicted new therapeutic targets. To this end, the fatty-acid-binding protein (FABP) superfamily has proposed multifunctional roles, including functions intersecting vaccine and drug therapy, such as immune modulation and anthelmintic sequestration. Research is hindered by a lack of understanding of the full FABP superfamily complement. Although discovery studies predicted FABPs as promising vaccine candidates, it is unclear if uncharacterized FABPs are more relevant for vaccine formulations. We have coupled genome, transcriptome, and EST data mining with proteomics and phylogenetics to reveal a liver fluke FABP superfamily of seven clades: previously identified clades I-III and newly identified clades IV-VII. All new clade FABPs were analyzed using bioinformatics and cloned from both liver flukes. The extended FABP data set will provide new study tools to research the role of FABPs in parasite biology and as therapy targets.

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.

Multi level ecological fitting: indirect life cycles are not a barrier to host switching and invasion.

Many invasive species are able to escape from coevolved enemies and thus enjoy a competitive advantage over native species. However, during the invasion phase, non-native species must overcome many ecological and/or physiological hurdles before they become established and spread in their new habitats. This may explain why most introduced species either fail to establish or remain as rare interstitials in their new ranges. Studies focusing on invasive species have been based on plants or animals where establishment requires the possession of preadapted traits from their native ranges that enables them to establish and spread in their new habitats. The possession of preadapted traits that facilitate the exploitation of novel resources or to colonize novel habitats is known as 'ecological fitting'. Some species have evolved traits and life histories that reflect highly intimate associations with very specific types of habitats or niches. For these species, their phenological windows are narrow, and thus the ability to colonize non-native habitats requires that a number of conditions need to be met in accordance with their more specialized life histories. Some of the strongest examples of more complex ecological fitting involve invasive parasites that require different animal hosts to complete their life cycles. For instance, the giant liver fluke, Fascioloides magna, is a major parasite of several species of ungulates in North America. The species exhibits a life cycle whereby newly hatched larvae must find suitable intermediate hosts (freshwater snails) and mature larvae, definitive hosts (ungulates). Intermediate and definitive host ranges of F. magna in its native range are low in number, yet this parasite has been successfully introduced into Europe where it has become a parasite of native European snails and deer. We discuss how the ability of these parasites to overcome multiple ecophysiological barriers represents an excellent example of 'multiple-level ecological fitting'.

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.

Prognostic significance of microsatellite alterations at 1p36 in cholangiocarcinoma.

AIM: To investigate loss of heterozygosity (LOH) and microsatellite instability (MSI) on the chromosomal region 1p36-pter in cholangiocarcinoma (CCA) patients and determine the association between microsatellite alterations and clinicopathological parameters. METHODS: Ten polymorphic microsatellite markers were determined for LOH and MSI using GS-3000 gel scan fragment autoanalyzer. RESULTS: Sixty-eight out of 90 cases (75.6%) showed LOH in one or more loci. LOH was found most frequently at D1S199 (40.0%), D1S507 (34.6%), D1S2845 (30.5%), and D1S2734 (30.1%). MSI was found in 34 of 90 cases (37.8%) at one or more loci. Fine mapping at 1p36 showed two distinctive regions of common loss, which were D1S2845 and the 25.5-cM region between D1S507 and D1S2734, indicating the existence of putative tumor suppressor genes that is likely to play important roles in the development of CCA. Patients with LOH at D1S234 showed less lymphatic invasion (P = 0.017), whereas patients with LOH at D1S2676 exhibited more lymphatic invasion than those without (P = 0.031). LOH at D1S2845 showed a significant correlation with nerve invasion (P = 0.029). Moreover, patients who demonstrated MSI at D1S228 showed a poor prognosis (P = 0.0026). CONCLUSION: Allelic loss plays a major role in microsatellite alterations at chromosome 1p36, which may contribute to carcinogenesis and pathogenesis of liver fluke related CCA and these alterations can be used as molecular prognostic indicators for CCA patients.

Natural infection of a horse with Fascioloides magna.

A 25-year-old Quarterhorse mare was euthanized for a variety of medical reasons. At necropsy, 7 liver flukes, identified as Fascioloides magna, were recovered from the liver. This is the first report of F. magna in a horse.

[Fascioliasis case in the patient with hepatitis A].

There is some portion of patients with clinically manifested acute viral hepatitis, which are seronegative to hepatitis A markers. They have to be differentiated with other patients with B, C, D hepatitis, mechanical jaundice, etc. Such clinical cases make physician to recall the parasitic diseases, such as fascioliasis, which affects hepatobiliary system, causes prolongation of cholestasis and dystrophic changes in the biliary tract and likely to cause liver cirrhosis. In the presented case the initial diagnosis was severe acute Hepatitis A (anti-HAV IgM+), though the peripheral blood examination showed moderate eosinophilia, ultrasound investigation revealed multiple sites of damage in the liver, which made us to consider fascioliasis, the latter was confirmed by the serological analysis. Appropriate medical treatment was effective and the state of the patient has improved.

Hiperendemicidad de fasciolosis humana en el Valle del Mantaro, Perú: Factores de riesgo de la infección por Fasciola Hepática / Hyperendemicity of human fasciolosis in the Mantaro Valley, Peru: Factors for infection with fasciole hepatica

Una muestra de 206 niños (entre 1 y 16 años de edad) en los distritos de Huertas (n=110) y Julcan (n=96) pertenecientes al Valle de Mantaro, Perú, fueron estudiados mediante una entrevista clínico-epidemiológica, pruebas serológicas y exámenes parasitológicos de heces para describir la prevalencia y analizar los factores de riesgo para la infección por Fasciola hepática (Fh). La prevalencia de Fh por exámenes de heces en Huertas (H) fue 28.3 por ciento y Julcan (J), 12.6 por ciento; empleando pruebas serológicas, las prevalencias fueron: para H, 36.3 por ciento y para J, 22.7 por ciento. El análisis univariado demostró una asociación significativa entre la infección por Fh y 3 variables en H: vivir cerca de acequias (OR=4.8; P menor 0.05) o cultivos (OR=2.88; P menor 0.05) y el hábito de beber emolientes (OR=2.92; P menor 0.05); y otras 3 variables en J: tener una habitación por casa (OR=21.0; P menor 0.05); y otras 3 variables en J: tener una habitación por casa (OR=21.0; P menor 0.05), eliminar las heces a campo abierto (OR=4.77; P menor 0.05), historia de ingesta de medicamentos antiparasitarios en los últimos 6 meses (OR=4.61; P menor 0.05). El análisis multivariado, aplicado a todos los datos reveló como posibles factores de riesgo para la infección por Fh: el hábito de beber emlientes (OR=5.16; P menor 0.05) y vivir ceca de acequias (OR=17.22; P menor 0.05) o cultivos (OR=8.13; P menor 0.05). De acuerdo a estos resultados uno de los más importantes y poco reconocido factor de riesgo es el hábito de beber emolientes. Debido a la importancia clínica de las fasciolasis humana, concluimos que su presencia en el Valle de Mantaro es un importante probelma de slaud pública, un diagnóstico precoz efectivo permitiría un tratamiento oportuno y evitaría el serio daño clínico de la infección crónica.

An experimental study on triclabendazole resistance of Fasciola hepatica in sheep.

The efficacy of triclabendazole in sheep experimentally infected with Fasciola hepatica was studied. Two groups of 12 lambs were infected with a susceptible (S) or a resistant (R) strain of F. hepatica. Eight weeks after infection, six lambs of each group (ST and RT) were treated with triclabendazole (10mg/kg). The other lambs were used as untreated controls (SC and RC). The parameters studied were: GLDH, gamma-GT, ELISA measuring antibodies against recombinant cathepsin-L(1) and eggs per gram faeces (epg). The lambs were slaughtered 16 weeks after infection and the number of flukes counted. The GLDH, gamma-GT levels and the OD value of the ELISA decreased as a result of the treatment in group ST. Patent infections were observed in all animals of groups SC, RT and RC. In group ST, occasionally a few eggs were found in five lambs. The percentage of flukes was 31.3 in SC and 37.6 in RC. In the treated groups ST and RT, the percentage of flukes was 0.06 and 33.6, respectively. These results corresponded to efficacies of 99.8% in the susceptible and 10.8% in the resistant strain. Since the resistant strain was isolated from a mixed cattle and sheep farm, it confirms the presence of triclabendazole resistance in the Netherlands.

Fascioloidiasis in game-ranched elk from Montana.

The distribution of Fascioloides magna in game-ranched elk and the potential for spread of the parasite through movement of infected animals was examined in Montana (USA). Fecal samples (n = 448) collected from captive elk on 29 game ranches were examined for eggs of F. magna by fecal sedimentation. Eggs were detected in elk on 5 ranches. This suggests that F. magna has been translocated by infected game-ranched elk. The wide distribution of snail intermediate hosts for F. magna in Montana indicates a potential to spread the parasite to other captive cervids domestic livestock or free-ranging wildlife.

[Bionomy, occurrence and importance of the giant liver fluke (Fascioloides magna), an important parasite of free-living ruminants]. / Bionómia, výskyt a význam cicavice obrovskej (Fascioloides magna), závazného parazita vol'ne zijúcich prezúvavcov.

The paper summarizes the most important knowledge of the giant liver fluke of ruminants, Fascioloides magna, with respect to its occurrence throughout the Northern Hemisphere, its fenotypic and genetic discriminative characteristics, its pathogenicity in various obligatory and facultative hosts and possible prophylaxis and medical control. The paper emphasizes the fact that new natural foci with heavy F. magna infections of deer regularly occur in Europe.

Use of bait containing triclabendazole to treat Fascioloides magna infections in free ranging white-tailed deer.

Triclabendazole-medicated corn bait was given to white-tailed deer (Odocoileus virginianus) on the Welder Wildlife Refuge, Sinton, Texas (USA), at a dose of 11 mg/kg body weight per deer per day for seven days, for control of Fascioloides magna. Medicated bait was offered for one week each during the winters of 1987, 1988, and 1989. Deer collected from treated areas, from baited control and from unbaited control areas were examined before the start of the study in 1987, and four weeks after the end of the baiting period in each of three years. Prior to the study, prevalence of fluke infection was 68%. After treatment with triclabendazole medicated corn, 13 (56%) of 23 deer collected were infected with flukes of which 15% had live parasites; this was evidence for therapeutic treatment. Of the deer collected in the baited and unbaited control areas, 63% and 80%, respectively, were infected only with live flukes. Prevalence of live flukes in deer was significantly (P < 0.05) lower in the treatment pasture than in the baited or unbaited control areas in each of the three years. Efficacy of the baiting system over the three years was 63% when comparing the treatment area and the baited control area.

Efficacy of two formulations of albendazole against liver flukes in cattle.

Albendazole (10 mg/kg of body weight) was administered as a drench suspension or as a feed additive to 24 cattle with naturally acquired infections of Fasciola hepatica and Fascioloides magna. Cattle were euthanatized 16 to 30 days after treatment, and the number of viable flukes was counted. Viable F hepatica and F magna were decreased by 91.4% and 70.6% for drench administration and by 82.9% and 71.9% for the feed additive treatment, respectively. There was no significant difference between the efficacy of the 2 formulations in decreasing viable fluke numbers, compared with untreated controls.

Epizootiology of fascioliasis in Montana.

During 1989-1990, the United States Department of Agriculture (USDA) meat inspection records were used to determine the distribution and incidence of liver flukes (Fasciola hepatica and Fascioloides magna) in Montana cattle. Of the cows and bulls slaughtered in USDA-inspected packing plants during a 12 month time period, 17.24% had livers that were condemned because of liver flukes. This was a 12% increase over USDA liver condemnations reported for 1973. Infected animals have been reported from 26 counties in Montana, mostly located in the south-central and western half of the state. Forty-nine percent of the 2.4 million cattle in Montana are raised in these counties. Lymnaeid snail species that may serve as intermediate hosts for Fasciola hepatica were found in most of the counties where liver flukes were reported. The principal vectors believed to be responsible for the transmission of Fasciola hepatica in Montana are species of the genus Fossaria. Stagnicola montanensis and Lymnaea stagnalis, which may serve as intermediate hosts for this parasite have also been collected. A known intermediate host for Fascioloides magna, Stagnicola caperata, was also found in several locations.

Experimental Fascioloides magna infections of mule deer (Odocoileus hemionus hemionus).

Six mule deer (Odocoileus hemionus hemionus) and one white-tailed deer (Odocoileus virginianus), approximately 5-mo-old, each were inoculated orally with 500 metacercariae of Fascioloides magna. All mule deer died from liver fluke infection between 69 and 134 days (mean = 114, SE = 9.9) after inoculation. Between 38 and 326 immature F. magna (mean = 102, SE = 45.5) were recovered from each deer at necropsy. Flukes were present in livers, lungs, and free in pleural and peritoneal spaces. Infection was characterized by necrotizing hepatitis, fibrosing peritonitis and pleuritis, and hematin pigment accumulation in liver, lung, and many other internal organs. Eggs of F. magna first were detected in feces of the white-tailed deer 28 wk after inoculation, and weekly thereafter until the healthy deer was euthanized at 31 wk. At necropsy, 205 F. magna, including 12 encapsulated mature and 193 nonencapsulated immature flukes were recovered from liver, lungs, and free in abdominal and thoracic spaces of the white-tailed deer. Based on these results, F. magna may be fatal to mule deer within 5 mo of infection. Like domestic sheep and goats, mule deer may be highly susceptible to infection, and it is unlikely mule deer can survive infection with large numbers of F. magna.

Efficacy of triclabendazole against natural infections of Fascioloides magna in wapiti.

Fourteen free-ranging adult wapiti (Cervus elaphus nelsoni) were captured in Banff National Park, Alberta (Canada) and held in captivity near Edmonton. A 24% suspension of triclabendazole at doses of 30 to 100 mg/kg body weight was drenched into the rumen of eight females and four males. Two male wapiti were used as untreated controls. Animals were killed and examined at 4 (n = 3), 6 (n = 4), or 8 (n = 4) wk after treatment. Efficacy was 90% against immature Fascioloides magna collected 4 wk after treatment and 98% against adult flukes collected 4, 6 or 8 wk after treatment. All 32 flukes recovered from control wapiti were active and apparently healthy. Treatment at 50 to 60 mg/kg is recommended against F. magna in wapiti. A protocol for treating infected wapiti is outlined.

Hepatic and pulmonary pathology of experimental Fascioloides magna infection in guinea pigs.

The guinea pig was used to study the pathology of Fascioloides magna, an important pathogen for sheep. Although flukes migrated freely through various tissues in infected guinea pigs, the most serious lesions occurred in the liver and lungs. The sequential development of lesions indicated that flukes first invaded the quadrate lobe of the liver and subsequently migrated to other liver lobes and tissues. Six weeks post-infection, there was a marked drop in the recovery of flukes from the liver along with a dramatic increase in pulmonary involvement. Much of the hepatic and pulmonary pathology in infected animals was secondary to extensive vascular lesions caused by migrating flukes. In the liver, vascular lesions predominantly involved the portal and hepatic veins. Thrombophlebitis and locally extensive necrosis, resembling infarction, were observed. Vascular lesions in the lungs occurred in the pulmonary arteries leading to thrombosis and haemorrhagic infarction. Discovery of a fluke in a pulmonary artery, along with the pattern of hepatic and pulmonary lesions, suggested that flukes probably used the cardiovascular system as a pathway for dissemination. Death in fluke-infested guinea pigs was most often associated with severe pulmonary lesions. The nature and distribution of fluke-induced lesions observed in this study demonstrate that the guinea pig is a suitable animal model for Fascioloides magna infection in sheep.

Survey of hepatic and pulmonary helminths of wild cervids in Alberta, Canada.

During the 1988 hunting season, livers and lungs from 263 mule deer (Odocoileus hemionus hemionus), 198 moose (Alces alces), 147 white-tailed deer (Odocoileus virginianus), and 94 wapiti (Cervus elaphus nelsoni) from Alberta (Canada) were collected for parasitological examination. Most of the samples (89%) were submitted by big game hunters throughout the province. Giant liver fluke (Fascioloides magna) was found in 9% of 22 yearling and 29% of 65 adult wapiti; 4% of 161 adult moose; and 2% of 97 adult white-tailed deer. The intensity of infection generally was low; however, one wapiti had over 600 flukes in the liver. Infections were restricted to alpine and montane regions in southwestern Alberta (97%) as well as boreal uplands of the Cypress Hills in southeastern Alberta (3%). Other parasites recorded included Taenia hydatigena cysts in liver of 61% of 191 moose and 14% of 247 mule deer. Dictyocaulus viviparus was found in lungs of 14% of 50 moose, 14% of 118 mule deer, 12% of 41 wapiti, and 6% of 54 white-tailed deer. Echinococcus granulosus cysts were found in lungs (and occasionally liver) of 37% of 51 moose. Incidental infections of Thysanosoma actinoides, Orthostrongylus macrotis, and Taenia omissa were recorded. Adult Dicrocoelium dendriticum were collected from liver of two wapiti, one mule deer, and one white-tailed deer from the Cypress Hills.

Domestic sheep as a rare definitive host of the large American liver fluke Fascioloides magna.

Two domestic sheep were inoculated orally with 250 metacercariae of Fascioloides magna. One sheep died 4 mo after inoculation and 22 immature F. magna were recovered. The second sheep remained healthy and was killed 8 mo after inoculation. Ten mature F. magna were recovered from hepatic parenchyma, and eggs of F. magna were observed in areas of the liver where flukes resided and from feces. Although F. magna is almost always lethal to sheep, this sheep was a rare definitive host.