Liver Fluke Fasciola Hepatica Research Articles

EPIDEMIOLOGY OF BOVINE FASCIOLIASIS: PREVALENCE AND HAEMATO-BIOCHEMICAL ALTERATIONS IN KATSINA ABATTOIR NORTH WESTERN NIGERIA

Bovine fascioliasis, caused by liver flukes Fasciola hepatica and Fasciola gigantica, significantly affects cattle health and production globally. This study evaluated the prevalence and haemato-biochemical changes associated with bovine fascioliasis in 134 cattle at Katsina Central Abattoir. Using the sedimentation technique, faecal samples revealed a 3.73% infection rate, with females showing a higher prevalence (4.55%) than males (2.17%), though not statistically significant (P > 0.05). The age group 2-3 years had the highest prevalence (25.00%), with significant differences among age groups (P < 0.05). Among breeds, Wadara had the highest prevalence (20.00%) compared to White Fulani (4.93%), with a significant difference (P < 0.05). Medium-conditioned cattle showed the highest prevalence (6.67%) versus good-conditioned ones (1.37%) with significant difference (P < 0.05). Infected cattle exhibited significant reductions (P < 0.05) in packed cell volume (PCV), haemoglobin (Hb), and total erythrocyte count (RBC), as well as decreases in white blood cell count (WBC), mean cellular volume (MCV), and mean cellular haemoglobin concentration (MCHC). Biochemical analysis indicated elevated alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), and total bilirubin (TB) levels (P < 0.05), with reduced albumin (ALB), total protein (TP), and conjugated bilirubin (CB) levels (P < 0.05). These results highlight the significant impact of bovine fascioliasis on cattle health in Katsina State, emphasizing the need for effective control strategies.

Mitochondrial genome of the fluke pond snail, Austropeplea cf. brazieri (Gastropoda: Lymnaeidae)

BackgroundLymnaeid snails of the genus Austropeplea are an important vector of the liver fluke (Fasciola hepatica), contributing to livestock production losses in Australia and New Zealand. However, the species status within Austropeplea is ambiguous due to heavy reliance on morphological analysis and a relative lack of genetic data. This study aimed to characterise the mitochondrial genome of A. cf. brazieri, an intermediate host of liver fluke in eastern Victoria.MethodsThe mitochondrial genome was assembled and annotated from a combination of second- and third-generation sequencing data. For comparative purposes, we performed phylogenetic analyses of the concatenated nucleotide sequences of the mitochondrial protein-coding genes, cytochrome c oxidase subunit 1 and 16S genes.ResultsThe assembled mt genome was 13,757 base pairs and comprised 37 genes, including 13 protein-coding genes, 22 transfer RNA genes and 2 ribosomal RNA genes. The mt genome length, gene order and nucleotide compositions were similar to related species of lymnaeids. Phylogenetic analyses of the mt nucleotide sequences placed A. cf. brazieri within the same clade as Orientogalba ollula with strong statistical supports. Phylogenies of the cox1 and 16S mt sequences were constructed due to the wide availability of these sequences representing the lymnaeid taxa. As expected in both these phylogenies, A. cf. brazieri clustered with other Austropeplea sequences, but the nodal supports were low.ConclusionsThe representative mt genome of A. cf. brazieri should provide a useful resource for future molecular, epidemiology and parasitological studies of this socio-economically important lymnaeid species.Graphical abstract

Early stage Fasciola hepatica infection in growing cattle: impact on weight gain and liver weight determined in flukicide efficacy studies.

Although the negative impact of liver fluke (Fasciola hepatica) infection on production and health in cattle is generally accepted, results of individual research have been variable, ranging from important negative impacts on the animal to minimal or no impact. To add information on the impact of F. hepatica infection in growing cattle, weight gain and liver weight of young experimentally infected animals from seven controlled efficacy studies were analyzed. In each study, fluke naïve animals were inoculated with approximately 450 to 500 F. hepatica encysted metacercariae, blocked on body weight and randomly assigned into one untreated group (controls) and groups which were administered an experimental flukicide when the flukes were 4 weeks old (migrating) and sacrificed 8 weeks thereafter (12 weeks after inoculation). Data of groups which demonstrated >90% reduction of fluke counts following treatment and groups left untreated (total 103 and 47 animals, respectively) were compared. There was a significant (p < 0.0001) negative association between fluke count and weight gain while fluke count and liver weight and fluke count and relative liver weight were positively associated (p < 0.0001). Over the 8-week post-treatment period, flukicide-treated cattle had almost 15% more weight gain than the controls (50.9 kg vs. 44.4 kg; p = 0.0003). Absolute and relative liver weight was significantly (p < 0.0001) lower in flukicide-treated compared to untreated cattle. Overall, this analysis provided evidence of a substantial negative effect of early (migrating) liver fluke infection on the growth of young cattle, likely due to pathology of the liver and associated reduction in its function as the central organ for bioenergy and protein metabolism.

Neoblast-like stem cells of Fasciola hepatica.

The common liver fluke (Fasciola hepatica) causes the disease fasciolosis, which results in considerable losses within the global agri-food industry. There is a shortfall in the drugs that are effective against both the adult and juvenile life stages within the mammalian host, such that new drug targets are needed. Over the last decade the stem cells of parasitic flatworms have emerged as reservoirs of putative novel targets due to their role in development and homeostasis, including at host-parasite interfaces. Here, we investigate and characterise the proliferating cells that underpin development in F. hepatica. We provide evidence that these cells are capable of self-renewal, differentiation, and are sensitive to ionising radiation-all attributes of neoblasts in other flatworms. Changes in cell proliferation were also noted during the early stages of in vitro juvenile growth/development (around four to seven days post excystment), which coincided with a marked reduction in the nuclear area of proliferating cells. Furthermore, we generated transcriptomes from worms following irradiation-based ablation of neoblasts, identifying 124 significantly downregulated transcripts, including known stem cell markers such as fgfrA and plk1. Sixty-eight of these had homologues associated with neoblast-like cells in Schistosoma mansoni. Finally, RNA interference mediated knockdown of histone h2b (a marker of proliferating cells), ablated neoblast-like cells and impaired worm development in vitro. In summary, this work demonstrates that the proliferating cells of F. hepatica are equivalent to neoblasts of other flatworm species and demonstrate that they may serve as attractive targets for novel anthelmintics.

Assessing the microbiota of the snail intermediate host of trematodes, Galba truncatula

BackgroundThe microbiome is known to play key roles in health and disease, including host susceptibility to parasite infections. The freshwater snail Galba truncatula is the intermediate host for many trematode species, including the liver and rumen flukes Fasciola hepatica and Calicophoron daubneyi, respectively. The snail-parasite system has previously been investigated. However, the specific interaction between the snail-associated microbiota and intra-snail developmental stages of trematodes has yet to be explored.MethodsGalba truncatula snails were collected from farms in Northern Ireland and trematode infection was diagnosed using PCR. High-throughput sequencing analysis of the bacterial 16S ribosomal DNA V3-V4 hypervariable regions was subsequently applied to characterise the microbiota of both uninfected and infected snails.Results We first showed that the snail harboured microbiota that was distinct for its environment. The microbiota of infected snails was found to differ significantly from that of uninfected snails. In particular, the bacterial genera Mycoplasma and Methylotenera were significantly more abundant in infected snails, while genera Sphingomonas and Nocardioides were predominantly associated with uninfected snails. ConclusionThese findings pave the way to future studies on the functional roles of bacteria in host-parasite relationships.Graphical

A survey for antibodies against Fasciola hepatica in cattle and sheep in Finland indicates a low level of exposure

BackgroundFasciolosis is a parasitic infection caused by the liver fluke Fasciola hepatica that can have a major economic impact on livestock industry. The prevalence of the disease has recently been increasing in many North European countries. The objective of this study was to determine the prevalence of antibody against F. hepatica in Finnish cattle herds and sheep flocks during 2019 by using a commercial enzyme-linked immunosorbent assay (ELISA). Randomly selected bulk tank milk samples were obtained from 660 dairy herds. Blood samples were collected at slaughterhouses from 1944 suckler cows from 309 herds and from 1120 sheep from 95 flocks.ResultsAntibodies against F. hepatica were found in 0.45% (95% confidence interval (CI): 0.15–1.33) of the dairy herds and 0.97% (95% CI: 0.33–2.82) of the suckler cow herds. The seropositive herds were located in eastern and central Finland. None of the sampled sheep flocks tested had antibodies against F. hepatica (95% CI: 0–3.89). The results of the assays were compared with meat inspection data received from the slaughterhouses. All positive herds also had liver condemnations due to F. hepatica based on the meat inspection reports.ConclusionsCompared to other North European countries, the prevalence of fasciolosis in Finland can be considered low, and according to meat inspection reports, there are no indications of the prevalence increasing in Finland.

Morphological and molecular characterization of Fasciola isolates from livestock in Golestan province, northern Iran.

Fascioliasis, caused by the liver flukes Fasciola hepatica and Fasciola gigantica, is a global zoonotic helminthic disease. The livestock and human are the final hosts of the parasites. Northern Iran is an important endemic region for fascioliasis. Few studies have been conducted on the characterization of Fasciola isolates from eastern regions of the Caspian littoral of the country. The aim of the present study was to identify F. hepatica, F. gigantica and intermediate/hybrid forms of Fasciola isolates from livestock in Golestan province, northern Iran, using morphometric and molecular tools. Livestock livers naturally infected with Fasciola spp. were collected from Golestan slaughterhouse during 2019-2020. The worms were morphometrically studied using a calibrated stereomicroscope. Genomic DNA was extracted from all samples, and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed on internal transcribed spacer (ITS1) region using Rsa1 restriction enzyme. All the isolates were then analysed by multiplex PCR on Pepck region. A total of 110 Fasciola isolates were collected from the infected livers, including 94 sheep, 12 cattle and 4 goats. Morphometric analysis of 61 adult Fasciola isolates indicated that, 44 and 17 isolates belonged to F. hepatica and F. gigantica, respectively. Eighty-one and 29 isolates belonged to F. hepatica and F. gigantica using ITS1-RFLP, respectively. However, Pepck Multiplex PCR indicated 72 F. hepatica, 26 F. gigantica and 12 intermediate/hybrid forms. All 12 hybrid isolates were found in sheep host. Two isolates were identified as F. gigantica using morphometry and F. hepatica using both molecular methods. The present study confirmed the existence of both F. hepatica and F. gigantica species and reported the first molecular evidence of hybrid Fasciola isolates in ruminants of Golestan province.

Coproscopical diagnosis of patent Fasciola hepatica infections in sheep – A comparison between standard sedimentation, FLUKEFINDER® and a combination of both

The liver fluke Fasciola hepatica is a highly pathogenic and zoonotic trematode with a cosmopolitan distribution. In livestock, infections may lead to significant economic losses if not diagnosed promptly and treated effectively. Particularly for small ruminants, the standard method for the detection of fluke infection is based on coproscopical methods such as the sedimentation method, which detects F. hepatica eggs in faecal samples. In this respect a recent innovative coproscopical approach to diagnose patent infections is the FLUKEFINDER® method, which relies on differential sieving before sedimentation. These two methods and a combination of both methods that allows larger amounts of faeces to be processed with the FLUKEFINDER® apparatus were compared, to assess which method is most appropriate to determine the prevalence and intensity of F. hepatica egg shedding. The methods were compared for their ability to recover eggs from ovine faecal samples containing different numbers of fluke eggs per gram (EPG) of faeces and diluting the samples further by mixing with faeces from uninfected sheep. To compare the specificity of the test procedures, positive and negative samples with a low EPG were analysed in parallel by an investigator blinded to the nature of the samples. Significant differences concerning the EPG outcome were found: The FLUKEFINDER® method demonstrated the highest EPG values (p < 0.001) in the undiluted samples as well as in all mixing levels, followed by the modified FLUKEFINDER® method. The standard sedimentation showed the lowest EPG values and the highest variability between technical replicates. The precision of the FLUKEFINDER® method and the modified FLUKEFINDER® method were significantly higher than the precision of the standard sedimentation as determined by comparison of variability between technical replicates. The highest raw egg counts were detected using the modified FLUKEFINDER® method. The FLUKEFINDER® method and the combined method showed a sensitivity of 100 % even at the lowest egg concentrations, whereas the sensitivity of the standard sedimentation was 98.1 % for the same set of samples (i.e. one false negative sample). In a separate investigation aiming to estimate the specificity no differences were found between the three methods: all protocols showed 100 % specificity and were able to correctly distinguish between truly positive and truly negative samples without any evidence of cross-contamination between positive and negative samples processed in parallel.

Multispecies helminth parasitism of grazing dairy cows in Germany and Austria, examined in the housing period

Helminth composition and burden data for dairy cows have not been reported for >40years for Germany and even less information is available for Austria. In the context of two recent studies, helminth parasitism was studied in 32 cows (23 from six farms in Bavaria and Tyrol; 9 from one farm in Saxony) from pasture-based dairy farms necropsied during the housing period. Helminths were enumerated and identified based on morphological characters (all helminths but rumen flukes) or molecular techniques (rumen flukes). Thirteen species of gastrointestinal nematodes and two species each of liver flukes (Fasciola hepatica, Dicrocoelium dendriticum) and rumen flukes (Calicophoron daubneyi, Paramphistomum leydeni) were recorded; no lungworms were recovered from any cow. Early fourth-stage (inhibited) larval Ostertagia species nematodes (210 to 140,600) were recovered from all cows, 31 each had adult Ostertagia ostertagi/Ostertagia lyrata (40 to 2020) and Trichostrongylus axei (10 to 53,400), 23 Oesophagostomum radiatum (1 to 242) and 20 Cooperia punctata (10 to 3330). Other nematodes present in descending order of prevalence were: Cooperia oncophora/Cooperia surnabada, Ostertagia leptospicularis/Ostertagia kolchida, Oesophagostomum venulosum, Bunostomum phlebotomum, Chabertia ovina, Nematodirus helvetianus, Trichostrongylus longispicularis, Haemonchus contortus and Aonchotheca bilobata. The cows from Bavaria and Tyrol harbored more total gastrointestinal nematodes than that from Saxony (geometric mean adult plus inhibited larval nematodes, 6510 vs. 2051, respectively). However, in both cohorts of cows abomasal nematodes accounted for ∼97% of the total nematode burden with inhibited larval Ostertagia species nematodes contributing over 70% of the total gastrointestinal nematode burden and∼96% of the Ostertagia species burden. Approximately 44%, 37% and 19% of the cows harbored <5000, 5000 to 10,000 or>10,000 total gastrointestinal nematodes, respectively. Fecal nematode egg and coproculture nematode larval counts significantly correlated with the cows' total adult nematode burden (rs=0.354, p<0.05, and rs=0.608, p<0.001, respectively). Although the magnitude of nematode burden to exert production effects on dairy cows is not well defined and may vary relative to several factors including nutritional supplementation, the level of mixed parasitism found in this investigation supports consideration of grazing dairy cows in helminth control measures, especially at the time of housing in autumn.

Prevalence, coinfection, and risk factors associated with Fasciola hepatica and other gastrointestinal parasites in cattle from the Peruvian Amazon.

Extensive cattle rearing is a major source of economy for the inhabitants of the Amazon region of Peru. Milk and meat production is generally affected by the prevalence of various parasites, including hepatic and gastrointestinal parasites, as these products provide ideal conditions for parasitic growth. This poses a serious public health threat. This study aimed to estimate the prevalence, coinfection, and risk factors associated with the liver fluke (Fasciola hepatica) and other gastrointestinal parasites in cattle from the Amazon region of Peru. Fecal samples obtained from 1450 bovine specimens were analyzed using flotation and sedimentation methods to identify parasites, including Eimeria spp., strongyle-type eggs (STEs), and F. hepatica. We collected information about the specimens, including age, sex, origin, breed, category, frequency of deworming, farm size, herd size, water sources, and rearing system by conducting simple inspections and interviewing owners. The data obtained were statistically evaluated using the Chi-square test (p < 0.05) to determine the association between the qualitative variables. We also calculated the odds ratio at a 95% confidence interval to identify the risk factors. We observed that F. hepatica, Eimeria spp., and STEs were 45.6%, 39.8%, and 35.3% prevalent, respectively. We found risk factors related to distomatosis in the animals from Huambo, where the drinking water sources are mainly streams, ditches, and rivers, while the specimens from Valle Chico were predisposed to coccidiosis. Further, the risk factors related to the presence of STEs in feces were age (61-90 months), origin (Valle Chico), herd size (<50 animals), and type of extensive rearing. Furthermore, significant coinfection was observed between Eimeria spp. and STEs. The high percentages of parasites in cattle observed were related to epidemiological factors, such as the origin of the sample, water sources, age, herd size, and extensive breeding. Similarly, the presence of STEs was a risk factor for contracting coccidiosis. Our future goals include investigating these parasites using a larger sample size and identifying more risk factors using more sensitive and specific diagnostic tests.

An evolutionary molecular adaptation of an unusual stefin from the liver fluke Fasciola hepatica redefines the cystatin superfamily

Fasciolosis is a worldwide parasitic disease of ruminants and an emerging human disease caused by the liver fluke Fasciola hepatica. The cystatin superfamily of cysteine protease inhibitors is composed of distinct families of intracellular stefins and secreted true cystatins. FhCyLS-2 from F.hepatica is an unusual member of the superfamily, where our sequence and 3D structure analyses in this study revealed that it combines characteristics of both families. The protein architecture demonstrates its relationship to stefins, but FhCyLS-2 also contains the secretion signal peptide and disulfide bridges typical of true cystatins. The secretion status was confirmed by detecting the presence of FhCyLS-2 in excretory/secretory products, supported by immunolocalization. Our high-resolution crystal structure of FhCyLS-2 showed a distinct disulfide bridging pattern and functional reactive center. We determined that FhCyLS-2 is a broad specificity inhibitor of cysteine cathepsins from both the host and F.hepatica, suggesting a dual role in the regulation of exogenous and endogenous proteolysis. Based on phylogenetic analysis that identified several FhCyLS-2 homologues in liver/intestinal foodborne flukes, we propose a new group within the cystatin superfamily called cystatin-like stefins.

Seroprevalence and coprological prevalence of liver fluke Fasciola hepatica in cattle and sheep from Santander department, Colombia.

Fasciola hepatica is a parasite with a worldwide distribution that affects several mammals, including humans, and is considered a public health problem. Therefore, the aim of this study was to determine the prevalence of Fasciola hepatica in humans, cattle and sheep, as well as to evaluate factors associated with the prevalence. A total of 185 serum samples from sheep, 290 from cattle, and 114 from humans were collected and processed using an in-house developed ELISA to detect IgG antibodies against F. hepatica. Additionally, 185 stool samples from sheep and 290 from cattle were examined using a Dennis sedimentation technique. Risk factors were analyzed using epidemiological surveys. The overall seroprevalence was 46.5% (86/185) in sheep, 32.5% (94/289) in cattle, and no humans tested positive for the infection. The coprological prevalence was 47.7% (86/180) in sheep and 33.7% (98/290) in cattle. Female gender and cattle living with alternate grazing management showed 2.5 and 6.5 times higher probability of infection, respectively. Bovines coexisting with sheep exhibited a higher risk of infection (odds ratio [OR]=4.3) compared to those without sheep. We concluded that F. hepatica in cattle and sheep has an endemic behavior, and therefore represents a problem of public health for rural communities.

Fasciolosis-An Increasing Challenge in the Sheep Industry.

Simple SummaryFasciolosis caused by Fasciola hepatica is a serious disease and a huge challenge in the sheep industry. The disease has several clinical manifestations including acute death, anemia, ill-thrift and loss of body condition. Climate change with milder temperatures and heavier rainfall will increase the risk of fasciolosis. Grazing management and treatment with flukicide are at present the only options to restrain F. hepatica infection. However, control possibilities are challenging, and resistance to flukicide drugs is increasing. Diagnostic improvements, targeted treatment and vaccines will hopefully increase animal health and welfare on fluke infested pastures in the future.The liver fluke Fasciola hepatica may cause severe infection in several mammalian species, including sheep and humans. Fasciolosis is a parasitic disease occurring worldwide in temperate climates and involves intermediate lymnaeid snails as vectors, in Europe the pond snail Galba truncatula in particular. In the sheep industry, the disease is a serious welfare and health problem. Fasciolosis is usually classified as acute, subacute or chronic according to the number and stage of flukes present in the liver, but with a considerable overlap. Acute disease, associated with a large number of migrating larvae, often results in sudden death due to acute and massive hemorrhage, while chronic fasciolosis is characterized by anemia, hypoalbuminaemia and weight loss. The management of fasciolosis is an increasing challenge in the sheep industry. Early diagnostic tests are limited. Protective immunity against liver flukes in sheep is low or lacking, and vaccines are not yet available. Treatment and control possibilities are challenging, and resistance to flukicide drugs is increasing. In addition, climate change with warmer and more humid weather will have a substantial effect on the establishment of both flukes and snails and will most likely increase the future distribution of F. hepatica.

Diagnosing Human Fascioliasis Using ELISA Immunoassays at a Tertiary Referral Hospital in Hanoi: A Cross-Sectional Study.

Infections with the zoonotic liver flukes Fasciola gigantica and Fasciola hepatica may result in severe disease in humans. In Vietnam, an emergence of fascioliasis cases has been observed from the late 1990s onwards. Various diagnostic tools are used in the country, but the agreement between these tools has not been critically evaluated. We aimed to describe the clinical presentation and diagnostic outcomes in fascioliasis patients in Vietnam. A retrospective, cross-sectional study was conducted on the medical records of a group of 145 patients diagnosed with fascioliasis at a tertiary referral hospital in Hanoi. Based on the review, sociodemographic background and clinical presentation were recorded. These patients all received standard routine serologic tests, including internal antibody (Ab)-ELISA, an enzyme-linked immunosorbent assay (ELISA), and commercial coproantigen (cAg)-ELISA. The majority of cases were between 30 and 59 years old (68.3%), and about half of them were male (51.0%). Upper quadrant and epigastric pain were the most commonly reported symptoms (61.4% and 35.2%, respectively). All but one patient had liver lesions upon ultrasound examination (99.3%), and eosinophilia was present in most of the patients (89.7%). A high number of patients were positive in the in-house and the commercial Ab-ELISA (95.9% and 87.4%, respectively), yet only a slight agreement was observed between the two tests (kappa coefficient, 0.06). A further 47.4% of cases were positive for the commercial cAg-ELISA, whereas stool microscopy indicated the presence of Fasciola spp. eggs in 25.7% of patients. The current study emphasizes the challenges related to the diagnosis of human fascioliasis in Vietnam.

Targeting Secreted Protease/Anti-Protease Balance as a Vaccine Strategy against the Helminth Fasciola hepatica.

The liver fluke Fasciola hepatica is an economically important global pathogen of humans and their livestock. To facilitate host invasion and migration, F. hepatica secretes an abundance of cathepsin peptidases but prevents excessive damage to both parasite and host tissues by co-secreting regulatory peptidase inhibitors, cystatins/stefins and Kunitz-type inhibitors. Here, we report a vaccine strategy aimed at disrupting the parasite’s protease/anti-protease balance by targeting these key inhibitors. Our vaccine cocktail containing three recombinant stefins (rFhStf-1, rFhStf-2, rFhStf-3) and a Kunitz-type inhibitor (rFhKT1) formulated in adjuvant Montanide 61VG was assessed in two independent sheep trials. While fluke burden was not reduced in either trial, in Trial 1 the vaccinated animals showed significantly greater weight gain (p < 0.05) relative to the non-vaccinated control group. In both trials we observed a significant reduction in egg viability (36–42%). Multivariate regression analyses showed vaccination and increased levels of IgG2 antibodies specific for the F. hepatica peptidase inhibitors were positive indicators for increased weight gain and levels of haemoglobin within the normal range at 16 weeks post-infection (wpi; p < 0.05). These studies point to the potential of targeting peptidase inhibitors as vaccine cocktails for fasciolosis control in sheep.

Liver fluke in beef cattle – Impact on production efficiency and associated greenhouse gas emissions estimated using causal inference methods

We aimed to estimate 1) the marginal effect of liver fluke (Fasciola hepatica) infection on productivity of Scottish beef cattle, and 2) the associated greenhouse gas emissions intensity (GHG EI). Data comprised 240,065 abattoir records from NE Scotland from 2014 to 2017, including the presence or absence of lesions typical of liver fluke in the liver at the time of slaughter, from which we inferred liver fluke infection status. The retrospective analysis of abattoir records to estimate marginal effects of an exposure is complicated by the multi-dimensional, clustered nature of the datasets, which result in confounding of potential causal factors with the exposure. Causal inference methods are required to identify and correct for variation in background exposure. We constructed directed acyclic graphs (DAGs) of observed variables, including the potential confounders, breed, sex, breeder, finisher, season of birth and year of birth. We then applied inverse probability weighting (IPW) to adjust for variation among exposure risk and applied a doubly robust generalized linear model (DRGLM) to the weighted observations to estimate the marginal effect of fluke on the growth rate of animals and total days from birth until slaughter. We compared these estimates with the results of linear mixed effects (LME) models with the same variables, treating breeder and producer as random effects. To estimate GHG EI, we applied IPCC tier-2 type GHG calculations to the marginal effects estimated from IPW with DRGLM. The IPW with DRGLM model estimated that animals with active fluke lesions (adult fluke seen on postmortem inspection) gained 17 (95 % CI 12–22) g/d less saleable beef than animals with no lesions and no visible fluke. Animals with active fluke lesions were 11 (95 % CI 6.5–15) d older at slaughter weight than animals with no lesions. Animals with historic lesions in which there was scarring of the liver but in which no adult fluke were seen showed a wide variation in effect estimates, consistent with some misclassification. The effect estimates from LME models suggested slightly lower effects of fluke on growth rate and days to slaughter but with overlapping 95 % confidence intervals. Calculation of the associated GHG emissions suggest the EI of meat from a herd with no fluke is approximately 1.5 % lower than the same herd with fluke. Sustainably controlling liver fluke would have additional production benefits not included in this estimate and could therefore have a much greater impact on GHG EI in practice than demonstrated here.

Evaluation of bioaccumulation of some heavy metals in liver flukes (Fasciola hepatica and Dicrocoelium dendriticum) and liver samples of sheep.

Different living organisms are used as applicable bioindicators to determine heavy metal pollutions. Recent studies have shown that helminths parasites can be used as efficient environmental sentinels. This study aimed to evaluate Fasciola hepatica and Dicrocoelium dendriticum as bioaccumulators of lead (Pb), chromium (Cr), cadmium (Cd), copper (Cu). For this work. A total of 50 samples (F. hepatica, D. dendriticum, and livers from the infected and uninfected sheep, each of 10 samples) were collected from sheep slaughtered in Tabriz abattoir. One gram of each sample was incinerated and analyzed by Flame Atomic Absorption Spectrometry. The analysis of samples showed that Pb, Cr and Cu values in F. hepatica were higher than those in D. dendriticum, but only the differences of Pb and Cu were significant. The values of heavy metals in F. hepatica were significantly higher than those in the infected livers (except for Cd), while in D. dendriticum, Cr and Cd were only higher. Based on metal levels in livers, it was found that bioconcentration factors (BCFs) of Cr, Pb and Cu for F. hepatica were much more than one, and BCFs of these three metals between two flukes were statistically significant. This study indicated that F. hepatica had a higher bioindicator potential than D. dendriticum to evaluate environmental pollutants by some metals.

Evaluation of Immunogenicity and Efficacy of Fasciola hepatica Tetraspanin 2 (TSP2) Fused to E. coli Heat-Labile Enterotoxin B Subunit LTB Adjuvant Following Intranasal Vaccination of Cattle.

Fasciolosis, caused by the liver flukes Fasciola hepatica and F. gigantica, is an economically important and globally distributed zoonotic disease. Liver fluke infections in livestock cause significant losses in production and are of particular concern to regions where drug resistance is emerging. Antigens of the F. hepatica surface tegument represent promising vaccine candidates for controlling this disease. Tetraspanins are integral tegumental antigens that have shown partial protection as vaccine candidates against other trematode species. The Escherichia coli heat-labile enterotoxin’s B subunit (LTB) is a potent mucosal adjuvant capable of inducing an immune response to fused antigens. This study investigates the potential of F. hepatica tetraspanin 2 extracellular loop 2 (rFhTSP2) as a protective vaccine antigen and determines if fusion of FhTSP2 to LTB can enhance protection in cattle. Cattle were immunised subcutaneously with rFhTSP2 mixed in the Freund’s adjuvant and intranasally with rLTB-FhTSP2 in saline, accounting for equal molar ratios of tetraspanin in both groups. Vaccination with rFhTSP2 stimulated a strong specific serum IgG response, whereas there was no significant serum IgG response following rLTB-FhTSP2 intranasal vaccination. There was no substantial antigen specific serum IgA generated in all groups across the trial. Contrastingly, after the fluke challenge, a rise in antigen specific saliva IgA was observed in both vaccination groups on Day 42, with the rLTB-FhTSP2 vaccination group showing significant mucosal IgA production at Day 84. However, neither vaccine group showed a significant reduction of fluke burden nor faecal egg output. These results suggest that intranasal vaccination with rLTB-FhTSP2 does elicit a humoral mucosal response but further work is needed to evaluate if mucosal delivery of liver fluke antigens fused to LTB is a viable vaccine strategy.

Prevalence and gross pathology of liver fluke in macropods cohabiting livestock farms in north eastern NSW, Australia, and diagnosis using cELISA.

Liver fluke (Fasciola hepatica) is a parasite of herbivores including wildlife. Macropods, such as Eastern grey kangaroo (Macropus giganteus) and Common wallaroo (Osphranter robustus), are frequently observed sharing grazing sites with domestic livestock. The impact of Macropods, as reservoirs of infection, on livestock production and risks to cross-species transmission are largely unknown. In Phase 1 of this study, liver and faecal samples were collected from 245 Macropods (181 Eastern grey kangaroos, 64 Common wallaroos) cohabiting livestock farms (n = 7) in the Northern Tablelands regions of New South Wales. Total fluke (TFC) and fluke eggs (FEC) were counted in the liver and faeces, respectively, to assess prevalence. Faecal antigens were also measured using the commercial Bio-X Diagnostic Monoscreen AgELISA Fasciola hepatica kit (cELISA) to assess suitability as a diagnostic tool. In Phase 2, Macropod faecal samples were collected from 60 livestock farms to conduct FEC and assess prevalence by region. Liver fluke was prevalent in 22% of Eastern grey kangaroo and 20% of Common wallaroos with prevalence as high as 45% in the Eastern grey kangaroo. Fluke burdens ranged from 1 to 122 flukes (mean = 9 flukes) with a FEC range of 0–195 eggs per gram (epg) of faeces (mean = 18 epg). Evidence of dead and live flukes trapped within fibrotic capsules confirms the ability of Macropods to resolve infections. cELISA proved highly specific (100%) and sensitive (98%) in liver fluke detection however fibrotic capsules observed in the liver may reduce the correlation of coproantigens with fluke burden. Phase 2 revealed that 27% of livestock farms had Macropods infected with liver fluke. Overall, this study confirmed Eastern grey kangaroo and Common wallaroo are susceptible hosts and potential reservoirs for liver fluke and, monitoring infections in Macropods would assist in livestock disease management.

Kangaroos and liver fluke: The role played in cross-species transmission and drug resistance

Australian livestock are challenged by liver fluke (Fasciola hepatica) in grazing regions endemic to the intermediate snail host. Liver fluke infests a wide range of herbivores including free-roaming wildlife such as kangaroos (Macropods). The role played by Macropods in cross-species transmission and as vectors for anthelmintic resistance is largely unknown. In Phase 1 of this study, liver fluke of Eastern grey kangaroo (Macropus giganteus Shaw, 1790) origin (Kangaroo isolate) were artificially infected in sheep to confirm establishment and cross-species transmission. In Phase 2, the efficacy of triclabendazole (TCBZ) was assessed in vivo against the Kangaroo isolate to identify any drug resistance. Forty (40) merino sheep were housed in pens and allocated to one of 4 groups (Groups 1–4). Groups 1 and 2 were artificially infected with a TCBZ resistant liver fluke isolate (Oberon) originating from sheep whilst Groups 3 and 4 were infected with the Kangaroo isolate (Phase 1). At 9 weeks post infection (wpi), sheep in Groups 2 and 4 were treated with 10 mg/kg TCBZ (Phase 2). Sheep were subsequently euthanased at 11 wpi to conduct total fluke counts (TFC) in the liver. Faecal samples were collected fortnightly to measure fluke egg counts and coproantigens. Individual blood samples were collected, concurrently with faecal sampling, to monitor haematocrit and plasma proteins levels. Liver fluke of kangaroo origin established to patent infections in sheep with similar establishment and pathogenicity to the Oberon isolate. TCBZ achieved an 86 % reduction in TFC (99.8 % - adult fluke, 0 % - immature fluke) in sheep with the Kangaroo isolate and a 28 % reduction in the Oberon isolate (37 % - adult, 0 % - immature fluke). An 89 % reduction in faecal coproantigens was observed in sheep with the Kangaroo isolate and no reduction in sheep with Oberon. This study confirmed cross-species transmission of liver fluke from a kangaroo to sheep. When cohabiting the livestock grazing environment, kangaroos may act as reservoirs for liver fluke and vectors for drug resistance within liver fluke endemic areas.