Fluke Burden Research Articles

Assessment of accuracy of liver fluke diagnostic tests using the gold standard of total worm counts

In many regions of New Zealand liver fluke is endemic, infecting most grazing ruminants, including cattle, sheep, and deer. Restricting the economic losses and welfare costs associated with liver fluke relies on accurately identifying those animals with a production limiting infection. This has proven a difficult goal and although several antemortem quantitative tests are available, including faecal egg counts (FEC), serum ELISA and copro-antigen ELISA, none can be considered a gold standard test of liver fluke infection. The accepted gold standard test for fascioliasis is the total fluke count, which is both laborious and can only be completed at post-mortem. This study aimed to compare the performance of four liver fluke diagnostic tests, against the results of a gold standard total fluke count test. Two groups of cattle were selected, 29 culled mixed age beef cows (MAC) and ten 30-month-old steers. The cattle were blood sampled and faecal sampled prior to slaughter and their whole livers recovered post slaughter at the abattoir. Liveweight was also recorded at slaughter. After collection, each liver was weighed, scored for gross pathology, then serum, faeces and livers were frozen at −20 °C for later analysis. Faecal egg counts and F. hepatica copro-antigen ELISA tests were completed on the faecal samples and total fluke counts were completed on the livers. Fasciola hepatica antibody concentration in serum samples were quantified using a commercial ELISA test. Poisson regression models were built to model the association between each diagnostic test and the total fluke count, and a linear regression model was built to examine the relationship between each diagnostic test and live weight at slaughter.The median fluke count was significantly higher in MAC than steers (p = 0.01), and F. hepatica eggs were present in 100% steers and 66% MAC. There was a significant effect of copro-antigen ELISA value on total fluke count (p < 0.0001), with a coproantigen ELISA value = 20.1 predicting 10 flukes and a value = 44.8 predicting 30 flukes. There was also a significant effect of FEC on total fluke count (p = 0.002) but the R-squared value for this model was lower. There was no association between liver fibrosis score or antibody ELISA test and total fluke count (p = 0.95, p = 0.73, respectively). There was a significant effect of total fluke count (p = 0.03) on liveweight at slaughter, with liveweight falling 20.4 kg for each unit increase in loge (total fluke count). There was no effect of FEC (p = 0.11), antibody ELISA (p = 0.55) or copro-antigen ELISA value (p = 0.16) on liveweight at slaughter. Taken together, these results show that the coproantigen ELISA test is the better test for estimating the true liver fluke burden and that the number of flukes in the liver has a negative effect on cattle live weights at slaughter.

Evaluation of Reproductive Histology Response of Adult Fasciola hepatica in Goats Vaccinated with Cathepsin L Phage-Exposed Mimotopes.

Fasciolosis, a globally re-emerging zoonotic disease, is mostly caused by the parasitic infection with Fasciola hepatica, often known as the liver fluke. This disease has a considerable impact on livestock productivity. This study aimed to evaluate the fluke burdens and faecal egg counts in goats that were administered phage clones of cathepsin L mimotopes and then infected with F. hepatica metacercariae. Additionally, the impact of vaccination on the histology of the reproductive system, specifically related to egg generation in adult parasites, was examined. A total of twenty-four goats, which were raised in sheds, were divided into four groups consisting of six animals each. These groups were randomly assigned. The goats were then subjected to two rounds of vaccination. Each vaccination involved the administration of 1 × 1013 phage particles containing specific mimotopes for cathepsin L2 (group 1: PPIRNGK), cathepsin L1 (group 2: DPWWLKQ), and cathepsin L1 (group 3: SGTFLFS). The immunisations were carried out on weeks 0 and 4, and the Quil A adjuvant was used in combination with the mimotopes. The control group was administered phosphate-buffered saline (PBS) (group 4). At week 6, all groups were orally infected with 200 metacercariae of F. hepatica. At week 22 following the initial immunisation, the subjects were euthanised, and adult F. hepatica specimens were retrieved from the bile ducts and liver tissue, and subsequently quantified. The specimens underwent whole-mount histology for the examination of the reproductive system, including the testis, ovary, vitellaria, Mehlis' gland, and uterus. The mean fluke burdens following the challenge were seen to decrease by 50.4%, 62.2%, and 75.3% (p < 0.05) in goats that received vaccinations containing cathepsin L2 PPIRNGK, cathepsin L1 DPWWLKQ, and cathepsin L1 SGTFLFS, respectively. Animals that received vaccination exhibited a significant reduction in the production of parasite eggs. The levels of IgG1 and IgG2 isotypes in vaccinated goats were significantly higher than in the control group, indicating that protection is associated with the induction of a mixed Th1/Th2 immune response. The administration of cathepsin L to goats exhibits a modest level of efficacy in inducing histological impairment in the reproductive organs of liver flukes, resulting in a reduction in egg output.

Evaluation of Th1/Th2, regulatory cytokines and transcriptional factor FoxP3 in sheep immunized with a partially protective and non-protective vaccine and challenged with Fasciola hepatica

Gene expression for Th1/Th2 cytokines (IL-4 and IFN-ɣ), regulatory cytokines (TGF-β and IL-10) and the transcriptional factor FoxP3 was analyzed in the liver and hepatic lymph nodes (HLN) from sheep immunized with partially protective and non-protective vaccine candidates and challenged with Fasciola hepatica. FoxP3 T cells were also evaluated by immunohistochemistry (IHQ). The most remarkable difference between the partially protected vaccinated (V1) group and the non-protected vaccinated (V2) group was a more severe expansion of FoxP3 T cells recorded by IHQ in both the liver and HLN of the V2 group as compared to the V1 group, whereas no differences were found between the V2 group and the infected control (IC) group. Similar results were recorded for FoxP3 gene expression although significant differences among V1 and V2 groups were only significant in the HLN, while FoxP3 gene expression was very similar in the V2 and IC groups both in the liver and HLN. No significant differences for the remaining cytokines were recorded between the V1 and V2 groups, but in the liver the V2 group shows significant increases of IFN-ɣ and IL-10 as compared to the uninfected control (UC) group whereas the V1 group did not. The lower expansion of FoxP3 T cells and lower increase of IFN-ɣ and IL-10 in the partially protected vaccinated group may be related with lower hepatic lesions and fluke burdens recorded in this group as compared to the other two infected groups. The most relevant change in regulatory cytokine gene expression was the significant increase of TGF-β in the liver of IC, V1 and V2 groups as compared to the UC group, which could be related to hepatic lesions.

Efficacy of a single-dose albendazole against lancet liver fluke Dicrocoelium dendriticum and liver enzymes activity in naturally infected sheep

Infections with D. dendriticum are distributed worldwide and mostly associated with ruminant livestock. Depending on the length and strength of the infection it can be manifested with losses in milk production, reductions in milk and wool quality, decreased weight gains, reproductive performance and poor carcass quality. The objective of this study was to determine the efficacy of albendazole (ABZ) against the lancet liver fluke Dicrocoelium dendriticum in naturally infected sheep using parasitological methods. Twenty-four sheep were divided into four groups: two untreated control groups (C1, C2) and two treated groups (T1, T2), with six animals in each group. The sheep in the treated groups were administered a single oral dose (15 mg/kg bwt) of ABZ suspension. After ABZ treatment the animals were slaughtered on Day 14 (groups C1, T1) and Day 30 (groups C2, T2) and were necropsied. Coprological therapeutic ABZ efficacy reached 92.4% on Day 14 (P < 0.001) and 88.5% on Day 30 (P < 0.001). On Day 30, the serum activities of hepatic and cholestatic enzymes including serological analysis of total protein concentration (TP) and protein fractions were evaluated. Significant decrease of aspartate aminotransferase (AST) (P < 0.01) and gamma-glutamyltransferase (GGT) (P < 0.05) activity by 36.9% and 34.6%, respectively, were detected for sheep in T2 group. These enzymes showed a strong positive correlation to fluke burden: AST (r = 0.654) and GGT (r = 0.768), respectively (P < 0.05). Additionally, the electrophoretic analysis of serum total protein and protein fraction concentrations revealed minimal hypoproteinemia and hyperalbuminemia after ABZ treatment. The decrease of liver enzyme activities and their correlation with fluke burden may indicate recovery of hepatocellular and biliary damage following the reduction of fluke burdens after ABZ therapy. A decline in AST and GGT activity could serve as a valuable adjunct bioindicator of liver damage and fluke reduction after treatment of dicrocoeliosis in naturally infected sheep.

Prevalence and associated risk factors assessment of bovine fasciolosis in the Imbo Region, Burundi

Fasciolosis is a zoonosis that limits the productivity of ruminants worldwide, but there is a lack of information on its occurrence in Burundi. Therefore, this study aimed to fill the information gap by determining the prevalence and risk factors associated with bovine fasciolosis in the Imbo Region of Burundi. Two prevalence studies were conducted in parallel in the five communes of the five provinces in the Imbo region. In the first study, a total of 426 fecal samples were collected from randomly selected cattle farms and microscopically examined to determine Fasciola egg burden. Survey data on cattle husbandry were collected from owners of these cattle and analyzed to determine the risk factors for bovine fasciolosis. In the second study, 467 cattle were randomly selected in abattoirs and their livers were examined postmortem to determine liver fluke burdens. Data were entered separately into Microsoft Excel and analyzed using R software. The overall prevalence of bovine fasciolosis was 47.7% (42.9–52.4, 95% CI) for microscopic examination and 33.2% (28.9–37.5, 95% CI) for postmortem examinations. The majority of positive cattle (60.6%) had light intensity infections as determined by eggs per gram of feces (epg). Postmortem examinations corroborated these results and indicated that 80% of cattle had light intensity infections. Chi-square analysis showed a statistical association with the presence of bovine fasciolosis and the age, sex, and origin of cattle and the practices of cattle owners (P < 0.05).

Fasciola hepatica antioxidant and protease-inhibitor cocktail recombinant vaccines administered five times elicit potent and sustained immune responses in sheep but do not confer protection

Our laboratory’s vaccine development strategy against the livestock parasite Fasciola hepatica centres around disrupting key biological processes by combining groups of antigens with similar/complementary functional actions into a single vaccine cocktail. In this study the focus was on antioxidant protein vaccines and a protease inhibitor vaccine aimed at disrupting the parasite’s ability to defend against oxidative stress and protease-inhibitor balance, respectively. Two combinations of recombinantly expressed antioxidants were assessed, namely peroxiredoxin (rFhPrx), thioredoxin (rFhTrx) and thioredoxin-glutathione reductase (rFhTGR) (Group 1) and rFhPrx, rFhTrx, and two superoxide dismutases (rFhSOD1 and rFhSOD3) (Group 2). The protease inhibitor vaccine cocktail included representatives of each of the key secreted protease inhibitor families, namely a Kunitz-type inhibitor (rFhKT1), a serpin (rFhSrp1) and a stefin, (rFhStf1) (Group 3). The vaccine combinations were formulated in adjuvant Montanide 61VG administered at five timepoints; two before experimental challenge with 60 F. hepatica metacercariae and three after infection. The vaccine combinations did not reduce the liver fluke burden, and only Group 2 displayed a marginal reduction in egg viability (8.2%). Despite previous results showing an effect of liver fluke vaccines on overall weight gain in infected animals, no significant (P value >0.05) impact on weight gain was observed in this study. Antibodies were elicited against all the vaccine antigens within the cocktails and were maintained at high levels to the end of the trial, due to our strategy of continuing vaccine administration after infection. However, these responses were not boosted by the challenge F. hepatica infection. A comparative analysis with previous vaccine data using a protease inhibitor vaccine found no repeat of the promising outcomes associated with this vaccine, indicating that the addition of rFhSrp1 to the vaccine cocktail did not improve vaccine efficacy. Assessment of liver pathology across the two trials using a modified liver enzyme score (glutamate dehydrogenase to platelet ratio) at eight weeks post infection suggests an association with liver fluke burden above 45 flukes, which could be used to predict liver pathology in future trials. The results reported in this study highlight the ambiguousness in liver fluke vaccine development and the difficulty in obtaining consistent and repeatable protection. This work stresses the need for repetition of trials and the use of sufficiently sized groups to assess vaccine efficacy with adequate statistical power.

Study of Prevalence of Paramphistomum cervi in domestic ruminants of Pokhara Lekhnath Metropolitan City, Kaski

Pramphistomum cervi has been a neglected but very crucial trematode parasite causing huge unseen economic loss in ruminant production globally. It has become an endemic species of fluke predominant in tropics and subtropics of Nepal. This study was aimed to estimate the overall prevalence of Pramphistomum cervi eggs in different domestic ruminant species of Pokhara Lekhnath Metropolitan City of Kaski district and to find the interaction of prevalence with other variables. A cross sectional study was performed to study the prevalence of Paramphistomum cervi eggs identified by Parasitological unit of Veterinary Hospital and Livestock Service Experts Centre, Kaski from January 2021 to April 2022. Out of 900 observations,842 observations with complete information were filtered out and included in our study. Statistical analysis was done with EpiInfo (Version 7.2.4.0) and graphical representation was done with MS Excel 2016. The overall prevalence of Pramphistomum cervi in different species of domestic ruminants was found to be 16.98%(143/842). The highest prevalence was seen in Cattle that was18.21% (45/247), followed by goats 16.66% (5/30) and buffaloes 16.46% (93/565). Seasonwise study of prevalence of Paramphistomum cervi in all the three ruminant species showed the highest overall infectivity during summer i.e 20.42% (88/431) followed by 13.38% (55/411) in winter and this difference was statistically significant (p-value 0.0065 at 95%CI). The higher prevalence of parasite was observed in adult lactating females of improved breeds. Thus, Paramphistomum cervi infection has become an important animal health issue to be addressed for safeguarding animal health and improvement of ruminant production in Pokhara Lekhnath Metropolitan City, Kaski. The biological intermediate host control measures in the pasture and routine screening of the parasite with routine deworming is essential to reduce the fluke burden among ruminants of Pokhara Metropolitan City in the future.

Proteomic analysis of exosome-like vesicles from Fasciola gigantica adult worm provides support for new vaccine targets against fascioliasis

BackgroundExtracellular vesicles (EVs) released by helminths play an important role in parasite-host communication. However, little is known about the characteristics and contents of the EVs of Fasciola gigantica, a parasitic flatworm that causes tropical fascioliasis. A better understanding of EVs released by F. gigantica will help elucidate the mechanism of F. gigantica-host interaction and facilitate the search for new vaccine candidates for the control and treatment of fascioliasis.MethodsTwo different populations of EVs (15k EVs and 100k EVs) were purified from adult F. gigantica culture media by ultracentrifugation. The morphology and size of the purified EVs were determined by transmission electron microscopy (TEM) and by the Zetasizer Nano ZSP high performance particle characterization system. With the aim of identifying diagnostic markers or potential vaccine candidates, proteins within the isolated 100k EVs were analyzed using mass spectrometry-based proteomics (LC–MS/MS). Mice were then vaccinated with excretory/secretory products (ESPs; depleted of EVs), 15k EVs, 100k EVs and recombinant F. gigantica heat shock protein 70 (rFg-HSP70) combined with alum adjuvant followed by challenge infection with F. gigantica metacercariae. Fluke recovery and antibody levels were used as measures of vaccine protection.ResultsTEM analysis and nanoparticle tracking analysis indicated the successful isolation of two subpopulations of EVs (15k EVs and 100k EVs) from adult F. gigantica culture supernatants using differential centrifugation. A total of 755 proteins were identified in the 100k EVs. Exosome biogenesis or vesicle trafficking proteins, ESCRT (endosomal sorting complex required for transport) pathway proteins and exosome markers, heat shock proteins and 14-3-3 proteins were identified in the 100k EVs. These results indicate that the isolated 100k EVs were exosome-like vesicles. The functions of the identified proteins may be associated with immune regulation, immune evasion and virulence. Mice immunized with F. gigantica ESPs, 15k EVs, 100k EVs and rFg-HSP70 exhibited a reduction in fluke burden of 67.90%, 60.38%, 37.73% and 56.6%, respectively, compared with the adjuvant control group. The vaccination of mice with F. gigantica 100k EVs, 15k EVs, ESP and rFg-HSP70 induced significant production of specific immunoglobulins in sera, namely IgG, IgG1 and IgG2a.ConclusionThe results of this study suggest that proteins within the exosome-like vesicles of F. gigantica have immunomodulatory, immune evasion and virulence functions. This knowledge may lead to new strategies for immunotherapy, vaccination and the diagnosis of fascioliasis.Graphical

A Study into the Identity, Patterns of Infection and Potential Pathological Effects of Rumen Fluke and the Frequency of Co-Infections with Liver Fluke in Cattle and Sheep

Rumen fluke (RF) are trematode parasites that have increased in prevalence within European ruminant livestock since the 1990s. Morbidity and mortality can result from the development of juvenile flukes in the duodenum, however, evidence for significant impacts of adult fluke in the rumen of the final host is equivocal. The presence of rumen fluke in Scotland had not previously been quantified, so the purpose of this study was to use historical coprological data collected between 2008 and 2018 at the School of Veterinary Medicine in Glasgow to evaluate the frequency of rumen fluke infection and to compare this with the presence of liver fluke. This analysis showed evidence of adult rumen fluke in 6.6% of samples submitted, with a substantial and significant increase in positive diagnoses from 2016, following which positive rumen fluke diagnoses equaled or slightly outnumbered those of liver fluke. A prospective post-mortem examination study was also carried out to determine the presence of rumen and liver fluke in cattle and sheep, to quantify adult rumen fluke burdens and to assess any pathological changes in the reticulorumen and proximal duodenum associated with infection. The presence of rumen fluke in post-mortem cases was 26.9% (n = 18/67), the majority (66.7%, n = 12/18) with burdens of less than 100 adult rumen flukes. There was no significant difference in mean ruminal papillar density and length in animals with and without adult rumen fluke and no significant gross pathology was observed in the rumen or reticulum. Examination of animals with adult rumen fluke provided no evidence of any consistent associations with acute or chronic inflammatory changes in the duodenum. All rumen fluke collected at necropsy were identified by PCR and sequencing as Calicophoron daubneyi.

Epidemiological tools to assess the spread of Fascioloides magna

The giant liver fluke (Fascioloides magna) has four continuously expanding focus endemics in Europe. The parasite is considered an invasive species in our continent and could affect the local host populations or, after host switching, it can infect other potential host species. Therefore it is important to track this alien species' presence. The authors compared the gold standard test with the effectiveness of two potential screening tests, the conventional sedimentation method and an illustrated guideline-based approach, which could be applicable in the field. The gold standard test was based on the necropsy detection of adult flukes in the liver tissue of hunted animals (N=319). Besides applying the linear regression, the sensitivity and specificity were determined in both approaches. The analysis showed that the shed egg number was moderately associated with the fluke burden (R2=0.5679; p&lt;0.0001) and the flukes' dry mass (R2=0.6016; p&lt;0.0001). The final results of sensitivity (100%; CI95%: 97.2 – 100) and specificity (96.3%; CI95%: 92.5 – 98.5) confirmed that the illustrated guideline-based approach is a capable method for monitoring the F. magna expansion in endemic areas.

Effect of rumen fluke on feed digestibility and methane emissions in sheep.

Thirty-six ewes aged 18months were assigned to one of three groups (n = 12) on the basis of faecal egg count (FEC) for rumen fluke; C: control (FEC = 0-5 epg), T: affected (FEC ≥ 6 epg) and treated, NT: affected (FEC ≥ 6 epg) and not treated. T ewes were treated with oxyclozanide 14days prior to the trial commencing. Ewes were fed grass silage ad libitum. Digestibility crates and respiration chambers were used to measure feed digestibility and methane production. Dry matter intake (DMI), feed digestibility and CH4/DMI did not differ (P > 0.05) between treatments. However, CH4/live weight (LW) was significantly greater (P < 0.001) for NT ewes, while that of C and T ewes were similar. This study indicates that a high rumen fluke burden can increase methane emissions but only when expressed on a body weight basis (per kg LW). As the link between rumen fluke infection and methane production has not previously been investigated within the literature, these findings demonstrate the need for further research to better understand these relationships.

Philophthalmus gralli in domestic waterfowl: An environmental study in an urban area from Brazil.

Philophthalmosis is a zoonotic ocular disease that affects mainly domestic and wild avian species in different parts of the world. However, aspects related to its epizoology in domestic animals are not completely known. In the present study, 47 Muscovy duck (Cairina moschata), 8 mallards (Anas platyrhynchos platyrhynchos) and 6 African geese (Anser cygnoides), free-living in an urban lake in Belo Horizonte, Minas Gerais, Brazil, were evaluated for eye fluke infection. The animals were manually contained, and the conjunctival sac and nictitating membrane were evaluated by direct ophthalmic examination (DOE), in which the fluke burden was estimated by a semiquantitative scale. Moreover, the conjunctival sac lavage (CSL) technique was applied for the search of parasite eggs. When possible, the detected trematodes were carefully removed for fixed for taxonomic identification. In order to characterize the parasite-host-environmental system, malacological collections were performed at the locality, and the collected intermediate host mollusks (Melanoides tuberculata) were evaluated for infection with larval philophthalmids. In addition, wild vertebrates found dead in the locality, including one common gallinule (Gallinula galeata) and two capybaras (Hydrochoerus hydrochaeris), were necropsied for the search of eye flukes. Samples from the developmental stages obtained from the hosts were subjected to morphological and molecular studies (amplification and sequencing of a partial region of the Nad-1 gene). In the DOE, the presence of eye flukes was verified in 67% (4/6), 31% (15/47) and 25% (2/8) in geese, Muscovy ducks and mallards, respectively. In most cases, the birds showed mild parasitism (1-5 trematodes), with no apparent eye damage, characterizing a subclinical infection. In only one goose, which had a high fluke burden (20 trematodes), it was possible to obtain eggs by CSL. Regarding the malacological survey, 73/4545 (1.6%) specimens of M. tuberculata evaluated were found infected with megalurous cercariae, compatible with representatives of the family Philophthalmidae. Additionally, 147 flukes were recovered from a specimen of G. galeata found dead at the lake. The morphological and molecular study of parasites obtained from different hosts evaluated in this study enabled the identification of Philophthalmus gralli, a species native from Asia and introduced in the Americas. Factors such as the wide distribution of the intermediate host and the participation of domestic and wild reservoirs in the parasite transmission chain may favor the occurrence of parasite spillover from wild to domestic birds. Furthermore, the occurrence of the life cycle of P. gralli in an urban waterbody serves as an alert to the possibility of human cases of this zoonotic eye parasite.

Bovine Natural Antibody Relationships to Specific Antibodies and Fasciola hepatica Burdens after Experimental Infection and Vaccination with Glutathione S-Transferase.

Fasciola hepatica is the causative agent of fasciolosis, a significant parasitic disease occurring worldwide. Despite ongoing efforts, there is still no vaccine to control liver fluke infections in livestock. Recently, it has been suggested that natural antibodies (NAbs) can amplify specific antibodies (SpAb) and have a direct killing effect, but it is unknown if this phenomenon occurs during parasitic helminth infection or targeted vaccination. NAbs are antibodies produced by the innate immune system, capable of binding antigens without prior exposure. This study explores the role of bovine NAbs, using the exogenous glycoprotein keyhole limpet hemocyanin (KLH), in response to F. hepatica infection and SpAb production after infection and vaccination. The cattle’s NAbs were differently influenced by parasite infection and vaccination, with an increase in KLH-binding IgG and IgM levels after infection and reduced KLH-binding IgM levels following vaccination. Underlying NAbs reacting to KLH showed no correlations to the final fluke burdens after experimental infection or vaccination. However, NAbs reacting to whole-worm extract (WWE) prior to infection were positively correlated to increased fluke burdens within the infected bovine host. Furthermore, after infection, the specific IgG reacting to WWE was positively reflected by the underlying NAb IgG response. Following subcutaneous vaccination with F. hepatica native glutathione S-transferase (GST), there was a non-significant 33% reduction in fluke burden. Vaccinated animals with higher underlying NAbs had a higher induction of vaccine-induced SpAbs, with trends observed between KLH-binding IgM and anti-GST IgG and IgM. Our findings provide a platform to allow further investigation to determine if NAb levels could mirror fluke-SpAb production for exploitation in a combined selective breeding and vaccination program. Additionally, this work suggests that liver fluke could possibly evade the host’s immune system by utilising surface-bound IgM NAbs.

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.

Sika Deer (Cervus nippon) are not "Dead-End Hosts" of the Giant Liver Fluke, Fascioloides magna (Bassi, 1875) Ward, 1917.

Fascioloides magna, although of North American origin, is well established in central Europe. Here it is expanding its geographic range and has been exposed to new potential final hosts including native and naturalized species. Based on their contribution to the propagation and transmission of F. magna, its final hosts have been grouped into three types including 'dead-end hosts' that comprise species in which flukes reach the liver but rarely mature and produce few eggs which are not excreted. Sika deer (Cervus nippon) were classified as dead-end hosts, this being quoted in the literature without robust substantiation. In an investigation on the occurrence of F. magna among sympatric wild ungulates in a recently established focus of the parasite in Germany, nine of 24 sika deer were found infected with up to seven F. magna in their livers, and six of the fluke-positive sika deer had Fascioloides eggs in their feces. Most flukes were recovered in pairs from fibrous capsules. Associated with the low fluke burden, gross pathology of the livers was generally mild. The presence of mature flukes in fibrous capsules, and passing of ova in the feces, demonstrates sika deer to be suitable definitive hosts of F. magna and to be of epidemiologic significance because of their implication in the transmission of the fluke.

Seasonality of salmonid parasites from flow‐through aquaculture in northern Germany: Emphasis on pathogenicity of Diplostomum spp. metacercaria

AbstractThe aim of the present study was to identify the parasite fauna of cultured rainbow trout (Oncorhynchus mykiss Walbaum, 1792) and Arctic charr (Salvelinus alpinus (L.)) from a flow‐through aquaculture system at Lake Tollense in northern Germany. The fish were sampled during different seasons in 2018 from an open freshwater raceway. For rainbow trout, 10 different parasite species were isolated, belonging to Monogenea (1), Cestoda (2), Crustacea (2) and Digenea (5), including the eye flukes Tylodelphys clavata Niewiadomska, 1984, Diplostomum pseudospathaceum and Diplostomum spathaceum (Rudolphi, 1819) Olsson, 1876, the latter representing a potential pathogenic species, which was molecularly identified and reported as a core species with prevalences of 80.0%–100.0% and high mean abundances of 42.8–55.0. Arctic charr only harboured T. clavata and D. spathaceum. Other taxa were found with low prevalence in the summer season exclusively, indicating strong seasonality of the detected parasites. Statistical correlation was positively tested for coherences of eye fluke burden and fish growth, weight and performance between seasons, recommending implementation of an enhanced aquaculture management for the surface water–dependent flow‐through aquaculture system at Lake Tollense.

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.

Analysis of daily variation in the release of faecal eggs and coproantigen of Fasciola hepatica in naturally infected dairy cattle and the impact on diagnostic test sensitivity

The liver fluke, Fasciola hepatica (F. hepatica) is a widespread parasite infection in dairy cattle in Victoria, South-eastern Australia. Robust diagnosis of fluke infection is needed in dairy cattle to identify sub-clinical infections which often go unnoticed, causing significant production losses. We tested the coproantigen ELISA (cELISA) and the FlukeFinder faecal egg count kit® on naturally infected cows in a fluke endemic region of Victoria. The aim of the study was to investigate the variation in the release of coproantigens and eggs into faeces over a 5-day period, at the morning (AM) and afternoon (PM) milkings, and to assess the impact of the timing of faecal sample collection on diagnostic test sensitivity. Ten cows were enrolled into the study based on positive F. hepatica faecal egg counts (LFEC) and faecal samples from the ten cows were collected twice daily, at the 7–9 AM and 4–6 PM milking, for five consecutive days. At the conclusion of the sampling period, the cows were euthanized and F. hepatica burden determined at necropsy. A moderate negative correlation between cow age and cELISA optical density (OD) was observed using data from all samples (R -0.63; 95 % CI -0.68 to -0.57). Over the 5-day sampling period, we observed within-animal variation between days for both the cELISA OD (2.6–8.9 fold) and LFEC (5–16 fold), with more variation in values observed in the PM samples for both tests. The correlation with total fluke burden was higher in the AM sampling using both the cELISA and LFEC (R 0.64 and 0.78, respectively). The sensitivity was 100 % for the cELISA using various cut offs from the literature (0.014 OD, 0.030 OD, and 1.3 % or 1.6 % of the positive control). The sensitivity of the FlukeFinder kit® (based on 588 faecal samples and not accounting for lack of independence in the data) was 88 % (95 % CI 85 %–90 %). Seventy one false negatives were recorded from the 588 LFEC tests all of which were observed in the cows with fluke burdens <14 flukes; 42 of the 71 false negative LFECs occurred in one individual cow which had the lowest burden of nine flukes. In dairy cows, the cut-off for production losses due to fasciolosis is estimated at> 10 fluke. Both the cELISA and the LFEC identified all cows that had burdens equal to or greater than this cut-off. Five of the ten cows also exhibited relatively high paramphistome egg counts.

Vaccination with cathepsin L phage-exposed mimotopes, single or in combination, reduce size, fluke burden, egg production and viability in sheep experimentally infected with Fasciola hepatica.

Fascioliasis is a worldwide emergent zoonotic disease that significantly constrains the productivity of livestock. In this study, fluke burdens, liver fluke size and biomass, faecal eggs counts, serum levels of hepatic enzymes and immune response were assessed in sheep vaccinated with peptide mimotopes of cathepsin L and infected with metacercariae. A total of 25 sheep were allocated randomly into five groups of five animals each, and experimental groups were immunised with 1×1013 filamentous phage particles of cathepsin L1 (CL1) (TPWKDKQ), CL2 (YGSCFLR) and mixtures of CL1+CL2 mimotopes, in combination with Quil A adjuvant, and wild-type M13KE phage in a two-vaccination scheme on weeks 0 and 4. The control group received phosphate-buffered saline. All groups were challenged with 300 metacercariae two weeks after the last immunisation and euthanised 16weeks later. The CL1 vaccine was estimated to provide 57.58% protection compared with the control group; no effect was observed in animals immunised with CL2 and CL1+CL2 (33.14% and 11.63%, respectively). However, animals receiving CL2 had a significant reduction in parasite egg output. Vaccinated animals showed a significant reduction in fluke length and width and wet weights. In the CL1 group, there was a significant reduction in the total biomass of parasites recovered. Egg development was divided into seven stages: dead, empty, unembryonated, cell division, eyespot, hatched and hatching. The highest percentage of developmental stages was detected for vaccinated sheep administered CL1+CL2 with cell division, and the lowest percentage was observed in the hatching stage. Furthermore, a significant difference in all developmental stages was observed between vaccinated animals and the control group (P<0.01). The levels of anti-phage total IgG in immune sera increased significantly at four weeks after immunisation and were always significantly higher for cathepsin L vaccine group than in the challenged control group. Total IgG was inversely and significantly correlated with worm burden in the CL1 group.