Programmed knockout mutation of liver fluke granulin, Ov-grn-1, attenuates virulence and impedes malignant transformation during chronic opisthorchiasis

Infection with the food-borne liver fluke Opisthorchis viverrini is the principal risk factor for cholangiocarcinoma (CCA; bile duct cancer) in the Lower Mekong River Basin countries including Thailand, Lao PDR, Vietnam, Myanmar and Cambodia. Using gene edited liver flukes in a hamster model of CCA involving concurrent infection and administration of sub-carcinogenic levels of nitrosamine, we explored the role of the fluke granulin-like growth factor Ov-GRN-1 in malignancy. We produced programmed gene knockout flukes (ΔOv-grn-1) by delivery of a CRISPR/Cas9/gRNA system by square wave electroporation. Targeted genome sequencing confirmed Cas9-catalyzed mutations in target parasite genes, and the rapid depletion of transcripts and the targeted proteins. Hamsters were infected with gene edited larval parasites and exposed to sub-carcinogenic levels of dimethyl nitrosamine in drinking water. Whereas Ov-grn-1 gene-edited parasites colonized the biliary tract of hamsters and developed into adult flukes, less hepatobiliary tract disease manifested during chronic infection with ΔOv-grn-1 worms in comparison to hamsters infected with control parasites. Specifically, immunohistochemical analysis of thin sections of hamster livers revealed markedly less fibrosis surrounding flukes and less global liver fibrosis as a result of infection with ΔOv-grn-1 worms, minimal biliary epithelial cell proliferation, and markedly fewer mutations of TP53 in biliary epithelial cells. Moreover, fewer hamsters developed high grade CCA when infected with ΔOv-grn-1 flukes compared to controls. The clinically-relevant pathophysiological phenotype of the livers of the hamsters confirmed a role for this parasite secreted growth factor in morbidity and malignancy during opisthorchiasis.

Csi-let-7a-5p delivered by extracellular vesicles from a liver fluke activates M1-like macrophages and exacerbates biliary injuries

Chronic infection with liver flukes (such as Clonorchis sinensis) can induce severe biliary injuries, which can cause cholangitis, biliary fibrosis, and even cholangiocarcinoma. The release of extracellular vesicles by C. sinensis (CsEVs) is of importance in the long-distance communication between the hosts and worms. However, the biological effects of EVs from liver fluke on biliary injuries and the underlying molecular mechanisms remain poorly characterized. In the present study, we found that CsEVs induced M1-like activation. In addition, the mice that were administrated with CsEVs showed severe biliary injuries associated with remarkable activation of M1-like macrophages. We further characterized the signatures of miRNAs packaged in CsEVs and identified a miRNA Csi-let-7a-5p, which was highly enriched. Further study showed that Csi-let-7a-5p facilitated the activation of M1-like macrophages by targeting Socs1 and Clec7a; however, CsEVs with silencing Csi-let-7a-5p showed a decrease in proinflammatory responses and biliary injuries, which involved in the Socs1- and Clec7a-regulated NF-κB signaling pathway. Our study demonstrates that Csi-let-7a-5p delivered by CsEVs plays a critical role in the activation of M1-like macrophages and contributes to the biliary injuries by targeting the Socs1- and Clec7a-mediated NF-κB signaling pathway, which indicates a mechanism contributing to biliary injuries caused by fluke infection. However, molecules other than Csi-let-7a-5p from CsEVs that may also promote M1-like polarization and exacerbate biliary injuries are not excluded.

Molecular characterization of Fasciola isolates collected from sheep, goats and cattle in Kisumu, Baringo and Narok Counties, Kenya

Fasciolosis is a neglected trematode infection of public health and veterinary importance caused by Fasciola gigantica and Fasciola hepatica. Molecular analysis using the internal transcribed spacers’ ITS-1 and ITS-2 of nuclear ribosomal DNA is useful in distinguishing Fasciola species. This study aimed to characterize liver flukes from sheep, goats and cattle using these genetic markers. Fifty nine adult Fasciola specimens were collected from livers of naturally infected sheep, goats and cattle at selected abattoirs in Kisumu, Baringo and Narok Counties. Sequence comparison of ITS-1 and ITS-2 sequences of Fasciola isolates from this study and sequences in Genbank was carried out. A maximum likelihood tree was constructed for phylogenetic analysis. Analysis of ITS-1 and ITS-2 rDNA sequences revealed that F. hepatica and F. gigantica caused infection in both cattle and sheep and in goats only F. gigantica caused infection. The sequenced PCR amplicons showed a close relationship between Fasciola species in this study with Fasciola isolates from other regions in the world. Phylogenetic analysis showed that sequences of F. hepatica are similar to the sequence from Spain, China and Tunisia obtained from GenBank. The sequences of F. gigantica in this study have similarity to the sequence from Iran and Burkina Faso. Data from this study provides information that serves as basis for further studies on the distribution of F. gigantica and F. hepatica in other localities in Kenya, and is also important in designing epidemiological and control programmes for zoonotic fascioliasis.

PSIII-6 Relationship of geographical location, cattle type, sex, and pen size to rates of liver abnormalities

Bovine liver abscess outcomes of individual animals (n = 989,296) were observed from 2013 to 2021 on a pen (n = 6,420) basis to quantify liver abscess prevalence and severity, as well as other liver abnormalities such as liver flukes. Cattle originated from 122 feedlots located in Arizona, Colorado, Idaho, Kansas, Minnesota, Missouri, Nebraska, Oklahoma, and Texas. Livers were audited at eighteen different beef abattoirs. Observational categories included sex (heifer, steer), cattle type (Holstein, beef-type), and pen count. Pen counts were grouped (< 40, 41–80, 81–160, 161–320, and >320) to quantify the association of pen size on liver abscess outcomes. Data were analyzed as a series of binomial proportions using generalized linear mixed models with fixed effects of observational categories (cattle type, feedyard location, pen size and sex). Cattle fed in Oklahoma or Texas exhibited greater (P < 0.01) frequency of liver flukes (3.2% vs 0.5%) than cattle fed in Colorado or Nebraska. Cattle fed in Colorado, Kansas, Missouri, Nebraska, Arizona, and Oklahoma exhibited fewer (P < 0.01) total abscesses (23.5%, 26.0%, 23.6%, 19.3%, 16.9%, and 18.1%) than cattle fed in Idaho, Minnesota, and Texas (36.4%, 36.4%, and 33.2%). Beef-type cattle were observed to exhibit fewer (P < 0.01) total abscesses (22.0% vs 41.1%) and fewer (P < 0.01) severe abscesses (10.2% vs 26.4%) than Holstein cattle. Likewise, native heifers exhibited fewer (P < 0.01) total abscesses (20.6% vs 23.3%) and fewer (P < 0.01) severe abscesses (9.3% vs 11.1%) than native steers. Abscess prevalence differed as a function of pen size. Both total abscess and severe abscess rates decreased (P < 0.01) as animal count within a pen increased (32.66%, 27.47%, 27.86%, 23.17%, and 20.33%; 17.20%, 14.48%, 14.80%, 11.45%, and 9.14%). These results suggest liver abscess prevalence and severity is affected by a wide range of factors.

Gastroenterology

Disorders of the mouth and pharynx?, Dyspepsia, dysphagia and reflux, Upper GI bleeding, Oesophageal varices, Acute abdominal pain, Acute diarrhea, Persistent diarrhea, Compendium of diarrhea-causing pathogens, Travelers’ diarrhea, Food poisoning, Intestinal flukes, Schistosomiasis (bilharzia), Soil-transmitted helminths (STH), Toxocariasis, Perianal complaints, Acute pancreatitis, Biliary disease, Liver flukes, Liver disease, Viral hepatitis, Alcohol and drug-induced hepatitis, Chronic liver disease and cirrhosis, Portal hypertension, Liver failure, Amoebic liver disease, Liver cancer, Hydatid disease

Rumen (Calicophoron/Paramphistomum spp.) and Liver Flukes (Fasciola hepatica) in Cattle—Prevalence, Distribution, and Impact of Management Factors in Germany

This study was carried out to determine the prevalence of rumen flukes on German cattle farms via the sedimentation technique, and to identify the rumen fluke species occurring in Germany. Additionally, the prevalence of patent Fasciola hepatica infections was determined. Furthermore, a short questionnaire was answered by the farmers. A prevalence of 5.5% and 9.5% was detected for rumen flukes and liver flukes, respectively. Coinfections occurred on 2.1% of farms. In northern Germany, the rumen fluke prevalence was higher than in southern Germany, while for liver fluke the distribution was reversed. Rumen flukes were mostly identified as Calicophoron daubneyi, but in four cases, sequencing revealed Paramphistomum leydeni for the first time in Germany. Grazing and feeding of fresh grass, as well as organic farming, were significantly associated with rumen and liver fluke occurrence. In contrast, suckler cow husbandry only had an influence on the occurrence of rumen flukes, but not liver flukes. Trematode eggs could be detected in both, farms with and without deworming. Since there were only a few studies about Paramphistomidosis in Germany, more attention should be paid to these parasitic diseases for animal welfare and animal health reasons.

Quantitative Monitoring of Selected Groups of Parasites in Domestic Ruminants: A Comparative Review

Parasites have had a significant impact on domestic ruminant health and production for a long time, but the emerging threat of drug resistance urgently requires an improved approach to parasite monitoring and control activities. The study reviewed the international literature to analyze the different proposals for the sampling approach and the quantitative estimation of parasite burdens in groups of animals. Moreover, the use of thresholds to decide when and which animal to treat was also investigated. The findings of the study highlighted the presence of a wide-ranging literature on quantitative monitoring for gastrointestinal nematodes (GIN), while more limited data were found for coccidia, and no specific indications were reported for tapeworms. Concerning liver flukes, bronchopulmonary nematodes (BPN) and permanent ectoparasites (lice and mange mites), the diagnostic process is usually aimed at the detection of the parasite rather than at the burden estimation. The main research gaps that need further investigation were also highlighted. For some groups of parasites (e.g., GIN and coccidia) the quantitative approach requires an improved standardization, while its usefulness needs to be confirmed for others (e.g., BPN and lice). The development of practical guidelines for monitoring is also encouraged.

In Vitro Assessment of Anthelmintic Activities of AgO Nanoparticle in Comparison to Closantel Against Liver Fluke Dicrocoelium Dendriticum

Background: Dicrocoeliasis is a rare Food-Born parasitic disease of the grazing herbivores as well humans, caused by Dicrocoelium dendriticum making severe pathological changes of the liver and bile systems, and therapeutic options for treatment are limited. With the appearance of drug resistance in liver flukes, there is a need to focus on alternative approaches to control helminth parasites of veterinary importance. Because of low-performance medications; drug delivery poses a great challenge for better treatment of Dicrocoeliasis. The current study aims to determine the anthelmintic properties of silver oxide nanoparticles (AgO) as a new method in dicrocoeliasis treatment, in vitro assay.Methods: The impacts of various concentrations of AgO nanoparticles (50-200 µg/ml) for 12-24 hours were compared with the Closantel, as the chemical drug. The anthelmintic efficacy was measured by scanning electron microscopy (SEM) technique.Results: SEM images of treated worms by AgO (200 µg/ml) showed severe damage, including complete loss of sensory papillae and destruction of prominent network structures and tegument vesicles. The mortality rates how the anthelmintic properties of AgO were highly relied on time and concentration, as far as increasing the time and concentration cause increasing the mortality rate. According to the MTT assay, the toxicity of AgO, at concentrations, 800 µg/ml is 8.7 %. Conclusions: Hence, it could be concluded that AgO NPs performed anthelmintic properties effects. To the best of our knowledge, no previous reports have assessed the effect of AgO NP on liver fluke D.dendriticum. Therefore, the present study provides a basis for future research on the control of this common trematode.

Seroprevalence of Major Pasture-Borne Parasitoses (Gastrointestinal Nematodes, Liver Flukes and Lungworms) in German Dairy Cattle Herds, Association with Management Factors and Impact on Production Parameters

Pasture-borne parasites adversely affect bovine health and productivity worldwide. In Europe, gastrointestinal nematodes, especially Ostertagia ostertagi, the liver fluke Fasciola hepatica and the lungworm Dictyocaulus viviparus represent the most important parasites of dairy cattle. The present study assessed exposure towards these parasites among 646 cattle herds in three parts of Germany during 2017–2019 via antibody detection in bulk tank milk (BTM). Overall, O. ostertagi levels indicative of production losses were detected in 41.2% (266/646; 95% confidence interval (CI): 37.4–45.1%) of BTM samples, while F. hepatica seroprevalence amounted to 14.9% (96/646; 95% CI: 12.2–17.9%). Only 2.3% (15/646; 95% CI: 1.4–3.9%) of samples were D. viviparus antibody-positive. Significantly lower O. ostertagi as well as F. hepatica seroprevalence was detected in dual-purpose breeds compared to high-performance breeds from the same region. Management factors related to parasite exposure included access to fresh grass and hay, silage quality and anthelmintic treatment. Furthermore, F. hepatica and O. ostertagi seropositivity was significantly associated with suboptimal herd-level body condition. Interestingly, the relationship between seropositivity and productivity differed between breed types. Negative impacts on milk yield were detected only in high-performance breeds, while O. ostertagi seropositivity was associated with a lower milk fat content in dual-purpose herds.