Prevalence and Intensity of Cardicola spp. Infection in Ranched Southern Bluefin Tuna and a Comparison of Diagnostic Methods

The parasitic blood flukes Cardicola forsteri and C. orientalis are an ongoing health concern for Southern Bluefin Tuna Thunnus maccoyii (SBT) ranched in Australia. In this study we compared the effect of treatment, company, and ranching year on blood fluke infections in ranched SBT. SBT were sampled during the 2018 and 2019 ranching seasons from praziquantel (PZQ) treated pontoons and untreated pontoons managed by two companies. Severity of infection was diagnosed by several criteria including adult fluke counts from hearts, egg counts from gill filaments and the use of specific quantitative polymerase chain reaction (qPCR) for detection of C. forsteri and C. orientalis ITS-2 DNA in SBT hearts and gills. PZQ treatment remains highly effective against C. forsteri infection. Prevalence and intensity of Cardicola spp. infection was lower in 2019 than 2018 for Company A in treated pontoons at week 12 and week 17 of ranching, and lower for Company A than Company B in untreated pontoons at month 5 of ranching. Results indicate re-infection may be less likely in the environment near Company A pontoons, and consistent years of treatment may have lowered the parasite load in the environment. qPCR demonstrated higher sensitivity when comparing diagnostic methods for C. forsteri in heart, and higher specificity when comparing diagnostic methods for Cardicola spp. in gills. Continuing to monitor blood fluke infections in ranched SBT can help to detect changes in drug efficacy over time and help industry to develop a best practice for treatment.

Anthelmintic Activity of Antioxidants: In Vitro Effects on the Liver Fluke Opisthorchis felineus

Currently, molecular parasitologists are searching for new agents against trematodiases. Redox metabolism is important for parasites as far as long-lived adult parasites inside a mammalian host are exposed to redox challenges. Antioxidants have been poorly studied as anthelmintic agents, in particular against the foodborne trematodes. Study of in vitro anthelmintic activity of nonenzymatic natural and synthetic antioxidants of various chemical structures was performed using standard motility and mortality assays against juvenile and adult Opisthorchis felineus worms. Promising agents have been found among both natural and synthetic compounds. The mitochondria-targeted antioxidant SkQ1 [10-(6′-plastoquinonyl)decyltriphenylphosphonium] in motility assays was as effective (half-maximal inhibitory concentration [IC50] 0.6–1.4 μM) as praziquantel (IC50 0.47–1.4 μM), and SkQ1 was significantly more effective than praziquantel in mortality assays. Moreover, extensive tegument damage of the adult fluke was revealed after SkQ1 treatment. Flavonoids manifested potency too, with IC50 values in a micromolar range (5.1–17.4 μM). Other natural and synthetic compounds tested against helminths were significantly less effective than praziquantel. Results of our study indicate that SkQ1 and flavonoids have high anthelmintic activities against the liver flukes. We propose that structure–activity relationship research might be worthwhile based on the structures of the most effective substances.

Characterization and localization of antigens for serodiagnosis of human paragonimiasis

Paragonimiasis is a foodborne trematode infection that affects 23 million people, mainly in Asia. Lung fluke infections lead frequently to chronic cough with fever and hemoptysis, and are often confused with lung cancer or tuberculosis. Paragonimiasis can be efficiently treated with praziquantel, but diagnosis is often delayed, and patients are frequently treated for other conditions. To improve diagnosis, we selected five Paragonimus kellicotti proteins based on transcriptional abundance, recognition by patient sera, and conservation among trematodes and expressed them as His-fusion proteins in Escherichia coli. Sequences for these proteins have 76–99% identity with amino acid sequences for orthologs in the genomes of Paragonimus westermani, Paragonimus heterotremus, and Paragonimus miyazakii. Immunohistology studies showed that antibodies raised to four recombinant proteins bound to the tegument of adult P. kellicotti worms, at the parasite host interface. Only a known egg antigen was absent from the tegument but present in developing and mature eggs. We evaluated the diagnostic potential of these antigens by Western blot with sera from patients with paragonimiasis (from MO and the Philippines), fascioliasis, and schistosomiasis, and with sera from healthy North American controls. Two recombinant proteins (a cysteine protease and a myoglobin) showed the highest sensitivity and specificity as diagnostic antigens, and they detected antibodies in sera from paragonimiasis patients with early or mature infections. In contrast, antibodies to egg yolk ferritin appeared to be specific marker for patients with adult fluke infections that produce eggs. Our study has identified and localized antigens that are promising for serodiagnosis of human paragonimiasis.

Transcriptome and secretome analysis of intra-mammalian life-stages of the emerging helminth pathogen, Calicophoron daubneyi reveals adaptation to a unique host environment.

Paramphistomosis, caused by the rumen fluke, Calicophoron daubneyi, is a parasitic infection of ruminant livestock which has seen a rapid rise in prevalence throughout Western Europe in recent years. Following ingestion of metacercariae (parasite cysts) by the mammalian host, newly-excysted juveniles (NEJs) emerge and invade the duodenal submucosa which causes significant pathology in heavy infections. The immature larvae then migrate upwards, along the gastrointestinal tract, and enter the rumen where they mature and begin to produce eggs. Despite their emergence, and sporadic outbreaks of acute disease, we know little about the molecular mechanisms used by C. daubneyi to establish infection, acquire nutrients and to avoid the host immune response. Here, transcriptome analysis of four intra-mammalian life-cycle stages, integrated with secretome analysis of the NEJ and adult parasites (responsible for acute and chronic disease respectively), revealed how the expression and secretion of selected families of virulence factors and immunomodulators are regulated in accordance with fluke development and migration. Our data show that whilst a family of cathepsins B with varying S2 sub-site residues (indicating distinct substrate specificities) are differentially secreted by NEJs and adult flukes, cathepsins L and F are secreted in low abundance by NEJs only. We found that C. daubneyi has an expanded family of aspartic peptidases, which is up-regulated in adult worms, although they are underrepresented in the secretome. The most abundant proteins in adult fluke secretions were helminth defence molecules (HDMs) that likely establish an immune environment permissive to fluke survival and/or neutralise pathogen-associated molecular patterns (PAMPs) such as bacterial lipopolysaccharide in the microbiome-rich rumen. The distinct collection of molecules secreted by C. daubneyi allowed the development of the first coproantigen-based ELISA for paramphistomosis which, importantly, did not recognise antigens from other helminths commonly found as co-infections with rumen fluke.

Occurrence, Morphometric, and Molecular Investigation of Cattle and Buffalo Liver Adult Fluke in Peninsular Malaysia Main Abattoirs

Fascioliasis is a parasitic disease of human and animal caused by Fasciola gigantica (F. gigantica) and Fasciola hepatica (F. hepatica). More than 700 million of grazing animals and over 180 million human population are at the risk of fascioliasis. This study was conducted in Banting, Ipoh, Shah Alam, and Taiping abattoirs within Peninsular Malaysia to determine the occurrence and identify the species of liver flukes, causing liver condemnation in cattle and buffaloes. Within a study period from January to December 2018, a total of 25 condemned bovine livers were collected from Banting, Ipoh, Shah Alam, and Taiping abattoirs and analyzed. Taiping abattoir had the highest occurrence of fascioliasis [14/1014 (1.38%)], and Shah Alam abattoir had the least occurrence [1/3377 (0.03%)]. From all the sampled livers, the average number of adult flukes recorded ranged from 1 to 83. A total of 440 adult flukes were studied morphometrically, using parameters such as body length (BL), body width (BW), cone length (CL), cone width (CL), body area (BA), body perimeter (BP), the distance between the ventral sucker and posterior end of body (VS-P), BL/BW ratio, BL/V-SP, and body roundness (BR). Furthermore, molecular analysis was conducted using PCR-RFLP methods to distinguish between F. gigantica and F. hepatica involving ITS1 primer and RsaI restriction enzyme. RFLP pattern with RsaI produced a consistent pattern of 360,100 and 60 bp fragments in F. hepatica, whereas F. gigantica worms had a profile of 360,170, and 60 bp in size. The morphometric and molecular analysis results indicated that cattle and buffaloes slaughtered at Banting, Shah Alam, Taiping, and Ipoh abattoirs were infected with F. gigantica.

Human case of Fasciola gigantica-like infection, review of human fascioliasis reports in Nepal, and epidemiological analysis within the South Central Asia

The diagnosis of a 22 year-old male patient from Kerabari, Morang District, Nepal led to the review of human fascioliasis cases and analysis of the epidemiological situation in that country not included in the WHO fascioliasis map. Symptom onset one month before egg detection and normal levels of ALT and AST did not agree with the 3–4-month migratory period of fascioliasis. A shorter acute phase may happen when the main biliary duct is reached by the migratory juveniles directly from the intestinal lumen. The causal agent was ascribed to F. gigantica-like worms after considering adult fluke morphology, altitude of the patient’s infection area, fasciolid characteristics in the neighbouring Bangladesh, and lymnaeid snail vector species known in Nepal and in the patient’s infection area. Previous reports of human infection by Fasciola in Nepal are reviewed. The patient in question proved to be the twelfth case and the first in whom a F. gigantica-like infection is reported. In Nepal, the wide geographical distribution of livestock fascioliasis, with high prevalences in buffaloes, cattle and goats, and the reports of Fasciola-infected schoolchildren close to the capital Kathmandu, give rise to concern on the situation in remote rural areas in a country where most of the population lives in rural areas. Moreover, the climate change impact in Nepal remembers Pakistan, where human fascioliasis emergence has been related to climate change and man-made irrigation. All in all, the present analysis suggests that human infection by Fasciola may be underestimated in Nepal.

Opisthorchis felineus Infection and Harmful Metals Bio-Concentration: A Pilot Study in Hamster Model

Nowadays, the question about the negative impacts of liver fluke on the host organism and mechanisms of this damage is open. The bio-concentration of some heavy and toxic metals in tissue of adult forms of Opisthorchis felineus and its accumulation in liver tissue and cardiac muscle in the Syrian Golden Hamster (Mesocricetus auratus) model was analyzed. It is outlined, that Opisthorchis felineus infection leads to accumulating aluminum in a liver tissue of the host. It was also found, that adult fluke bio-concentrates iron and aluminum in their tissue. The shortage of such essential elements as Mn, Zn and Сu in infected organism is discussed.

Biochemical Characterization and Differential Expression of a 16.5-Kilodalton Tegument-Associated Antigen from the Liver Fluke Fasciola hepatica

ABSTRACTA cDNA encoding a 16.5-kDa protein termed FhTP16.5 was identified by immunoscreening of a cDNA library fromFasciola hepaticaadult flukes using pooled sera from rabbits infected withF. hepaticafor 4 weeks. Quantitative reverse transcriptase PCR (qPCR) analysis revealed that FhTP16.5 is not expressed in unembryonated eggs. It is poorly expressed in miracidia and highly expressed at the juvenile and adult stages; however, significant differences were found between the expression levels of FhTP16.5 in juveniles versus adult flukes. Recombinant FhTP16.5 was expressed at high levels inEscherichia coli, purified by affinity chromatography, and used to raise anti-FhTP16.5 polyclonal antibodies in rabbits. Immunoblot analysis using the anti-FhTP16.5 IgG antibody identified FhTP16.5 in crude and tegumental extracts and in excretory-secretory products ofF. hepatica. The protein was not detected in crude extracts ofSchistosoma mansoniorSchistosomajaponicum. Antibodies to FhTP16.5 were detected in the sera of rabbits at 3 to 12 weeks ofF. hepaticainfection as well as in the sera of humans with chronic fascioliasis; these findings suggest that FhTP16.5 could be a good antigen for serodiagnosis of fascioliasis. Immunohistochemistry demonstrated that FhTP16.5 localizes to the surface of the tegument of various developmental stages and in parenchymal tissues of the adult fluke. Such specific localization makes FhTP16.5 an attractive target for immunoprophylaxis or chemotherapy.

Using lectins to identify hidden antigens in Fasciola hepatica

Fasciola hepatica is the causative agent of fascioliasis, one of the most economically important helminth diseases of livestock worldwide. Traditionally, fascioliasis has been controlled by the strategic use of fasciolicidal drugs, but the emergence of resistant parasites has spurred an interest in developing vaccines as an alternative means of control. Most vaccine studies to date have evaluated conventional antigens, which are exposed to the host's immune system during the course of a natural infection. ‘Hidden’ antigens have proven to be effective vaccine candidates in other parasite species, most notably the blood-feeding nematode parasite, Haemonchus contortus, and tend to be expressed in the intestine or gut of the parasite. Fasciola hepatica is known to ingest large quantities of blood and may be vulnerable to this approach. Most, if not all, of the candidate antigens identified thus far have been membrane-bound glycoproteins which were solubilized by detergents. Here, we have attempted to employ lectins to select gut-associated glycoproteins from complex mixtures of somatic extracts of adult F. hepatica. We have conducted a comprehensive lectin-binding screen on adult histological sections with a panel of 16 fluorescently labelled lectins. Seven of the lectins bound to molecules within the gastrodermis but also bound to a range of other tissues. Within the gut tissues, jacalin and peanut lectins bound selectively to the gut lamellae and gastrodermal cells, respectively. These lectins were then used to isolate proteins from the integral membrane protein component of the adult fluke. Both lectins showed selectivity for relatively simple subsets of proteins compared to the original crude extracts.