Gross examination and histopathological evaluation At six time points after infection (indicated above), ani- mals in each infected group were sacrificed, their livers were harvested and examined for pathological lesions and the presence of the flukes. Parasite eggs were recov- ered by filtering bile fluid through a 0.15 mm pore size mesh. Fasciolagiganticainfection was confirmed by ob- serving gross pathological lesions, associated with flukes in the livers and/or by the presence of flukes and eggs in the bile ducts. Samples of liver tissue (~8 g) showing pathological lesions were collected from each animal. Tissue samples were resuspended in 10% PBS-buffered formalin solution overnight, then dehydrated in alcohol, rinsed in xylene, and embedded in paraffin. 3 μm sec- tions of paraffin-embedded tissue were mounted onto glass slides, and stained with hematoxylin and eosin (H&E). Stained tissue sections were examined micro- scopically at 400× magnification and imaged using a Zeiss Axio Imager manual upright research microscope. Additional liver tissue samples for RNA extraction were collected and kept in RNA store buffer (Tiangen
Tropical fasciolosis is regarded as one of the most important helminthic infections of ruminants in Asia and Africa. Throughout most of its geographical range, Fasciolagigantica is of great importance as a parasite in sheep. The aim of this study was to detect seroprevalence of fascioliasis in sheep in Khouzestan province. In this study, due to the importance of livestock diseases in Khozestan province, total of 519 sheep from different areas in Khouzestan province were randomly bleeded, and their sera were preserved at 20°C. All sera were evaluated for anti- Fasciolagigantica excretory-secretory antigen by an in-house ELISA test. The results obtained from sheep show that the prevalence rate is 15.2 % (prevalence in female and male sheep was 14.3% and 25%, respectively). The prevalence rate in the age group of 4-5 years was higher than other groups, and it was lower in the age group of 2-3 years. than other groups, in Baghmalek the prevalence rate was higher area than other areas of the province. According to the results and the importance of this disease and its high prevalence, it is necessary to use appropriate strategies for controlling, preventing and treating it in the sheep in Khouzestan.
Eighteen buffaloes, 8–10-month-old, were purchased from a water buffalo farm in Guangxi Zhuang Autono- mous Region, PR China. Animals were randomly divided into two groups: (i) nine for the non-infected control group and (ii) nine for the infected group. Each group was further subdivided into 3 subgroups, each of 3 buf- faloes. To rule out any prior infection with F. gigantica, faecal examination and ELISA testing using IgG and IgM antibodies against F. gigantica were performed . Also, after an acclimatization period of 2 weeks, all buf- faloes were treated with triclabendazole 5% w/v oral sus- pension in order to eliminate any liver flukes. After four weeks of the withdrawal time, nine buffaloes were in- fected orally with 500 viable metacercariae per animal, whereas control animals were mock-inoculated with 0.85% NaCl solution without metacercariae . At 3, 42 and 70 days post-infection (dpi), three animals from each of the infected and control groups were sacrificed and their livers were collected and stored at -80 °C until analysis. Liver was selected because it is the target organ and preferable habitat of F. gigantica flukes in the defini- tive host (buffaloes) and in the mean time it performs many vital physiological, metabolic and immunological functions in the body . At necropsy, whole blood samples of all animals were collected aseptically into tubes without anticoagulant and were separated by cen- trifugation for collection and testing of the sera. Fasciolagiganticainfection was also confirmed by observing gross pathological lesions associated with the presence of adult flukes in the liver of infected animals.
KEGG pathway analysis identified several pathways involved in immune response, receptor signaling, can- cers and some diseases. Among these, immune response- related pathways have caught our particular interest. The pronounced DNA hypermethylation of TLR2, TLR4 and IL-12B (the most functional unit of IL-12), and the high level of hydroxymethylation of TNF in treated DCs were enriched in multiple pathways. Studies have shown that TLR4-dependent IL-12 productions play crucial roles in promoting DC maturation and the pro-inflammatory immune response to eliminate pathogens from the infec- tion . TNF, which can be mediated by both TLR2- dependent and TLR2-independent pathways [42–44], is known to induce a semi-mature phenotype and a lim- ited activation of DCs . In agreement with the pre- diction of (h)MeDIP-Seq, our qRT-PCR analysis showed that the mRNA levels of TLR2, TLR4 and IL-12B were downregulated, and TNF transcription was increased. This may contribute to the suppression of buffalo DC maturation. It is known that liver fluke infection utilizes their ESPs to weaken immune cells and reduce the Th1- type response through disrupting TLR signaling . Fasciolagiganticainfection in buffaloes has shown to suppress the TLR4 signaling pathway [47, 48]. Similarly, the expression of TLR2 and TLR4 in immune cells could be suppressed by ESPs of F. hepatica [9, 10, 49]. In addi- tion, the NF-κB signaling pathway enriched by several hypermethylated genes (including TLR4) may indicate a Fig. 7 Comparative sketch of the relative fold changes between (h)
Human sera. Sera were obtained from serum banks of different departments of the Faculty of Medicine, Khon Kaen University (1992 to 2003). Each serum was aliquoted and stored at ⫺70°C until used. The study protocol was approved by the Human Research Ethics Committee of Khon Kaen University. Informed consent was obtained from all adult participants, parents, or legal guardians. Thirteen serum samples were obtained from parasitologically confirmed F. gi- gantica cases. Each of these was confirmed on the basis of removing F. gigantica adult worms during cholecystectomy, T-tube choledochostomy, or other bile duct operations. To evaluate potential cross-reactivity, 209 serum samples from indi- viduals with parasitic diseases other than fasciolosis were included. These sam- ples were obtained from parasitologically confirmed cases of paragonimiasis (n ⫽ 25), opisthorchiasis (n ⫽ 25), ascariasis (n ⫽ 14), hookworm infection (n ⫽ 12), strongyloidiasis (n ⫽ 30), capillariasis philippinensis (n ⫽ 12), gnathostomiasis (n ⫽ 13), angiostrongyliasis (n ⫽ 9), malaria (n ⫽ 14; eight with Plasmodium falciparum, five with P. vivax, and one mixed infection with P. falciparum and P. vivax), cysticercosis (n ⫽ 5), trichinosis (n ⫽ 17), echinostomiasis (n ⫽ 5), taeniasis (n ⫽ 4), trichuriasis (n ⫽ 4), amoebiasis (n ⫽ 3), and giardiasis (n ⫽ 2). Other parasitoses sera were obtained from cases with mixed parasitic infections (n ⫽ 15; four patients infected with Trichuris trichiura and Ascaris lumbricoides; three patients infected with T. trichiura and hookworm; four patients infected with T. trichiura, A. lumbricoides, and hookworm; one patient infected T. tri- chiura, Strongyloides stercoralis, and hookworm; three patients infected with T. trichiura, A. lumbricoides, S. stercoralis, and hookworm). Thirty-two serum sam- ples were obtained from patients who were residents of areas endemic for Opisthorchis viverrini and whose pathological features were compatible with a diagnosis of bile duct cancer or cholangiocarcinoma (nine of them were proven opisthorchiasis-associated cholangiocarcinoma). Negative control sera were ob- tained from 42 healthy adults who had stool examinations performed at the time of the blood collections using the formalin-ether concentration method (11), and no evidence for intestinal parasitic infections was found. Human sera were pooled as positive and negative reference sera by combining equal volumes of proven fasciolosis and healthy control sera, respectively.
because our infections were only carried out through five snail generations and the answer to this question would need the infection of a greater number of genera- tions. Two perhaps complementary hypotheses may be proposed to comment on these changes in successive generations of infected L. fuscus and R. balthica. The first would be to admit decreased genetic variation in host resistance through selection of snails with lowest resistance and/or a series of bottlenecks during the five successive generations of snails. This first hypothesis is based on the protocol used for the two lymnaeids, with the selection of offspring coming from already infected parents. However, another hypothesis, based on a pro- gressive decrease in the number of the snail ganglionic neurons through the successive generations of infected snails originating from already infected parents, cannot be excluded. This interpretation is supported with the report by Szmidt-Adjidé et al.  on the numbers of neurons in the model G. truncatula/F. hepatica. Accord- ing to these authors, many neurons in the dorsal lobes of cerebroid ganglia and in pedal ganglia of G. trunca- tula disappeared during snail infection and lost their function. If this neuronal death also exists in the corre- sponding ganglia of infected L. fuscus and R. balthica, the surviving neurons would gradually secrete a lower quantity of neuromediators  through successive gen- erations of infected snails. As a consequence, this de- crease could induce a progressive change in the mechanisms of the snail immune system response to F. hepatica and also a progressive lifting of bottleneck exerted by the snail on larval development of the parasite.
The expected PCR products of 456bp were produced in all morphologically like F. gigantica samples using ITS2 gene primers inde- pendently from the different geographical ori- gins and the different host species. Restriction fragment length polymorphism (RFLP) pat- terns of parasitic flukes were obtained after digestion of the PCR products with pagI en- zyme, in order to differentiate two fluke spe- cies (Fig. 2B). Based on sequence differences of amplified portions of the ITS2 gene be- tween F. gigantica and F. hepatica, the restriction enzyme pagI was expected to have one cutting site on the ITS2 PCR amplicons from F. gigan- tica. Accordingly, all of the ITS2 gene PCR amplicons of 456 bp from F. gigantica flukes were cut by the pagI endonuclease to frag- ments of 165 and 291bp. None of ITS2 PCR products from F. hepatica like samples was di- gested by the restriction enzyme treatment (Fig. 2B). Based on RFLP analysis, all samples confirmed to be F. gigantica (n=14) were sub- jected to PCR amplification of ND1 gene. The expected PCR products of 534 bp were produced in all reactions from F. gigantica sam- ples (Fig. 2A). In the current study, sequenc- ing results of the ND1 PCR products were used to characterize the genotypic diversity of the F. gigantica flukes obtained from endemic
infection rates than the traditional methods (9). In this study, the presence of the developmen- tal stages of F. gigantica in L. auricularia snails from North West of Iran was confirmed by using the PCR-RFLP analysis. This analysis has been used for discriminating the Fasciola species from different parts of the world by targeting diverse gene regions including 18SrDNA, ITS1, 5.8SrDNA, ITS2 and 28SrDNA (15, 16, 21-24). Ribosomal DNA is available in high copy number and contains variable sequences flanked by more conserved sections. It is, therefore, considered as a suita- ble genetic marker for genotyping, intraspe- cific variations and phylogenetic studies of organisms at different taxonomic levels (21, 23). Rokni et al. (24) could distinguish F. gigan- tica isolates from buffalos of Khuzestan, South West Iran by RFLP analysis of the ITS1 re- gion. They also suggested that digestion of ITS1 is a reliable method for differentiating F. gigantica from its congener species, F. hepatica. Mahami-Oskouei et al. (16) by restriction di- gestion of the ribosomal DNA spanning ITS1, 5.8SrDNA and ITS2 discriminated F. gigantica and F. hepatica isolated from sheep and cattle of three Iranian provinces. It was also realized from this study that the restriction enzyme AvaII is able to generate restricted DNA fragments from 28SrDNA based on which F. gigantica originating from distinct geographical locations can unambiguously be distinguished from the rest of Fasciola species. In a similar study, Saki et al. (25) using the digestion pat- tern of 28SrDNA generated by AvaII enzyme verified the infection with F. gigantica in the liver of different livestock from southwest Iran.
The spatial predictive model of rumen fluke infection was based on both surveillance data and ovine survey data, both collected between October 2014 and February 2015. Farm GPS locations of submitted samples to the RVL were extracted and the ovine survey data (which already included location information) were merged. Overall, presence or absence of infection from 477 herds and 351 flocks were included in the model. All cattle herds plus 61 sheep flocks belonged to the RVLs dataset, while the remaining 290 sheep flocks’ data derived from the cross-sectional sheep survey. Since veterinary advice in Ireland is not to treat routinely for rumen fluke infec- tions, we hypothesised that faecal examination data gave a truthful representation of infection. While the sensitiv- ity of the sedimentation technique for the detection of rumen fluke infection is unknown, it is assumed to be similar if not higher (due to the lack of routine treat- ment and therefore higher numbers of eggs per gram expected, as well as the fact that eggs are not secreted in the gall-bladder) to that for fasciolosis, which produces eggs of similar size and shape. The sensitivity for detect- ing F. hepatica infection using the sedimentation method can range 43 to 64% in cattle depending on the amount of faeces employed, but is thought to be higher in sheep due to lower variability in egg shedding and smaller volume of faeces produced. Specificity of the sedimentation method is generally estimated to exceed 95% [64, 65].
The proportion of cattle fasciolosis, paramphistomosis and schistosomosis were found higher in old (35.1%, 48.6% and 32.4% respectively) than young (16.5%, 37.6% and 28.4%) and adult (23.1%, 37.8% and 24.8%) cattle. There was no a statistical significance variation in the prevalence of single trematode parasitic infections between age groups of cattle (P-value > 0.05). Generally, the proportion of paramphistomosis was found highest in all age groups of cattle in the area. However, the prevalence of cattle fasciolosis was lowest in young cattle although the proportion of paramphistomosis was more or less the same in both young and adult cattle (37.6% and 37.8% respectively). There was no a concomitant infection with all Fasciola, Paramphistomum and Schistosoma in old cattle (Table 3). The current study indicated that there was no a statistical significant difference between mixed infection of trematodes and all associated risk factors apart from address of cattle (P-value > 0.05).
Results: The FgRab10 gene (618 bp), encodes 205-amino-acid residues with a molecular mass of ~23 kDa, had complete nucleotide sequence homology with F. hepatica Ras family protein gene (PIS87503.1). The rFgRab10 protein specifically cross-reacted with anti-Fasciola antibodies as shown by Western blot and immunofluorescence analysis. This protein exhibited multiple effects on goat PBMCs, including increased production of cytokines [interleukin-2 (IL-2), IL-4, IL-10, transforming growth factor beta (TGF- β ) and interferon gamma (IFN- γ )] and total nitric oxide (NO), enhancing apoptosis and migration of PBMCs, and promoting the phagocytic ability of monocytes. However, it significantly inhibited cell proliferation. Homology modelling revealed 63% identity between rFgRab10 and human Rab10 protein (Uniprot ID: P61026). Protein interaction network analysis revealed more stabilizing interactions between Rab proteins geranylgeranyltransferase component A 1 (CHM) and Rab proteins geranylgeranyltransferase component A 2 (CHML) and rFgRab10 protein. Gene Ontology analysis identified RabGTPase mediated signaling as the most represented pathway.
There are several methods for detecting and studying proteins which among them Double Dimension Electrophoresis is common and practical technique. Isoelectric Focusing Elec- trophoresis (IEF) separates proteins based on their isoelectric point. In this method, proteins move to one pole and at the isoelectric point accumulate in the special point of the gel (4). Subsequently, electrophoresis would be con- tinued using SDS-PAGE as second dimension. In this step, the proteins separate based on their molecular weight and after staining dif- ferent proteins appear as spots in the gel (5). SDS-PAGE technique has been used to com- pare ES and Somatic extract in F. hepatica and F. gigantica (6). Double Dimension Electro- phoresis (2DE) and Mass Spectrometry were used for comparison of proteome between ES products of F. hepatica in vitro and in vivo (7).
In another experiment, given that resistant individuals had always been challenged with Cuban isolates of F. hepatica, we exposed for the first time, susceptible and resistant P. columella from Cuba to two allopatric liver fluke isolates from the Caribbean (short distance) and Europe (large distance), and compared their infection outcomes with those of the sympatric Cuban isolate. The theory of local adaptation predicts that parasites perform better on their local (sympatric) hosts rather than foreign (allopatric) hosts . However, exceptions exist (e.g. [14, 19]), thus a differential exposure of a host population to an “ unknown ” entity (i.e. allopatric para- sites) might result in differential outcomes and can test for different patterns of susceptibility or even resistance. Given the geographical isolation between the parasite iso- lates used, genetic differences are expected and should ac- count for variations in compatibility with the snail host, particularly between the Cuban and the European isolates. With this experimental approach, it is likely that a higher infection success from exposing susceptible P. columella to sympatric F. hepatica would be observed, while the resulting outcome with resistant populations could give clues concerning the specificity of their resistance. If no infection occurs in resistant P. columella then we might hypothesize that this phenotypic response is not restricted only to local (Cuban) parasites but it has a broader or even global scale.
During acute phase of F. hepatica infection, larva migration, hemorrhages, infiltration with eosinophils, lymphocytes, and macrophages, fibrosis, and necrotic areas have been reported . Here, we have reported exacerbated liver damage in PD-L2 KO mice as denoted by hepatocyte enlargement, cytoplasmic vacuolization, and regions of cellular infiltration and foci of necrosis, hemorrhage and fibrosis. All these features might be associated with the unregulated Th1-type specific immune response developed by PD-L2 KO mice after F. hepatica infection. Consistent with these data, it has been reported that during HBV infection, the virus-specific Th1-type response is critical in the pathogenesis of fulminant hepatitis . It has been demonstrated that during T. cruzi infection pro-inflammatory Th1-type cytokines as TNF can exert deleterious effects on tissues , however we found similar levels of TNF in sera from PD-L2 KO and WT mice (data not shown).
Although E. granulosus (s.l.) is able to infect a wide range of mammalian hosts, the metacestode stage has a different capacity to produce protoscoleces, the stage in- fective to the definitive host. For unknown reasons, the parasite can, in some animals, produce protoscoleces in- side the cyst, generating a fertile hydatid cyst, but other animals with cystic echonococcosis possess cysts without protoscoleces called infertile hydatid cysts [8, 9]. Since cyst fertility is associated with the size of the hydatid cyst , there is a subset of hydatid cysts that are too small to be classified as either fertile or infertile. The cel- lular and molecular mechanisms involved in the process of cyst fertility remain unknown . In cattle, hydatid cyst fertility status ranges from 0 to 96% in different parts of the world [11–22] and infection with F. hepatica is common in many parts of the world. However, there are no studies on relationships between E. granulosus (s.l.) and F. hepatica in co-infections.
The parasite Fasciola hepatica is a major cause of economic loss to the agricultural commu- nity worldwide as a result of morbidity and mortality in livestock, including cattle. Cattle are the principle reservoir of verocytotoxigenic Escherichia coli O157 (VTEC O157), an impor- tant cause of disease in humans. To date there has been little empirical research on the interaction between F. hepatica and VTEC O157. It is hypothesised that F. hepatica, which is known to suppress type 1 immune responses and induce an anti-inﬂammatory or regu- latory immune environment in the host, may promote colonisation of the bovine intestine with VTEC O157. Here we assess whether it is statistically feasible to augment a prospective study to quantify the prevalence of VTEC O157 in cattle in Great Britain with a pilot study to test this hypothesis. We simulate data under the framework of a mixed-effects logistic regression model in order to calculate the power to detect an association effect size (odds ratio) of 2. In order to reduce the resources required for such a study, we exploit the fact that the test results for VTEC O157 will be known in advance of testing for F. hepatica by restricting analysis to farms with a VTEC O157 sample prevalence of >0% and <100%.
Other factors, which we were unable to measure in this study, should also be considered in any future study. For example, the age of cattle were not recorded and the previous BTB history of the premises was not known; a significant correlation between the age of the animal and the likelihood of it being BTB infected has been documented (de la Rua Domenech et al., 2006). In chapter 5, the stage of lactation is significantly correlated with F. hepatica exposure and this may be due to the time lapsed since last dry period when a flukicidal dose is often given. With time since last dry period we would expect that, assuming equal risk of exposure to metacercariae, the risk of infection would increase with time and season. Type of grazing; zero grazing, housing for winter and diet will all impact upon this risk in different ways and were not measured in this study (Urquhart et al., 1996). A further consideration is that other infectious diseases may also affect the outcome of the SICCT test. Bovine Viral Diarrhoea virus (BVDv) is a common pathogen of cattle that has been shown to reduce lymphocyte proliferation in response to PPD-A and PPD-B and the IFNγ assay in BTB infected animals co-infected with BVDv. This virus has an immunosuppressive effect on the bovine host immune system that could last for over 3 weeks in acute infections (Charleston et al., 2001). Calves experimentally infected with BVDv also shed significantly higher amounts of M. bovis indicating the potential for silent BTB transmission with this co-infection as well.
readings of ELISA in both stool and serum samples. Other studies have demonstrated that coproantigens are correlated with Fasciola egg counts  and the parasite burden [31,39,40]. As the fecal egg count is presumably dependent on the number of flukes in the host, one can postulate that stool antigen level in patients infected with Fasciola are directly related to the number of adult parasites. Therefore, the absence of coproantigen in one of our patients may be due to a very light parasite bur- den and consequently undetectable levels of antigens in stools [14,18]. On the other hand, Ubeira et al.  reported that there was no correlation between number of ova/gm stool and coproantigens levels measured by ELISA.