Gigantica Infection Research Articles

Detection of Fasciola gigantica infection by using nano-structured porous silicon

Detection of Fasciola gigantica infection by using nano-structured porous silicon

Combination Vaccines of Fasciola gigantica Saposin-like Protein-2 and Leucine Aminopeptidase.

Saposin-like protein-2 (SAP-2) and leucine aminopeptidase (LAP) are major proteins involved in the digestive process of Fasciola gigantica (Fg). Both SAP-2 and LAP are highly expressed in F. gigantica; therefore, they could be vaccine candidates for fasciolosis. The aims of this study are (1) to observe the tissue expression of F. gigantica SAP-2 (FgSAP-2) and F. gigantica LAP (FgLAP) in F. gigantica by indirect immunofluorescence technique under confocal microscopy and (2) to test the vaccine potentials of individual and combined recombinant (r) FgSAP-2 and rFgLAP against F. gigantica in Imprinting Control Region (ICR) mice (n = 10 per group). By indirect immunofluorescence-confocal microscopy, FgSAP-2 and FgLAP were localized in the caecal epithelium but at different sites: FgSAP-2 appeared in small granules that are distributed in the middle and lower parts of the cytoplasm of epithelial cells, while FgLAP appeared as a line or zone in the apical cytoplasm of caecal epithelial cells. For vaccine testing, the percent protection of combined rFgSAP-2 and rFgLAP vaccines against F. gigantica was at 80.7 to 81.4% when compared with aluminum hydroxide (alum) adjuvant and unimmunized controls, respectively. The levels of IgG1 and IgG2a in the sera were significantly increased in single and combine vaccinated groups compared with the control groups. Vaccinated mice showed reduced liver damage when compared with control groups. This study indicates that the combined rFgSAP-2 and rFgLAP vaccine had a higher vaccine potential than a single vaccine. These results support the further testing and application of this combined vaccine against F. gigantica infection in farmed livestock animals.

Secondary infection of Fasciola gigantica in buffaloes shows a similar pattern of serum cytokine secretion as in primary infection.

As a natural host of Fasciola gigantica, buffalo is widely infected by F. gigantica. Its impact on buffalo production has caused great losses to the husbandry sector, and repeat infection is non-negligible. In buffaloes experimentally infected with F. gigantica, primary and secondary infection have yielded the same rate of fluke recovery, indicating a high susceptibility of buffalo to F. gigantica, which contributes to the high infection rate. Determining the immunological mechanism of susceptibility will deepen the understanding of the interaction between F. gigantica and buffalo. Here, we explored the immune response of buffaloes against primary and secondary F. gigantica infection, with a focus on cytokines' dynamics explored through serum cytokine detection. Buffaloes were assigned to three groups: group A (noninfected, n = 4), group B (primary infection, n = 3), and group C (secondary infection, n = 3). Group B was infected via oral gavage with 250 viable F. gigantica metacercariae, and group C was infected twice with 250 metacercariae at an interval of 4 weeks. The second infection of group C was performed simultaneously with that of group B. Whole blood samples were collected pre-infection (0 weeks) and at 1-6, 10, and 12 weeks after that. The serum levels of seven cytokines (IFN-γ, IL-4, IL-5, IL-10, IL-13, TGF-β, and IL-17) were simultaneously determined using ELISA and further analyzed. In the present study, no significant changes in Th1-type cytokines production were detected in early infection, both in primary and secondary infections, while the Th2-type response was strongly induced. A comparison of primary and secondary infection showed no significant difference in the cytokine secretion, which may indicate that the re-infection at 4 weeks after primary infection could not induce a robust adaptive immune response. The full extent of interaction between buffalo and F. gigantica in re-infection requires further study.

Proteomic analysis of Fasciola gigantica excretory and secretory products (FgESPs) co-immunoprecipitated using a time course of infected buffalo sera.

Widespread Fasciola gigantica infection in buffaloes has caused great economic losses in buffalo farming. Studies on F. gigantica excretory and secretory products (FgESP) have highlighted their importance in F. gigantica parasitism and their potential in vaccine development. Identifying FgESP components involved in F. gigantica-buffalo interactions during different periods is important for developing effective strategies against fasciolosis. Buffaloes were assigned to non-infection (n = 3, as control group) and infection (n = 3) groups. The infection group was orally administrated 250 metacercariae. Sera were collected at 3, 10, and 16 weeks post-infection (wpi) for the non-infection group and at 0 (pre-infection), 1, 3, 6, 8, 10, 13, and 16 wpi for the infection group. FgESP components interacting with sera from the non-infection and infection groups assay were pulled down by co-IP and identified using LC-MS/MS. Interacting FgESP components in infection group were subjected to Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathway and gene ontology (GO) functional annotation to infer their potential functions. Proteins of FgESP components identified in the non-infection group at 3, 10, and 16 wpi accounted for 80.5%, 84.3%, and 82.1% of all proteins identified in these three time points, respectively, indicating surroundings did not affect buffalo immune response during maintenance. Four hundred and ninety proteins were identified in the infection group, of which 87 were consistently identified at 7 time points. Following GO analysis showed that most of these 87 proteins were in biological processes, while KEGG analysis showed they mainly functioned in metabolism and cellular processing, some of which were thought to functions throughout the infection process. The numbers of specific interactors identified for each week were 1 (n = 12), 3 (n = 5), 6 (n = 8), 8 (n = 15), 10 (n = 23), 13 (n = 22), and 16 (n = 14) wpi, some of which were thought to functions in specific infection process. This study screened the antigenic targets in FgESP during a dense time course over a long period. These findings may enhance the understanding of molecular F. gigantica-buffalo interactions and help identify new potential vaccine and drug target candidates.

Seasonal dynamics of Fasciola gigantica transmission in Prafi district, Manokwari Regency, West Papua, Indonesia.

Indonesia's farming practices are a perfect setting for establishing an infection with Fasciola gigantica which can result in economically detrimental. The objectives of the current study were to describe and analyze the transmission dynamics of fasciolosis (F. gigantica) in Prafi District, to provide information on effective control strategies and to identify risk factors associated with fasciolosis in cattle. Fecal samples were purposively collected from the rectum of 60 beef cattle in Prafi District, Manokwari Regency, West Papua Province, Indonesia. The samples were collected once a month for 8 months from April 2019 to November 2019. Furthermore, the samples were taken from two rearing system groups: 30 stall cattle and 30 cattle in a palm oil garden. The presence of F. gigantica eggs in the fecal samples was examined using a modified Danish Bilharziasis Laboratory technique-sedimentation. Meanwhile, the antigenic diagnosis of Fasciola in the fecal samples was analyzed using an enzyme-linked immunosorbent assay kit to perform an indirect sandwich assay on feces. Snails were collected from an irrigation canal, rice field, and palm oil garden canal around Prafi District. These snails were examined for infection with cercariae larvae of F. gigantica by cercarial shedding and crushing techniques. The peak occurrence of F. gigantica infection was identified in August (65.00%) and the lowest in June (35.00%). The highest prevalence of fasciolosis in cattle was recorded in August and November (90.00%) and the lowest was in May (40.00%). Moreover, the highest prevalence of fasciolosis in cattle exposed to the palm oil garden was recorded in April (53.33%) and the lowest prevalence of F. gigantica infection was recorded in June (23.33%). In total, 2046 snails were screened by the cercarial shedding and crushing method; of these, 426 (20.82%) were found to be positive for trematode cercariae. The prevalence of F. gigantica infection in Lymnaea spp. snails was highest in November (47.46%) and lowest in April (9.28%). The current study shows that beef cattle grown in two types of rearing systems in Prafi District were infected with F. gigantica during the dry and rainy season. It was revealed that Lymnaea spp. are common snails found in and around Prafi District, and can act as intermediate hosts with an infective stage of trematode. The transmission to cattle was highly effective, despite the short activity period, the low infection rate of snails, and the incidental anthelminthic treatment.

Development of a colloidal gold immunochromatographic strip for the rapid detection of antibodies against Fasciola gigantica in buffalo

BackgroundFasciola gigantica, a tropical liver fluke, infects buffalo in Asian and African countries, causing significant economic losses and posing public health threats. The diagnostic of buffalo fascioliasis caused by F. gigantica is vital in fascioliasis control and preventation. The 22nd gel filtration chromatography fraction of F. gigantica Excretory-Secretory Products (FgESP), namely Fasciola 22 (F22), which was used as a diagnostic antigen in indirect ELISA, has demonstrated great potential for fascioliasis diagnosing. In the absence of rapid diagnostic methods, the use of a colloidal gold immunochromatographic strip based on F22 was applied to detect F. gigantica infection in buffalo.MethodsIn the present study, the 22nd gel filtration chromatography fraction of FgESP (F22) was used as an antigen to establish the colloidal gold-based immunochromatographic strip (ICS). The nitrocellulose membrane was incubated with F22 at the test line (T line) and goat anti-mouse secondary antibody at the control line (C line). The mouse anti-buffalo secondary antibody 2G7 conjugated to colloidal gold particles was used as the detection system for line visualization. The strip was assembled and developed by optimizing reaction conditions. The sensitivity, specificity, stability, and early diagnostic value of the strip were evaluated employing buffalo-derived sera.ResultsAn immunochromatographic strip for the rapid detection of antibodies against F. gigantica-FgICS was developed. The strip demonstrated high sensitivity and specificity. Sensitivity tests confirmed positive results even when the positive reference serum was diluted 4,096 times. Except for one Schistosoma japonicum-positive serum that tested positive via FgICS, specificity tests confirmed no cross-reactivity with other positive sera of Schistosoma japonicum and Babesia bovis. The strip remained stable after storage at 4°C for up to 3 months. In infected buffalo, antibodies could be detected as early as 14–21 days post-infection. The detection of 17 positive sera yielded an 82.4% positive rate via FgICS vs. a 100.0% positive rate via ELISA based on FgESP. For FgICS, the 95% confidence interval of sensitivity was 84.8–95.4%, while specificity was 4.2–14.7%.ConclusionThe immunochromatographic strip FgICS developed in this study provides a simple and rapid method of F. gigantica antibody detection and infected buffalo monitoring in the field.

Effect of primary and secondary Fasciola gigantica infection on specific IgG responses, hepatic enzyme levels and weight gain in buffaloes

Buffaloes, as highly susceptible definitive hosts of Fasciola gigantica, suffer from a high infection rate of fasciolosis, which causes enormous economic losses. Repeat infection is responsible for this high rate; thus, elucidating the protective immunity mechanism in repeat infection is decisive in fasciolosis prevention. Herein, a secondary experimental infection model was established to preliminarily reveal the protective immunity that occurs in repeat infection. In brief, animals were assigned to three groups: group A (uninfected control), group B (primary infection) and group C (secondary infection). Buffaloes were autopsied 20weeks post-infection for measurements of the recovered flukes and hepatic examination. In addition, the detection of specific antibody (IgG) responses to F. gigantica excretory-secretory product (FgESP) throughout the whole period and weight gain throughout the first 4months as a percentage (%) of the starting weight were also determined. The serum hepatic enzyme gamma glutathione transferase (GGT) levels were monitored to assess hepatic damage throughout the study period. Infection establishment was compared between group B and group C. Similar specific IgG patterns were observed between group B and group C, and hepatic damage was more severe in group C than group B. Significant differences in weight gain as a percentage of the start weight were observed between group A and group B at the 3rd and 4th months postprimary infection, while significant differences were not observed between group A and group C or group B and group C. Our results suggest that challenge infection cannot induce resistance against F. gigantica in buffaloes, which is consistent with the protective immunity against Fasciola hepatica reinfection observed in sheep and goats.

Characterization of the Pathology, Biochemistry, and Immune Response in Kunming (KM) Mice Following Fasciola gigantica Infection.

As a putative model of Fasciola gigantica infection, detailed data in Kunming (KM) mice infected with F. gigantica are lacking. In this study, KM mice were orally infected with 15 metacercaria for 8 weeks. Macroscopic and microscopic changes, serum biochemistry, cytokine responses, and changes in parasite-specific immunoglobulin G (IgG) antibody levels were monitored at 1, 3, 5, 7, and 8 weeks post-infection (wpi), respectively. The serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) increased after infection, while that of albumin (ALB) decreased, which was positively correlated with the degree of liver damage. Between 5 and 7 wpi, the mice showed symptoms of anemia and weight loss, possibly caused by the decrease of alkaline phosphatase (ALP). Moreover, the changing tendencies of the levels of globulin (GLB) and parasite-specific IgG antibody were similar, suggesting a potential correlation between GLB production and adaptive immune response in the host. Coordinated variations in interferon gamma (IFN-γ) and interleukin 4 (IL-4) indicated a mixed T helper 1 (Th1)/Th2 cellular immune response. Furthermore, the serum IgG antibody increased after infection and peaked at 5 wpi, and it was positively correlated with the average parasite burdens. The worms collected from mice were approximately 1 cm in length at 8 wpi, their digestive and reproductive systems were well developed, and no eggs were found in the uterus. To the best of our knowledge, this is the first report describing detailed histological, biochemical, and immunological indices in KM mice infected with F. gigantica, which provides basic information on KM mice against infection with F. gigantica.

Hematological, immunological, and polyamines alterations in the concomitant occurrence of Fasciola gigantica and hepatic leiomyoma in cattle

No previous studies have investigated the polyamines alterations during fascioliasis due to F. gigantica in ruminants. This study was therefore carried out to find out the possible relationship between the extent of liver destruction and leiomyoma and some hematological and immunological parameters and polyamines alterations in F. gigantica infection. Fifty cattle with liver fascioliasis and fifteen healthy cattle were selected for the study. For the histopathological study, liver tissue samples were stained with hematoxylin and eosin (H&E) and Masson's Trichrome methods. The leiomyoma suspected specimens were immunohistochemically stained for smooth muscle actin and desmin. Different hematological parameters were investigated in infected and non-infected animals.Furthermore, levels of putrescine, spermidine, and spermine were measured in homogenized liver samples. Serum IL-4 and TNF-α levels were also evaluated. By histological examination, the lesions were noted in all the infected specimens. These lesions were varied from leiomyoma, chronic catarrhal cholangitis, arteriosclerosis, telangiectasia, and fresh migratory tunnels filled with RBC and eosinophils. Comparison of hemogram results between infected and non-infected groups revealed a significant decrease in red blood cell counts (RBC), mean corpuscular hemoglobin concentration (MCHC), and platelet count (PLT) in infected animals. Also, a significant elevation in mean corpuscular volume (MCV) concentration was detected in infected animals. The putrescine and spermine levels of the infected animals were significantly higher than the non-infected animals. Although spermidine was increased in infected livers, its elevation was not significant. Based on the results, the level of IL-4 and TNF-α was not significantly changed in infected animals. In conclusion, the concurrent occurrence of leiomyoma and fascioliasis due to F. gigantica and polyamines elevation (putrescine and spermine) is reported for the first time. The role of polyamines in the concurrent occurrence of leiomyoma and fascioliasis is an area for future research.

ASSESSMENT OF FASCIOLA GIGANTICA INFECTION IN THE LIVER OF CATTLE SLAUGHTERED IN IKPOBA/OKHA AREA COUNCIL, EDO STATE, NIGERIA

This study investigated the prevalence and intensity of Fasciola gigantica infection in the liver of cattle slaughtered in three abattoirs located in Ikpoba/Okha Local Government Area of Edo State, Nigeria. It also estimated the economic implications of the condemned liver. A total of 17,325 cattle were examined in the three abattoirs over a period of 252 days (approximately 69 cattle/day) spanning over the wet and dry seasons; 1,683 cattle were infected with an overall prevalence of 9.71%. In Abattoir A, 11774 cattle were examined with 1069 (9.08%) prevalence. In Abattoirs B and C, 4352 and 1199 cattle, respectively, were examined and 563 (12.94%) and 51 (4.25%) were respectively infected. From the three abattoirs, 155 (0.89%) livers were considered unfit for human consumption due to F. gigantica infection. For the wet season, 1062 (10.89%) cattle livers were infected while 621 (8.20%) were infected during the dry season. The mean intensity of infection during the wet season was 37.80 and 30.42 during the dry season. The financial loss due to infected liver condemnation was at N803,160 (1,947.050USD) showing that liver condemnation arising from F. gigantica infection is a major cause of financial loss to cattle farmers and dealers. There is, therefore, need for adequate veterinary inspections for early detection and treatment of the disease. There should be more meat inspection, especially in private/individual abattoirs.

High-throughput sequencing of Fasciola spp. shows co-infection and intermediate forms in Balochistan, but only Fasciola gigantica in the Punjab province of Pakistan.

Fasciola gigantica and Fasciola hepatica are digenetic trematodes causing fasciolosis in ruminants. The host and geographical distribution of both Fasciola species are influenced by environmental and climatic conditions favouring survival and development of free-living stages and intermediate hosts, and livestock management practices. The aim of the present study was to describe the host distribution of the two Fasciola species in buffalo, cattle, goats, and sheep in the Balochistan and Punjab provinces of Pakistan. 359 flukes were collected from a total of 32 livers from the four livestock species. Deep amplicon sequencing of the internal transcribed spacer region 2 of ribosomal DNA (rDNA ITS-2) and mitochondrial nicotinamide adenine dinucleotide dehydrogenase 1 (mtDNA ND-1) loci confirmed co-infection of F. hepatica and F. gigantica in Balochistan and single species F. gigantica infection in Punjab. In Balochistan, co-infections and hybrids of both Fasciola species were identified in cattle, with more F. hepatica detected than F. gigantica. However, F. hepatica was the only species identified in goats, and F. gigantica was the only species identified in buffalo. In Punjab, all flukes were confirmed as F. gigantica in each of the four livestock species. Overall, the results indicate differences in the host and geographical distribution of F. gigantica and F. hepatica, and provide useful knowledge for the development of control strategies for livestock and humans.

Identification of Fasciola spp. based on ITS-2 reveals the Fasciola gigantica infection in buffaloes in Nanning city, South China

Fasciolosis is harmful to ruminant husbandry worldwide. Given the superficial survey on Fasciolosis infection in Guangxi, the main buffalo breeding area in China, an in-depth investigation in the infection of buffaloes in Nanning, the capital of Guangxi Zhuang Autonomous Region, with Fasciola (Platyhelminthes: Trematoda: Digenea) species will provide a theoretical support for the control and prevention of Fasciolosis infection in buffaloes. Five water buffalo livers were collected from an abattoir in Nanning every 2 weeks from June 2018 to April 2019, and a total of 101 livers were obtained. All livers were then dissected to observe the liver lesions caused by the flukes. Afterwards, Fasciola spp. collected from Fasciolosis-infected livers were numbered and measured. Then, the livers infected with more than 3 flukes were marked, and 3 flukes were picked from each liver according to their morphological differences, such as body length (BL), body maximum width (BW) and length-width ratio (BL/BW). Moreover, these Fasciola spp. worms were selected for molecular biological analysis. The second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA (rDNA) was amplified by polymerase chain reaction (PCR) and sequenced. Finally, sequential and phylogenetic analyses were also performed. The infection rate was 38.6 % according to anatomic examination, and the livers infected by Fasciola spp. were damaged seriously. The principal manifestations were the enlargement of the liver and protrusion of the bile ducts. In some cases, the bile duct wall became inflamed and rough, in which some sediment such as phosphate could be easily found. After dissection, 1243 Fasciola spp. flukes were collected from 39 out of 101 livers. The morphometric measurements obtained from the present study showed that the BL/BW ranged from 1.42–10.25. However, it might vary considerably among different geographical locations and could not be used as an accurate method for the identification of Fasciola spp.. Analysis of the ITS-2 sequences showed that 83 out of 87 flukes had 100 % homology with each other, and the other 4 flukes with 99.3 % homology possessed a nucleotide polymorphism. A unique position (271) was detected in flukes in Nanning isolates. Phylogenetic analysis indicated that all the flukes were Fasciola gigantica, and no Fasciola hepatica or the intermediate form was found in this study.

The combined recombinant cathepsin L1H and cathepsin B3 vaccine against Fasciola gigantica infection.

Protections against Fasciola gigantica infection in mice immunized with the individual and combined cathepsin L1H and cathepsin B3 vaccines were assessed. The vaccines comprised recombinant (r) pro-proteins of cathepsin L1H and B3 (rproFgCatL1H and rproFgCatB3) and combined proteins which were expressed in Pichia pastoris. The experimental trials were performed in ICR mice (n=10 per group) by subcutaneous injection with 50μg of the recombinant proteins combined with Alum or Freund's adjuvants. At two weeks after the third immunization, mice were infected with 15F. gigantica metacercariae per mouse by oral route. The percents of protection of rproFgCatL1H, rproFgCatB3 and combined vaccines against F. gigantica were approximately 58.8 to 75.0% when compared with adjuvant-infected control. These protective effects were similar among groups receiving vaccines with Alum or Freund's adjuvants. By determining the levels of IgG1 and IgG2a in the immune sera, which are indicative of Th1 and Th2 immune responses, it was found that both Th1 and Th2 humoral immune responses were significantly increased in vaccinated groups compared with the control groups, with higher levels of IgG1 (Th2) than IgG2a (Th1). Mice in vaccinated groups showed reduction in liver pathological lesions when compared with control groups. This study indicates that the combined rproFgCatB3 and rproFgCatL1H vaccine had a high protective potential than a single a vaccine, with Alum and Freund's adjuvants showing similar level of protection. These results can serve as guidelines for the testing of this F. gigantica vaccine in larger economic animals.

Dysregulation of hepatic microRNA expression in C57BL/6 mice affected by excretory-secretory products of Fasciola gigantica.

The excretory-secretory products released by the liver fluke Fasciola gigantica (FgESPs) play important roles in regulating the host immune response during the infection. Identification of hepatic miRNAs altered by FgESPs may improve our understanding of the pathogenesis of F. gigantica infection. In this study, we investigated the alterations in the hepatic microRNAs (miRNAs) in mice treated with FgESPs using high-throughput small RNA (sRNA) sequencing and bioinformatics analysis. The expression of seven miRNAs was confirmed by quantitative stem-loop reverse transcription quantitative PCR (qRT-PCR). A total of 1,313 miRNAs were identified in the liver of mice, and the differentially expressed (DE) miRNAs varied across the time lapsed post exposure to FgESPs. We identified 67, 154 and 53 dysregulated miRNAs at 1, 4 and 12 weeks post-exposure, respectively. 5 miRNAs (miR-126a-3p, miR-150-5p, miR-155-5p, miR-181a-5p and miR-362-3p) were commonly dysregulated at the three time points. We also found that most of the DE miRNAs were induced by FgESPs in the mouse liver after 4 weeks of exposure. These were subjected to Gene Ontology (GO) enrichment analysis, which showed that the predicted targets of the hepatic DE miRNAs of mice 4 weeks of FgESPs injection were enriched in GO terms, including cell membrane, ion binding, cellular communication, organelle and DNA damage. KEGG analysis indicated that the predicted targets of the most downregulated miRNAs were involved in 15 neural activity-related pathways, 6 digestion-related pathways, 20 immune response-related pathways and 17 cancer-related pathways. These data provide new insights into how FgESPs can dysregulate hepatic miRNAs, which play important roles in modulating several aspects of F. gigantica pathogenesis.

Milk As a New Diagnostic Tool for Rapid Detection of Fascioliasis in Dairy Goats Using Excretory/Secretory Antigen

Fascioliasis is a serious livestock illness of particular importance for dairy goats; the objectives of this study were to describe effects of F. gigantica on milk composition and to use this information to estimate economic damages linked with Fasciola spp. infections. Furthermore, the study sought to standardize the use of milk instead of serum for early diagnosis of fascioliasis in dairy goats. One-hundred samples of goat milk along with corresponding blood samples were obtained at random from flocks in Cairo and Giza Governorates. The ELISA and DOT-ELISA were performed in both serum and milk of dairy goats. Total mesophilic count (mean value) was 2.12 × 106 ± 1.63 × 105CFU/ml in enzyme-linked immunosorbent assay (ELISA) positive samples and 1.46 × 104 ± 8.58 × 102CFU/ml in ELISA-negative samples. The mean values were significantly different (P < 0.05). The mean values of percentages of fat, SNF, protein, salts, lactose, pH, and MSCC/ml in ELISA-positive samples were 2.3 ± 0.17, 8.21 ± 0.63, 3.08 ± 0.18, 0.90 ± 0.06, 3.64 ± 0.28, 6.93 + 0.53, and 1.18 × 106 ± 9.07 × 104cells/ml, respectively. A significant difference (P < 0.05) between the mean values of two composition parameters, i.e., percent of fat and MSCC/ml in ELISA-positive and -negative samples, for Fasciola gigantica was observed. The antigen used for the diagnosis of F. gigantica was excretory/secretory (E/S) antigen. The dilutions of (E/S) concentrations after checkerboard titration for indirect ELISA were 20μg/ml protein and for dot-ELISA, 300ng/μl. Sera dilution was 1:100 in the two tests, and milk dilution was 1:50 for indirect ELISA, and 1:25 for dot-ELISA. The two tests were performed using known F. gigantica positive and negative goat sera and known rat hyper immunized negative and positive sera against E/S antigen of F. gigantica as well as known sera for paramphistomes without F. gigantica infection. The cutoff values in indirect ELISA were 0.45 for sera and 0.35 for milk. The application of different serological technique in goat farms reveals a good test in rapid diagnosis of fascioliasis especially the uses of dot ELISA when using the milk instead of the serum.

Fasciola gigantica excretory-secretory products (FgESPs) modulate the differentiation and immune functions of buffalo dendritic cells through a mechanism involving DNMT1 and TET1

BackgroundFasciola gigantica infection threatens the health of both humans and animals in the world. The excretory/secretory products (ESPs) of this fluke has been reported to impair the activation and maturation of immune cells. We have previously shown the influence of F. gigantica ESPs (FgESPs) on the maturation of buffalo dendritic cells (DCs). However, the underlying mechanisms remain unclear. The objective of this study was to investigate the potency of FgESPs in shifting the differentiation and immune functions of buffalo DCs.MethodsBuffalo DCs were incubated with FgESPs directly or further co-cultured with lymphocytes in vitro. qRT-PCR was employed to determine the gene expression profile of DCs or the mixed cells, and an ELISA was used to measure cytokine levels in the supernatants. Hoechst and Giemsa staining assays, transmission electron microscopy, caspase-3/7 activity test and histone methylation test were performed to determine DC phenotyping, apoptosis and methylation. To investigate the mechanism involved with DNA methylation, a Co-IP assay and immunofluorescent staining assay were performed to observe if there was any direct interaction between FgESPs and DNMT1/TET1 in buffalo DCs, while RNAi technology was employed to knockdown DNMT1 and TET1 in order to evaluate any different influence of FgESPs on DCs when these genes were absent.ResultsqRT-PCR and ELISA data together demonstrated the upregulation of DC2 and Th2/Treg markers in DCs alone and DCs with a mixed lymphocyte reaction (MLR), suggesting a bias of DC2 that potentially directed Th2 differentiation in vitro. DC apoptosis was also found and evidenced morphologically and biochemically, which might be a source of tolerogenic DCs that led to Treg differentiation. In addition, FgESPs induced methylation level changes of histones H3K4 and H3K9, which correlate with DNA methylation. Co-IP and immunofluorescent subcellular localization assays showed no direct interaction between the FgESPs and DNMT1/TET1 in buffalo DCs. The productions of IL-6 and IL-12 were found separately altered by the knockdown of DNMT1 and TET1 in DCs after FgESPs treatment.ConclusionsFgESPs may induce the DC2 phenotype or the apoptosis of buffalo DCs to induce the downstream Th2/Treg response of T cells, possibly through a DNMT1- or TET1-dependent manner(s).

Cloning and Expression of Fasciola gigantica Cathepsin-B Recombinant Proteins

Fasciola gigantica cathepsin (cysteine) proteases are potential diagnostic antigens for animal and human fasciolosis. These include cathepsin-L proteases that have been exploited in the diagnosis of animal fasciolosis. However, no scientific data on the diagnostic potential of F. gigantica cathepsin B proteases is available. Therefore, three recombinant antigens of F. gigantica viz. cathepsin (cat) B-1, cat B-2 and cat B-3 were expressed in prokaryotic expression system. The recombinant antigens were purified under denaturing conditions by Nickel affinity chromatography and an optimal level of the recombinant proteins was obtained. These recombinant proteins will further be evaluated for their potential in the early prepatent diagnosis of F. gigantica infection in domestic ruminants.

Serodiagnosis of Fasciola gigantica Infection in Buffaloes with Native Cathepsin-L Proteases and Recombinant Cathepsin L1-D

Serodiagnosis of Fasciola gigantica natural infection in buffaloes with recombinant cathepsin L1-D and native cathepsin-L protease antigens. The recombinant cat L1-D antigen was expressed in prokaryotic expression system and native cathepsin-L proteases were purified by alcoholic fractionation from adult F. gigantica flukes. Buffaloes (n =325) were screened for anti-Fasciola antibodies with the above antigens in immunoglobulin-G-enzyme linked immunosorbent assay (IgG-ELISA). The recombinant cat L1-D antigen showed positive reactivity with 101/122 necropsy positive animals but 21/122 necropsy confirmed positive animals were negative in this ELISA (sensitivity 82.8%). However, 30/203 (14.8%) necropsy negative animals for Fasciola were seropositive with specificity of 85.2%. With native cat-L protease, 104/122 necropsy confirmed positive animals were ELISA positive but 18/122 necropsy positive animals were seronegative, thereby depicting the sensitivity of 85.2%. But ELISA with this antigen showed 27/203 (13.3%) necropsy negative animals as positive (specificity 86.7%). Comparative evaluation of both the antigens showed that they are suitable for serodiagnosis of F. gigantica infection in buffalo herds.

Genetic diversity and multiplicity of infection in Fasciola gigantica isolates of Pakistani livestock

Fasciola spp. are responsible for over 3 billion US dollars of production loss annually in livestock and cause widespread zoonotic disease. Nevertheless, understating of the emergence and spread of the trematode species is poor. The multiplicity of F. gigantica infection and its spread is potentially influenced by multiple factors, including the abundance of suitable intermediate hosts, climatic conditions favouring the completion of the parasite's lifecycle, and translocation of infected animals, or free-living parasite stages between regions. Here we describe the development of a ‘tremabiome’ metabarcoding sequencing method to explore the numbers of F. gigantica genotypes per infection and patterns of parasite spread, based on genetic characteristics of the mitochondrial NADH dehydrogenase 1 (mt-ND-1) locus. We collected F. gigantica from three abattoirs in the Punjab and Balochistan provinces of Pakistan, and our results show a high level of genetic diversity in 20 F. gigantica populations derived from small and large ruminants consigned to slaughter in both provinces. This implies that F. gigantica can reproduce in its definitive hosts through meiosis involving cross- and self-breeding, as described in the closely related species, Fasciola hepatica. The genetic diversity between the 20 populations derived from different locations also illustrates the impact of animal movements on gene flow. Our results demonstrate the predominance of single haplotypes, consistent with a single introduction of F. gigantica infection in 85% of the hosts from which the parasite populations were derived. This is consistent with clonal reproduction in the intermediate snail hosts.

Occurrence of Fasciola gigantica and Paramphistomum spp Infection in Aceh Cattle

Fasciola gigantica and Paramphistomum spp. are trematode helminth causing severe economic losses in cattle farming in Aceh Province, Indonesia. This study was conducted to examine the correlation between the prevalence of F. gigantica and Paramphistomum spp infections with the body condition and sex of Aceh cattle. In total, 103 cattle (50 males and 53 females) from an abattoir in Banda Aceh were used. The body condition score was recorded and the number of fluke eggs in feces was examined coproscopically. The results showed that F. gigantica prevalence was 41% and 72% in females and males, respectively, whereas, the prevalence of Paramphistomum spp in females and males was 81% and 72%, respectively. The average number of fasciola eggs was 2.55 eggs/ g feces and 2.75 eggs/ g feces in females and males, respectively, The average number of Paramphistomum spp eggs was 127.6 eggs/g feces and 36.8 eggs/ g feces in males, and female respectively. Based on the Body Condition Score (BCS), the prevalence of both trematodes was higher in the skinny cattle (BSC 2 and 3). This study established that the infection of Fasciola in BCS 3 was higher than BCS 2 and 4. Male cattle were more susceptible to infection than females.