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

Background: Fasciola 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. Methods: In 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. Results: An 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%. Conclusion: The 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 20 weeks 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 4 months 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.

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.

High-quality reference genome of Fasciola gigantica: Insights into the genomic signatures of transposon-mediated evolution and specific parasitic adaption in tropical regions.

Fasciola gigantica and Fasciola hepatica are causative pathogens of fascioliasis, with the widest latitudinal, longitudinal, and altitudinal distribution; however, among parasites, they have the largest sequenced genomes, hindering genomic research. In the present study, we used various sequencing and assembly technologies to generate a new high-quality Fasciola gigantica reference genome. We improved the integration of gene structure prediction, and identified two independent transposable element expansion events contributing to (1) the speciation between Fasciola and Fasciolopsis during the Cretaceous-Paleogene boundary mass extinction, and (2) the habitat switch to the liver during the Paleocene-Eocene Thermal Maximum, accompanied by gene length increment. Long interspersed element (LINE) duplication contributed to the second transposon-mediated alteration, showing an obvious trend of insertion into gene regions, regardless of strong purifying effect. Gene ontology analysis of genes with long LINE insertions identified membrane-associated and vesicle secretion process proteins, further implicating the functional alteration of the gene network. We identified 852 predicted excretory/secretory proteins and 3300 protein-protein interactions between Fasciola gigantica and its host. Among them, copper/zinc superoxide dismutase genes, with specific gene copy number variations, might play a central role in the phase I detoxification process. Analysis of 559 single-copy orthologs suggested that Fasciola gigantica and Fasciola hepatica diverged at 11.8 Ma near the Middle and Late Miocene Epoch boundary. We identified 98 rapidly evolving gene families, including actin and aquaporin, which might explain the large body size and the parasitic adaptive character resulting in these liver flukes becoming epidemic in tropical and subtropical regions.