As a severely harmful zoonotic disease, toxoplasmosis has complex clinical symptoms with atypical lesions, thus making it difficult for diagnosis in a timely manner. Therefore, developing an appropriate technique to diagnose and prevent toxoplasmosis has become a subject of many studies. In this study, a colloidal gold immunochromatographic test strips for the diagnosis of Toxoplasma gondii was established on the basis of the optimization of antigenic epitopes of GRA1 and GRA7 genes. Western blot and ELISA results showed that expressed rGRA exhibited specifically reaction with Toxoplasma gondii-positive mouse serum. This strip could be used to diagnose the toxoplasmosis of mouse. Moreover, compared with ELISA and PCR, GICA strip exhibited similar sensitivity in the diagnosis of Toxoplasma gondii in pigs. Here, we preliminarily established a diagnostic method for rapidly detecting toxoplasmosis, which may provide technical support for large-scale screening of Toxoplasma gondii.
Background Schistosomiasis japonica is a severe zoonosis. Domestic animals are the primary source of infection and play an important role in disease transmission. Surveillance and diagnosis play key roles in schistosomiasis control; however, current techniques for the surveillance and diagnosis of the disease have limitations. In this study, we developed a novel fluorescence immunochromatographic assay (FICA) strip to detect anti-Schistosoma japonicum antibodies in host serum. Methods A FICA strip was developed for the diagnosis of Schistosoma japonicum in domestic animals. Streptococcus protein G (SPG) and soluble egg antigen (SEA) were transferred onto a nitrocellulose (NC) membrane to form the control line (C) and the test line (T), respectively. With fluorescence activity as well as binding activity to multispecies IgG, the recombinant protein rSPG-RFP was expressed and employed as an antibody indicator in the FICA strips. Results The dual gene fusion plasmid was verified by PCR and restriction enzyme digestion. The expressed recombinant protein was 39.72 kDa in size, which was consistent with the predicted molecular weight. The western blot results showed binding activity between rSPG-RFP and IgGs from different hosts. Fluorescence microscopy also showed the fluorescence activity of the protein present. The affinity constant (Ka) values of rSPG-RFP with rabbit, donkey, mouse and goat IgG were 1.9 × 105, 4.1 × 105, 1.7 × 105 and 5.4 × 105, respectively. Moreover, based on the recombinant protein, the test strip for detecting S. japonicum in buffaloes could distinguish positive from negative serum. The lower limit of detection of the FICA strip was 1:10,000. Compared with ELISA, the FICA strips exhibited similar results in the diagnosis of infection in clinical bovine serum samples, with a kappa value of 0.9660 and P < 0.01. The cross-reactivities of the FICA strips with Haemonchus contortus and Schistosoma turkestanicum (30.15% and 91.66%, respectively) were higher than those of ELISA (26.98% and 87.5%, respectively). Conclusions Based on the rSPG-RFP protein that we developed, strip detection can be completed within 15 min. Heightened sensitivity allows the strip to accurately identify schistosome antibodies in serum. In conclusion, this method is convenient, feasible, rapid and effective for detecting S. japonicum.
Background The existing diagnostic techniques for detecting schistosomiasis turkestanica, such as aetiological assays, identify infection by parasitic worms via the incubation of miracidia from faeces or observing eggs under microscopy. However, they are limited in the diagnosis of low-grade and prepatent infections, which lead to a high misdetection rates. Therefore, a new method for parasite diagnosis with increased sensitivity is urgently needed. Methods Goats in Nimu County (Tibet, China) infected with Schistosoma turkestanicum in an epidemic area were selected according positivity for the infection by faecal examination. Adult worms were collected, eggs were extracted by the sodium hydroxide (NaOH) erosion method, and soluble worm antigen preparation (SWAP) and soluble egg antigen (SEA) were isolated. The best coating concentration of the antigens and the best degree of dilution for serum were determined by square array experiments, and the optimal blocking solution and serum diluents were selected. The specificity, sensitivity and crossover of the ELISA method were determined using 48 samples of goat sera positive for S. turkestanicum, 100 samples of goat sera negative for S. turkestanicum, and 54 samples of buffalo sera positive for S. japonicum. Serological assays were established with samples from goats naturally grazed in a rural area of Nimu County, Tibet Province, by using the indirect ELISA method for the diagnosis of schistosomiasis, and faeces were collected for miracidia hatching. The sensitivity of the two detection methods was compared. Results Eggs of S. turkestanicum were distributed in the host duodenum and small intestine. Eggs in the host intestinal wall were extracted by the NaOH erosion method, which provided intact
Currently the diagnosis of schistosomiasis is mainly determined by observing the presence of eggs in host stool samples. Because of the overwhelming number of impurities in the stool, eggs are rarely observed. Therefore, the stool hatching method is used to observe the miracidia in the water. However, the miracidia of Schistosoma japonicum are small and difficult to detect, and missed detection is likely to occur when the infection level is low. In this study, recombinant streptococcal protein G-enhanced green fluorescent protein (rSPG-EGFP) was expressed, purified, and used as a fluorescence staining reagent for miracidia. A preliminary miracidium fluorescence staining method based on antigen and antibody bindingwas established by combining recombinant protein staining with the stool hatching method. The stool hatching method was used to collect the miracidia of S. japonicum, and Schistosoma-positive serum and the recombinant protein were mixed to assess the feasibility of fluorescence staining of miracidia. The miracidia of S. japonicum and Schistosoma turkestanicum were incubated with S. japonicum-positive serum and S. turkestanicum-positive serum, respectively, to identify miracidia species. When the fluorescence staining method was used to observe living miracidia, the miracidiawere labelled by the recombinant protein, and their motility status was not affected.
Background Schistosomiasis is an important zoonotic parasitic disease, which remains a major public concern in china. However, the detection of schistosomiasis in the field is still based on the traditional faecal hatching method, which is tedious and time-consuming. Therefore, method for detecting schistosomiasis in the field needs to be improved. Methods New Zealand rabbits artificially infected with S. japonicum cercariae were used as animal models to study the deposition characteristics of Schistosoma japonicum eggs. The distributions of eggs in the intestinal wall at 42 d and 60 d post-infection were compared. The distributions of eggs in rabbit faecal samples were also observed. Goat faeces were used to compare the conventional faecal hatching method and the simplified direct immersion faecal hatching method. Results The distribution of eggs in the intestinal wall in the animal model at 42 d post-infection was as follows: the number of eggs per gram (EPG) was the highest (42780.13 ± 4789.81 eggs/g) in the rectum. The caecum had the largest proportion (42.97%) of eggs deposited. At 60 d post-infection, the rectum still had the highest EPG (117868.20 ± 67232.80 eggs/g). However, instead of the caecum, the lower colon had the largest proportion (64.90%). Moreover, 42.20% of eggs occupied the periphery of rabbit faeces. In the comparison between the conventional faecal hatching method and the simplified direct immersion faecal hatching method, the direct faecal hatching method was simpler, and the results were similar to those of the conventional faecal hatching method. Conclusion The deposition characteristics of eggs and their distributions in faecal samples suggest that the direct faecal hatching method can be used to simplify routine faecal hatching detection.
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