Nested-PCR assay for detection of Schistosoma japonicum infection in domestic animals.

BACKGROUND: Schistosomiasis japonica is a common zoonosis. Domestic animals are the primary source of infection and play an important role in disease transmission. The prevalence and infectivity of this disease in domestic animals in China have significantly decreased and, for this reason, diagnostics with a higher sensitivity have become increasingly necessary. It was reported that polymerase chain reaction (PCR)-based methods could be used to detect schistosome infection in humans and animals and presented a high sensitivity and specificity. The present study aimed to develop a PCR-based method for detection of Schistosoma japonicum infection in domestic animals. METHODS: A specific nested-PCR assay was developed to detect S. japonicum infection in domestic animals via amplification of a 231-bp DNA fragment of retrotransposon SjR2. The developed assay was first used in sera and dry blood filter paper (DBFP) from goats and buffaloes at different time points of infection. Then, 78 DBFPs from 39 artificially-infected bovines at 14 and 28 days post-infection and 42 DBFPs from schistosome-negative bovines from the city of Huangshan in the Anhui province were used to evaluate the diagnostic validity. Furthermore, this assay was used to detect S. japonicum infection in domestic animals in Dongzhi and Wangjiang counties. RESULTS: The expected PCR product was detected in eggs and adult worms of S. japonicum and blood samples from S. japonicum-infected goats and water buffaloes, but not from Fasciola and Haemonchus contortus worms. The nested-PCR assay could detect the target S. japonicum DNA in DBFPs from goats and buffaloes after day 3 post-infection. The sensitivity in buffaloes at 14 and 28 days post-infection was 92.30% (36/39) and 100% (39/39), respectively. The specificity was 97.60% (41/42). The positivity rates in Dongzhi and Wangjiang counties were 6.00% and 8.00% in bovines and 22.00% and 16.67% in goats, respectively. The positivity rates in goats in both counties were higher than those in bovines with a significant difference in Dongzhi County but not in Wangjiang County (P < 0.05 and P = 0.23, respectively). CONCLUSIONS: Our results suggest that the developed nested-PCR assay may be used for the diagnosis of S. japonicum infection in domestic animals, and the control of S. japonicum infection in goats should be paid more attention.

[Distribution characteristics of deposited eggs and pathological changes in viscera of New Zealand white rabbits infected with Schistosoma japonicum at different time].

OBJECTIVE: To study the distribution characteristics of deposited eggs and pathological changes in the viscera of animal infected with Schistosoma japonicum at different time. METHODS: New Zealand white rabbits were infected artificially with quantitative S. japonicum miracidia, then the distribution characteristics and the hatchability of schistosome eggs as well as the pathological changes of the corresponding viscera of the rabbits 42 and 60 d post-infection were observed and compared. RESULTS: On the 42nd day post-infection, among all the viscera observed, the percentage of eggs deposited, the number of eggs per gram and the hatchability were the highest in the liver, while on the 60th day post-infection, the tissues and organs with the highest values of the above 3 indexes were the liver, rectum and upper section of the small intestine, respectively. From 42 day to 60 day post-infection, the liver of infected rabbits became swelling, hardening and lost elasticity, the color changed from black to dark grey, and egg nodules gradually appeared in the different sections of the small intestine, and also the mucosal hyperemia, edema and egg nodules were seen in the colon, cecum and rectum. The lesion levels tended to be correlated with the deposition of eggs. CONCLUSION: The amount and the density as well as the hatching rate of deposited eggs of S. japonicum in the viscera of infected rabbits at different time are different, and the lesion level in the host is correlated with the deposition of eggs.

[Effect of praziquantel transdermal delivery on infection of Schistosoma japonicum of mice].

OBJECTIVE: To evaluate the effect of transdermal agent of PZQ on infection of Schistosoma japonicum in different developmental stages and the early period of re-infection. METHODS: All Kunming mice in the experiment groups were infected with 40 +/- 2 Schistosoma japonicum cercariae. The mice which were infected once and re-infected were treated by abdominal transdermal agent of praziquantel. Control groups were set for all of the experiment groups. All of the mice were dissected 42 d after the infection, and the worm reduction rate, egg reduction rate and miracidium hatching reduction rate were calculated. In addition, the dynamic changes of IgG antibody in sera of the mice were detected by ELISA in different time of primary infection and re-infection. RESULTS: The worm reduction rates of 1, 7, 14, 21 and 28 d after the infection were 48.9%, 0%, 28.8%, 84.3% and 70.2%, respectively, and those of 1 d and 14 d after re-infection were 85.6% and 90.8%, respectively. After the primary infection, the specific IgG level gradually increased with the prolongation of time, and the ratio of P/N of specific anti-ESA of IgG was significantly raised after re-infection. CONCLUSION: The transdermal agent of praziquantel is effective to Schistosoma japonicum at developmental stages, and the effect to schistosomula at early period of re-infection is more significant.

[Phylogenetic analysis of CO I gene of Oncomelania snails from project of afforestation for schistosomiasis control in marshland endemic regions].

OBJECTIVE: To investigate the genetic difference of cytochrome oxidase I (CO I ) of Oncomelania snails from the project of afforestation for schistosomiasis control in marshland regions, so as to explore the effects of different ecological environments. METHODS: The snails were collected from 3 different areas, Anqing, Tongling, Wuwei, i.e. the upstream, midstream and downstream regions along the Yangtz River in Anhui Province. Genomic DNA was extracted from the snails, and CO I gene fragments were amplified by PCR, then purified and sequenced. The sequences were edited by using Blast. The CO I genes of O. h. minima and Biomphalaria glabrata were used as the reference of exogenous gene. The genetic distances of the various regions were calculated by the Kimura method and phylogenetic trees were constructed with UPGMA and the NJ method of MEGA (3.1) software. RESULTS: The amplified CO I gene of the snail was a fragment about 700 bp including 2 primers in length. There were little genetic diversity among the different areas, the identities were higher than or equal to 98%. The genetic distances indicated that the distance between the projects of afforestation and woodland in Anqing was 0.003, while Tongling was 0.019, Wuwei was 0.007. The distances among the three projects of afforestation were 0.003-0.012. The two phylogenetic trees were constructed by the methods of UPGMA and NJ respectively, which took on very similar topo-structure in which isolates of Biomphalaria glabrata located in one clade and all the others in the other one. In the other one clade, O. H. minima located in one clade. There was little genetic diversity among Anqing, Tongling, Wuwei clusters. The afforestations of Anqing and Wuwei clustered into one group, while the woodlands of Anqing and Wuwei appeared as another group. CONCLUSION: There is a little genetic diversity of the snail cytochrome oxidase I (CO I ) in different ecological environments among the upstream, midstream and downstream regions along the Yangtz River in Anhui Province.

[Longitudinal observation of epidemic dynamics of schistosomiasis in bovine in two mountainous endemic regions].

OBJECTIVE: To understand the endemic situation dynamics of schistosomiasis in domestic animals (mainly bovine) in mountainous endemic regions, so as to provide the reference for evaluating the control effect and improving control strategy. METHODS: Two representative pilots (Renmei and Dacang) in mountainous schistosomiasis endemic regions were selected for survey. The schistosome infection status of bovine was investigated by the miracidium hatching method, the pasture of bovine were investigated by home visiting, and the distributions of wild feces and Oncomelania snails, and the snail schistosome infection status were also investigated in April and September every year. RESULTS: The schistosome infection rates of bovine reduced by 98.4% and 93.8% in two pilots in 2007 compared with those in 1993, and the infection intensities also showed a decline trend. The infection rate of wild faces was 0 in Renmei pilot since 1995, while in Dacang pilot, the infection rate of wild feces fluctuated in 2007, and the intensities of living snails and infected snails showed a declined trend. CONCLUSIONS: Due to the special natural environment of mountainous endemic regions, there is a dot-like or band-like distribution of endemic areas. The strengthening of schistosomiasis examination and chemotherapy will rapidly reduce endemic situation. However, to completely interrupt the transmission of schistosomiasis, we should emphasize environmental modification and domestic animal management.

[Experiment of praziquantel rectal administration in treatment of schistosomiasis in mice].

OBJECTIVE: To evaluate the efficiency of rectal administration of praziquantel in the treatment of schistosomiasis in mice. METHODS: Forty mice were divided into 4 groups. Each mouse was infected with Schistosoma japonicum cercariae 40 +/- 2. Forty-two days after the infection, the mouse was rectally administered with different doses of praziquantel. In the first, second and third group, each mouse was given 100, 200 mg/kg, and 400 mg/kg single dose of praziquantel, and the fourth group was a blank control group. One week after the administration, all the mice were sacrificed and the worm reduction rate, reduction rate of liver eggs, and matching reduction rate were calculated. RESULTS The worm reduction rate and matching reduction rate were 57.63% and 76.60% respectively in the 200 mg/kg group, and 49.15% and 51.06% respectively in the 400 mg/kg group, which were better than those in the 100 mg/kg group. CONCLUSION: Rectal administration of praziquantel has good efficiency in the treatment of schistosomiasis in mice; therefore, it provides a new option for the prevention and control of animal schistosomiasis.

Studies of impact on physical fitness and working capacity of patients with advanced Schistosomiasis japonica in Susong County, Anhui Province.

To assess the impact level on physical fitness and working capacity in patients with advanced Schistosomiasis japonica, a field study was carried out. According to the records of patients with advanced schistosomiasis in Susong County, Anhui Province, 48 advanced cases without other serious chronic diseases from endemic areas in two townships and 56 healthy individuals from non-endemic area, served as control group with matched ages between 40 and 70 years and matched sex were investigated with questionnaire, anthropometric measure and hemoglobin level. The impairment level of the liver was measured by ultrasonography and physical fitness was measured by the Step test in the case and control groups. All situations including lifestyle, working, socio-economic status and residing environment was similar in the case and control groups. Average height and weight was significantly lower in the case group than in the control group (height = 156.29 and 159.41 cm; weight = 50.72 and 53.92 kg; respectively, all P < 0.05). Thirteen individuals (28.3%) in the case group had moderate reduction of working capacity or even unable to work, but only seven (12.7%) individuals in the control group had moderate reduction of working capacity and all in the control group were able to work (P < 0.01). In the past 1 year, the average working days lost was 4.11 days in the case group and 0.86 day in the control group (P < 0.01). Both groups differed significantly in symptoms of abdominal pain, diarrhea and weakness (all P < 0.05). Twenty-one cases (43.8%) had grade II impairment of the liver and eight cases (16.7%) had grade III impairment of the liver in the case group, whereas seven individuals (12.7%) had grade II impairment of the liver in the control group (P < 0.01), as assessed by ultrasound. The hemoglobin levels and the power of gripping in the case group were significantly lower than those in the control group (Hb = 111.06 and 122.27 g/l; grip = 303.83 and 344.20 N, respectively, all P < 0.01). Physical fitness scores showed the control group (score: 71.84) was significantly fitter than the case group (score: 61.09, P < 0.01). Compared with the control group, the physical fitness of the case group reduced by 15%. The results showed that physical fitness and working capacity were reduced in advanced cases. Although most of the cases were treated and had reached a status of 'clinical cure', the impact on physical fitness and working capacity still existed.