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Objective To evaluate the immunoprotective effect of active immunization with recombinant peptidyl-prolyl cis-trans isomerase from Babesia microti against B. microti infection in mice. Methods Female BALB/c mice at 6 weeks of age, each weighing approximately 20 g, were divided into the recombinant protein immunization group, the infection control group and the normal control group, of 25, 18, 15 mice in each group, respectively. Mice in the recombinant protein immunization group were given active immunization with recombinant BmPPIase protein, and 18 mice with the highest antibody titers were intraperitoneally injected with 100 μL of B. microti-infected whole blood 2 weeks after the last immunization. Mice in the infection control group were intraperitoneally injected with 100 μL of B. microti-infected whole blood, while 15 mice in the normal control group received no treatment. Blood samples were collected from mice in the recombinant protein immunization group and the infection control group on days 0 to 30 post-immunization for detection of B. microti infection, and blood samples were collected on days 0, 7, 14, 21, and 28 post-immunization for routine blood tests with a blood cell analyzer and for detection of serum cytokines using cytometric bead array. Results Anti-BmPPIase antibodies were detected in 25 mice in the recombinant protein immunization group 2 weeks after the last immunization, with titers of 5 × 103 to 8 × 104. B. microti infection rate peaked in mice in both the recombinant protein immunization and the infection control group on day 7 post-immunization, with positive infection rates of 13.3% and 50.0%, and there were significant differences between the two groups in terms of B. microti infection rate on days 3 (χ2= 113.18, P < 0.01), 5 (χ2 = 475.22, P < 0.01), 7 (χ2 = 465.98, P < 0.01) and 9 post-infection (χ2= 18.71, P < 0.01), while the B. microti infection rate tended to be 0 in both groups on day 11 post-immunization. Routine blood tests showed higher red blood cell counts [(5.30 ± 0.50) × 1012 to (9.87 ± 0.24) × 1012 counts/L)] and hemoglobin levels [(89.67 ± 22.80) to (148.60 ± 3.05) g/L)] in the recombinant protein immunization group than in the infection control group on days 0 to 28 post-immunization. Cytometric bead array detected higher serum interferon-γ [(748.59 ± 17.56) to (3 858.28 ± 1 049.10) fg/mL], tumor necrosis factor-α [(6 687.34 ± 1 016.64) to (12 708.13 ± 1 629.79) fg/mL], interleukin (IL)-6 [(611.05 ± 75.60) to (6 852.68 ± 1 554.00) fg/mL] and IL-17a [(167.68 ± 185.00) to (10 849.27 ± 355.40) fg/mL] and lower IL-10 levels [(247.65 ± 138.00) to (18 787.20 ± 2 830.22) fg/mL] in the recombinant protein immunization group than in the infection control group during the study period. Conclusions Recombinant BmPPIase protein induces up-regulation of interferon-γ, tumor necrosis factor-α and presents a high immunoprotective activity against B. microti infection in mice, which is a potential vaccine candidate protein.
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Echinococcosis is a zoonotic parasitic disease caused by Echinococcus infections, and this disorder may cause fibrosis of multiple vital organs, which may further progress into cirrhosis. Early-stage hepatic fibrosis is reversible, and unraveling the mechanisms underlying hepatic fibrosis induced by Echinococcus infections is of great significance for the prevention and treatment of early-stage hepatic fibrosis. Recently, the studies pertaining to hepatic fibrosis associated with Echinococcus infections focus on cytokines and immune cells. This review summarizes the advances in the mechanisms underlying host immune cells- and cytokines-mediated hepatic fibrosis in humans or mice following Echinococcus infections.
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Objective To investigate the epidemiological characteristics and identify the risk factors of Giardia lamblia infections among patients with colorectal cancer in Henan Province. Methods A cross-sectional study was performed for questionnaire surveys among colorectal cancer patients in Henan Cancer Hospital during the period from March to July, 2021. Patients’ stool samples were collected, and the triosephosphate isomerase (tpi) gene of G. lamblia was amplified in stool samples using nested PCR assay to characterize the parasite genotype. Univariate analysis and multivariate logistic regression analyses were employed to identify the risk factors of G. lamblia infections among colorectal cancer patients. Results A total of 307 colorectal cancer patients were investigated, including 176 males (57.3%) and 131 females (42.7%). PCR assay detected 8.1% [95% confidential interval (CI): (0.056, 0.117)] prevalence of G. lamblia infections among the study subjects, and there was no significant difference in the prevalence between men [9.1%, 95% CI: (0.057, 0.143)] and women [6.9%, 95% CI: (0.037, 0.125)] (χ2 = 0.495, P = 0.482). In addition, there was no age-specific prevalence of G. lamblia infections among the participants (χ2 = 1.534, P = 0.675). Multivariate logistic regression analysis identified use of septic tanks [odds ratio (OR) = 3.336, 95% CI: (1.201, 9.267)], daily use of well water [OR = 3.042, 95% CI: (1.093, 8.465)] and raising livestock [OR = 3.740, 95% CI: (1.154, 12.121)] as risk factors of G. lamblia infections among colorectal cancer patients, and the prevalence of abdominal pain was significantly greater in colorectal cancer patients with G. lamblia infections than in those without infections (P = 0.017). Among the 25 patients with G. lamblia infections, assemblage A was characterized in 24 (96.0%) cases and assemblage B in one case (4.0%). Conclusions The prevalence of G. lamblia is high among colorectal cancer patients in Henan Province, and assemblage A is the dominant genotype of G. lamblia. Use of septic tanks, daily use of well water and raising livestock are risk factors of G. lamblia infections among patients with colorectal cancer.
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Objective To construct a cDNA library of Sparganum mansoni and immunoscreen antigen candidates for immunodiagnosis of sparganosis mansoni. Methods Total RNA was extracted from S. mansoni, and reversely transcribed into cDNA, which was ligated into the phage vector. These recombinant vectors were packaged in vitro to construct the SMART cDNA library of S. mansoni. Then, the cDNA library was immunoscreened with sera from patients with sparganosis mansoni to yield positive clones. The inserted fragments of positive clones were sequenced and subjected to homology analyses, and the structure and functions of the coding proteins were predicted. Results The SMATR cDNA library of S. mansoni was successfully constructed. The titer of the cDNA library was 6.25 × 106 pfu/mL, with a recombinant efficiency of 100%, and the mean length of the inserted fragments in the library was larger than 1 100 bp. A total of 12 positive clones were obtained by immunoscreening, and were categorized into Sm-I (Sm60-1), Sm-II (Sm58-1), Sm-III (Sm20-1) and Sm-IV (Sm22-3), with 1 134, 1 063, 883 bp and 969 bp long inserted fragments. Their coding proteins were highly homologous with the Spirometra erinaceieuropaei antigenic polypeptide, cytoplasmic antigen, ribosomal protein S4-like protein and unnamed protein product, respectively. Conclusions A SMART cDNA library of S. mansoni has been successfully constructed and 4 categories of positive clones have been identified, which provides a basis for further studies on diagnostic antigens for sparganosis mansoni.
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Human sparganosis is a food-borne parasitic disease caused by the plerocercoids of Spirometra species. Clinical diagnosis of sparganosis is crucial for effective treatment, thus it is important to identify sensitive and specific antigens of plerocercoids. The aim of the current study was to identify and characterize the immunogenic proteins of Spirometra erinaceieuropaei plerocercoids that were recognized by patient sera. Crude soluble extract of the plerocercoids were separated using 2-dimensional gel electrophoresis coupled with immunoblot and mass spectrometry analysis. Based on immunoblotting patterns and mass spectrometry results, 8 antigenic proteins were identified from the plerocercoid. Among the proteins, cysteine protease protein might be developed as an antigen for diagnosis of sparganosis.
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Objective To evaluate the effects of intravenous injection of different blood components containing Babesia microti on B. microti infection in mice. Methods Healthy mice were infected with B. microti, and then blood samples were collected from the mouse orbit to prepare whole blood, serum-free blood components and pure red blood cells containing B. microti. Twenty seven BALB/c mice were divided into three groups, including the whole blood group, the serum-free blood component group and the pure red blood cell group, of 9 mice in each group, and then, each group was divided into three subgroups, of 3 mice in each subgroup, which were injected with 100 μL of blood components containing B. microti at concentrations of 9.00, 0.90, 0.09 B. microti parasites/μL (900, 90, 9 B. microti parasites) via the tail vein, respectively. Blood samples were collected from the mouse tail tip every other day since one day post-injection to prepare thin blood smears. Following Giemsa staining of blood smears, B. microti infection was identified in red blood cells using microscopy. Results Following injection of 900 B. microti parasites, B. microti was identified in the peripheral blood in the whole blood group and the serum-free blood component group 3 days post-injection, and the density of B. microti parasites started to increase 15 days post-injection and peaked 21 days post-injection, with 2.21% and 1.76% rates of B. microti infection in red blood cells, respectively. Subsequently, the density of B. microti parasites declined, and the percentage of B. microti infection in red blood cells tended to be 0 31 days post-injection. During the study period, no B. microti was found in the peripheral blood in the pure red blood cell group. Following injection of 90 B. microti parasites, B. microti was identified in the peripheral blood in the whole blood group 3 days post-injection, and the density of B. microti parasites increased 15 days post-injection and peaked 21 days post-injection, with a 1.35% rate of B. microti infection in red blood cells, while the percentage of B. microti infection in red blood cells tended to be 0 31 days post-injection. During the study period, no B. microti was detected in the peripheral blood in the serum-free blood component group or the pure red blood cell group. Following injection of 9 B. microti parasites, no B. microti was detected in the peripheral blood in the whole blood group, the serum-free blood component group or the pure red blood cell group. Conclusion Blood components and dose of B. microti parasites may affect intravenous injection of B. microti injection in mice, and transfusion of blood components may case a risk of Babesia infection.
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Objective To evaluate the effects of intravenous injection of different blood components containing Babesia microti on B. microti infection in mice. Methods Healthy mice were infected with B. microti, and then blood samples were collected from the mouse orbit to prepare whole blood, serum-free blood components and pure red blood cells containing B. microti. Twenty seven BALB/c mice were divided into three groups, including the whole blood group, the serum-free blood component group and the pure red blood cell group, of 9 mice in each group, and then, each group was divided into three subgroups, of 3 mice in each subgroup, which were injected with 100 μL of blood components containing B. microti at concentrations of 9.00, 0.90, 0.09 B. microti parasites/μL (900, 90, 9 B. microti parasites) via the tail vein, respectively. Blood samples were collected from the mouse tail tip every other day since one day post-injection to prepare thin blood smears. Following Giemsa staining of blood smears, B. microti infection was identified in red blood cells using microscopy. Results Following injection of 900 B. microti parasites, B. microti was identified in the peripheral blood in the whole blood group and the serum-free blood component group 3 days post-injection, and the density of B. microti parasites started to increase 15 days post-injection and peaked 21 days post-injection, with 2.21% and 1.76% rates of B. microti infection in red blood cells, respectively. Subsequently, the density of B. microti parasites declined, and the percentage of B. microti infection in red blood cells tended to be 0 31 days post-injection. During the study period, no B. microti was found in the peripheral blood in the pure red blood cell group. Following injection of 90 B. microti parasites, B. microti was identified in the peripheral blood in the whole blood group 3 days post-injection, and the density of B. microti parasites increased 15 days post-injection and peaked 21 days post-injection, with a 1.35% rate of B. microti infection in red blood cells, while the percentage of B. microti infection in red blood cells tended to be 0 31 days post-injection. During the study period, no B. microti was detected in the peripheral blood in the serum-free blood component group or the pure red blood cell group. Following injection of 9 B. microti parasites, no B. microti was detected in the peripheral blood in the whole blood group, the serum-free blood component group or the pure red blood cell group. Conclusion Blood components and dose of B. microti parasites may affect intravenous injection of B. microti injection in mice, and transfusion of blood components may case a risk of Babesia infection.
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Objective To understand the changes in body weight,spleen weight and complete blood cells in BALB/c mice infected with Babesia microti.Methods For the infection group,six weeks old BALB/c mice were injected intraperitoneally with a dose of 100μL of B.microti infected blood(20％RBC infection rate,each mouse).For the determination of the progres-sion of B.microti infection up to 28 days of the infection,the microscopic visualization of thin blood smears of tail blood stained with Giemsa staining was performed in the infection group.The experiment was carried out at different intervals on days 0,7,14,21,and 28 after the infection,respectively.The mice were sacrificed,and spleens were collected and weighed,and the body weight of the mice was also determined.The blood cells of the mice were analyzed by using Mindray BC-5300 Vet animal automatic hematology analyzer.Results On the first day after the infection,B.microti was visualized in RBC of the infection group.The significantly highest infection rate(55％)appeared on the seventh day of the infection,and then steadily decreased;the mice attained the latent infection phase on the 28th day post-infection,when the parasite could not be visualized in the pe-ripheral blood.The mice in the infected group acquired a significantly lowest body weight on the 7th day of the infection,and then gradually returned to normal.The weight of the spleen was the significantly highest on the 14th day of the infection,and then consistently decreased.On the 28th day of infection,the spleen weight was still higher than that of the control group.There were no significant changes in the number of white blood cells(WBC),lymphocytes,and eosinophils in the infected mice;and altered levels were all within the normal mouse reference range.The number of red blood cells,hemoglobin,and platelet count in the infected mice were decreased to the lowest level when the B.microti infection rate achieved to the highest,and then gradu-ally returned to the normal levels.Conclusions B.microti infection can cause body weight loss,splenic weight gain,and re-duction in the number of erythrocytes and platelets in whole blood of the mice.Besides,the whole blood cell analyzer has a diag-nostic significance in the identification of babesiosis.
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Objective To predict the monthly reported echinococcosis cases in China with the autoregressive integrated mov-ing average(ARIMA)model,so as to provide a reference for prevention and control of echinococcosis. Methods SPSS 24.0 software was used to construct the ARIMA models based on the monthly reported echinococcosis cases of time series from 2007 to 2015 and 2007 to 2014,respectively,and the accuracies of the two ARIMA models were compared. Results The model based on the data of the monthly reported cases of echinococcosis in China from 2007 to 2015 was ARIMA(1,0,0)(1,1, 0)12,the relative error among reported cases and predicted cases was-13.97%,AR(1)=0.367(t=3.816,P<0.001),SAR (1)=-0.328(t=-3.361,P=0.001),and Ljung-Box Q=14.119(df=16,P=0.590).The model based on the data of the monthly reported cases of echinococcosis in China from 2007 to 2014 was ARIMA(1,0,0)(1,0,1)12,the relative error among reported cases and predicted cases was 0.56%,AR(1)=0.413(t=4.244,P<0.001),SAR(1)=0.809(t=9.584, P<0.001),SMA(1)=0.356(t=2.278,P=0.025),and Ljung-Box Q=18.924(df=15,P=0.217).Conclusions The different time series may have different ARIMA models as for the same infectious diseases.It is needed to be further verified that the more data are accumulated,the shorter time of predication is,and the smaller the average of the relative error is.The estab-lishment and prediction of an ARIMA model is a dynamic process that needs to be adjusted and optimized continuously accord-ing to the accumulated data,meantime,we should give full consideration to the intensity of the work related to infectious diseas-es reported(such as disease census and special investigation).
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Fascioliasis is a foodborne zoonotic parasitic disease. We report 4 cases occurring in the same family, in whom diagnosis of acute fascioliasis was established after series of tests. One case was hospitalized with fever, eosinophilia, and hepatic lesions. MRI showed hypodense changes in both liver lobes. The remaining 3 cases presented with the symptom of stomachache only. Stool analysis was positive for Fasciola eggs in 2 adult patients. The immunological test and molecular identification of eggs were confirmed at the National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, Shanghai, China. The results of serological detection were positive in all the 4 patients. DNA sequencing of PCR products of the eggs demonstrated 100% homology with ITS and cox1 of Fasciola hepatica. The conditions of the patients were not improved by broad-spectrum anti-parasitic drugs until administration of triclabendazole.
Subject(s)
Adult , Humans , Asian People , China , Diagnosis , Eggs , Eosinophilia , Fasciola , Fasciola hepatica , Fascioliasis , Fever , Immunologic Tests , Liver , Magnetic Resonance Imaging , Ovum , Parasitic Diseases , Polymerase Chain Reaction , Sequence Analysis, DNAABSTRACT
We described 4 human infection cases of zoonotic fish-tapeworm, Diphyllobothrium nihonkaiense, identified with morphological and molecular characters and briefly reviewed Chinese cases in consideration of it as an emerging parasitic disease in China. The scolex and mature and gravid proglottids of some cases were seen, a rosette-shaped uterus was observed in the middle of the mature and gravid proglottids, and the diphyllobothriid eggs were yellowish-brown in color and displayed a small knob or abopercular protuberance on the opposite end of a lid-like opening. The average size of the eggs was recorded as 62–67×42–45 μm. The parasitic materials gathered from 4 human cases were morphologically identified as belonging to the genera Diphyllobothrium and Adenocephalus. The phylogenetic analysis based on the nucleotide sequences of cytochrome c oxidase subunit 1 gene of the etiologic agents confirmed that the 4 cases were D. nihonkaiense infection. The finding of 4 additional D. nihonkaiense cases suggests that D. nihonkaiense might be a major causative species of human diphyllobothriasis in China. A combined morphological and molecular analysis is the main method to confirm D. nihonkaiense infection.
Subject(s)
Humans , Asian People , Base Sequence , China , Diphyllobothriasis , Diphyllobothrium , Eggs , Electron Transport Complex IV , Methods , Ovum , Parasitic Diseases , UterusABSTRACT
Objective To construct cDNA entry library and cDNA expression library of Armillifer agkistrodontis (A.) nymphs and make a preliminary immunoscreening for the cDNA expression library.Methods The nymphs were collected from the Kunming mice infected experimentally with A.agkistrodontis eggs and the total RNA were extracted from the nymphs using TRIzol Reagent.After purifying the mRNA,the synthesized cDNAs were cloned into the donor vector pDONR222 by BP reaction of Gateway technology and the recombinants were transformed into the DH10B cells by electroporation,the cDNA entry library was obtained.Next,the expression vector pDEST17 was ligated with entry clones by LR reaction,and the recombinants were transformed into the BL21 (DE3) cells.Hence,the cDNA expression library was constructed.Then,the expression library was immunoscreened with the mixed sera of mice infected with A.agkistrodontis,and the insertions of positive clones were sequenced.After that,the open reading frame(ORF) of positive slone sequence,the homology of the screened genes and their encoded proteins were analyzed by Finder and BLAST (basic local alignment search tool) program of National Center of Biotechnology Information(NCBI),and the discovered new genes were submitted into the GenBank.Besides,the physico-chemical properties,secondary structure and B cell epitopes of encoded proteins were also analyzed by bioinformatics software.Results The average titer and total clones of the cDNA entry library were 1.45 × 105 CFU/ml(colony-forming unit,CFU) and 1.74 × 106 CFU,respectively,and the range of fragment length of the inserted cDNA was between 0.2-4.0 kb,with an average of 1.4 kb.The total clones of cDNA expression library were 1.00 × 105 CFU,and the fragment length of the inserted cDNA was between 0.3-2.2 kb,with an average of 1.0 kb.Five positive clones,coded S1,S5,A1,D1 and F1,respectively,were obtained through preliminary immunoscreening.The sequence and homology of the five positive clones were sequenced and analyzed by BLAST program.No significant similarities were found in pentastomida species,which meant that they were all novel genes of A.agkistrodontis.The gene sequences were submitted to GenBank,with the accession number from JQ180451 to JQ180455.Also,results obtained by bioinformatics software showed that the predictive encoding proteins were all potential to be valuable recombinant diagnostic antigens.Conclusions The cDNA library of A.agkistrodontis nymphs is successfully constructed,and five new genes of A.agkistrodontis are discovered.The establishment of cDNA library and the discovery of the new genes will lay a foundation for further studying the gene functions and screening the immunodiagnostic antigens.
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Objective The aim of the study was to investigate the epidemic situation,clinical symptom,diagnosis and epidemiological characteristics of human Fasciola gigantica infection in Dali,Yunnan province.It will also provide a scientific basis for fasciolosis control and prevention.Methods Epidemic data were collected and patient's clinical signs and symptoms were studied.Serum soluble antigen of Fasciola gigantica of patients and part of family members and health people in the same village was detected using enzyme-linked immunosorbent assay (ELISA) and the eggs of Fasciola gigantica in stool were observed under microscope.Sequencing and PCR amplification of Fasciola gigantica eggs had been done.Sequencing results were analyzed using basic local alignment search tool (BLAST) program of the U.S.National Center for Biotechnology Information (NCBI) and the similarity of the two in the sequence of nucleic acid was compared.Furthermore,patients were experimentally given orally therapeutic doses of Triclabendazole 10 mg·kg-1·d-1 daily for 2 days,and kept in the hospital for observation for one week.Moreover,host and vector were investigated in the surrounding ditches of Dali prefecture and Limnaea peregra snail samples were collected.All the snails were squashed by glass sheet in order to detect the cercarie.Cow dung and sheep manure was collected in the Limnaea peregra distribution environment,and the eggs in the feces were checked by microscope after washing and precipitation.Results All the 26 patients had a continued hyperpyrexia with distinct alimentary system symptoms of nausea,vomiting,stomachache,abdominal distension as well as hepatomegaly,sensitive to percussion,different levels of liver damage detected by CT.All the patients had an eaten history of raw Herba Houttuyniae and other aquatic plants,and the course of the disease was similar,with the same epidemiological characteristics.ELISA detection was used in the 26 patients,family members and other healthy population,the results of all the 26 patients were positive(100.0％,26/26) ; the positive rates of the 57 family members and other health people of the same village were 31.6％ (18/57) and 17.1％ (6/35),respectively.The results of sequencing and BLAST program showed that the pathogen was Fasciola gigantica with the similarity between 99％-100％.PCR amplification also confirmed that the eggs were Fasciola gigantica eggs with an approximately 1000 bp band on agarose gel.After treatment with Triclabendazole,body temperature of the patients dropped to normal and symptoms improved markedly.Moreover,329 Limnaea peregra snails were collected including 5 ones with redia and one-tailed cercariae which were preliminary identified as the larva of Fasciola gigantica.There were also eggs of Fasciola gigantica detected in one stool of cattle and one of goat.Conclusions Eating raw food is the leading cause of the onset of the disease.Triclabendazole is the drug of choice to treat Fasciolasis.Health education should be strengthened by government and disease prevention and control departments in order to make the local residents to understand the potential hazard of eating raw aquatic vegetable and drinking unboiled water,which is the key to prevent the occurrence of the disease.