Ziqiang Cheng

Development of colloidal gold-based immunochromatographic assay for rapid detection of Mycoplasma suis in porcine plasma.

A one-step immunochromatographic assay using gold nanoparticles coated with polyclonal antibody (pAb) against Mycoplasma suis (M. suis) was developed in this study for the detection of M. suis in porcine plasma. The colloidal gold was prepared by the reduction of gold salt with sodium citrate coupled with pAb against M. suis. The pAb was produced by immunizing the BALB/c mice with recombinant MSG1 (rMSG1) protein from M. suis expressed in Escherichia coli. The optimal concentrations of the capture antibody and the coating antibody were 12 μg/ml and 1.5 mg/ml, respectively, and that of the blocking buffer was 1% bovine serum albumin. The lower detection limit of the immunochromatographic assay test was 100 ng/ml with visual detection under optimal conditions of analysis. Classical swine fever virus, porcine reproductive and respiratory syndrome virus, swine pneumonia mycoplasma, swine toxoplasma, and porcine parvovirus were used to evaluate the specificity of the immunochromatographic strips. No cross-reaction of the antibodies with other related swine pathogens was observed. This qualitative test based on the visual evaluation of the results did not require any equipment. The assay time for M. suis detection was less than 10 min, suitable for rapid detection at the grassroots level. The one-step colloidal gold immunochromatographic strips that we developed had high specificity and sensitivity. Therefore, this method would be feasible, convenient, rapid, and effective for detecting M. suis in porcine plasma.

Detection of Babesia divergens using molecular methods in anemic patients in Shandong Province, China

Babesiosis (piroplasmosis) is a zoonotic disease caused by an intraerythrocytic protozoan transmitted by Ixodes ticks. The aim of this study was to detect Babesia spp. infection using molecular methods in 377 blood samples from anemic patients. Sequence analysis showed that the 18S rRNA gene was 439 bases long by polymerase chain reaction (PCR) amplification and that the PCR products from the samples had an identical sequence (named Taian China, HM355854). BLAST search showed that the sequence was identical to the 18S rRNA sequences of Babesia divergens. The 18S rRNA sequence for Toxoplasma gondii was included as the outlier for phylogenetic analysis by using the program MEGA4.0 software. The results showed that the 18S rRNA gene sequences obtained from the present study was most closely related to B. divergens Switzerland (DQ312439) with 98.4% similarity (differing only by seven bases). The phylogenetic analysis also revealed that this sequence closely resembled B. divergens strains from other countries and belonged to the same clade. This is the first report of a human being infected by B. divergens in China.

Maternal antibody induced by recombinant gp85 protein vaccine adjuvanted with CpG-ODN protects against ALV-J early infection in chickens.

In this study, the efficacy of a recombinant protein vaccine encoding the gp85 gene from the subgroup J avian leukosis virus (ALV-J) co-administered with cytosine-phosphate-guanine oligodeoxynucleotide (CpG-ODN) or Freunds adjuvants was investigated for the protection against early ALV-J infection in chickens. The gp85 gene from ALV-J was amplified using polymerase chain reaction (PCR), and the recombinant protein was expressed in Escherichia coli. The purified recombinant protein was injected intramuscularly into the breeder hens along with CpG-ODN or Freunds adjuvants, and the antibodies in the serum were assayed regularly post inoculation. The fertilized eggs from the vaccinated hens were hatched, the hatched chickens were challenged with 10(2.2) 50% tissue culture infective dose (TCID50) ALV-J on 1 day, and the maternal antibodies in the hatched chickens were examined regularly before and after the challenge. The viremia was determined weekly, and a histopathological analysis of the immunosuppressive lesions was performed. The results suggest that the gp85 recombinant protein was successfully prepared and was inoculated with CpG-ODN adjuvant into breeder hens to induce serological antibody against ALV-J in the hens and in the hatched chickens. The positive maternal antibodies in the hatched chickens provided effective protection for most chickens against viremia and dramatically decreased the number of immunosuppressive lesions; these protective effects were better than those of the gp85 recombinant protein plus Freunds adjuvant. The data will provide a scientific basis for the application of the ALV-J subunit vaccine to control ALV-J infection in chicken flocks.

Liposomes containing recombinant gp85 protein vaccine against ALV-J in chickens

To study the potential of liposome vaccines in the clinical prevention of ALV-J, the effect of recombinant gp85 protein of subgroup J avian leukosis virus (ALV-J) entrapped by liposomes in chickens against ALV-J infection was investigated in this paper. A recombinant plasmid (PET28a-gp85) containing the PET28a vector and gp85 gene was constructed and then expressed in Rosetta (DE3) cells with 0.5mM IPTG to produce recombinant gp85 proteins that could be entrapped by liposomes through reverse-phase evaporation. The chickens were inoculated intramuscularly either once or twice with the liposomes or with Freunds adjuvant emulsion containing recombinant gp85 protein. Sixty chickens were raised to one week old for the first inoculation and to three weeks old for the second inoculation. Chickens raised to five weeks old were challenged with a 10(2.4) 50% tissue culture infective dose (TCID50) of ALV-J. Blood samples were collected from each chicken at weekly intervals for serum antibody and viremia analyses. Changes in serum antibodies showed that positive serum antibodies (S/P value >0.6) could be induced in all groups regardless of the frequency of inoculation but improved significantly in the twice-inoculated groups. As well, high levels of antibodies emerged earlier in the Freunds adjuvant groups but persisted longer in the liposome groups. Detection of viremia indicated that the liposomes provide better protection against ALV-J than Freunds adjuvant emulsion and that this protection is directly influenced by serum antibody levels. Overall, this study reveals the potential of liposome vaccines containing recombinant gp85 protein in the clinical prevention of ALV-J.

Liposomes containing recombinant E protein vaccine against duck Tembusu virus in ducks

To obtain an effective vaccine candidate against duck Tembusu viral (DTMUV) disease which causes egg-drop and great economical loss in the Chinese duck industry, liposome vaccines containing recombinant E protein were prepared and assessed in this study. The recombinant plasmid (PET28a-E) was constructed and transformed into BL21 (DE3) cells to produce E proteins. The recombinant E proteins were purified and entrapped by liposomes through reverse-phase evaporation. Eighty-four cherry valley ducks were randomly divided into seven groups and inoculated intramuscularly at one- or seven-day-old with liposomes-E protein or Freunds adjuvant-E protein vaccine. Blood samples were collected from the first week to the tenth week for serum antibody, plasma for viremia, as well as oropharyngeal and cloacal swabs for virus shedding analyses after being challenged with a 10(2.4) 50% tissue culture infective dose (TCID50) of duck Tembusu virus. Results showed that serum antibody level of the liposomes vaccine was higher than the Freunds adjuvant vaccine, and inoculating twice was superior to once; furthermore, the viremia and virus shedding tests also proved that the liposomes vaccine can provide complete protection against DTMUV challenge. These results demonstrated that the liposomes-E protein vaccine could be used as a potential candidate vaccine to prevent DTMUV infection in ducks.

Serological survey of canine dirofilariosis in Chongqing, Kunming, Nanchang, Fuzhou, Guangzhou, Shenzhen, and Nanning in Southern China.

The present study conducts a serological survey on the presence of canine dirofilariosis in domestic dogs using an enzyme-linked immunosorbent assay (ELISA) kit. A total of 310 household dogs (166 females and 144 males) in Chongqing, Kunming, Nanchang, Fuzhou, Guangzhou, Shenzhen, and Nanning in Southern China were examined. Of the 310 dogs, 42 (13.5%) were seropositive for dirofilariosis. No statistically significant difference was observed in terms of sex in the seroprevalence of dirofilariosis using the ELISA kit. The positive rates for dirofilariosis were 6.6% in the 0-1-year-old group, 13.8% in the 1-4-year-old group, and 21.6% in the less than 4-year-old group. The statistical analysis revealed that significant differences were observed in the 1-4-year-old group (P=0.037, OR=0.441, 95% CI=0.170-1.144) and less than 4-year-old group (P<0.001, OR=0.256, 95% CI=0.095-0.693). In the regional comparison, the shoreline city Shenzhen (18.8%) had a significantly higher prevalence than urban and mountain areas (P<0.05, OR=0.310, 95% CI=0.066-1.445). In conclusion, Dirofilaria immitis infection in domestic dogs was present in Chongqing, Kunming, Nanchang, Fuzhou, Guangzhou, Shenzhen, and Nanning. Therefore, heartworm treatment and/or chemoprophylaxis for the captured domestic dogs are necessary in these areas. To the best of our knowledge, the present study is the first using serological methods to examine D. immitis infection in domestic dogs in Mainland China in the recent years.

Seroprevalence of Toxoplasma gondii in Dogs in Shandong, Henan, and Heilongjiang Provinces, and in the Xinjiang Uygur Autonomous Region, People's Republic of China

abstract:  Seroprevalence of Toxoplasma gondii infection was determined in sera of 632 dogs (551 pets, 81 strays) from Shandong, Henan, and Heilongjiang Provinces, and in the Xinjiang Uygur Autonomous Region, Peoples Republic of China (PRC), using the indirect hemagglutination assay (cutoff titer 1∶64 or higher); 11.1% were seropositive. The seroprevalence in stray dogs and in ≥3-yr-old dogs was significantly higher (P < 0.05) than that in household dogs and in <3-yr-old dogs. There were no significant differences in terms of gender, breed, or locality (P ≥ 0.05). The results indicate that T. gondii infections are common in dogs in PRC.

Influence of REV and ALV-J co-infection on immunologic function of T lymphocytes and histopathology in broiler chickens.

Abstract The aim of present investigation is to study the effect of single- and co-infection with REV and ALV-J on T lymphocytes bioactivities and histopathology in broiler chickens. The bioactivities of blood and spleen T lymphocytes including lymphoproliferation responses, cytotoxicitic responses, and histopathology of spleen were detected in broiler chickens singly- or co-infected with REV and ALV-J at different days post inoculation and the virus expressions in spleen of infected broiler chickens were detected with immunofluorescence assay (IFA). The results indicated that blood and spleen T lymphocytes proliferation responses and cytotoxicity in broilers infected with REV or/and ALV-J were inhibited in the whole observed period compared with controls. In the co-infected chickens they were highly inhibited than in the single-infected. The histopathology of spleen in infected chickens at 17 and 37 d post inoculation (dpi) indicated that cell interium increased, the numbers of lymphocytes decreased, and the regrowth were destroyed or decreased, especially more significantly at 17 than at 37 dpi. The different numbers of virus were detected in spleen lymphocytes in REV-infected and/or ALV-J-infected chickens. In the spleen of co-infected chicken, both REV and ALV-J were detected and the total numbers of viruses were more than in chickens singly-infected with REV or ALV-J. Thus, the co-effect of REV and ALV-J caused more immunosuppression on T lymphocytes bioactivities in broiler chickens than single-effect of ALV-J or REV, which contributed to the sever histopathology and the product of tumor cells. This study will be helpful for understanding the effect of co-infection with many viruses and control them in poultry.

Effects of chromium picolinate on the viability of chick embryo fibroblast.

Chromium picolinate (CrPic), which is used as a nutritional supplement and to treat type 2 diabetes, has gained much attention because of its cytotoxicity. This study evaluated the effects of CrPic on the viability of the chick embryo fibroblast (CEF) using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, morphological detection, and flow cytometry. The results show that lower concentrations of CrPic (8 and 16 μM) did not damage CEF viability (p > 0.05). However, higher CrPic concentrations (400 and 600 μM) indicated a highly significant effect on the production of intracellular reactive oxygen species, alteration of mitochondrial membrane potential, intracellular calcium ion concentration, and the apoptosis rate (p < 0.01), contrary to lower CrPic concentrations (8 and 16 μM) and control group. Moreover, apoptotic morphological changes induced by these processes in CEF were confirmed using Hoechst 33258 staining. Cell death induced by higher concentrations of CrPic was caused by an apoptotic and a necrotic mechanism, whereas the main mechanism of oxidative stress-induced mitochondrial dysfunction was apoptotic death.

Electrochemical immunosensor with nanocellulose-Au composite assisted multiple signal amplification for detection of avian leukosis virus subgroup J

A sensitive sandwich-type electrochemical immunosensor was developed for the detection of avian leukosis virus subgroup J (ALV-J), which benefitted from multiple signal amplification involving graphene-perylene-3,4,9,10-tetracarboxylic acid nanocomposites (GR-PTCA), nanocellulose-Au NP composites (NC-Au) and the alkaline phosphatase (ALP) catalytic reaction. GR-PTCA nanocomposites on glassy carbon electrodes served as the immunosensor platform. Due to their excellent electrical conductivity and abundant polycarboxylic sites, the GR-PTCA nanocomposites allowed fast electron transfer and good immobilization of primary antibodies, thereby affording a strong immunosensor signal in the presence of ALV-J. The detected signal could be further amplified by the introduction of NC-Au composites as a carrier of secondary antibodies (Ab2) and by harnessing the catalytic properties of Au and ALP. Under optimized testing conditions, the electrochemical immunosensor displayed excellent analytical performance for the detection of ALV-J, showing a linear current response from 102.08 to 104.0 TCID50/mL (TCID50: 50% tissue culture infective dose) with a low detection limit of 101.98 TCID50/mL (S/N = 3). In addition to high sensitivity, the immunosensor showed very good selectivity, reproducibility and operational stability, demonstrating potential application for the quantitative detection of ALV-J in clinical diagnosis.