[Immunoreactivity of chimeric proteins carrying poliovirus epitopes on the VP6 of rotavirus as a vector].

Rotavirus and poliovirus continue to present significant risks and burden of disease to children in developing countries. Developing a combined vaccine may effectively prevent both illnesses and may be advantageous in terms of maximizing compliance and vaccine coverage at the same visit. Recently, we sought to generate a vaccine vector by incorporating multiple epitopes into the rotavirus group antigenic protein, VP6. In the present study, a foreign epitope presenting a system using VP6 as a vector was created with six sites on the outer surface of the vector that could be used for insertion of foreign epitopes, and three VP6-based PV1 epitope chimeric proteins were constructed. The chimeric proteins were confirmed by immunoblot, immunofluorescence assay, and injected into guinea pigs to analyze the epitope-specific humoral response. Results showed that these chimeric proteins reacted with anti-VP6F and -PV1 antibodies, and elicited antibodies against both proteins in guinea pigs. Antibodies against the chimeric proteins carrying PV1 epitopes neutralized rotavirus Wa and PV1 infection in vitro. Our study contributes to a better understanding of the use of VP6-based vectors as multiple-epitope delivery vehicles and the epitopes displayed in this form could be considered for development of epitope-based vaccines against rotavirus and poliovirus.

Immunogenicity and antigenicity of a recombinant chimeric protein containing epitopes of poliovirus type 1.

To design a vaccine that simultaneously prevents both rotavirus (RV) and poliovirus (PV), a PV type 1 (PV1) chimeric protein using RV VP6 as a vector (VP6F) was constructed, expressed in Escherichia coli expression system and characterized by SDS-PAGE, Western blot, immunofluorescence assay and neutralization test. The results showed that the chimeric protein reacted with anti-VP6F and anti-PV1 antibodies and elicited production of serum antibodies against the chimeric protein in guinea pigs. Antibodies against the chimeric protein neutralized RV Wa and PV1 infection in vitro. The results provided a relevant possibility of developing novel approaches in the rational design of vaccines effective against both RV and PV.

First Report of Bacterial Soft Rot of Horn Lian (Typhonium giganteum) Caused by Pectobacterium carotovorum subsp. carotovorum in Jilin Province of China.

Horn lian (Typhonium giganteum) is a perennial herb of the family Aracea and is commonly used for expelling phlegm and as an antispasmodic treatment. In August 2012, horn lian grown in Changchun, Jilin Province of China, exhibited soft rot disease with ~60% incidence and experienced great losses. Water-soaked and dark green lesions on leaves expanded along main veins. Semitransparent, water-soaked, and sunken lesions on stems expanded rapidly and caused the whole plant to collapse with a foul smell. Nine representative strains were isolated from infected leaves and stems on nutrient agar (NA) medium after 36 h incubation at 28°C (1). Colonies were round, shiny, grayish white, and convex on NA medium. All strains were gram-negative, non-fluorescent on King's B medium (KB), facultatively anaerobic, motile with three to six peritrichous flagella (observed by electron transmission microscope), positive for catalase and pectolytic activity test on potato slices, but negative for oxidase, urease, and lecithinase. Strains grew at 37°C and in yeast salts broth medium containing 5% NaCl. They also liquefied gelatin and reduced nitrate, but did not reduce sucrose. Strains were also negative for starch hydrolysis, malonate utilization, gas production from glucose and indole. Results were variable for the Voges-Proskauer test. The strains utilized sucrose, arabinose, fructose, D-galactose, D-glucose, inositol, lactose, D-mannose, D-mannitol, melibiose, rhamnose, salicin, trehalose, maltose, raffinose, glycerol, D-xylose, and cellobiose as carbon sources, but not melezitose, α-CH3-D-gluconate, sorbitol, or dulcitol. Species identity was confirmed by molecular characterization of one of the nine strains, DJL1-2. DNA GC content indicated by high performance liquid chromatography (HPLC) was 51.7%. The 16S rDNA sequence (KC07897) of DJL1-2 showed 99% identity to that of a Pectobacterium carotovorum subsp. carotovorum (Pcc) strain (CP001657) and the sequence of the 16S-23S rDNA spacer region (KJ623257) was 93% similar to that of another known strain of Pcc (CP003776). As a result, the strains were identified as Pcc (2). Pathogenicity of the nine strains was evaluated by spraying 1 ml of bacterial cell suspension (108 CFU/ml) onto healthy leaves and injecting 0.1 ml of cell suspension into stems of 3-year-old horn lian plants with a sterile pipette tip. Three seedlings were used for each strain and sterilized water served as negative controls. Pcc SMG-2 reference strain (from milk thistle) was also inoculated into horn lian leaves and stems. Inoculated plants were covered with plastic bags for 24 h in a greenhouse at 28 to 30°C. After 72 h, water-soaked lesions similar to the naturally infected plants were observed on leaves and stems inoculated by the nine isolated strains and Pcc SMG-2, while negative control plants remained symptomless. Biochemical tests and 16S rDNA sequence analysis confirmed that the re-isolated bacteria were Pcc. To our knowledge, this is the first report of Pcc causing bacterial soft rot of horn lian in Changchun, Jilin Province, China. References: (1) Z. D. Fang. Research Method of Phytopathology. China Agricultural Press, 1998. (2) N. W. Schaad, et al. Laboratory Guide for Identification of Plant Pathogenic Bacteria, 3rd ed. American Phytopathological Society, St. Paul, MN, 2001.

Alternaria brassicicola Causes a Leaf Spot on Isatis indigotica in China.

Chinese woad (Isatis indigotica) is a biennial herb in the Brassicaceae that is widely cultivated in China. Extracts from the roots and leaves have potential pharmaceutical use for treatment of flu, encephalitis, measles, hepatitis, and mumps (2). In June 2012, a leaf spot was observed on 1-year-old plants of I. indigotica in the medicinal garden of Jilin Agricultural University, Changchun, Jilin Province, China. More than 50% of the leaves and 100% of the plants in the garden were symptomatic. In the initial stage of infection, irregular to circular, dark gray spots, each surrounded by a chlorotic halo, appeared on leaves. The spots ranged from pinpoint to 5 mm in diameter. Some spots enlarged and coalesced, forming concentric rings. Black, sunken, fusiform lesions were observed on the petioles. Lesions gradually dried and exhibited a shot-hole appearance, and entire infected leaves desiccated. Small pieces of infected leaves and petioles were surface-disinfested in 75% ethanol for 60 s, rinsed thrice in sterilized distilled water, dried, and plated on potato dextrose agar. Olive-green mycelium developed after 2 days of incubation at 25°C, turned dark green, and covered the petri dish 10 days later. The periphery of each colony was gray and velvety. On potato carrot agar medium, conidia formed on branched chains. Conidiophores arose singly or in clusters, were straight or flexuous, separated, and measured 6.8 to 26.7 × 3.1 to 11.9 µm Conidia on host plant tissues were olivaceous, cylindrical or inverted clavate, and 25.8 to 65.2 × 10.9 to 18.3 µm Larger conidia were cylindrical or obclavate, and smaller conidia were oval. Transverse and longitudinal septa of conidia ranged from 3 to 10 and from 0 to 7 µm, respectively. A very small conidial beak or no beak was observed on each conidium. On the basis of these morphological characteristics,the fungus was identified as Alternaria brassicicola (3). A PCR assay with the ITS4 and ITS5 primers was used to amplify DNA extracted from each of four isolates (1). The sequence (567 bp) of isolate Sl-8 was submitted to GenBank (Accession No. KF531832), and showed 100% similarity to that of an A. brassicicola isolate (AF392985.1), confirming the species identification. Pathogenicity assays with 10 single-conidium isolates were done by spraying a conidial suspension (1 × 106 conidia/ml) of each isolate, or sterilized water for the control treatment, onto healthy leaves and petioles of five 3-month-old plants of I. indigotica. Inoculated and control plants were enclosed in plastic bags for 48 h. After 7 days, symptoms on inoculated plants were similar to those on the original diseased plants, while control plants remained symptomless. Re-isolation from inoculated plants produced mycelial colonies with morphological characteristics of A. brassicicola, fulfilling Koch's postulates. No fungus was isolated from control plants. A. napiformis and A. brassicae have been reported as causal agents of Alternaria leaf spot on I. indigotica in China (3). To our knowledge, however, this is the first report of A. brassicicola as a pathogen on I. indigotica in China. References: (1) N. L. Glass and G. C. Donaldson. Appl. Environ. Microbiol. 61:1323, 1995. (2) A. J. Li et al. Flora Reipublicae Popularis Sinicae Tomus 33, 1998. (3) T. Y. Zhang. Alternaria. Pages 99-100 in: Flora Fungorum Sinicorum, 2003.

First Report of Bacterial Soft Rot of Milk Thistle (Silybum marianum) Caused by Pectobacterium carotovorum subsp. carotovorum in Jilin Province of China.

Milk thistle (Silybum marianum) is an annual or biannual plant of the Asteraceae family that produces the hepaprotectant silymarin. In 2012, almost all milk thistle grown in the medicinal herbal garden of Jilin Agricultural University (Changchun, Jilin Province, China) exhibited symptoms of a previously undetected soft rot disease. Initial symptoms on stems appeared as tan, semitransparent, and water-soaked, then became sunken. The rotted lesions expanded rapidly and inner stem tissues were rotten with a foul smell. Eventually, the whole plant became black, then collapsed and died. Economic losses were significant as the seed crop was almost completely lost. Nine bacterial strains were isolated from tissues on nutrient agar (NA) medium after 36 h incubation at 28°C (1). Colonies of the nine strains were round, shiny, grayish white, and convex on NA medium. All strains were gram-negative, non-fluorescent, facultatively anaerobic, motile with two to four peritrichous flagella (observed by electron transmission microscope), positive for catalase and potato rot, but negative for oxidase and lecithinase. Strains grew at 37°C and in yeast salts broth medium containing 5% NaCl. They also liquefied gelatin. Strains were also negative for starch hydrolysis, malonate utilization, gas production from glucose, and indole. Results were variable for the Voges-Proskauer test and production of H2S from cysteine. The strains utilized esculin, fructose, D-galactose, D-glucose, inositol, lactose, D-mannose, D-mannitol, melibiose, rhamnose, salicin, trehalose, D-xylose, and cellobiose as carbon sources, but not melezitose, α-CH3-D-gluconate, sorbitol, or starch. Glycerol and maltose were only weakly utilized. Species identity was confirmed by molecular analysis of one of the strains, SMG-2. HPLC indicated a DNA GC content of 50.55%. The 16S rDNA sequence (KC207898) of SMG-2 showed 99% sequence identity to that of a Pectobacterium carotovorum subsp. carotovorum strain (DQ333384) and the sequence of the 16S-23S rDNA spacer region (KJ415377) was 95% similar to that of another known strain of P. carotovorum subsp. carotovorum (AF232684). Based on biochemical and physiological characteristics (2), as well as 16S rDNA gene analysis, the strains were identified as P. carotovorum subsp. carotovorum. Pathogenicity of the nine strains was evaluated by depositing a bacterial suspension (108 CFU/ml) on wounded stems (made with a disinfected razor blade) of 3-month-old milk thistle plants. Three plants were inoculated with each strain and three plants were treated with sterilized water as negative controls. Inoculated plants were covered with plastic bags for 24 h in a greenhouse at 28 to 30°C. After 48 h, the plants inoculated with bacteria showed similar symptoms as the naturally infected plants, while control plants remained symptomless. The symptoms observed on inoculated stems were rotten and sunken tissues. Bacteria were re-isolated from the inoculated plants and confirmed to be identical to the original strains based on 16S rDNA sequence analysis. To our knowledge, this is the first report of P. carotovorum subsp. carotovorum causing bacterial soft rot of milk thistle in Changchun, Jilin Province, China. References: (1) Z. D. Fang. Research Method of Phytopathology. China Agricultural Press (In Chinese), 1998. (2) N. W. Schaad et al., eds. Laboratory Guide for Identification of Plant Pathogenic Bacteria, 3rd ed. American Phytopathological Society, St. Paul, MN, 2001.

First Report of Phoma rhei as a Pathogen of Rheum rhabarbarum in China.

Rheum rhabarbarum L., rhubarb, is a perennial herb planted mainly in Hebei, Hubei, Shanxi, Heilongjiang, and Jilin provinces as well as Inner Mongolia, China. The plant grows about 1,000 meters above sea level (4), and is used widely in China to treat constipation and gout. From June to September 2012, a leaf spot was observed on R. rhabarbarum in the medicinal garden of Jilin Agricultural University, Changchun, Jilin Province, causing significant effects on the leaves of all infected plants. In the early stage of disease development, small red lesions were visible on infected leaves, which subsequently developed into irregularly shaped or circular necrotic spots, each with a light colored center, pink-red alternating concentric rings, and surrounded by a chlorotic halo. Some lesions became perforated in the center. Lesions ranged from 1 to 15 mm in diameter. Extensive spotting resulted in general browning and yellowing of entire leaves. As lesions enlarged and coalesced, some leaves died from the margin inwards. Lesions on the stem were fusiform and sunken. Small pieces of diseased leaves and stems were surface-disinfested in 75% ethanol for 60 s, rinsed twice in distilled water, dried, and plated on potato dextrose agar (PDA). A Phoma species was isolated that produced a gray or dark gray colony after 5 to 7 days. The isolate was transferred to oatmeal agar (OA) (3). Pycnidia were dark brown to black, globose to subglobose, and 121 to 354 × 100 to 262 µm. Conidia were ellipsoidal or reniform, colorless, unicellular, and 3.8 to 6.5 × 1.7 to 4.1 µm. On the basis of these characteristics, the fungus was identified as Phoma rhei (1). A PCR assay with the ITS4 and ITS5 primers was used to amplify the internal transcribed spacer (ITS) region of ribosomal DNA (rDNA) (2). The amplified product (567 bp) was sequenced and the sequence submitted to GenBank (Accession No. KF531831). The ITS sequence exhibited 99% identity to that of a P. rhei isolate in GenBank (GU237743.1), confirming the morphological identification. Pathogenicity of eight isolates on rhubarb was confirmed by spraying a spore suspension (1 × 106 spores/ml) produced on PDA on the leaves of each 6-year-old R. rhabarbarum (cv. Boyedahuang) plant. Each isolate was inoculated onto five plants, and five plants were sprayed similarly with sterilized water as a control treatment. The plants were then covered with plastic bags for 48 h, and kept in a greenhouse (20 to 30°C with a 12-h photoperiod/day). Initial symptoms on inoculated leaves were observed 3 to 4 days after inoculation, while the control plants remained healthy. Re-isolations from lesions on the inoculated leaves, using the same protocol as the original isolations, produced fungal colonies with the same morphological characteristics as the original isolates of P. rhei, but no fungi were re-isolated from the control plants. This fungus has been found on R. rhaponticum in New Zealand (1), but to our knowledge this is the first report of P. rhei on R. rhabarbarum in China. References: (1) G. H. Boerema et al. Phoma Identification Manual. Diffferentiation of Specific and Infra-Specific Taxa in Culture. CABI Publishing. Wallingford, UK, 2004. (2) D. E. L. Cooke et al. Mycol. Res. 101:667, 1997. (3) Z. D. Fang. Research Method of Phytopathology. China Agricultural Press (In Chinese), 1998. (4) A. J. Li et al. Flora Reipublicae Popularis Sinicae. Tomus 25:171, 1998.

[The experimental study on optimal cell density and formation time of tissue engineered autologous cartilage].

OBJECTIVE: This paper aims to investigate the suitable cell density and the best formation time of tissue engineered autologous cartilage and to provide theoretical basis and parameters for clinical application. METHODS: The chondrocytes isolated from mini swines' ears were mixed with injectable biocompatible matrix (Pluronic), and the density of cell suspensions were 10, 20, 30, 40, 50, 60, 70 x 10(4)/ml. The chondrocyte-polymer constructs were subcutaneously injected into the abdomen of autologous swine. The specimens were observed grossly and histologically after 6 weeks, and investigated the suitable cell density. Then the chondrocyte-polymer constructs with suitable cell density were transplanted into the abdomen of autologous swine and evaluated grossly and histologically in 1, 3, 6, 9, 15 weeks after transplantation to investigate the best formation time of tissue engineered cartilage. RESULTS: The experiments demonstrated that the tissue engineered autologous cartilage was similar to the natural cartilage on animals with normal immune system in histological characteristics. The optimal chondrocyte density is 50 x 10(6)/ml, and the proper harvest time is the sixth week. CONCLUSION: With tissue engineering skills, we have identified the optimal chondrocyte density and the proper harvest time.

[The clinical analysis of 39 cases of oral mucosal hyperplasia]

This study was reviewing in analysed the clinical,histopathological and treatment features of 39 cases suffered in oral mucosal hyperplasia.It definited the sorts of disease which may be malignant changed and the histopathological grading criteria of hyperplasis.The different treatment methods were administered according to the different grade of the malignant change.The results of the post-treatment observation showed that the drug method only be fit for the cases suffered slight hyperplasia without nuclei differentiation.

[Primary non-Hodgkin's lymphoma on parotid gland clinicopathologic and immunohistochemical study.].

A clinico-pathologic and immunohistochemical study of 20 cases of primary non-Hodgkin's lymphoma on the parotid gland was made.The result showed 45% of the cases originated from B cells,white 40% from T cells,the rest 15% from the small lymphocytes.Immunohistochemical study was made on 11 cases,the result of immunophenotype was in concordance with the histopathologic diagnosis in 10 cases,except in one case which was initially diagnosed as plasmocytoid lymphocytic lymphoma,but the immunmophenotype suggested that it was T cell type of malignant lymphoma(ML).Follow-up data can be collected in 16 cases(80%).Three of the 16 cases(18.75%) died of the tumor within one year,they were diagnosed initially as B cleaved cell,lymphoplastic cell and polymorphic T cell type of ML,each in 1 case.Other cases survived from 15 months to 9.25 years,among them 2 were diagnosed as low-grade ML(both of small lymphocyte type),7 cases as middle-grade ML(5 of B cleaved cell,2 of B mixed type),and 4 cases as high-grade ML (2 of clear T cell,2 of polymorphic T cell type).They were treated by surgical removal and combined western and traditional Chinese medicine,their prognosis was quite good.

[Comparative study on healing process between vascularized and non-vascularized autogenous bone grafts]

This study made a histological comparison (light microscope and transmission electron microscope) between vascularized bone graft (VBG) and non-vascularized bone graft (NVBG) in mandible of dog.The study showed:the healing process of VBG was the same as that of bone fracture.The "creeping substitution" process of NVBG was imbued with the inflammation induced by dead bone.There was no significat difference on the bone union between VBG and NVBG perhaps for the recipient region was better vascularized.

Immune function in transplanted small intestine. Total secretory IgA production and response against cholera toxin.

Secretory IgA is the dominant immunoglobulin produced in the small intestine and one important component of the local defense against dietary and infectious agents present in the gut lumen. The effect of small intestine transplantation on total production of sIgA and on the response to a newly presented antigen, cholera toxin, was determined in a rat segmental heterotopic intestinal transplant model. Lewis x Brown Norway F1 (LBNF1) allografts in Lewis hosts made normal amounts of sIgA, when compared with LBNF1 Thiry-Vella loops or LBNF1 isografts. In contrast, the allografts failed to make a significant specific sIgA response when immunized with cholera toxin at days 0 and 7 following transplantation. This failure was not the result of surgical manipulation, as isografts made normal amounts of specific sIgA directed against cholera toxin. Cyclosporine immunosuppression delayed, but did not prevent, the secretion of specific antibody in isografts. This failure to respond to a new antigen may have important implications for the safety of small bowel transplantation.