Evaluation of biofilm of Aeromonas hydrophila for oral vaccination of Channa striatus.

Our laboratory has developed a biofilm oral vaccine of Aeromonas hydrophila, which has given significantly higher antibody agglutination titre and protection in herbivorous carps and omnivorous walking catfish compared to that with free cell vaccine. Against this background, in the present study A. hydrophila biofilm oral vaccine was evaluated in Channa striatus, a carnivorous fish model. The fish was fed with biofilm (BF) and free cell (FC) of A. hydrophila vaccine at 10(10) cells/g fish/day for 20 d. Serum antibody production monitored with a monoclonal antibody based ELISA for 60 day post vaccination. Significantly higher antibody titre was recorded with BF compared to that with FC. Furthermore, BF vaccinated fish upon challenge with A. hydrophila at 10(9) cfu/ml had significantly higher relative per cent survival (88) than that with FC (29.6).

Monoclonal antibody based immunodot for specific detection of proteins of the shrimp Penaeus species.

Frozen shrimp continued to be the single largest item of export from India in terms of value accounting for about 44% of the total marine export earnings. Headless, peeled frozen shrimp is a common and dominant item in the market and there is need for differentiating peeled Penaeus sp from Metapenaeus, Parapenopsis and Macrobrachium sp as consumer preference and price vary. Furthermore, there is need to find out original species used in value addition of shrimp products. Hence, it is essential for development of simple and consumer friendly technique for the identification of shrimp and their products in the market. Two monoclonal antibodies (MAbs) C-15 (IgG3) and C-52 (IgG2a) reacting with 65 and 47 kD proteins of Penaeus monodon respectively in the Western blot were selected. In epitope analysis by immunodot, the two MAbs reacted and recognized specific proteins of P. monodon, Fenneropenaeus indicus and Littopenaeus vannamei and not that of Metapenaeus, Parapenopsis, Macrobrachium rosenbergii, crabs and fishes. The immunodot required 120 min for completion. The sensitivity of the immunodot to detect proteins of P. monodon was 0.225 mg with MAb C-15 and 0.028 mg with MAb C-52. The MAb based immunodot developed, could be used for identifying and differentiating meat of P. monodon, F. indicus, and L. vannamei from that of Metapenaeus, Parapenopsis, M. rosenbergii, crabs and fishes.

Multilineage potential and proteomic profiling of human dental stem cells derived from a single donor.

Dental tissues provide an alternative autologous source of mesenchymal stem cells (MSCs) for regenerative medicine. In this study, we isolated human dental MSCs of follicle, pulp and papilla tissue from a single donor tooth after impacted third molar extraction by excluding the individual differences. We then compared the morphology, proliferation rate, expression of MSC-specific and pluripotency markers, and in vitro differentiation ability into osteoblasts, adipocytes, chondrocytes and functional hepatocyte-like cells (HLCs). Finally, we analyzed the protein expression profiles of undifferentiated dental MSCs using 2DE coupled with MALDI-TOF-MS. Three types of dental MSCs largely shared similar morphology, proliferation potential, expression of surface markers and pluripotent transcription factors, and differentiation ability into osteoblasts, adipocytes, and chondrocytes. Upon hepatogenic induction, all MSCs were transdifferentiated into functional HLCs, and acquired hepatocyte functions by showing their ability for glycogen storage and urea production. Based on the proteome profiling results, we identified nineteen proteins either found commonly or differentially expressed among the three types of dental MSCs. In conclusion, three kinds of dental MSCs from a single donor tooth possessed largely similar cellular properties and multilineage potential. Further, these dental MSCs had similar proteomic profiles, suggesting their interchangeable applications for basic research and call therapy.

Development and standardization of a monoclonal antibody-based rapid flow-through immunoassay for the detection of Aphanomyces invadans in the field.

A monoclonal antibody-based flow-through immunoassay (FTA) was developed using a nitrocellulose membrane placed on the top of adsorbent pads enclosed in a plastic cassette with a test zone at the center. The FTA could be completed within 10 min. Clear purple dots against a white background indicated the presence of Aphanomyces (A.) invadans. The FTA limit of detection was 7 µg/mL for A. invadans compared to 56 µg/mL for the immunodot. FTA and polymerase chain reaction (PCR) could detect A. invadans in fish tissue homogenates at a 10(-11) dilution compared to a 10(-8) dilution by immunodot. In fish suffering from natural cases of epizootic ulcerative syndrome (EUS) collected from Mangalore, India, FTA and PCR could detect A. invadans in 100% of the samples compared to 89.04% detected by immunodot. FTA reagents were stable and produced expected results for 4 months when stored at 4~8°C. This rapid test could serve as simple and cost-effective on-site screening tool to detect A. invadans in fish from EUS outbreak areas and in ports during the shipment of live or frozen fish.

Epitope analysis of white spot syndrome virus of Penaeus monodon by in vivo neutralization assay employing a panel of monoclonal antibodies.

A panel of six monoclonal antibodies (MAbs) against the major envelope proteins VP18, VP26 and VP28 of white spot syndrome virus (WSSV) was evaluated for neutralization of the virus in vivo in Penaeus monodon. WSSV stock diluted to 1 x 10⁻6 resulting in 100% mortality on 12 day post injection (dpi) was used as optimum infectious dose of virus for challenge. Constant quantity (100 µg/ml) of MAbs C-5, C-14, C-33, C-38, C-56 and C-72 was incubated separately with WSSV (1 x 10⁻6 dilution) at 27 °C for 90 min and injected to shrimp. WSSV infection was neutralized by the MAbs C-5, C-14 and C-33 with a relative percent survival (RPS) of 60, 80 and 60 on 12 dpi, respectively compared to 100% mortality in positive control injected with WSSV alone. MAbs C-38, C-56 and C-72 could neutralize WSSV infection with RPS on 12 dpi of 40, 30 and 30, respectively. Shrimp injected with WSSV (1 x 10⁻6 dilution) incubated with panel of the MAbs at 100 µg/ml separately were subjected to nested PCR analysis at 0, 8, 12, 24, 36, 48 and 72 hour post injection (hpi) to provide further evidence for neutralization. MAbs C-5, C-14 and C-33 showed delay in WSSV positivity by 24 and 48 hpi by 2nd and 1st step PCR, respectively. MAbs C-38, C-56 and C-72 showed WSSV positivity by 12 and 24 hpi by 2nd and 1st step PCR, respectively. Shrimp injected with WSSV alone showed WSSV positivity by 8 and 12 hpi by 2nd and 1st step PCR, respectively. The study clearly shows that infectivity of WSSV could be delayed by MAbs C-14, C-5 and C-33.

Evaluation of immune response and resistance to diseases in tiger shrimp, Penaeus monodon fed with biofilm of Vibrio alginolyticus.

Immune response in juvenile tiger shrimp, Penaeus monodon fed with biofilm (BF) and free cells (FC) of Vibrio alginolyticus was studied by evaluating the hemocyte count, phenoloxidase activity and antibacterial activity. The above immune responses were higher in BF fed shrimp than that in FC fed or control shrimp. Among the different doses of BF of V. alginolyticus tested, 10(9) cfu g(-1) shrimp day(-1) for two weeks could evoke higher immune response. BF fed shrimp were more resistant to injection challenge with V. alginolyticus and whitespot syndrome virus (WSSV) with significantly higher RPS compared to that with FC fed and control shrimp. Better resistance was also reflected by rapid clearance of V. alginolyticus and WSSV from the hemolymph as confirmed by immunodot and histopathology.

Evaluation of an immunodot test to manage white spot syndrome virus (WSSV) during cultivation of the giant tiger shrimp Penaeus monodon.

A monoclonal antibody-based immunodot test was compared to a polymerase chain reaction (PCR) assay for managing white spot syndrome virus (WSSV) on shrimp farms at Kundapur and Kumta situated in Udupi and Uttar Kannada Districts, respectively, of Karnataka on the west coast of India. Of 12 grow-out farms in Kundapur, 6 (F1 to F6) yielded shrimp samples that were negative for WSSV by both immunodot test and 1-step PCR from stocking to successful harvest. Samples from the other 6 farms (F7 to F12) were positive for WSSV by both immunodot test and 1-step PCR at various times post stocking, and their crops failed. In the 2 farms at Kumta (F13, F14), immunodot and 1-step PCR results were both negative, and harvests were successful. In contrast to 1-step PCR results, farms F5, F6, F13, and F14 gave positive results for WSSV by 2-step PCR, and they were successfully harvested at 105 d post stocking. Our results indicate that an inexpensive immunodot assay can be used to replace the more expensive 1-step PCR assay for disease monitoring.

The incidence of Proteus mirabilis infection increases in patients on treatment but does not trigger disease activity.

The aim of this study is to determine whether treatment increases the levels of anti-Proteus antibodies (APA) in patients with rheumatoid arthritis (RA). The blood samples of 32 patients suffering from RA who were recruited in our previous study and continued to participate in our follow-up study were collected after 1 year. Their first and follow-up samples were analysed for the presence of IgG isotype and total immunoglobulins (IgG+IgA+IgM) against Proteus mirabalis (PM) using enzyme-linked immunosorbent assay with two kinds of antigen preparations: whole bacteria and sodium dodecyl sulphate (SDS) lysed bacterial extract. All patients were treated with methotrexate and hydroxychloroquine with adequate dose of non-steroidal anti-inflammatory drug. After 1 year, 11 patients were in clinical remission [erythrocyte sedimentation rate (ESR) less than 30 mm/h and C-reactive protein (CRP) less than or equal to 10 mg/l], while the rest of the 21 were in the state of active disease. Correlation and Student's t test were used for statistical analysis. APA titres were significantly elevated in patients after 1 year of therapy. However, the rise was not different between patients who were in clinical remission and those in the state of active disease. APA titre increases in the treatment of RA, and the probable mechanisms are discussed.