Ratchanee Hongprayoon

Biological and molecular characterization of tospoviruses in Thailand

Twenty-eight isolates of tospoviruses associated with tomato, pepper, cucurbits, peanut, and Physalis plants collected from fields in different regions of Thailand were characterized. On the basis of N gene and protein sequence relationships, three tospoviruses were identified, namely Watermelon silver mottle virus (WSMoV), Capsicum chlorosis virus (CaCV), and Melon yellow spot virus (MYSV). CLUSTAL analysis of selected N protein sequences showed different isolates of CaCV in three distinct clades. Based on necrosis symptoms on tomato and their 93% identity to CaCV isolates in the other two clades, CaCV-TD8, CaCV-AIT and CaCV-KS16-Thailand tomato tospovirus were designated as CaCV-tomato necrosis strain. A phylogenetic tree based on the 413-amino-acid Gc fragment of the CaCV-Pkk isolate supported the existence of three distinct CaCV clades. Vigna unguiculata produced concentric rings useful for discriminating the Thai CaCV peanut isolates from tomato or pepper isolates. By using reverse transcription polymerase chain reaction with species-specific primers, the three tospoviruses could be detected in mixed infections in watermelon and Physalis, as well as in the bodies of thrips vectors, Thrips palmi and Scirtothrips dorsalis, collected from fields.

Novel and Highly Specific Monoclonal Antibody to Acidovorax citrulli and Development of ELISA-Based Detection in Cucurbit Leaves and Seed

A novel monoclonal antibody (MAb) specific to the seedborne bacterium Acidovorax citrulli was produced. MAb 11E5 reacted specifically with 19 strains of A. citrulli but not with three closely related bacteria in the family Comamonadaceae (i.e., A. facilis, Comamonas acidovorans, and C. testosteroni) and another seven phytopathogenic bacteria. Moreover, this MAb detected a strain of A. citrulli that was not detected by a commercial enzyme-linked immunosorbent assay (ELISA)-based kit and a commercial immunochromatographic strip test. In Western blot analysis, MAb 11E5 reacted with an A. citrulli protein of a molecular mass >170 kDa. MAb 11E5 was employed to develop two sandwich ELISA systems: MAb captured-sandwich ELISA (MC-sELISA) and polyclonal antibody captured-sandwich ELISA (PC-sELISA). MC-sELISA was 10 times more sensitive than PC-sELISA for detection of A. citrulli in cucurbit leaf and seed extracts. The detection limit of the MC-sELISA was 5 × 104 CFU/ml. Detection of A. citrulli in naturally infected cucurbit leaves, fruit, and seed was also feasible using MC-sELISA. The newly established MCsELISA provides another alternative for specific detection of A. citrulli in cucurbits and can be applied for routine field inspection.

The production of anti-Vibrio harveyi egg yolk immunoglobulin and evaluation of its stability and neutralisation efficacy

In order to replace the antibiotic treatment for control of Vibrio harveyi, a causal agent of luminous disease in Black tiger shrimp, anti-V. harveyi IgY was produced and showed its potential in our preliminary study. However, for further use as feed additive, the IgY stability should be evaluated. The titre of specific IgY was enhanced with an immunostimulant, C-phosphate guanosine oligodeoxynucleotide (CpG-ODN). IgY was stable at natural pH while the activity was decreased below pH 4 or above pH 10. The supplementation of 30% sorbitol significantly enhanced the IgY stability at tested temperatures in excess of 70°C. The activity of IgY remained at 9 and 94% after 4 h incubation in simulated gastric and intestinal fluids, respectively. A storage temperature at −20°C was found to be the best condition. IgY at the concentrations of 1, 5 and 10 mg/ml efficiently inhibited V. harveyi in vitro within 24 h after exposure.

Efficient amplification of light and heavy chain variable regions and construction of a non-immune phage scFv library

Non-immune phage scFv library is one of the most attractive resources for therapeutics, diagnostics and basic research. As a matter of fact, quality of the library is limited by inefficient PCR cloning of antibody genes using degenerated primers. PCR using this type of primers is difficult to optimize conditions for efficient amplification, and therefore causes loss of antibody diversities. To overcome this problem, we described a novel two-step amplification of Vκ and VH genes with newly designed primer sets. Initially, we amplified Vκ and VH genes from their signal sequences to the joining region to keep antibody diversity as large as possible. Thereafter, highly degenerated primers were used to amplify the Vκ and VH genes from the framework region 1 to the joining region. The Vκ and VH genes from the second PCR then were linked by PCR overlapping extension to generate the scFv library. Fifteen clones from the library were randomly picked and sequenced, and the diversity of full-length scFvs was confirmed. Expression capability of clones in the library was 80% after confirmation using colony hybridization. The results demonstrated the efficiency of this strategy and the primer sets for construction of the scFv library.

Exposure assessment of aflatoxins in Thai peanut consumption

Abstract Aflatoxins (AFs) and aflatoxin B1 (AFB1) intake through consumption of peanuts and peanut products were estimated from 60 samples. The samples were collected from 2 retail and 2 wholesale markets in Bangkok during November 2013–January 2014. The results showed that 80% of raw peanut samples were contaminated with AFs while 100% of roasted and ground peanuts samples were contaminated. The highest concentration of AFs was found in ground peanuts at 362.48 ng g−1 resulting in the highest mean concentration (68.22 ng g−1). According to a food consumption database, the average intake of AFs was estimated at 0.49, 0.40 and 2.13 ng/kg bw/day for raw, roasted and ground peanuts, respectively. The potential risk for cancer was estimated at 0.01–0.12 cancer/year/100,000 persons. The results suggest that the current situation of aflatoxins contamination in peanuts and peanut products (especially in ground peanuts) has an adverse effect on the health of the Thai population.