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Khon Kaen Agriculture Journal, 50, 3, 819-829

A triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) and an immunocapture reverse transcription-polymerase chain reaction (IC-RT-PCR) for pepper mild mottle virus (PMMoV) detection were developed based on monoclonal and polyclonal antibodies produced in this study. Production of anti-PMMoV monoclonal antibodies (MAbs) was achieved using the hybridoma technique and a recombinant protein of the PMMoV coat protein as the antigen. Production of a polyclonal antibody (PAb) against the recombinant coat protein of PMMoV was carried out in a New Zealand White rabbit. The PAb was specific to five tobamoviruses consisting of PMMoV, tobacco mosaic virus (TMV), odontoglossum ringspot virus (ORSV), tomato mosaic virus (ToMV) and ribgrass mosaic virus (RMV). Two TAS-ELISA protocols were developed based on the characteristics of the antibodies produced. One protocol, based on MAb TOBA1, could detect five tobamoviruses (TMV, PMMoV, ORSV, ToMV, and RMV), four of which infect peppers. In the other protocol, TOBA10 was used to specifically detect PMMoV. We also successfully developed an IC-RT-PCR procedure using TOBA1. The sensitivity of the IC-RT-PCR (1:2,000,000) was much higher than TAS-ELISA (1:5120) for detection of the virus in plant sap. We anticipate these assays to facilitate the screening process of tobamovirus-resistant pepper cultivars in breeding programs.

Khon Kaen Agriculture Journal, 48, 5, 954-965

Thai Agricultural Research Journal, 37, 2, 139-150

Thai Agricultural Research Journal, 36, 2, 141-152

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.

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.

Melon yellow spot virus (MYSV) is one of the most devastating plant viruses in the genus Tospovirus and adversely affects productivity, and quality of cucumber and melon. The locally made polyclonal antibody against nucleocapsid (N) protein of Thai isolate of MYSV has been previously used for the diagnosis of tospovirus species in serogroup IV as well as MYSV, therefore, causing problems for specific MYSV diagnosis. In this research, polyclonal antibody against MYSV- non structural (NSs) protein was produced and evaluated for use in MYSV diagnosis. The N and NSs genes were directly amplified by RT-PCR from MYSV-infecting melon and cloned. Analysis of N gene indicated MYSV infection in diseased melon (designated as isolate MYSV-SUT). The cloned NSs gene of MYSV-SUT is composed of 1,410 nucleotides which encoded for 470 amino acid residues. In vitro protein expression of NSs gene inserted in pQE80L expression vector yielded a recombinant 6xHis-NSs polypeptide. The rabbit polyclonal antibodies against the expressed 6xHis-NSs protein (PAb-MYSV-NSs) had titer up to 1:12,800 when tested with 3 μg/ml of the 6xHis-NSs protein by DAC-ELISA. This antibody reacted positively to crude sap from MYSV-infected melon and cucumber leaf tissue by nitrocellulose ELISA (NCM-ELISA), and not to crude sap of symptomatic plants infected with Watermelon silver mottle virus (WSMoV), Capsicum chlorosis virus (CaCV) and Tomato necrotic ring spot virus (TNRV) isolates from Thailand. However, crude sap of MYSV-infected melon from fields did not react with PAb-MYSV-NSs in the DAC-ELISA conditions used in this study.

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.

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.

Sugarcane disease surveys were conducted from 2010 to 2014 at major sugarcane growing areas in 5 provinces (Nakhon Pathom, Kanchanaburi, Udon Thani, Khon Kaen, Nakhon Ratchasima) and germplasm collection fields. Random samples of the virus-like sugarcane leaves obtained from the surveyed areas suggested yellow streak mosaic symptoms. Direct antigen coating ELISA using locally produced SCSMV antiserum, revealed widespread incidence of SCSMV in the major sugarcane growing areas and the germplasm collection fields, ranging from 43.48-90.91% and 54.17-100% respectively. The virus isolate from sugarcane in Kamphaeng Saen, Nakhon Pathom, designated as THA-NP3, was characterized by genomic sequencing. Complete genome of THA-NP3 (JN163911) contained 9,781 nucleotides, excluding 3¢ Poly (A) tail which encoded a polyprotein of 3,130 amino acid residues comprising 10 functional proteins, namely P1, HC-Pro, P3, 6K1, CI, 6K2, NIa-VPg, NIa-Pro, NIb and CP. Sequence comparisons revealed that THA-NP3 showed 97.84% nucleotide identity to JP2 (JF488065) from China and 81.39-97.78% nucleotide identities to other recorded SCSMV sequences. Detection for the presence of CP gene by RT-PCR indicated 1094 bp containing 846 bp of the CP coding region. Analysis of the CP gene revealed genetic variation of 58 Thai SCSMV isolates, 86.17-100% nucleotide identities among them and 85.70-99.29% nucleotide identities to SCSMV isolates from other countries. Recombination events existed in the CP coding regions between two distinct sub-populations, the germplasm isolates and the farmers’ field isolates. These results suggested the incidence of SCSMV variants between the farmers’ fields and the germplasm collection fields.

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 V(kappa) and V(H) genes with newly designed primer sets. Initially, we amplified V(kappa) and V(H) 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 V(kappa) and V(H) genes from the framework region 1 to the joining region. The V(kappa) and V(H) 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.

Egg yolk immunoglobulin (IgY) is an alternative treatment for the prevention of gastrointestinal infections in pigs, cows, chickens and fish. In our previous report, we preliminarily proved that anti-V. harveyi IgY had effective potential to control luminous disease in black tiger shrimp. However,IgY activity may be reduced or destroyed by gastric conditions, particularly low pH and digestive enzymes. Therefore, it is necessary to find an effective method to preserve the therapeutic function of IgY antibodies during gastric passage. Chitosan-alginate microcapsules have been developed to protect IgY from gastric inactivation. The processing factors included different forms of chitosan and alginate, while a CaCl2 concentration and encapsulation medium was investigated. The optimum results were obtained under the following conditions: High Mw chitosan concentration 0.2% (w/v), medium viscosity alginate concentration 2% (w/v), CaCl2 concentration 0.5% (w/v). The stability of IgY in simulated gastric fluid (SGF, pH 1.2) was greatly improved by encapsulation in chitosan-alginate microcapsules, and retained greater than 90% activity after 2 h exposure to SGF. Less than 10% IgY was released upon the microcapsules’ exposure to SGF for 2 h, and more than 80% IgY was released upon the microcapsules’ exposure to simulated intestinal fluid (SIF, pH 6.8) for 16 h.