Penicillium setosum, a member of the Penicillium section Lanata-divaricata, is known for its ability to produce high levels of patulin. However, there is a notable lack of information regarding the influence of environmental factors on the growth and patulin production in fruit juice of this species. In this study, we investigated the impacts of temperature (25, 30, 35, or 40 °C), initial pH value (3, 5, or 7), and incubation time (2, 4, or 6 days) on the growth and patulin production in pineapple juice of the selected P. setosum strain HR9-5. The modified Gompertz model was applied to estimate the maximum growth rate and lag time prior to growth. This strain had the highest growth rate with the shortest lag time within the temperature range 30–35 °C, with an initial pH range of 3–5. Furthermore, it had the greatest biomass dry weight and patulin production at 35 °C with an initial pH of 3, and at 25 °C with an initial pH of 7, respectively. The growth rate and lag time prior to growth of P. setosum HR9-5 were significantly influenced by the initial pH, whereas the biomass dry weight was strongly affected by temperature and incubation time. Additionally, all factors and their interactions had a significant impact on patulin production. This study provided the first report on the effect of environmental factors on growth and patulin production by P. setosum in pineapple juice. These findings can contribute to formulating effective prevention strategies to control the growth of P. setosum and subsequent patulin production in pineapple and other fruit juices.

Heat-resistant molds (HRMs) are important spoilage fungi of heat-processed fruit products worldwide. Ascospores of HRMs are widely distributed in the soil in which fruits are grown and are often found associated with raw fruit materials. To date, there is little available information on the distribution of HRMs in the soil and on their heat resistance. Thus, this study determined the presence and characterized the heat resistance of HRMs in soil samples from pineapple and sugarcane fields in Thailand. HRMs were detected in all soil samples, and the most dominant species was Aspergillus with 50-99.2% relative abundance. Other isolates, in descending order of frequency, were Penicillium, Talaromyces, Hamigera, and Paecilomyces. Then, 100 representative HRM isolates were identified based on a combination of morphological characteristics and ITS sequences. They were classified into 5 genera and 24 species. The heat resistance of ascospores aged 30 days produced by selected HRMs was qualitatively determined in a glucose-buffered solution. Based on their log reductions after heat shock at 75°C for 30 min, they were classified as less, moderately, or highly heat-resistant ascospores. HRMs belonging to A. chevalieri, A. denticulatus, A. siamensis, A. laciniosus, A. fennelliae, A. spinosus, Paec. niveus, H. pallida, and T. macrosporus produced high heat-resistant ascospores. In addition, soil physicochemical properties significantly influenced the prevalence of HRMs, depending on the fungal genus. The thermal resistance of ascospores was significantly and positively correlated to available phosphorus, whereas it was negatively correlated to soil pH. The results of this study confirmed the presence of HRMs in soils and potential HRM contamination, especially in fruits growing in acidic or high-nutrient soils, or both.

The contamination and spoilage of heat-treated fruit juices by heat-resistant mold ascospores present significant challenges to the food industry. Understanding effective strategies to mitigate this contamination is vital for ensuring the shelf-life and microbial safety of heat-treated fruit juices. This study investigated the thermal resistance of ascospores from different heat-resistant mold species, including Aspergillus laciniosus, A. chevalieri, A. denticulatus, A. siamensis, Hamigera pallida, and Talaromyces macrosporus, isolated from pineapple and sugarcane field soils. Ascospores inactivation kinetics in pineapple juice under heat treatment (75–97 °C) were analyzed using log-linear and Weibull models. Among these species, A. laciniosus displayed the highest heat resistance (δ-value: 104.59 min at 85 °C), while A. siamensis exhibited the lowest (δ-value: 3.39 min at 80 °C). Furthermore, A. laciniosus, the most heat-resistant species, showed notable tolerance to sanitizers. The most effective inactivation was achieved using 1.0% (w/v) sodium hypochlorite for 15 min. Chlorine dioxide, however, was generally ineffective and even activated dormant ascospores in some cases. The combination of hot water (65 °C for 5 min) with sanitizer increased ascospore reduction in most species but did not achieve the 3-log reduction required by the European Standard N13697. This study revealed a correlation between ascospore resistance to heat and chlorine dioxide, offering significant findings for practical inactivation strategies.

The recombinant, modified leucine-rich repeat protein rhKU_Sej_LRR_2271 has been suggested as a candidate for leptospiral vaccine development since it was predicted to be a transmembrane protein containing leucine-rich repeat motifs and immunogenic epitopes. The immunogenic epitopes showed binding affinities with lower IC50 values than peptides of known antigenic proteins, e.g., LipL32. Moreover, this protein was immunoreactive with hyperimmune sera against several serovars. In this study, we aimed to develop a lateral flow strip test using the rhKU_Sej_LRR_2271 protein for the detection of anti-leptospiral IgG in dogs. The lateral flow assay was performed with 184 dog plasma samples and evaluated with a culture method, 16S ribosomal RNA gene (rss) analysis real-time PCR, and LipL32 ELISA. The culture method failed to detect leptospires in the dog blood samples. Six of nine symptomatic dogs gave positive results with the real-time PCR assay. The lateral flow assay and LipL32 ELISA gave positive results with 59 and 50 dogs, respectively. The sensitivity, specificity, and accuracy of the rhKU_Sej_LRR_2271 lateral flow strip test were 70.00, 82.09, and 78.80%, respectively, when compared with LipL32 ELISA. There was a significant association between the LipL32 ELISA and the rhKU_Sej_LRR_2271 lateral flow assay. The rhKU_Sej_LRR_2271 lateral flow strip test has therefore demonstrated a good potential to detect anti-leptospiral IgG in dogs.

Domestic and stray dogs can be frequently infected by Leptospira, and thus may represent a source for transmission of this zoonotic disease in Thailand. Here, we have used peptides derived from a recombinant leucine-rich repeat (LRR) protein of Leptospira, rKU_Sej_LRR_2012M, for the development of an indirect enzyme-linked immunosorbent assay (ELISA) aimed at detecting antibodies against Leptospira interrogans, L. borgpetersenii, and L. biflexa, the three major seroprevalences in Thai dogs. The rKU_Sej_LRR_2012M protein is recognized by hyperimmune sera against several leptospiral serovars. The epitope peptides of the rKU_Sej_LRR_2012M showed binding affinities with lower IC50 values than peptides of known antigenic protein LipL32. Four peptides, 2012-3T, 2012-4B, 2012-5B and pool 2012-B, were specifically recognized by rabbit hyperimmune sera against nine serovars from three Leptospira spp. The indirect peptide-based ELISAs with these four peptides were evaluated with the LipL32 ELISA by using a receiver–operator curve (ROC) analysis. All peptides had an area under the curve of ROC (AUC) greater than 0.8, and the sum of sensitivity and specificity for each peptide was greater than 1.5. The degree of agreement of 2012-3T and pool 2012-B and 2012-4B and 2012-5B peptides were in moderate-to-good levels with kappa values of 0.41–0.60 and 0.61–0.80, when compared with LipL32, respectively. This finding would suggest an excellent capability of the 2012-4B and 2012-5B peptide-based ELISAs assay for the diagnosis of canine leptospiral infections.

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.

In-house immunoaffinity column for aflatoxins (AFs) detection is firstly developed by Kasetsart University since 2011 i.e. the first IAC prototype, KU-AF01. Detection of aflatoxin B1 (AFB1) up to 200 ng is considered significantly efficient, but the production cost is considered high. The high cost was due to the cost of monoclonal antibody specific to aflatoxin and its supporting materials i.e. CNBr-activated sepharose 4B. Therefore, this study was aimed to improve the efficiency of in-house immunoaffinity colums by replacing CNBr-activated sepharose 4B with other supporting materials and to lower the ratio of antibody to supporting materials.  Response surface methodology (RSM) was applied to determine the optimum alternative supporting materials and the ratio. Results revealed that when compared with CNBr-activated sepharose 4B, all materials tested could recover higher than 80% AFB1, except the activated CH sepharose 4B. Results also indicated that the optimum ratio was 0.4 mg anti-aflatoxin monoclonal antibody to 204 µL CNBr-activated sepharose 4B. CNBr-activated sepharose 4B was considered to provide the best precision in recovering AFB1. KU-AF02 increased the ability to detect AFB1 to 500 ppb. The recovery of AFs in the reference materials using KU-AF02 was more than 96% successful, with HorRat value range of 0.34 – 0.76. The reference materials used were 2 levels of AFs in peanuts, i.e. 47.68 and 72.14 ng/g. In terms of quality control in IAC production, there were no significant differences among the 5 batches produced (p > 0.05). KU-AF02 demonstrated stable and constant percent recovery at 4 oC for up to 12 months (tested with standard 200 ppb AFB1). This study indicated that KU-AF02 could lower the ratio of supporting materials to the antibody and that KU-AF02 has high stability and has good ability for cleaning up AFs. Therefore, KU-AF02 is recommended to be used as an in-house immunoaffinity column for aflatoxin detection in Thailand.

Aflatoxins are cancer-causing chemicals produced primarily by Aspergillus flavus and A. parasiticus. Aflatoxin B1 (AFB1) is the most commonly found aflatoxin in improperly stored staple commodities such grain and feed. Its presence in the food supply, can be carried over to animal products such as meat, liver, kidney, pig blood and milk. A specific and sensitive detection method is required for preliminary screening of these samples. This research sought to develop a detection kit for total aflatoxin by immunochromatographic technique using monoclonal antibody (MAb) from the hybridoma cell line 4G6. The experiments were conducted at the Serology and Diagnostic Laboratory, Kasetsart University, Kamphaeng Saen Campus, Nakhon Pathom province during 2014-2016. The MAb is composed of IgG2b isotype and lambda light chain. Its specificity recognized four aflatoxins including AFB1, AFB2, AFG1 and AFG2 with cross reactivity at 100%, 89.2%, 82.6%, and 72.7%, respectively by direct competitive enzyme-linked immunosorbent assay (dcELISA). In vitro propagation of the hybridoma was carried out using an Integra CELLine Culture System and the antibody was purified by affinity column chromatography. The conjugate probe was prepared by comparing two sizes of colloidal gold particles at 20 and 40 nm in diameter for the conjugation with the MAb. The MAb conjugate with 40 nm colloidal gold was selected and sprayed onto the conjugate release pad (CRP). The target cut-off value for the developed immunochromatographic strip (ICS) was 20 ng/mL according to a regulation limit in Thailand. The study on the appropriate conditions for this strip showed that aflatoxin B1 conjugated to bovine serum albumin (AFB1-BSA) and goat antimouse immunoglobulin (GAM) should be immobilized at the test line and control line at the same concentrations of 0.25 mg/mL. The testing sample was extracted with 70% methanol and further diluted 1:4 with Tris buffer saline with 0.05% Tween-20 (TBST) before application on the sample application pad (SAP) and the reaction could be visualized within 15 min. The analysis of 5 naturally contaminated corn samples (n=7) indicated that 2 samples contained  20 μg/kg and 3 samples contained  20 μg/kg. Five samples, analyzed by dcELISA, showed contamination levels at <4, 9.6, 19.9, 10.5 and 39.7 μg/kg which delivered a good correlation to the results from ICS analysis.

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.

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.

<p class="zhengwen"><span lang="EN-GB">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-<em>V. harveyi</em> IgY had effective potential to control luminous disease in black tiger shrimp. However,</span><span lang="EN-GB">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. </span></p>

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.

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.

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.

Cucumber mosaic virus (CMV) causes serious problems in economically important crops, especially members of the Solanaceae and Cucurbitaceae families. Serological detection of this virus by specific antibodies is required as a control measure as well as for quarantine investigation to ensure any components used for agricultural propagation, especially commercial seeds, are disease free. However, the selection of recombinant antibodies by phage display nowadays presents a real challenge to provide the antibodies that are urgently needed. In this research, an anti-CMV single-chain variable fragment (scFv) was constructed using a phage display system. Both heavy (VH) and kappa light chain variable (Vk) genes were amplified by RT-PCR from the hybridoma cell line CM2, secreting a monoclonal antibody (MAb) specific to both serogroup I and II of CMV. The VH and Vk amplified products, approximately 400 bp in length, were joined by a PCR overlapping extension method to generate the scFv gene. A recombinant phagemid pCANTAB5E harboring the scFv gene was constructed and transformed into Escherichia coli TG1. The bacterial transformants were rescued by helper phage M13 to produce phage-displayd scFv and the screening for CMV-specific scFv was carried out by ELISA. Three positive, recombinant clones (2C1, 6A1 and 1D4) which gave high signal-to-noise in ELISA were utilized in order to produce soluble antibodies. Western blotting and DNA sequencing were performed to characterize the scFv products. The result showed that all clones were identical and able to bind CMV of both subgroups. DNA comparisons showed that all the VH belonged to the J558.32 subgroup and JH2, while Vk belonged to Vk genes and JK2.

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.

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) is a major problem faced by pork producers in Thailand. In order to properly solve PRRS problems in the field, a rapid diagnostic method is required for the differentiation of North American and European isolates of the PRRSV. This paper reports on a Reverse Transcription-nested PCR (RT-nPCR) method for the detection and differentiation of