Tippawan Jantafong

Background and Aim: Porcine reproductive and respiratory syndrome virus (PRRSV) remains a major economic threat to the global swine industry, causing reproductive losses and severe respiratory illness. Accurate and cost-effective diagnostic tools are essential for timely detection and genetic monitoring, particularly in resource-limited settings. This study aimed to (i) establish a nested reverse transcription polymerase chain reaction (RT-PCR) assay targeting the open reading frame 7 (ORF7) gene to detect and differentiate PRRSV-1 and PRRSV-2, and (ii) refine the genetic classification of PRRSV-2 strains circulating in Thailand through ORF5-based phylogenetic analysis. Materials and Methods: A nested RT-PCR assay was developed using external primers for general PRRSV detection and internal primers specific to PRRSV-1 and PRRSV-2. Analytical specificity was assessed against modified-live vaccines, clinical isolates, and heterologous swine viruses (swine influenza virus and foot-and-mouth disease virus). Diagnostic accuracy was evaluated using 96 clinical serum samples and compared with a commercial real-time RT-PCR kit. To confirm genotyping capability, ORF7-positive samples underwent ORF5 sequencing and phylogenetic analysis. In addition, 386 complete ORF5 sequences (2000–2023) from Thai isolates and global references were analyzed using maximum likelihood methods to refine lineage and sublineage classification. Results: The nested ORF7 RT-PCR assay demonstrated high specificity without cross-amplification and achieved 100% con­cordance with real-time RT-PCR, confirming its diagnostic reliability. Among the clinical samples, PRRSV-1, PRRSV-2, and mixed infections were successfully detected. Sequencing confirmed strain identities and revealed close similarity with both endemic and vaccine-related strains. Phylogenetic analysis classified Thai PRRSV-2 strains into five lineages (L1, L5, L8, L9, L10) and five sublineages (L1I, L5A, L8C, L8E, L9D). Notably, this study is the first to report sublineages L8C and L9D in Thailand, while also documenting a lineage shift from L8E to L10 as the predominant circulating strain. Conclusion: The integration of nested ORF7 RT-PCR with ORF5-based phylogenetic analysis provides a sensitive, affordable, and reliable diagnostic platform for PRRSV detection and genetic classification. These findings enhance understanding of PRRSV-2 diversity in Thailand, highlight emerging sublineages, and underscore the importance of continuous molecular surveillance to inform vaccine strategies and disease control policies. Keywords: molecular diagnostics, nested reverse transcription polymerase chain reaction, open reading frame 5 phylogeny, open reading frame 7, porcine reproductive and respiratory syndrome virus, Thailand.

Background and Aim: Porcine reproductive and respiratory syndrome (PRRS), caused by PRRS virus (PRRSV), is a global issue that affects Thai swine as well. In Thailand, PRRSV-2 predominates over PRRSV-1. The origin of PRRSV-1 transmission remains undiscovered. This study traced the source of infected pigs responsible for disease transmission among three pig-fattening farms and analyzed the spread of PRRSV-1. Materials and Methods: A total of 696 swine samples from breeding and pig-fattening farms in Thailand were screened for PRRSV using open reading frames (ORF7) reverse transcription polymerase chain reaction (RT-PCR). Positive samples were identified as PRRSV-1 using ORF5 RT-PCR. The analysis included the study of nucleotide homology, GP5 amino acid sequences, and N-linked glycosylation patterns to assess the spread of PRRSV-1 across these farms. Results: Genetic examination identified 28 PRRSV-1-positive samples, of which 13 were chosen as representatives. These strains were categorized into three groups based on breeding farm pig houses and showed distinct distribution patterns across pig-fattening farms. Group 1 included piglets transferred from pig house A to Nakhon Pathom, Chonburi, and Sa Kaeo. Groups 2 and 3 showed transfers from pig houses F and H to Chonburi and Sa Kaeo farms. All 13 PRRSV-1 strains were categorized into PRRSV-1 subtype 1/clade H. N-linked glycosylation analysis revealed that nearly all PRRSV-1 strains exhibited a conserved glycosylation pattern at amino acid positions N37, N46, and N53. This pattern is consistent with the glycosylation profile of the previous Thai PRRSV-1 subtype 1/clade H. Conclusion: The present study highlights the persistent presence of PRRSV-1 in Thai swine, which leads to sporadic outbreaks. The molecular genetic analysis identified three primary strain groups dispersed throughout the pig production system, emphasizing the importance of regular monitoring for new PRRSV strains in this herd. Understanding the PRRSV-1 distribution in swine farms is vital for veterinarians. This knowledge supports strategies for eradicating the virus and managing swine health effectively in Thailand. Keywords: porcine reproductive and respiratory syndrome virus-1, swine production system, Thailand, virus distribution.

Background and Aim: The flinders technology associates (FTA) card is a cotton-based cellulose membrane impregnated with a chaotropic agent that inactivates infectious microorganisms, lyses cellular material, and fixes DNA and/or RNA within the fiber matrix. However, little is known about the effectiveness of these cards for detecting RNA viruses in animals. This study aimed to evaluate the sensitivity of RNA virus detection using conventional reverse-transcription polymerase chain reaction (RT-PCR) on FTA cards. Materials and Methods: A highly virulent Newcastle disease virus (NDV) and an avian influenza virus (AIV) with low pathogenicity were propagated using chicken embryonic eggs. Three days after inoculation, the allantoic fluid was harvested, stored at –80°C, and the stock virus was tested for virus titration. African horse sickness virus (AHSV) was obtained from a live attenuated vaccine that was dissolved and stored at –80°C. For sample preparation, each stock virus was 10-fold serially diluted and each dilution was inoculated onto an FTA card, followed by drying in a Class II safety cabinet. Both the stock virus and infected FTA card were genomically isolated using an extraction kit, FTA purification kit, and extraction kit with Tris-EDTA (TE) buffer. The target genome was then detected by one-step RT-PCR for NDV and AIV, and two-step RT-PCR for African horse sickness, including gel electrophoresis for the detection of specific nucleic acids. Results: The detection limit of stock AIV was compared on FTA cards, using the FTA purification kit, and with TE buffer with an extraction kit. The corresponding results were 1.47, 1.17, and 2.18 log10 EID50, respectively, while for NDV the results were 4.13, 4.83, and 4.84 log10 ELD50. Finally, detection limit of stock AHSV and AHSV on the FTA card extracted using TE buffer with an extraction kit were 4.30 and 4.01 log10 plaque-forming units, respectively. Conclusion: This study demonstrated that the detection limit or sensitivity of all tested RNA viruses on FTA cards did not differ when compared with those of the stock virus and in both methods for RNA isolation on FTA cards. These cards are suitable for collecting and transporting samples infected with RNA viruses, particularly AIV, NDV, and AHSV. Flinders technology associates cards also provide hazard-free samples, a reliable source of RNA for molecular characterization, and sufficient quantity for diagnostic applications based on nucleic acid-based detection.

Aim: This study aimed to evaluate the bactericidal and virucidal activity of food additive grade calcium hydroxide (FdCa(OH)2) under various concentrations, organic material conditions, and exposure duration including its stability. Materials and Methods: The FdCa(OH)2 powder as well as the 0.17% and 3% solutions were evaluated for bacteria and virus inactivating efficacies against Salmonella infantis (SI), Escherichia coli, Newcastle disease virus (NDV), and avian influenza virus (AIV), in the absence or presence of organic materials. In addition, the stability of FdCa(OH)2, was also examined using wet-dry conditions and under sunlight. Results: The FdCa(OH)2 powder could inactivate both NDV and AIV in the absence and presence of organic materials within a 3 min exposure period. The bactericidal efficacy using solution form revealed that 0.17% and 3% of FdCa(OH)2 could inactivate SI in the absence and presence of organic materials within 3 min of exposure. However, 3% of FdCa(OH)2 inactivated E. coli both with and without organic materials within 3min, while 0.17% required 5 min to be efficacious. The virucidal efficacy also showed that 0.17% FdCa(OH)2 could inactivate NDV in the absence and presence of organic materials within 10 min and 30 min, respectively. However, AIV inactivation was achieved within 30 sec under all conditions. In addition, under wet and dry conditions, FdCa(OH)2 powder demonstrated high efficacy when re-suspended at least 16 times for NDV and 7 times for AIV. Simultaneously, the FdCa(OH)2 powder retained its efficacy under the sunlight during up to 4 months for NDV and at least 6 months for AIV. Conclusion: The present study indicates that FdCa(OH)2 powder and solutions could inactivate SI, E. coli, NDV, and AIV while retaining good stability under challenging environmental conditions. Finally, the FdCa(OH)2 is safe for consumers because it is of food additive grade and can be useful as an alternative disinfectant, especially for biosecurity enhancement on and around poultry farms.

Aim: The present study was examined the virucidal activity comparison between fresh charcoal ash (FCA) and slaked lime (SL) against avian influenza virus (AIV) and Newcastle disease virus (NDV), using powder and liquid forms, either in the absence or presence of organic materials. In addition, both FCA and SL were evaluated for the persistence of virucidal activity in wet and dry conditions and stability of the solution. Materials and Methods: Two hundred milligrams of FCA or SL powders were mixed with 100 μl of AIV or NDV in the absence of organic material or 33% of organic materials. In the same time, 400 μl of 1%, 5%, or 10% solution samples were mixed with 100 μl of each virus and then incubated at room temperature for an indicated time. After that, the mixed solution was stop activity of sample using 500 μl of 1M Tris-HCl pH 7.2. Each treatment was titrated onto Madin-Darby canine kidney cells or chicken embryo fibroblasts for AIV or NDV, respectively, for determining the efficacy of viral inactivation. In addition, the stability of powder under the wet-dry condition and solution stability under room temperature was examined. Results: The results demonstrated that the FCA and SL in powder form could inactivate AIV and NDV even in the absence or presence of organic materials. In the liquid form, 5% and 10% of FCA could inactivate AIV and NDV either in the absence or presence of organic materials. Alongside, 1%, 5%, and 10% of SL could inactivate both viruses. 10% of FCA solution could inactivate virus at a shortest time when compared with other concentrations. In addition, the efficacy of wet-dry conditions of FCA was limited when compared with SL. On the other hand, it is demonstrated that the FCA solution was more stable and kept at room temperature longer than SL. Conclusion: The FCA may, hence, be used as an alternative virucide, while applying it to prevent spreading of poultry disease on commercial chicken farms and also backyard chickens, especially in developing countries, including in rural areas of Thailand.

This study was to determine the effectiveness of the fourth-generation quaternary ammonium compounds, didecyl dimethyl ammonium bromide (DDAB), on the efficacy of bacterial and viral decontamination against pathogens commonly found in livestock industry including Salmonella infantis (SI), Escherichia coli, and avian influenza virus