Duck enteritis virus and Pasteurella multocida are major duck pathogens that induce duck plague and fowl cholera, respectively, in ducks and other waterfowl populations, leading to high levels of morbidity and mortality. Immunization with live attenuated DEV vaccine containing P. multocida outer membrane protein H (OmpH) can provide the most effective protection against these two infectious diseases in ducks. We have recently reported the construction of recombinant DEV expressing P. multocida ompH gene using the CRISPR/Cas9 gene editing strategy with the goal of using it as a bivalent vaccine that can simultaneously protect against both infections. Here we describe the findings of our investigation into the systemic immune responses, potency and clinical protection induced by the two recombinant DEV-ompH vaccine constructs, where one copy each of the ompH gene was inserted into the DEV genome at the UL55-LORF11 and UL44-44.5 intergenic regions, respectively. Our study demonstrated that the insertion of the ompH gene exerted no adverse effect on the DEV parental virus. Moreover, ducklings immunized with the rDEV-ompH-UL55 and rDEV-ompH-UL44 vaccines induced promising levels of P. multocida OmpH-specific as well as DEV-specific antibodies and were completely protected from both diseases. Analysis of the humoral and cellular immunity confirmed the immunogenicity of both recombinant vaccines, which provided strong immune responses against DEV and P. multocida. This study not only provides insights into understanding the immune responses of ducks to recombinant DEV-ompH vaccines but also demonstrates the potential for simultaneous prevention of viral and bacterial infections using viral vectors expressing bacterial immunogens.

Duck enteritis virus (DEV) and Pasteurella multocida, the causative agent of duck plague and fowl cholera, are acute contagious diseases and leading causes of morbidity and mortality in duck. The NHEJ-CRISPR/Cas9-mediated gene editing strategy, accompanied with the Cre–Lox system, have been employed in the present study to show that two new sites at UL55-LORF11 and UL44-44.5 loci in the genome of the attenuated Jansen strain of DEV can be used for the stable expression of the outer membrane protein H (ompH) gene of P. multocida that could be used as a bivalent vaccine candidate with the potential of protecting ducks simultaneously against major viral and bacterial pathogens. The two recombinant viruses, DEV-OmpH-V5-UL55-LORF11 and DEV-OmpH-V5-UL44-44.5, with the insertion of ompH-V5 gene at the UL55-LORF11 and UL44-44.5 loci respectively, showed similar growth kinetics and plaque size, compared to the wildtype virus, confirming that the insertion of the foreign gene into these did not have any detrimental effects on DEV. This is the first time the CRISPR/Cas9 system has been applied to insert a highly immunogenic gene from bacteria into the DEV genome rapidly and efficiently. This approach offers an efficient way to introduce other antigens into the DEV genome for multivalent vector.

Fowl cholera is caused by the bacterium Pasteurella multocida, a highly transmissible avian ailment with significant global implications, leading to substantial economic repercussions. The control of fowl cholera outbreaks primarily relies on vaccination using traditional vaccines that are still in use today despite their many limitations. In this research, we describe the development of a genetically engineered herpesvirus of turkeys (HVT) that carries the OmpH gene from P. multocida integrated into UL 45/46 intergenic region using CRISPR/Cas9-NHEJ and Cre-Lox system editing. The integration and expression of the foreign cassettes were confirmed using polymerase chain reaction (PCR), indirect immunofluorescence assays, and Western blot assays. The novel recombinant virus (rHVT-OmpH) demonstrated stable integration of the OmpH gene even after 15 consecutive in vitro passages, along with similar in vitro growth kinetics as the parent HVT virus. The protective efficacy of the rHVT-OmpH vaccine was evaluated in vaccinated ducks by examining the levels of P. multocida OmpH-specific antibodies in serum samples using ELISA. Groups of ducks that received the rHVT-OmpH vaccine or the rOmpH protein with Montanide™ (SEPPIC, Paris, France) adjuvant exhibited high levels of antibodies, in contrast to the negative control groups that received the parental HVT or PBS. The recombinant rHVT-OmpH vaccine also provided complete protection against exposure to virulent P. multocida X-73 seven days post-vaccination. This outcome not only demonstrates that the HVT vector possesses many characteristics of an ideal recombinant viral vaccine vector for protecting non-chicken hosts, such as ducks, but also represents significant research progress in identifying a modern, effective vaccine candidate for combatting ancient infectious diseases.

Duck Plague Virus (DPV), an alphaherpesvirus, causes significant morbidity and mortality in waterfowl and remains a persistent threat to duck farming across Asia. This study reports the first complete genomic characterization of DPV isolates from Thailand including a commercial vaccine strain (DPVac) and two contemporary field isolates (DPV7 and DPV8). Using Oxford Nanopore Technologies (ONT) and hybrid Illumina-ONT sequencing, we successfully assembled complete genomes ranging from 160,511 to 163,789 bp. The assembled genomes showed high sequence identity to the European reference strain DEV 2085. Comparative genomic analysis revealed structural differences in the UL and US regions. Phylogenetic reconstruction based on core genome single-nucleotide polymorphisms (SNPs) placed DPV7 within a clade of recent Chinese field strains, while DPV8 clustered closely with vaccine-associated lineages from Germany, India, and Bangladesh. Notably, SNP analysis identified multiple virulence-associated mutations uniquely present in DPV7. These mutations, absent in DPVac and DPV8, were located within or near genes involved in viral replication (UL54), host immune evasion (UL41, UL14), viral entry (UL44, UL8), intracellular trafficking (US3, US8), and virulence modulation (LORF3). These findings suggest that DPV7 may be undergoing adaptive evolution under immune pressure, potentially compromising vaccine effectiveness. Our results underscore the critical need for continuous molecular surveillance and functional studies to evaluate the impact of emerging DPV variants. The complete genome sequences reported herein provide a valuable resource for future research on DPV evolution, diagnostics, and vaccine development in endemic regions.

Toxoplasmosis is a significant parasitic zoonosis caused by Toxoplasma gondii. Among livestock animals, small ruminants, especially sheep and goats, are the most infected. This infection is a leading cause of abortion, resulting in considerable economic losses for goat breeders. The present study aimed to assess the prevalence of T. gondii infection in goats in northern Thailand, with an emphasis on its potential zoonotic transmission to humans. Polymerase chain reaction (PCR) targeting the T. gondii dihydrofolate reductase-thymidylate synthase (Tgdhfr-ts) gene was employed for molecular detection. This represents the first report of T. gondii molecular detection in blood samples from small ruminants in Thailand by PCR. A total of 176 meat goat blood samples were analyzed, yielding an 8.52% (15/176) positivity rate for T. gondii DNA. The selected DNA sequences from the positive T. gondii DNA displayed a high degree of nucleotide sequence homology with the reference Tgdhfr-ts sequence. Phylogenetic analysis revealed a single clade alongside other T. gondii strains, showing no differentiation based on genotype. This study contributes to the understanding of T. gondii epidemiology and provides a foundation for future strategies to control and manage T. gondii transmission in livestock populations.

Metastasis-related disease is a major cause of death in canine mammary tumours (CMTs). Immunotherapy has been investigated due to the less successful outcomes of systemic therapy. This study aims to examine the expression of Programmed Cell Death Ligand-1 (PD-L1) in canine mammary tumours in dogs of Chiang Mai, Thailand, and determine the relationship between the level of mRNA expression and clinicopathologic characteristics. A total of 28 CMT samples were collected at the Small Animal Hospital, Chiang Mai University. Quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) and western blot assays were performed. The results revealed that all CMTs in this study expressed PD-L1 mRNA and PD-L1 protein. The mean relative mRNA expression showed no significant differences between groups categorized by age, tumour size, or histopathological findings. However, the mean relative mRNA expression in tumours with a TNM stage >3 was significantly lower compared to those with TNM stage ≤2. In conclusion, this study investigates the expression of PD-L1 mRNA and PD-L1 protein, particularly in malignant CMTs. The findings strongly support the potential for developing effective immunotherapy methods targeting the PD-1/PD-L1 pathway for advanced CMTs in the future. For further conclusive assessment, future studies should focus on refining immunotherapy strategies for CMT cases expressing PD-L1.