Lumpy skin disease virus (LSDV) affects cattle and causes significant economic damage. The live vaccine derived from an attenuated strain is effective but is associated with mild disease and skin lesions in some vaccinated cattle. Moreover, recombinant LSDV strains, particularly one with wild-type field and vaccine strains, have recently emerged and spread throughout Asian countries. A cost-effective LSDV typing method is required. We developed a multi-locus real-time PCR with a high-resolution melting (HRM) assay to differentiate between the wild-type, vaccine, and dominant Asian recombinant strains. Based on a multiple alignment analysis, we selected three target genes for the HRM assay, ORF095, ORF126, and ORF145, in which there are insertions/deletions and nucleotide substitutions between wild-type and vaccine strains, and designed primer sets for the assay. Using the synthetic DNA encoding these genes for the two strains, it was shown that the PCR amplicons intercalated with a saturating fluorescent dye could clearly differentiate between wild-type and vaccine strains in the HRM analysis for all three target genes. Further, using clinical samples, our method was able to identify recombinant strains harboring the wild-type ORF095 and ORF145 and the vaccine strain ORF126 genes. Thus, our HRM assay may provide rapid LSDV typing.

Spotty liver disease (SLD) affects free-range laying hens, leading to mortality and reduced egg production. Campylobacter species, including Campylobacter hepaticus, have been associated with SLD cases worldwide. However, the cause of SLD-like lesions found in broilers in Japan still remains unclear. The present study aimed to investigate the involvement of Campylobacter spp. in broiler SLD by conducting microbiological, molecular biological, serological, histopathological, and immunohistopathological examinations using specimens of liver, bile, cecum, and serum from SLD-like and non-SLD chickens. C. jejuni was predominantly isolated and detected in approximately 40% of both non-SLD livers and SLD-like livers, with no significant difference between them. However, C. hepaticus was neither isolated nor detected in this study. Gross and histopathology revealed multifocal necrotizing hepatitis, suppurative granulomatous hepatitis, and cholangiohepatitis. Hepatitis stages are classified as no hepatitis, subclinical, acute, and chronic hepatitis. C. jejuni was more frequently present in acute-stage SLD-like livers, and high IgG antibody levels were noted in both subclinical and SLD-like-affected chickens, indicating C. jejuni infection. Immunohistochemical examination also supported these findings. The findings suggest that C. hepaticus was not involved in SLD-like in broilers in Japan, but C. jejuni translocation from the intestines to the liver may be a contributing factor.

Spotty liver disease (SLD) affects free-range laying hens, leading to mortality and reduced egg production. Campylobacter species, including a novel strain, Campylobacter hepaticus, have been associated with SLD cases worldwide. However, the cause of SLD like lesions found in broilers in Japan is still remains unclear. The present study aimed to investigate the involvement of Cam-pylobacter spp. in broiler SLD by conducting microbiological, molecular, serological, histopatho-logical, and immunohistopathological examinations using specimens of liver, bile, cecum, and serum from SLD-like and non-SLD chickens. C. jejuni was predominantly isolated and detected in approximately 40% of both non-SLD livers and SLD-like livers, with no significant difference between them. While C. hepaticus was neither isolated nor detected in this study. Gross and his-topathology revealed multifocal necrotizing hepatitis, suppurative granulomatous hepatitis, and cholangiohepatitis. Hepatitis stages classified as no hepatitis, subclinical, acute, and chronic hepatitis. C. jejuni was more frequently present in acute-stage SLD-like livers, and high IgG an-tibody levels were noted in both subclinical and SLD-like-affected chickens, indicating C. jejuni infection. Immunohistochemical also supported these findings. The findings suggest that C. he-paticus was not involved in SLD-like in broilers in Japan, but C. jejuni translocation from the in-testines to the liver may be a contributing factor.

High pathogenicity avian influenza viruses (AIV) are a constant worldwide threat to animals and humans. The ongoing spread of the H5N1 virus underlines the pressing need for enhanced monitoring and containment strategies. Here, we report the development and validation of a novel workflow for investigating AIV genes in environmental water. This approach integrates three key components: concentration via QuickConc, extraction using a COPMAN kit, and detection through reverse transcription-preamplification-quantitative PCR. Spike experiments demonstrated that our workflow exhibits a 100-fold increase in sensitivity for AIV gene detection, compared to a combination of hemagglutination assay, QIAamp RNA Blood Mini, and one-step RT-qPCR as a control method. The efficacy of this approach was further corroborated by the successful identification of influenza M and H5 genes in field samples, specifically from surface water volumes not exceeding 500 mL collected from a body of water frequented by migratory birds. This workflow represents a significant advancement in environmental AIV surveillance, potentially augmenting our capacity to monitor and track AIV dynamics in wild bird populations using aquatic ecosystems.