Influenza A viruses (IAVs) belonging to the goose/Guangdong (Gs/GD)-lineage H5Nx remain a major concern for the global poultry industry, wildlife, and humans. The hemagglutinin (HA) protein is the dominant antigenic epitope carrier within IAV, which in turn triggers substantial immunogenic responses in the infected host. The current study describes the development and validation of a highly sensitive competitive H5 ELISA (cELISA) based on a novel monoclonal antibody developed in mice immunized with inactivated virus H5N1 (A/Turkey/ON/6213/1966). The cELISA is capable of detecting the H5 antibody response to a wider range of H5-subtype viruses belonging to both North American and Eurasia lineages, including the Gs/GD H5Nx from clade 2.3.4.4b that is currently causing the highly pathogenic avian influenza outbreaks in Eurasia, Africa, and Latin and North America. The developed H5 cELISA provides a specific, sensitive, and species-independent serological assay for the rapid detection of H5 antibodies. The assay is more robust and more sensitive than the hemagglutination inhibition assay, which is the “Gold standard”. The assay can be used in serological diagnosis, serosurveillance, and vaccine monitoring of serum samples collected from different species of animals.

The early detection of classical swine fever (CSF) remains a key challenge, especially when outbreaks are caused by moderate and low-virulent CSF virus (CSFV) strains. Oral fluid is a reliable and cost-effective sample type that is regularly surveilled for endemic diseases in commercial pig herds in North America. Here, we explored the possibility of utilizing oral fluids for the early detection of CSFV incursions in commercial-size pig pens using two independent experiments. In the first experiment, a seeder pig infected with the moderately-virulent CSFV Pinillos strain was used, and in the second experiment, a seeder pig infected with the highly-virulent CSFV Koslov strain was used. Pen-based oral fluid samples were collected daily and individual samples (whole blood, swabs) every other day. All samples were tested by a CSFV-specific real-time RT-PCR assay. CSFV genomic material was detected in oral fluids on the seventh and fourth day post-introduction of the seeder pig into the pen, in the first and second experiments, respectively. In both experiments, oral fluids tested positive before the contact pigs developed viremia, and with no apparent sick pigs in the pen. These results indicate that pen-based oral fluids are a reliable and convenient sample type for the early detection of CSF, and therefore, can be used to supplement the ongoing CSF surveillance activities in North America.

African swine fever (ASF) continues to spread, threatening the global swine industry and endangered swine species. Sri Lanka is a tropical island situated south of India in the Indian Ocean. Here, we report the first detection of ASF in Sri Lanka. In September 2024, increased pig mortality was reported across the country, with initial confirmation of porcine reproductive and respiratory syndrome (PRRS). Despite vaccination for PRRS, the mortalities continued to increase and therefore, tissue samples collected from dead pigs were subjected to ASF real-time PCR. ASFV genomic material was detected in most of the samples. The real-time PCR-positive samples were then subjected to genotyping by partial genome sequencing. All p72 and p54 sequences were found to be aligned with ASFV genotype II viruses, and CD2v sequences were found to be aligned with ASFV serogroup 8 viruses. The real-time PCR-positive samples were inoculated onto primary porcine leukocytes for virus isolation, and a selected number of tissues collected from dead pigs were subjected to histopathology. Histopathological studies revealed widespread loss of lymphocytes together with inflammation and extensive staining of ASFV antigens in tissue samples. Hemadsorption (HAD)-positive isolates were obtained from seven clinical samples, and three of them were subjected to whole-genome sequencing. Phylogeographic analysis of the whole-genome sequences showed that the virus is closely related to ASFV strains circulating in China and Hong Kong.

Despite intensive eradication efforts, classical swine fever (CSF) remains endemic across South America, Europe, Asia, and the Caribbean, highlighting the need for more effective surveillance and detection methods. Reverse-transcription real-time polymerase chain reaction (RRT-PCR) is the fastest, and most sensitive assay for detecting CSF virus (CSFV) genomic material. Previously, we demonstrated that spleen swabs outperformed spleen homogenates for the detection of ASFV genomic material by RRT-PCR. In this study, we compared CSFV genome detection in paired spleen homogenates and spleen swabs generated using 49 frozen and 33 fresh spleen samples collected from experimentally inoculated pigs with acute infection. The results show that the CSFV genome detection in spleen swabs is comparable to that in spleen homogenates. The study also demonstrated that the CSFV genomic material can be detected in spleen swabs during early CSFV infections, and the viruses can be successfully isolated from the swabs. The use of spleen swabs instead of spleen tissue homogenates for CSF detection will reduce labor, decrease costs associated with reporting, and increase the diagnostic throughput.