In March 2024, highly pathogenic avian influenza virus (HPAIV) clade 2.3.4.4b H5N1 infections were reported in dairy cows in Texas, USA¹. Rapid dissemination to more than 380 farms in 14 states followed². Here we provide results of two independent clade 2.3.4.4b experimental infection studies evaluating the oronasal susceptibility to and transmission of a US H5N1 bovine isolate, genotype B3.13 (H5N1 B3.13), in calves, and the susceptibility of lactating cows following direct mammary gland inoculation of either H5N1 B3.13 or a current EU H5N1 wild bird isolate, genotype euDG (H5N1 euDG). Inoculation of the calves resulted in moderate nasal replication and shedding with no severe clinical signs or transmission to sentinel calves. In dairy cows, infection resulted in no nasal shedding, but severe acute infection of the mammary gland with necrotizing mastitis and high fever was observed for both H5N1 isolates. Milk production was rapidly and markedly reduced and the physical condition of the cows was severely compromised. Virus titres in milk rapidly peaked at 10⁹ 50% tissue culture infectious dose (TCID₅₀) per ml, but systemic infection did not ensue. Notably, the adaptive mutation E627K emerged in the viral polymerase basic protein 2 (PB2) after intramammary replication of H5N1 euDG. Our data suggest that in addition to H5N1 B3.13, other HPAIV H5N1 strains have the potential to replicate in the udder of cows and that milk and milking procedures, rather than respiratory spread, are likely to be the primary routes of H5N1 transmission between cattle.

Canine Pyometra, also known as cystic endometrial hyperplasia complex, is a common reproductive issue in bitches. This study aimed to identify associated risk factors, hematological variation, bacteria involved, and the most potent anti-bacterial against bacterial isolates of canine pyometra. Forty-five bitches of different habitats, breeds, and ages infected with pyometra were included in the study. The samples were cultured to isolate bacteria associated with the pyometra and antibiotic sensitivity was done for each bacterial isolates to get antibiogram. The study findings showed that potential risk factors such as age group, medroxyprogesterone acetate administration, and changes in the white blood cells parameters were significantly associated (P < 0.05) with the type of pyometra. Closed cervix pyometra in dogs showed significantly higher prevalence of clinical signs including depression, vomiting, abdominal enlargement, and fever compared to the open cervix pyometra. Low levels of red blood cells, pack cell volume, and hemoglobin indicated that the pyometra-infected dogs were more likely to have normocytic, normochromic, and non-regenerative anemia. Pyometra was attributed to an increase in AST (Aspertate aminotransferase), ALT (Alanine transaminase), ALP (Alkaline phosphatase), BUN (Blood Urea Nitrogen), and Creatinine while a decrease in serum albumin. Of the all bacterial isolates, E. coli (35.55%) was the most common pathogen isolated from canine pyometra, followed by Pseudomonas spp. (26.66%). E coli and Pseudomonas spp. were susceptible to Imipenem, Amikacin, and Gentamicin while highly resistant to Ampicillin and Erythromycin. Imipenem, Amikacin, and Gentamicin were the most sensitive antibiotics, while Ampicillin and Erythromycin were the most resistant antibiotics for the bacterial strain isolated from canine pyometra. Multidrug resistant was observed in 26 of the isolated bacteria, indicating acquired resistance due to improper and uncontrolled use. Hence early diagnosis and close monitoring of antimicrobial susceptibility before therapeutic intervention is indispensable in preventing the global threat of antimicrobial resistance.

Aflatoxins (AFs), secondary metabolites produced by fungi, pose significant health risks, especially to children and elderly individuals. In developing countries such as Nepal, the tropical climate promotes fungal growth, leading to elevated levels of AF in animal feed and milk. In this study, we aimed to investigate the occurrence of aflatoxin M1 (AFM1) in dairy milk from the Kathmandu District and to assess husbandry practices contributing to contamination. We collected 84 milk samples, including raw milk from farms, retailers’ milk, and packet milk, and analyzed them using the competitive enzyme-linked immunosorbent assay (c-ELISA) technique. We also interviewed farmers to gather information on feeding and storage practices. All the collected milk samples were contaminated with AFM1, with 97.6% of the samples exceeding the European Union (EU) maximum permissible limit of 50 ppt (0.05 μg/kg). The majority (98.5%) of the farms included paddy straw, and all farms (100%) included concentrate in their feed regimens. Only half (52%) of the farms had proper storage facilities. Straw was mostly stored in sacks outdoors or left open in a shed, while concentrates were stored in a closed room or shed. This study reveals very high levels of AFM1 contamination in the milk samples, presenting a serious public health issue, and recommends comprehensive surveillance and further investigations across the country, especially given the limited research and literature available.

The open reading frame 3a (ORF3a) is a protein important to the pathogenicity of SARS-CoV-2. The cytoplasmic domain of ORF3a has three canonical tyrosine-based sorting signals (160YNSV163, 211YYQL213, and 233YNKI236), and a previous study has indicated that mutation of the 160YNSV163 motif abrogated plasma membrane expression and inhibited ORF3a-induced apoptosis. Here, we have systematically removed all three tyrosine-based motifs and assessed the importance of each motif or combination of motifs in trafficking to the cell surface. Our results indicate that the 160YNSV163 motif alone was insufficient for ORF3a cell-surface trafficking, while the 211YYQL213 motif was the most important. Additionally, an ORF3a with all three YxxΦ motifs disrupted (ORF3a-[ΔYxxΦ]) was not transported to the cell surface, and LysoIP studies indicate that ORF3a but not ORF3a-[ΔYxxΦ] was present in late endosome/lysosome fractions. A growth-curve analysis of different SARS-CoV-2 viruses expressing the different mutant ORF3a proteins revealed no significant differences in virus replication. Finally, the inoculation of K18hACE-2 mice indicated that the SARS-CoV-2 lacking the three YxxΦ motifs was less pathogenic than the unmodified SARS-CoV-2. These results indicate that the tyrosine motifs of ORF3a contribute to cell-surface expression and SARS-CoV-2 pathogenesis

Highly pathogenic avian influenza (HPAI) is a transboundary and zoonotic viral disease affecting poultry and wild birds in many countries worldwide. Globally, HPAI outbreaks have led to the death or culling of hundreds of millions of birds over the past two decades and have caused nearly 1000 confirmed human H5N1 infections, with a case fatality rate of approximately 50%. Asia and Europe remain among the most affected regions, with recurrent outbreaks linked to intensive poultry production, live bird markets, and migratory bird pathways. In Nepal, HPAI has been reported since 2009, with more than 320 outbreaks recorded and over 2.7 million birds lost, alongside one confirmed human fatality. Control measures rely largely on stamping out, movement restrictions, and surveillance; however, gaps in farm-level biosecurity, informal cross-border poultry trade, and limited vaccination use continue to sustain vulnerability. Strengthened multisectoral coordination under a One Health framework, integrating veterinary and public health surveillance, molecular monitoring, community awareness, and risk-based biosecurity enforcement, is essential to reduce the impact of HPAI and mitigate future zoonotic and pandemic risks.

Aflatoxins (AF), secondary metabolites produced by fungi, pose significant health risks, especially to children and adults. In developing countries like Nepal, the tropical climate promotes fungal growth, leading to high levels of AF in animal feed and milk. In this study, we investigated the incidence of aflatoxin M1 (AFM1) contamination in 84 milk samples collected from the Kathmandu district using the competitive enzyme-linked immunosorbent assay (c-ELISA) technique. We also interviewed farmers to gather information on feeding and storage practices. . All the collected milk samples were contaminated with AFM1, with 97.6% of the samples exceeding the European Union (EU) maximum permissible limit of 50 ppt. The majority (98.5%) of the farms included paddy straw, and all farms (100%) included concentrate in their feed regimen. Only half (52%) of the farms had proper storage facilities. Straw was mostly stored in sacks outdoors or left open in a shed, while concentrates were stored in a closed room or shed. The study reports very high contamination levels of AFM1 in the milk samples, presenting a serious public health issue, and recommends comprehensive surveillance and further investigation across the country, especially given the limited research and literature available.