In March 2025, a backyard poultry flock in southern Illinois experienced acute illness with near-complete mortality (100 of 105 birds). The premises, about 2 miles from a large reservoir within the Mississippi Flyway, kept multiple avian species, cattle, and several domestic animals including cats and dogs. The primary veterinarian submitted remains of six birds and one rat for necropsy. Initial testing of a chicken sample followed USDA NAHLN qRT-PCR guidance and was presumptive positive; NVSL confirmed the isolate as HPAI H5N1 clade 2.3.4.4b.
Following confirmation, state and federal agencies were notified and the farm was quarantined and monitored for 4 months. Subsequent specimens from a 13-year-old domestic shorthaired cat and a feral rat found dead on the farm were submitted for diagnostic evaluation.
The University of Missouri VMDL performed postmortem examinations on the original avian submissions and reported gross lesions including hepatitis, pulmonary hemorrhage, and pulmonary edema in birds. Additional testing of birds for Mycoplasma spp. and avian paramyxovirus was negative. The rat submitted with the birds had bronchopneumonia and meningoencephalitis noted at necropsy; no further testing was initially performed on that rat.
Specimens from the cat and a second feral rat were sent to the University of Illinois VDL. The laboratory screened lung tissue by qRT-PCR following NAHLN protocols. The cat lung tested presumptive positive for influenza A (Ct 21.2) and was subtyped as H5; NVSL confirmed H5N1 clade 2.3.4.4b. The rat lung was negative on initial screening, but NVSL subsequently detected H5N1 in a tracheal swab from that animal.
Follow-up IAV qRT-PCR testing was performed on a panel of tissues: 12 samples from the cat and 15 samples from the rat. A Ct cutoff of <45 was used to classify IAV-positive results. All 12 cat tissue samples were positive (Ct range 10.9–31.1). Eleven of 15 rat tissues were positive (Ct range 17.1–39.4). Brain tissues had the lowest Ct values (highest viral RNA levels) in both animals: cat brain Ct 10.9 and rat brain Ct 17.1.
Both animals had positive respiratory tract swabs (nasal and tracheal; Ct 22.1–29.5). Kidney tissues were IAV-positive in both species (cat Ct 20.9; rat Ct 34.6) and rat urine tested positive (Ct 32.0). Cat feces contained detectable nucleic acid (Ct 28.6). A second lung sample from the rat gave a higher Ct (39.4). Rat feces, bone marrow, and testis tested negative when sampled.
The distribution of viral RNA across multiple organs, including central nervous system tissues, respiratory tract, and excreta, indicates systemic viral dissemination in these mammals on the infected premises.
Whole-genome sequencing was performed on three cat samples (brain, heart, nasal swab) and one rat brain sample using an amplicon-based enrichment approach and Illumina MiSeq sequencing; assemblies were generated on the Galaxy platform. Complete sequences for all eight viral genome segments were recovered and deposited (GenBank accession nos. provided in the source appendix). Sequence data from the chicken isolate from the same outbreak were included in comparative analyses.
Phylogenetic analysis showed the chicken, cat, and rat strains clustered together with other avian and mammalian HPAIV H5N1 sequences. The outbreak virus was designated genotype D1.1. Notably, sequences from the chicken, cat, and rat all harbored the polymerase basic 2 E627K substitution, a mutation associated with mammalian adaptation. Additional amino acid changes—neuraminidase G392D and nucleoprotein K372E—were present in the cat and rat sequences but were not found in the chicken isolate. A synonymous polymerase basic 2 C1552T change was observed uniquely in the rat strain.
Cat samples shared identical consensus sequences across specimens tested.
Histologic examination documented pulmonary and neurologic lesions consistent with influenza infection. The cat showed bronchointerstitial pneumonia and nonsuppurative meningoencephalitis. Immunohistochemistry (IHC) demonstrated influenza immunoreactivity in distal airway epithelium and in cerebrocortical glial cells and neurons.
The rat had background alveolitis in the lung and nonsuppurative periventricular encephalitis in the brain. IHC revealed influenza antigen in brain tissue. These findings align with the high viral RNA loads detected in neural tissues and support systemic infection with neural tropism in these mammals.
Following confirmation of HPAI H5N1 in poultry and detection of the virus in on-premises mammals, the Illinois Department of Agriculture and the USDA quarantined and monitored the farm for four months. The source article did not report human cases, details about human exposure assessments, or broader surveillance outcomes beyond the described animals.
The detection of HPAI H5N1 RNA in multiple tissues of a domestic cat and two rats, the presence of the PB2 E627K mammalian-adaptive mutation in isolates from poultry and mammals, and phylogenetic clustering with avian strains indicate spillover from infected birds to mammals at this backyard farm. The findings suggest that small mammals present on affected premises could contribute to local virus transmission dynamics and warrant consideration in outbreak investigations and control measures. Specific management actions beyond quarantine, results of contact tracing, and any further epidemiologic follow-up were not detailed in the source article.