兽研摘要
2025年，在美国伊利诺伊州的一个家禽群中检测到高致病性甲型H5N1禽流感病毒。对该农场猫和大鼠样本进行的定量逆转录PCR、测序和组织病理学检查显示多个组织呈阳性，且与一株禽源分离株序列同源性高。小型哺乳动物可能促进H5N1传播。
关键词：高致病性禽流感病毒，流感，病毒，呼吸道感染，人兽共患病，H5N1，猫，大鼠，家禽，美国
当前高致病性禽流感（HPAI）A(H5N1) 2.3.4.4b分支病毒的泛动物疫病于2020年在欧洲出现，到2022年已蔓延至欧洲、非洲和北美（1–4）。跨大西洋传入北美最早于2021年12月被记录，此后该病毒在野生和家养鸟类中持续存在，已在奶牛中检测到，并偶有其他哺乳动物报道（5）。水禽在海鸟在病毒传播中发挥了同等作用，鉴于其与家禽养殖场活动范围重叠的可能性更高，这令人担忧（2,6）。我们描述了美国伊利诺伊州一个后院家禽群中暴发的HPAI H5N1疫情，随后蔓延至该场所的家养和野生哺乳动物。
研究
2025年3月12日，伊利诺伊州南部一位拥有小型后院家禽群的养殖户在禽群发生急性疾病且几乎全部死亡（100/105只禽）后，将6只动物的遗体带到其初级兽医处进行剖检检查。该农场距密西西比迁徙通道内一个25,000英亩的水库湖约2英里，饲养有4只火鸡（Meleagris gallopavo）、1只鹅（Anser sp.）、20只珍珠鸡（Numida spp.）、80只鸡（Gallus gallus domesticus）、17头牛、15只猫和2只狗。由于怀疑为HPAI病毒（HPAIV），这6只动物（1只鸡、1只火鸡、1只鹅、2只珍珠鸡和1只大鼠（Rattus norvegicus））被送往密苏里大学兽医医学诊断实验室（VMDL；美国密苏里州哥伦比亚）进行死后检查。VMDL按照美国农业部（USDA）国家动物卫生实验室网络（NAHLN；https://www.aphis.usda.gov/labs/nahln）的指南，采集并评估了新鲜和福尔马林固定组织，并对1份鸡样本进行了甲型流感病毒（IAV）定量逆转录PCR（qRT-PCR）检测。该样本按NAHLN指南检测为推定阳性，VMDL将其亚型鉴定为H5流感。VMDL随后将样本送往国家兽医服务实验室（NVSL；美国爱荷华州艾姆斯）进行确认检测，结果证实为HPAIV H5N1 2.3.4.4b分支病毒。
对禽类进行的鸡毒支原体、滑液支原体和禽副黏病毒的进一步PCR检测均为阴性。剖检结果报告家禽出现肝炎、肺出血和肺水肿，大鼠出现支气管肺炎和脑膜脑炎。未对大鼠或其他鸟类进行额外检测。在报告农场出现HPAIV后，通知了州和联邦机构。伊利诺伊州农业部和USDA对该农场实施了隔离，并监测了4个月。
2025年3月16日，初级兽医从该农场采集了一只13岁家养短毛猫和一只野鼠的遗体。与伊利诺伊州农业部和USDA协作，猫和大鼠标本于3月19日被送往伊利诺伊大学兽医诊断实验室（VDL；美国伊利诺伊州厄巴纳）。VDL进行了大体剖检并采集了新鲜和福尔马林固定组织。VDL按照NAHLN指南，通过qRT-PCR对猫和大鼠的肺组织进行了IAV筛查。猫肺检测为推定阳性（循环阈值[Ct]值21.2），亚型鉴定为H5；NVSL确认其为H5N1 2.3.4.4b分支病毒。大鼠肺组织初筛为阴性，但NVSL对气管拭子样本进行了后续检测，结果为H5N1阳性。
此外，我们对猫的12份组织样本和大鼠的15份组织样本进行了后续IAV qRT-PCR检测，并将Ct值<45视为IAV阳性。猫的全部12份组织样本均为IAV阳性（Ct值10.9–31.1），大鼠15份组织中有11份为IAV阳性（Ct 17.1–39.4）（图1）。两种动物的脑组织病毒载量最高（猫Ct 10.9；大鼠Ct 17.1）。值得注意的是，两种动物的气管和鼻腔拭子样本均为IAV阳性（Ct范围22.1–29.5）。大鼠的第二份肺样本Ct值为39.4。两种动物的肾脏组织均为IAV阳性（猫Ct 20.9；大鼠Ct 34.6），大鼠尿液也检测为IAV阳性（Ct 32.0）。猫粪便中的核酸Ct值为28.6，但大鼠粪便中未检测到核酸。
[图：后院家禽暴发期间猫和大鼠中HPAIV A(H5N1)的定量逆转录PCR结果，U]
我们采用基于扩增子的富集方案，在MiSeq平台（Illumina，https://www.illumina.com）上对猫的3份样本（脑、心脏和鼻拭子）和大鼠的1份脑样本进行了全基因组测序。我们使用Galaxy平台（7）进行序列组装。所有样本均返回了8个片段的完整序列（GenBank登录号PX687012–27）（附录表）。此外，我们在分析中纳入了VMDL提交至NVSL的鸡样本中HPAIV H5N1毒株25-009367-001的序列数据（GenBank登录号PV616546–53）。系统发育树分析显示，鸡、大鼠和猫的毒株与其他禽类和哺乳动物HPAIV聚集在一起（图2；附录图1–8）。此次疫情中的所有3种动物均携带与哺乳动物适应性相关的聚合酶碱性蛋白2 E627K突变（8）。所有猫样本共享相同的共识序列。我们在猫和大鼠样本中发现了2个氨基酸突变——神经氨酸酶G392D和核蛋白K372E，但在鸡的IAV毒株中未发现。此外，我们在大鼠毒株中注意到1个同义聚合酶碱性蛋白2 C1552T突变，而在猫和鸡毒株中未见到该突变。此次疫情病毒的基因型为D1.1（9）（附录）。
[图：猫和大鼠中高致病性甲型禽流感(H5N1)病毒血凝素基因序列的系统发育树]
剖检时大体检查显示猫和大鼠肺部有多灶性红色斑驳，大鼠另有右前腹侧肺实变。猫的组织病理学发现包括多灶性支气管间质性肺炎、轻度多灶性非化脓性脑膜脑炎和急性肾上腺皮质坏死（图3，A–C面板）。我们在大鼠肺组织的弥漫性肺泡炎背景中注意到严重的腹侧前部支气管肺炎（未显示）（图3，D面板），可能与其他细菌感染有关。大鼠中可能与病毒感染相关的其他病变包括脑组织中轻度非化脓性室周脑炎（图3，E面板）和心肌炎。猫肺中IAV的免疫组化显示气道顶端上皮免疫反应性（图3，A面板插图），脑组织显示多灶性胶质细胞和神经元免疫反应性（图3，C面板）。我们还注意到大鼠脑内神经元有轻度室周免疫反应性（图3，F面板）。
[图：高致病性禽流感暴发期间发现死亡的猫和大鼠病变的组织病理学和免疫组化]
结论
我们描述了一株H5N1毒株，该毒株在美国伊利诺伊州一个后院农场导致鸡群高死亡率和小型哺乳动物致死性疾病。我们的结果表明，自然感染后猫和大鼠组织中均发生了广泛的全身性H5N1病毒播散。大鼠鼻甲和气管相对于肺优先表达α2,3-连接的唾液酸受体，这可能解释了上呼吸道组织中较低的Ct值和细菌性支气管肺炎的存在，并可能导致病毒排毒增加（10）。
此前已证明猫粪便和尿液可携带H5N1病毒（11,12），我们的结果证实了这一点。此前已有报道大鼠在农业环境中携带HPAIV，并且在当前动物流行病中也有检出（13,14）。在大鼠尿液和肾脏组织中检测到HPAIV提示啮齿动物尿液可能是潜在的传播媒介，值得进一步研究。大鼠栖息于许多农场，可捕食鸟类，并可能作为受感染禽类与易感哺乳动物宿主之间的中间环节（15）。这种共存对商业家禽和养猪封闭式农场尤其具有威胁性，因为这些农场尽管采取了严格的生物安全措施，仍经常存在啮齿动物种群。我们研究的一个局限性是，由于生物安全限制，未对组织或尿液进行病毒分离。
尽管已证明猫参与HPAIV传播，但我们的发现表明需要进一步研究以阐明啮齿动物的作用。本研究提高了对野生动物–家畜界面小型哺乳动物的认识，并强调了大鼠在流感向商业家畜生产传播中的潜在作用。
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致谢
我们感谢Megan Sherman在免疫组化染色和材料信息方面的贡献。我们也感谢允许对其动物进行进一步研究的动物主人。我们感谢NVSL对此次疫情的确认支持。
S.M.S.部分获得了美国农业部国家食品与农业研究所兽医服务资助计划的支持（项目资助号2021-70024-35413）。
作者简介
Storms博士是美国德克萨斯州大学城德克萨斯A&M兽医医学诊断实验室的兽医科学家。她的研究兴趣集中在流感病毒传播和诊断开发。
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Abstract
In 2025, highly pathogenic avian influenza A(H5N1) virus was detected in a poultry flock in Illinois, USA. Quantitative reverse transcription PCR, sequencing, and histopathology on cat and rat samples from the farm showed multiple positive tissues and high sequence identity to an avian isolate. Small mammals might contribute to H5N1 transmission.
Keywords: Highly pathogenic avian influenza virus, influenza, viruses, respiratory infections, zoonoses, H5N1, cat, rat, poultry, United States
The current panzootic of highly pathogenic avian influenza (HPAI) A(H5N1) clade 2.3.4.4b virus emerged in Europe in 2020 and by 2022 had spread throughout Europe, Africa, and North America (1–4). Trans-Atlantic introduction into North America was first documented in December 2021 and has since persisted in wild and domestic birds, has been detected in dairy cattle, and has been reported sporadically in other mammals (5). Waterfowl have played an equal role to seabirds in virus spread, which is concerning given their higher likelihood of overlapping ranges with poultry farms (2,6). We describe an HPAI H5N1 outbreak in a backyard poultry flock in Illinois, USA, with subsequent spillover into domestic and wild mammals on the premises.
The Study
On March 12, 2025, a farmer in southern Illinois with a small backyard poultry flock brought remains of 6 animals to their primary veterinarian for a necropsy workup after acute illness and near-complete mortality (100/105 birds) of the flock. The farm, ≈2 miles from a 25,000-acre reservoir lake within the Mississippi Flyway, housed 4 turkeys (Meleagris gallopavo), 1 goose (Anser sp.), 20 Guinea fowl (Numida spp.), 80 chickens (Gallus gallus domesticus), 17 cattle, 15 cats, and 2 dogs. Because HPAI virus (HPAIV) was suspected, the 6 animals, a chicken, a turkey, a goose, 2 Guinea fowl, and a rat (Rattus norvegicus), were sent to the University of Missouri Veterinary Medical Diagnostic Laboratory (VMDL; Columbia, MO, USA) for postmortem examination. VMDL collected and evaluated fresh and formalin-fixed tissues and performed influenza A virus (IAV) quantitative reverse transcription PCR (qRT-PCR) on 1 chicken sample, according to guidelines from the US Department of Agriculture (USDA) National Animal Health Laboratory Network (NAHLN; https://www.aphis.usda.gov/labs/nahln). The sample tested presumptive positive per NAHLN guidelines, and VMDL subtyped it as H5 influenza. VMDL then forwarded the sample to the National Veterinary Services Laboratories (NVSL; Ames, IA, USA) for confirmatory testing, which verified HPAIV H5N1 clade 2.3.4.4b virus.
Further PCR testing of the avian species for Mycoplasma gallisepticum, M. synoviae, and avian paramyxovirus were all negative. The necropsy findings reported hepatitis, pulmonary hemorrhage, and pulmonary edema in the poultry species and bronchopneumonia and meningoencephalitis in the rat. No additional testing was performed on the rat or other bird species. Upon report of HPAIV on the farm, state and federal agencies were notified. The Illinois Department of Agriculture and USDA quarantined the farm and monitored it for 4 months.
On March 16, 2025, the primary veterinarian collected the remains of a 13-year-old domestic shorthaired cat and a second feral rat from the farm. In conjunction with Illinois Department of Agriculture and USDA, the cat and rat specimens were sent to the University of Illinois Veterinary Diagnostic Laboratory (VDL; Urbana, IL, USA) on March 19. VDL performed gross necropsy and collected fresh and formalin-fixed tissues. VDL screened lung tissues from the cat and rat for IAV by qRT-PCR, following NAHLN guidelines. The cat lung tested presumptive positive (cycle threshold [Ct] value 21.2), subtyped as H5; NVSL confirmed H5N1 clade 2.3.4.4b virus. The rat lung tissue was negative upon initial screening, but NVSL conducted follow-up testing on tracheal swab samples, which were H5N1-positive.
In addition, we performed follow-up IAV qRT-PCR testing on 12 tissue samples from the cat and 15 tissue samples from the rat and considered Ct values <45 IAV-positive. All 12 cat tissue samples were IAV-positive (Ct values 10.9–31.1), and 11/15 rat tissues were IAV-positive (Ct 17.1–39.4) (Figure 1). Brain tissues of both animals had the highest viral loads (cat Ct 10.9; rat Ct 17.1). Of note, tracheal and nasal swab samples from both animals were IAV-positive (Ct range 22.1–29.5). A second lung sample from the rat returned a Ct of 39.4. Kidney tissues from both species were IAV-positive (cat Ct 20.9; rat Ct 34.6), and urine from the rat also tested IAV-positive (Ct 32.0). Nucleic acid in feces from the cat had a Ct value of 28.6, but no nucleic acid was detected in rat feces.
[图：Quantitative reverse transcription PCR results from HPAIV A(H5N1) in cat and rats during outbreak in backyard poultry, U]
We performed whole-genome sequencing on 3 samples from the cat (brain, heart, and nasal swab) and 1 brain sample from the rat by using an amplicon-based enrichment protocol and sequencing on a MiSeq platform (Illumina, https://www.illumina.com). We used the Galaxy platform (7) for sequence assembly. All samples returned complete sequences for 8 segments (GenBank accession nos. PX687012–27) (Appendix Table). In addition, we included sequence data of the HPAIV H5N1 strain 25-009367-001 (GenBank accession nos. PV616546–53) from the chicken sample submitted by VMDL to NVSL in the analysis. Phylogenetic tree analysis revealed that the chicken, rat, and cat strains clustered together with other avian and mammal HPAIV (Figure 2; Appendix Figures 1–8). All 3 animal species from the outbreak contained the polymerase basic 2 E627K mutation that is associated with mammalian adaptation (8). All cat samples shared identical consensus sequences. We found 2 amino acid mutations, neuraminidase G392D and nucleoprotein K372E, in the cat and rat samples but not in the IAV strain from the chicken. In addition, we noted 1 synonymous polymerase basic 2 C1552T mutation in the rat strain that we did not see in cat and chicken strains. The genotype of the outbreak virus was D1.1 (9) (Appendix).
[图：Phylogenetic tree of hemagglutinin gene sequences from highly pathogenic avian influenza A(H5N1) virus in cat and rats d]
Gross examination during necropsy showed multifocal red mottling of the cat and rat lungs and additional right cranioventral lung consolidation in the rat. Histopathology findings in the cat included multifocal bronchointerstitial pneumonia, mild multifocal nonsuppurative meningoencephalitis, and acute adrenocortical necrosis (Figure 3, panels A–C). We noted severe cranioventral bronchopneumonia (not shown) amongst a background of diffuse alveolitis in the rat lung tissue (Figure 3, panel D), likely related to a bacterial infection. Additional lesions in the rat possibly related to viral infection included mild nonsuppurative periventricular encephalitis in brain tissue (Figure 3, panel E) and myocarditis. Immunohistochemistry for IAV in the cat lung showed apical airway epithelial immunoreactivity (Figure 3, panel A, inset), and brain tissue showed multifocal glial and neuronal immunoreactivity (Figure 3, panel C). We also noted mild periventricular immunoreactivity in neurons in the rat brain (Figure 3, panel F).
[图：Histopathology and immunohistochemistry of lesions in a cat and rat found dead during outbreak of highly pathogenic avia]
Conclusions
We describe a strain of H5N1 that resulted in high mortality in a poultry flock and fatal illness in small mammals on a backyard farm in Illinois, USA. Our results show that extensive systemic H5N1 virus spread occurred in both cat and rat tissues after natural infection. Rat nasal turbinates and trachea preferentially expressed α2,3-linked sialic acid receptors relative to lung, likely explaining the lower Ct values in upper respiratory tissues and presence of bacterial bronchopneumonia, potentially leading to increased viral shedding (10).
Cat feces and urine have previously been shown to harbor H5N1 virus (11,12), which our results corroborate. Rats have previously been reported to harbor HPAIV in agricultural settings and have been detected in the current epizootic (13,14). Detection of HPAIV in rat urine and kidney tissue suggests that rodent urine could be a potential transmission medium and warrants further investigation. Rats reside on many farms, can predate birds, and might serve as an intermediate step between infected avian species and susceptible mammalian hosts (15). That coexistence is particularly threatening for commercial poultry and swine confinement farms, which frequently harbor rodent populations despite strict biosecurity efforts. A limitation of our study was that no virus isolation was performed on tissues or urine due to biosafety constraints.
Although cats have been shown to participate in HPAIV transmission, our findings indicate that further investigation is needed to clarify the role of rodents. This study raises awareness of small mammals at the wildlife–livestock interface and highlights the potential role of rats in influenza transmission to commercial livestock production.
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Acknowledgments
We thank Megan Sherman for her contributions regarding immunohistochemical staining and materials information. We also thank the animal owners who allowed further investigation of their animals for this study. We thank NVSL for their confirmatory support for this outbreak.
S.M.S. was partially supported by the Veterinary Services Grant Program from the USDA’s National Institute of Food and Agriculture (project award no. 2021-70024-35413).
Biography
Dr. Storms is a veterinarian-scientist at Texas A&M Veterinary Medical Diagnostic Laboratory, College Station, Texas, USA. Her research interests center around influenza virus transmission and diagnostic development.
Footnotes
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References
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Associated Data
Supplementary Materials
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