Avian Influenza Virus (H5N1) Was Not Detected Among Dairy Cattle and Farm Workers in Pakistan

IF 4.3 4区 医学 Q1 INFECTIOUS DISEASES
Aftab Ahmed, Samia Saqlain, Arslan Rasool, Shaban Muhammad, Sajid Umar
{"title":"Avian Influenza Virus (H5N1) Was Not Detected Among Dairy Cattle and Farm Workers in Pakistan","authors":"Aftab Ahmed,&nbsp;Samia Saqlain,&nbsp;Arslan Rasool,&nbsp;Shaban Muhammad,&nbsp;Sajid Umar","doi":"10.1111/irv.13317","DOIUrl":null,"url":null,"abstract":"<p>Influenza A virus (IAV) is a zoonotic pathogen that poses a significant challenge to avian and public health [<span>1</span>]. Migratory waterfowl, such as ducks and geese, are the main reservoir of IAVs and spread IAVs to other domestic birds and animals. Cross-species transmission of IAV is generally rare, but it can occur, particularly if there are mutations in the virus's genetic code that allow it to attach to receptors in a new host or if a reassortment event occurs when two different influenza viruses infect the same host simultaneously and exchange genetic material. The Avian Influenza A(H5N1) virus, commonly known as “bird flu,” remains primarily a virus that affects birds, but it has on occasion infected humans. A relatively low number of sporadic human infections with A(H5N1) has been reported over the years. A total of 26 sporadic human cases from January 2022 through April 2024 [<span>2</span>] from eight countries indicates ongoing transmission from birds to humans. These cases highlight the potential for this virus to cause severe illness and death in humans. On rare occasions, A(H5N1) has crossed over to some nonavian species especially when there has been close contact with infected birds [<span>3-5</span>]. Cattle, like other mammalian species, are generally susceptible to specific types of IAV and Influenza D virus. Typically, A(H5N1) is not a common infection in cattle. Surprisingly, numerous sporadic cases of A(H5N1) have been reported recently from dairy cattle in multiple states of the United States [<span>5, 6</span>]. The emerging bovine H5N1 virus is novel to the cattle industry. Unlike other mammals, H5N1 grows in the udder of the dairy cows and does not seem to cause respiratory disease in cattle. Bovine H5N1 virus has undergone a specific adaptation in an enzyme called polymerase allowing better replication inside cow udder. A dairy farm worker with conjunctivitis was also confirmed positive highlighting fresh concerns of bovine H5N1 virus to human health [<span>5, 6</span>]. The dairy worker might have encountered this virus during the milking process or through hand-to-eye contact. Further genetic changes within polymerase enzyme or other genome segments of bovine H5N1 virus could allow for faster adaptation and may even support cattle-to-cattle or cattle to human transmission [<span>5, 6</span>]. Together, these reports highlight that H5N1 has potential to evolve and become a serious threat to human health. There is no data bovine H5N1 virus from Pakistan. Therefore, this study was designed to monitor the prevalence of IAVs and potential spillover of novel H5N1 among cattle and farm workers in Punjab province of Pakistan. Human subject research was approved by Institutional Review Board (IRB) at Duke Kunshan University, China (2024SU040).</p><p>As part of the Influenza D virus surveillance study, we collected nasal washes (<i>n =</i> 117) and nasal swab samples (<i>n =</i> 376) from farm workers and dairy cattle respectively from January 2023 to March 2024 at six commercial dairy cattle farms located in different cities (Lahore, Kasur, Sahiwal, Multan, Layyah, and Bahawalpur) within Punjab province (Figure 1). These farms were visited twice a month to collect nasal wash and nasal swab samples as described previously [<span>7</span>]. In addition, we also collected milk samples (<i>n =</i> 243) during milking process in sterile tubes from different farms in April 2024 to detect bovine H5N1 virus. A commercial RNA extraction Kit (Cat#9766, Takara, Dalian, China) was used to extract viral RNA from samples following the manufacturer's recommendations [<span>1</span>]. To validate the integrity of reagents and the extraction process, we included positive and negative controls. Extracted samples were screened for H5N1 via one step RT-PCR using HiScript II One Step RT-PCR Kit (Vazyme, Nanjing, China). Specific primers were used to target hemagglutination (HA) gene [<span>8</span>]. Positive and negative controls were added in each PCR run to confirm the results. All positive controls showed successful amplification while negative controls did not yield amplification during PCR reactions.</p><p>No evidence for H5N1 infection was found in any sample type of cattle and farm workers in this study. Assuming our samples were representative of cattle and farm workers in Punjab province during the study time period, H5N1 did not appear to be endemic in Punjab cattle population. These findings could also be linked to strict biosecurity measures at these farms. The inclusion of more cattle farms in sampling plan in other provinces of Pakistan might reverse our observations about H5N1 prevalence. Despite the limitations of small sample size, small geographical area and duration of the study, our study is still valuable as it provided preliminary data with novel methodology that can prompt larger and more comprehensive studies.</p><p>To date, bovine H5N1 virus which is currently circulating in US dairy cattle poses a low risk to humans. However, animal farm workers and veterinarians need to be vigilant as they are at higher risk of infection. Appropriate biosecurity measures should be practiced while performing routine work at dairy farms to prevent interspecies transmission of bovine H5N1 virus. Additional sporadic human infections are anticipated because H5N1 has wide circulation in wild and domestic poultry and has potential to evolve rapidly if they are left unchecked. US has been a main exporter of dairy cattle to Pakistan. Therefore, dairy cattle herds must be tested for bovine H5N1 virus before shipment to Pakistan as a preventive measure. It is expected that mammalian host range and zoonotic transmission of IAV will expand due to globalization, intensive trade, and climate change. Therefore, it is critical to exercise proper biosecurity practices at animal farms. Keeping in mind the zoonotic potential and current wave of bovine H5N1 virus among US dairy cattle herds, a One Health approach is urgently required for regular monitoring of IAV in wild birds, domestic poultry, mammals, and people worldwide to determine genetic changes and public health risk.</p><p><b>Aftab Ahmed:</b> conceptualization, methodology, investigation. <b>Samia Saqlain:</b> investigation, formal analysis. <b>Arslan Rasool:</b> conceptualization, methodology, investigation. <b>Shaban Muhammad:</b> investigation, validation, visualization. <b>Sajid Umar:</b> conceptualization, methodology, investigation, validation, formal analysis, supervision, funding acquisition, project administration, writing–original draft, writing–review and editing.</p><p>The authors declare no conflicts of interest.</p>","PeriodicalId":13544,"journal":{"name":"Influenza and Other Respiratory Viruses","volume":null,"pages":null},"PeriodicalIF":4.3000,"publicationDate":"2024-05-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1111/irv.13317","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Influenza and Other Respiratory Viruses","FirstCategoryId":"3","ListUrlMain":"https://onlinelibrary.wiley.com/doi/10.1111/irv.13317","RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"INFECTIOUS DISEASES","Score":null,"Total":0}
引用次数: 0

Abstract

Influenza A virus (IAV) is a zoonotic pathogen that poses a significant challenge to avian and public health [1]. Migratory waterfowl, such as ducks and geese, are the main reservoir of IAVs and spread IAVs to other domestic birds and animals. Cross-species transmission of IAV is generally rare, but it can occur, particularly if there are mutations in the virus's genetic code that allow it to attach to receptors in a new host or if a reassortment event occurs when two different influenza viruses infect the same host simultaneously and exchange genetic material. The Avian Influenza A(H5N1) virus, commonly known as “bird flu,” remains primarily a virus that affects birds, but it has on occasion infected humans. A relatively low number of sporadic human infections with A(H5N1) has been reported over the years. A total of 26 sporadic human cases from January 2022 through April 2024 [2] from eight countries indicates ongoing transmission from birds to humans. These cases highlight the potential for this virus to cause severe illness and death in humans. On rare occasions, A(H5N1) has crossed over to some nonavian species especially when there has been close contact with infected birds [3-5]. Cattle, like other mammalian species, are generally susceptible to specific types of IAV and Influenza D virus. Typically, A(H5N1) is not a common infection in cattle. Surprisingly, numerous sporadic cases of A(H5N1) have been reported recently from dairy cattle in multiple states of the United States [5, 6]. The emerging bovine H5N1 virus is novel to the cattle industry. Unlike other mammals, H5N1 grows in the udder of the dairy cows and does not seem to cause respiratory disease in cattle. Bovine H5N1 virus has undergone a specific adaptation in an enzyme called polymerase allowing better replication inside cow udder. A dairy farm worker with conjunctivitis was also confirmed positive highlighting fresh concerns of bovine H5N1 virus to human health [5, 6]. The dairy worker might have encountered this virus during the milking process or through hand-to-eye contact. Further genetic changes within polymerase enzyme or other genome segments of bovine H5N1 virus could allow for faster adaptation and may even support cattle-to-cattle or cattle to human transmission [5, 6]. Together, these reports highlight that H5N1 has potential to evolve and become a serious threat to human health. There is no data bovine H5N1 virus from Pakistan. Therefore, this study was designed to monitor the prevalence of IAVs and potential spillover of novel H5N1 among cattle and farm workers in Punjab province of Pakistan. Human subject research was approved by Institutional Review Board (IRB) at Duke Kunshan University, China (2024SU040).

As part of the Influenza D virus surveillance study, we collected nasal washes (n = 117) and nasal swab samples (n = 376) from farm workers and dairy cattle respectively from January 2023 to March 2024 at six commercial dairy cattle farms located in different cities (Lahore, Kasur, Sahiwal, Multan, Layyah, and Bahawalpur) within Punjab province (Figure 1). These farms were visited twice a month to collect nasal wash and nasal swab samples as described previously [7]. In addition, we also collected milk samples (n = 243) during milking process in sterile tubes from different farms in April 2024 to detect bovine H5N1 virus. A commercial RNA extraction Kit (Cat#9766, Takara, Dalian, China) was used to extract viral RNA from samples following the manufacturer's recommendations [1]. To validate the integrity of reagents and the extraction process, we included positive and negative controls. Extracted samples were screened for H5N1 via one step RT-PCR using HiScript II One Step RT-PCR Kit (Vazyme, Nanjing, China). Specific primers were used to target hemagglutination (HA) gene [8]. Positive and negative controls were added in each PCR run to confirm the results. All positive controls showed successful amplification while negative controls did not yield amplification during PCR reactions.

No evidence for H5N1 infection was found in any sample type of cattle and farm workers in this study. Assuming our samples were representative of cattle and farm workers in Punjab province during the study time period, H5N1 did not appear to be endemic in Punjab cattle population. These findings could also be linked to strict biosecurity measures at these farms. The inclusion of more cattle farms in sampling plan in other provinces of Pakistan might reverse our observations about H5N1 prevalence. Despite the limitations of small sample size, small geographical area and duration of the study, our study is still valuable as it provided preliminary data with novel methodology that can prompt larger and more comprehensive studies.

To date, bovine H5N1 virus which is currently circulating in US dairy cattle poses a low risk to humans. However, animal farm workers and veterinarians need to be vigilant as they are at higher risk of infection. Appropriate biosecurity measures should be practiced while performing routine work at dairy farms to prevent interspecies transmission of bovine H5N1 virus. Additional sporadic human infections are anticipated because H5N1 has wide circulation in wild and domestic poultry and has potential to evolve rapidly if they are left unchecked. US has been a main exporter of dairy cattle to Pakistan. Therefore, dairy cattle herds must be tested for bovine H5N1 virus before shipment to Pakistan as a preventive measure. It is expected that mammalian host range and zoonotic transmission of IAV will expand due to globalization, intensive trade, and climate change. Therefore, it is critical to exercise proper biosecurity practices at animal farms. Keeping in mind the zoonotic potential and current wave of bovine H5N1 virus among US dairy cattle herds, a One Health approach is urgently required for regular monitoring of IAV in wild birds, domestic poultry, mammals, and people worldwide to determine genetic changes and public health risk.

Aftab Ahmed: conceptualization, methodology, investigation. Samia Saqlain: investigation, formal analysis. Arslan Rasool: conceptualization, methodology, investigation. Shaban Muhammad: investigation, validation, visualization. Sajid Umar: conceptualization, methodology, investigation, validation, formal analysis, supervision, funding acquisition, project administration, writing–original draft, writing–review and editing.

The authors declare no conflicts of interest.

Abstract Image

巴基斯坦奶牛和农场工人中未检测到禽流感病毒 (H5N1)
甲型流感病毒(IAV)是一种人畜共患病原体,对禽类和公共卫生构成重大挑战[1]。鸭和鹅等迁徙水禽是 IAV 的主要传播源,并将 IAV 传播给其他家禽和动物。IAV 的跨物种传播一般比较罕见,但也有可能发生,特别是当病毒的遗传密码发生突变,使其能够附着在新宿主的受体上,或者当两种不同的流感病毒同时感染同一宿主并交换遗传物质时,就会发生重配事件。俗称 "禽流感 "的甲型 H5N1 禽流感病毒主要影响鸟类,但有时也会感染人类。多年来,人类零星感染甲型 H5N1 病毒的报告数量相对较少。从 2022 年 1 月到 2024 年 4 月,8 个国家共发现 26 例人类零星感染病例[2],这表明病毒仍在从鸟类向人类传播。这些病例凸显了这种病毒可能导致人类重病和死亡。在极少数情况下,甲型 H5N1 病毒会传播到一些非鸟类物种,尤其是在与受感染鸟类有密切接触的情况下[3-5]。牛与其他哺乳动物一样,通常对特定类型的 IAV 和 D 型流感病毒易感。通常情况下,甲型 H5N1 流感病毒在牛中并不常见。令人惊讶的是,最近美国多个州报告了多起奶牛感染甲型 H5N1 病毒的零星病例 [5,6]。对于养牛业来说,新出现的牛 H5N1 病毒是一种新病毒。与其他哺乳动物不同,H5N1 生长在奶牛的乳房中,似乎不会引起牛的呼吸道疾病。牛 H5N1 病毒对一种称为聚合酶的酶进行了特殊调整,从而能够更好地在奶牛乳房内复制。一名患有结膜炎的奶牛场工人也被确诊为阳性,这凸显了牛 H5N1 病毒对人类健康的新担忧[5, 6]。该奶牛场工人可能在挤奶过程中或通过手眼接触感染了这种病毒。牛 H5N1 病毒聚合酶或其他基因组片段的进一步基因变化可能使病毒更快适应,甚至可能支持牛对牛或牛对人的传播 [5,6]。这些报告突出表明,H5N1 有可能进化并严重威胁人类健康。目前还没有来自巴基斯坦的牛 H5N1 病毒数据。因此,本研究旨在监测 IAV 的流行情况以及新型 H5N1 病毒在巴基斯坦旁遮普省的牛和农场工人中的潜在传播。人体研究已获得中国昆山杜克大学机构审查委员会(IRB)的批准(2024SU040)。作为 D 型流感病毒监测研究的一部分,我们于 2023 年 1 月至 2024 年 3 月期间在旁遮普省内不同城市(拉合尔、卡苏尔、萨希瓦尔、木尔坦、莱亚和巴哈瓦尔布尔)的六个商业奶牛场分别采集了农场工人和奶牛的鼻腔清洗液样本(n = 117)和鼻拭子样本(n = 376)(图 1)。按照之前的描述[7],我们每月两次前往这些农场采集鼻腔清洗液和鼻拭子样本。此外,我们还在 2024 年 4 月用无菌试管从不同牧场采集了挤奶过程中的牛奶样本(n = 243),以检测牛 H5N1 病毒。我们按照生产商的建议[1],使用商用 RNA 提取试剂盒(Cat#9766,Takara,中国大连)从样本中提取病毒 RNA。为了验证试剂和提取过程的完整性,我们加入了阳性和阴性对照。使用HiScript II一步式RT-PCR试剂盒(Vazyme公司,中国南京)通过一步式RT-PCR对提取的样本进行H5N1病毒筛查。使用针对血凝素(HA)基因的特异引物[8]。在每次 PCR 运行中加入阳性和阴性对照以确认结果。所有阳性对照均成功扩增,而阴性对照在 PCR 反应中未扩增。假设我们的样本代表了研究期间旁遮普省的牛和农场工人,那么 H5N1 似乎并未在旁遮普省的牛群中流行。这些发现也可能与这些农场严格的生物安全措施有关。将更多的养牛场纳入巴基斯坦其他省份的采样计划可能会扭转我们对 H5N1 流行情况的观察。尽管存在样本量小、地域范围小和研究持续时间长等局限性,但我们的研究仍然很有价值,因为它以新颖的方法提供了初步数据,可促进更大规模和更全面的研究。然而,畜牧场工人和兽医需要提高警惕,因为他们受感染的风险较高。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
求助全文
约1分钟内获得全文 求助全文
来源期刊
CiteScore
7.20
自引率
4.50%
发文量
120
审稿时长
6-12 weeks
期刊介绍: Influenza and Other Respiratory Viruses is the official journal of the International Society of Influenza and Other Respiratory Virus Diseases - an independent scientific professional society - dedicated to promoting the prevention, detection, treatment, and control of influenza and other respiratory virus diseases. Influenza and Other Respiratory Viruses is an Open Access journal. Copyright on any research article published by Influenza and Other Respiratory Viruses is retained by the author(s). Authors grant Wiley a license to publish the article and identify itself as the original publisher. Authors also grant any third party the right to use the article freely as long as its integrity is maintained and its original authors, citation details and publisher are identified.
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
确定
请完成安全验证×
copy
已复制链接
快去分享给好友吧!
我知道了
右上角分享
点击右上角分享
0
联系我们:info@booksci.cn Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。 Copyright © 2023 布克学术 All rights reserved.
京ICP备2023020795号-1
ghs 京公网安备 11010802042870号
Book学术文献互助
Book学术文献互助群
群 号:481959085
Book学术官方微信