The impact of influenza A H5N1 virus infection in dairy cows

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Frye, Matthew MacLachlan, Ana Rita Rebelo, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6101018/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 15 Jul, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract We investigated the impact of influenza A-H5N1 virus infection in a dairy herd. Clinical disease, which lasted for about three weeks, was recorded in 20.0% (777/3,876) of the adult cows. Milk losses of ~900 kg per cow were recorded in affected cows during a 60 day-post-outbreak period. Seroprevalence was 89.4% (570/637) in the herd, with 76.1% (485/637) of seropositive animals being subclinically infected. Clinically affected cows presented an increased risk of death (6 times) and of premature herd removal (3.6 times), when compared to non-clinical cows. Economic losses due to decreased milk production, mortality and early herd removal were estimated at $950 per clinically affected cow for a total cost of approximately $737,500 for the herd during the observation period. Our results demonstrate a long-lasting production impact and significant financial consequences of HPAI H5N1 virus infection to dairy farms. Biological sciences/Microbiology/Virology/Influenza virus Biological sciences/Microbiology/Clinical microbiology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Highly pathogenic avian influenza virus (HPAI) H5Nx virus has been circulating in wild bird populations worldwide since 1996. These viruses cause severe disease and high mortality in domestic poultry 1 . In 2014, HPAI H5N2 virus was introduced through migratory wild birds in the United States of America (USA), resulting in a large outbreak that lasted approximately two years (2014–2015) and caused the deaths or culling of over 41 million birds, leading to over $ 1.1 billion in economic losses due to direct mortality, market distortions for poultry products, and international trade restrictions 2 . In February of 2022, the HPAI H5N1 clade 2.3.4.4b virus was introduced in domestic poultry in the USA, causing an unprecedented outbreak that has now lasted more than three years. As of February 15, 2025 the outbreak had led to the deaths or culling of over 159 million birds 3 , which makes the current HPAI H5N1 outbreak the most economically costly animal disease outbreak in the history of the country 4 . The current circulating HPAI H5N1 clade 2.3.4.4b virus has also spilled over and caused fatalities in more than 28 species of mammals. In March 2024, HPAI H5N1 virus clade 2.3.4.4b genotype B3.13 was detected in lactating dairy cattle in multiple farms in Texas (TX) and subsequently spread to several other states 5 , 6 . As of February 15, 2025 there have been 973 confirmed herds in 17 US states in dairy cattle, including two additional documented spillover events of HPAI H5N1 virus clade 2.3.4.4b genotype D1.1 7 . Clinically, HPAI H5N1 virus infection in lactating dairy cows presents with a decrease in feed intake and rumination time, and a pronounced decrease in milk production, with milk appearing abnormal and resembling colostrum or mastitic milk. Affected animals may develop fever and mild respiratory signs including clear nasal discharge. Increased mortality has been reported by some affected farms 5 . The most important pathophysiological impact of HPAI H5N1 infection in dairy cows is associated with the virus tropism and replication in milk secreting epithelial cells in the mammary gland, which results in severe mastitis and degeneration and necrosis of infected cells 5 , 6 , 8 . Importantly, the effect of HPAI H5N1 infection in milk production appears to extend beyond the clinical phase of the disease. Here we studied the impact of an HPAI H5N1 outbreak in a dairy herd with approximately 3,876 adult cows. We investigated risk factors associated with clinical disease and the consequences of infection on production parameters of the herd. Additionally, based on observed milk losses, mortality, and premature herd removal, we estimated the economic impact of the HPAI H5N1 outbreak in the target farm. Results Clinico-epidemiological characteristics and outcomes of influenza infection in the dairy herd. To assess the impact of HPAI on dairy cows, we analyzed production and clinical data from a free-stall dairy farm (n = 3,876 cows; Extended Data Fig. 1 ) from Ohio (OH) that experienced an HPAI H5N1 virus outbreak in the spring of 2024 following transportation of 42 apparently healthy lactating cows from a farm in TX 1 . A summary of the OH farm herd demographics and production parameters in non-clinical and clinical animals is presented in Table 1 . An analysis of individual animal and herd level data collected for 91 days (March 8 to June 7, 2024), a period that encompasses pre- and post-outbreak data was performed. The data were obtained from the herd management software DairyComp 305 (Valley Ag Software, Tulare, CA) and through the AfiCollar® monitoring system (Afimilk® Ltd., Kibbutz Afikim, Israel). The first clinical influenza case in this herd was detected on March 21, 2024, and HPAI H5N1 virus diagnosis was confirmed by laboratory testing via real-time reverse transcriptase PCR (rRT-PCR) on March 29, 2024. At the beginning of the outbreak, there were 3,876 cows on the premises; 3,433 were lactating and 443 non-lactating. A total of 777 of 3,876 (20.0%) cows were diagnosed with clinical influenza by farm personnel based on production parameters (drop in milk production) and clinical signs (e.g. inappetence, apathy and decreased rumination time) recorded for each cow with the AfiCollar® monitoring system. Clinical diagnosis was followed by identification and segregation of sick animals to the hospital pen which is adjacent to pens used for housing of healthy nonlactating cows (Extended Data Fig. 1 ). Of the 777 clinical influenza cows, 776 were lactating and 1 was in the dry period, with most affected cows being at mid-to-late stages of lactation (100–200 [284/777, 36.6%] or > 200 [310/777, 39.9%] days in milk, respectively) and at the second (343/777, 44.1%) or greater (274/777, 35.3%) lactation (Table 1 ). Table 1 Descriptive characteristics of cows with and without a clinical influenza diagnosis on a Midwest dairy farm, including non-affected cows on the day of the first clinical diagnosis in the herd (March 21, 2024), and affected cows when they were diagnosed by farm personnel between March 21 and April 13, 2024 (n = 3,876). Item Cows without clinical Influenza diagnosis (n = 3,099) % (number) Cows diagnosed with clinical Influenza (n = 777) % (number) Lactation stage 0-100 days in milk 32.4% (1,004) 23.4% (182) 100–200 days in milk 29.5% (914) 36.6% (284) >200 days in milk 23.8% (739) 39.9% (310) Dry period 14.3% (442) 0.1% (1) Lactation number 1 34.6% (1,073) 20.6% (160) 2 34.6% (1,072) 44.1% (343) 3 ≥ 30.8% (954) 35.3% (274) Breed Holstein 39.4% (1,220) 40.3% (313) Jersey 21.9% (679) 21.0% (163) Crossbreed 38.7% (1,200) 35.3% (301) Milk production (Kg/ day) a 34.6 ± 10.1 36.2 ± 9.2 Rumination (Minutes/ day) a 403 ± 87 409.0 ± 80.1 Somatic cell count (cells/mL; log) b 4.3 ± 1.2 4.1 ± 1.2 a Average week 3/8/2024-3/15/2024 through AfiCollar® monitoring system (Afimilk, Israel). b Measured on last dairy herd improvement monthly test. Cows were diagnosed with clinical influenza between March 21 and April 13, 2024, with the peak disease incidence being observed on March 31, 2024, when 121 new cases were identified among 3,876 cows at risk in the herd (Fig. 1 a). The clinical phase of the disease lasted, on average, 7.9 ± 9.3 days, and cows stayed in the hospital pen for an average of 5.1 ± 9.3 days ( Extended Data Table 1 ; Fig. 1 b, and c ). Importantly, 53 of the 777 (6.8%) clinical influenza cows died or had to be euthanized within 13.6 ± 15.1 days from clinical diagnosis, while another 245 influenza affected cows (31.6%) were removed from the herd within 20.6 ± 15.4 days from clinical diagnosis ( Extended Data Tables 2 and 3; Fig. 1 d and e ). Table 2 Economic loss estimates incurred from milk production losses and replacement of animals that died or were removed prematurely from the herd. Conditional probability % above baseline Expected milk loss Expected replacement cost Total per clinical cow Total cost for the herd Died 6.7% 5.6% $ 13 $ 166 Sold 32.9% 23.9% $ 99 $ 448 Stayed 60.4% NA $ 221 NA x 776 Total 100.0% NA $ 335 $ 615 $ 950 a $ 737,510 b a Total cost per cow due to milk losses and animal replacement cost. b Total cost of the oubtreak to the target farm ($950 x 776 clinically affected cows). Risk factors associated with clinical influenza and its impact on cow mortality and herd removal. To identify potential risk factors associated with clinical influenza, we investigated the association of several parameters including days in milk (DIM), parity, breed, baseline milk production, and baseline somatic cell count (SCC) with clinical influenza cases. Importantly, we found that DIM and parity were associated with a greater risk of clinical influenza (Type III P-value 200 DIM (HR: 1.79 [1.47, 2.17]), when compared to cows between 0 and 100 DIM (referent) (Fig. 2 ) . Additionally, multiparous cows had an increased risk of exhibiting signs of clinical influenza when compared to primiparous cows (2nd parity: HR: 1.81 [1.49, 2.20]; 3rd parity or greater: HR: 1.85 [1.46, 2.33]). In contrast, breed, baseline milk production, and SCC were not associated with the risk of new clinical influenza (P > 0.18) (Fig. 2 ). We also evaluated the impact of clinical influenza on mortality and herd removal. Notably, compared to cows without clinical influenza, cows diagnosed with clinical disease presented an increased risk of death (relative risk [RR] [95% CI]: 6.0 [4.0, 9.1) and of being removed from the herd (RR [95% CI]: 3.6 [3.2, 4.2]) ( Extended Data Table 3 ). Impact of influenza on rumination and milk production. We evaluated the effect of clinical influenza on rumination and daily milk production in the affected herd. Rumination time during the pre-clinical period (March 8 to March 15, 2024) was higher (average 8 minutes per day; 409 ± 80 minutes/day) for cows that were clinically affected with H5N1 virus when compared to cows that were not clinically affected (403 ± 87 minutes/day; Table 1 ; Fig. 3 a). We observed a pronounced decrease in rumination time (average 160 minutes/day; range: -168, -151 minutes/day) in clinically affected animals which reached its lowest point on April 2, 2024, 12 days after the first clinical case was diagnosed in the herd (March 21, 2024) (Fig. 3 a). In the last 10 days of the clinical outbreak (April 3 to 13, 2024) rumination time in clinical cows increased; however, they remained slightly lower in clinically affected animals for at least another 30 days (11 minutes/day on average; range: -37, -7, on May 13, 2024) when compared to non-clinical animals (Fig. 3 a) Our analysis showed that before the diagnosis of the first influenza case (March 21, 2024), cows that were clinically affected produced between 0.2 to 0.7 kg more milk per day than the cows that were not clinically affected (Fig. 3 b). After the clinical diagnosis of the first case in the herd and throughout the clinical outbreak (March 21 to April 13, 2024), cows diagnosed with clinical influenza showed a pronounced reduction in milk production when compared to non-clinically affected cows (Fig. 3 b). This became evident at the herd level 5 days (on March 26, 2024) after the first clinical case and reached its lowest point 15 days post first clinical case (April 6, 2024), with an average reduction in milk production of 21.9 kg (95% CI: -22.7, -21.0) in affected animals (Fig. 3 b). Milk production remained lower in clinically affected cows, with a marked reduction compared to non-affected animals, which ranged between − 14.3 and − 8.3 kg/day during the entire post-clinical phase lasting for at least 77 days (until June 7, 2024) in which the herd was monitored (Fig. 3 b). These results demonstrate a long-lasting impact of HPAI H5N1 virus infection on the productivity of clinically affected dairy cows. Decrease in rumination and milk production precede clinical disease. To determine how early clinically affected animals present a decrease in rumination and milk production, we estimated adjusted daily means for rumination time (minutes/day) and milk production (Kg/day) for the clinically affected cows in the herd using mixed linear regression modeling. To validate this approach, the same modeling was applied to the 38 clinically affected cows in the herd that were confirmed to be HPAI H5N1 positive by real time polymerase chain reaction (RT-PCR). Our analyses revealed that rumination time starts decreasing around 7 days prior to clinical diagnosis, returning to pre-outbreak lengths within 14 days (Fig. 4 a). Importantly, analysis of the adjusted daily rumination time means in cows with confirmed laboratory diagnosis of HPAI H5N1 infection corroborated the results obtained in animals with clinical diagnosis only, with similar reduction in rumination time kinetics and magnitude being observed (Fig. 4 a and b ). When investigating daily milk production in cows diagnosed with clinical influenza only (Fig. 4 c), we observed that adjusted means for milk production in affected cows ranged from 35.5 to 36.3 kg/day during − 20 to -7 days of the first clinical diagnosis in the farm. Milk production started dropping considerably 5 days before diagnosis and reached its lowest point 2 days after clinical influenza diagnosis, with affected cows producing only 11.6 ± 0.4 kg/day (Fig. 4 c). Milk production then increased in the next 14 days post-diagnosis ranging between 20.8 and 24.0, but it remained significantly lower in clinically affected animals when compared to their milk yields prior to the HPAI H5N1 virus outbreak (Fig. 4 c and d ). Notably, analysis of adjusted means for daily milk production for clinically affected cows that were confirmed to be HPAI H5N1 positive by RT-PCR testing mirrored the results observed in animals clinically diagnosed (Fig. 4 c and d ). A similar pattern for adjusted daily rumination times and milk production was observed for clinically affected cows at different DIM, parities, and breeds ( Extended Data Fig. 2 ) Sero surveillance indicates a high rate of subclinical HPAI H5N1 virus infection in dairy cattle. To determine the seroprevalence of HPAI H5N1 virus infection in the herd, we assessed the presence of serum antibodies using ELISA and virus neutralization (VN) assays. For this, serum samples from 637 cows including 595 lactating and 42 dry cows that were at the farm during the clinical phase of the outbreak (March 21 to April 13, 2024) were collected on June 20, 2024 (105 days after the first clinical case was reported). Serum was collected blindly from approximately 25% of the cows on each pen. Initial testing of the samples using a multi-species influenza A nucleoprotein-based ELISA (ID Screen® Influenza A Antibody Competition Multi-species, Innovative Diagnostics, Grabels, France) revealed the presence of antibodies to influenza A in 553 of 637 animals (Fig. 5 a, Extended Data Table 4 ). Testing of these samples with an HPAI H5N1 virus neutralization (VN) assay at a 1:8 dilution revealed a slightly higher number (570 of 637; 89.4% seroprevalence) antibody positive animals. These VN results were confirmed by a titration VN assay, in which all 570 samples presented neutralizing antibody titers ranging between 8 and 2,056 ( Supplementary Table 1 ; Fig. 5 c). Importantly, HPAI H5N1 specific neutralizing antibodies were detected in lactating (553/595; 92.9%) and dry cows (17/42; 40.5%) with 67 of the sampled cows remaining seronegative. These results indicate broad exposure to HPAI H5N1 virus in animals that were in the farm at the time of the outbreak. Next, we investigated the association of clinical influenza with the serological status of the animals. Of the 637 cows tested, 85 (13.3%) were clinically positive and seropositive for HPAI H5N1 (i.e., CP and SP), and 1 (0.1%) animal was clinically positive but seronegative (i.e., CP and SN). Notably, 485 (76.1%) seropositive animals were clinically negative (i.e., CN and SP), indicating a large proportion of subclinically infected animals in the herd. The remaining 67 cows (10.5%) were clinically and serologically negative (i.e., CN and SN). The associations between clinical-serological diagnosis, DIM, lactation number, and breed, are presented in Extended Data Table 4 . Because of the pronounced impact on rumination and milk production that we observed in clinically affected animals (Fig. 3 a and b ), we also investigated the association between serological status and rumination time and daily milk production. Daily average rumination time was similar between CN and SP and CN and SN cows throughout the study period (Fig. 5 d). When evaluating pre-influenza milk production in relation to the animal’s serological status (SN vs. SP) post-outbreak, we noted that cows that seroconverted (SP) including clinically affected (CP and SP) and subclinically affected (CN and SP) cows produced on average 1.61 and 2.06 kg more milk per day when compared to non-infected cows (CN and SN), respectively (Fig. 5 e). Following the detection of influenza, a rapid decrease in milk production (average reduction of 15.50 kg/day (95% CI: [-19.37, -11.64]) was observed in clinically affected cows that seroconverted to HPAI H5N1 (CP and SP). In contrast, subclinically infected cows (CN and SP) maintained pre-outbreak milk production levels throughout the study period (Fig. 5 e). These results demonstrate a strong association between clinical disease and decrease in milk production. Economic impact of HPAI H5N1 virus in dairy cow production. To improve our understanding of the economic impact of an HPAI H5N1 virus outbreak to dairy producers, we estimated the costs incurred due to milk production losses, mortality and premature animal removal from the herd targeted in our study. We focused our analysis on a 67-day time frame including 7 days prior to and 60 days after the first clinical influenza diagnosis in the herd. We found that the milk production per cow decreased by a cumulative 901.2 kg per cow during the targeted period (average 18.8 kg per day), when compared to milk production between days − 21 and − 8 of the influenza diagnosis. Milder losses of 44.1 kg (or 6.3 kg per day) were observed in the 7 days prior to influenza diagnosis, which brings the total milk production loss per cow to 945.3 kg in the 67-day study period. Based on the average nominal (not adjusted for inflation) price ( $ 21.50) the producer received for 45.4 kg of milk (100 pounds) between March and May 2024 (outbreak period) 9 , and the total milk loss per cow (945.3 kg), we estimated that the economic loss due to decreased milk production alone per cow clinically affected that stayed in the herd was approximately $ 222. We also estimated the cost of milk losses incurred due to mortality and premature herd removal (selling) of lactating cows. As shown in Fig. 6 clinically affected cows that stayed in the herd produced more milk than affected animals that died or were sold. In contrast, those that died or were sold did not contribute to milk losses past their removal date. We found that the probability-weighted expected cost of milk losses at the time of clinical diagnosis from cows that stayed in the herd, died, or were sold were approximately $ 222, $ 14, and $ 99 per affected cow, respectively (Table 2 ). To determine the losses associated with clinical influenza, we estimated the increase in the risk of mortality and herd removal using adjusted risks, removing background mortality and sales from non-clinical cows ( Extended Data Table 3 ). We found that clinical influenza diagnosis (prevalence of 20.0%, n = 777), increased the mortality risk by 5.5% and herd removal risk by 22.9% (n = 3,662), above baseline compared to non-clinical cows ( Extended Data Table 3 ). The average cost associated with influenza deaths above background mortality rates was then estimated at $ 166 per clinically affected cow, based on the number of deaths attributed to influenza (n = 43) and the cost of a springer replacement ( $ 3,000/head, based on producer’s records). The cost of replacing the cows removed early from the herd attributable to clinical influenza diagnosis (n = 177) was partially offset by the average price ( $ 1,123) that the producer received for each cow removed and sold to slaughter, resulting in a net replacement cost of $ 1,877 per cow. We estimated the cost attributable to clinical influenza due to early removal and replacement of affected cows above background sale rates to be $ 448 per clinically affected cow. Therefore, considering costs associated with decreased milk production, mortality and replacement of dead animals and animals removed early from the herd, we estimated the total cost of the influenza outbreak to be $ 932 per clinically affected cow, amounting to a total of approximately $ 737,500 for the 776 affected lactating cows in our study during the 67-day period in which we monitored the herd for losses. Discussion Here we investigated the impact of an HPAI H5N1 virus outbreak in a dairy herd and showed that introduction of the virus in the herd resulted in milk production losses in clinically affected animals lasting up to 60 days post diagnosis. We demonstrated that the economic losses from the HPAI H5N1 outbreak during this period were striking, including decreased milk production and compounded by even higher costs associated with mortality and premature replacement of clinically affected cows. Using individual animal data obtained from the farm management system, we found that 20.0% of the animals at risk in the herd were clinically affected by HPAI H5N1 virus, with the first clinical case being observed ~ 2 weeks (13 days) after the introduction of apparently healthy lactating cows from an affected farm from TX into the herd 5 . New clinical cases were recorded daily in the herd during a 3-week period with the peak incidence occurring about 10 days after the first clinical case was diagnosed in the herd. The two major clinical indicators of H5N1 influenza A viral clinical infection were associated with decreased rumination time and milk production. Our analysis demonstrates that at the herd level, declines in rumination time and daily milk production occur within 7 days of identifying the first clinically affected animal. However, when examining adjusted individual animal means for rumination time (minutes/day) and milk production (kg/day) in relation to when each animal was diagnosed with clinical influenza by farm personnel, we observed that both parameters begin to decline approximately 5 days before clinical diagnosis. Therefore, farms utilizing monitoring systems should closely track individual cow rumination times and milk production, as decreases in these parameters can serve as early warning indicators of influenza A H5N1 virus introduction into the herd. The risk of clinical disease differed by parity and lactation stage, with a higher risk observed in multiparous cows compared to first-lactation cows. Although reports from a USDA survey performed on affected farms suggested this outcome 10 , results showed here represent the first epidemiological study using cow-level longitudinal data confirming a higher risk of infection in cows with greater number of lactations. The reasons behind these findings could be related to higher exposure and/or susceptibility; however, this requires further examination. The risk of clinical disease in dry cows was negligible (~ 0.1%), while the risk of clinical influenza diagnosis increased as lactation progressed. These results suggest an association between cumulative exposure to the milking process and the risk of clinical disease. They also support the notion that transmission of HPAI H5N1 could be occurring during the milking process 11 , as suggested by the high concentrations of the virus in milk 5 and the expression of viral receptors in mammary gland tissue 12 , 13 . However, the presence of seropositive dry cows, including one clinical case, which were not milked during the outbreak period, suggest that other transmission routes (i.e. respiratory route) may also be involved, and nonlactating cattle may present with more subtle clinical signs, or remain subclinical. The most remarkable findings of our study were the magnitude and duration of reduced milk production in clinically affected cows. Within two weeks from the first detection of a clinical H5N1 influenza A virus case in the herd milk production in clinically affected animals decreased by nearly 73% (~ 35 kg/day to 10 kg/day). These findings can be partially explained by the sharp drop in rumination time, compromising milk production 14 . Additionally, the abrupt and long-term drop in milk production could be a direct result of the virus replication in milk secreting epithelial cells in the mammary gland, which results in necrosis and destruction of these cells 5 . The observed reduction in milk production represents a pronounced decrease in milk yield, even when compared to other common bacterial clinical mastitis, in which milk losses up to 18 kg have been reported 15 – 17 . Future studies, evaluating milk production in subsequent lactations in affected cows will be critical to determine whether regeneration of the mammary gland epithelium that occurs during the dry period is sufficient to re-establish pre-infection milk yields in clinically affected H5N1 cows. Notably, cows with clinical influenza A H5N1 mastitis in our study did not reach their pre-infection milk yields during the remainder of the period studied after the onset of the disease which resulted in a cumulative loss of 901.2 kg of milk per cow during the 60 days post diagnosis. This persistent milk loss could be overlooked when only examining herd-level milk production where, after the initial introduction of H5N1 poorly performing cows are replaced and the bulk tank recovers. In our investigation, we were able to prospectively follow individual cows with daily milk production recording allowing us to estimate the individual cow milk losses with some granularity. In turn, this allowed us to more accurately estimate the economic losses due to lost milk production when a cow experiences a case of influenza. The seroprevalence in the target study was estimated to be 89.4% (570/637) in animals that were on the farm during the clinical phase of the outbreak (March 19 to April 11, 2024) suggesting a high transmission efficiency of the virus among cows. Importantly, of the 570 seropositive animals 463 (83.7%) were not clinically affected by influenza A H5N1, indicating a large proportion of subclinical infections. Although the precise mechanism of transmission of HPAI H5N1 virus in dairy cattle remains unknown, this is consistent with infections with other influenza A viruses which can quickly spread through susceptible mammalian populations including in humans, dogs and swine 18 , 19 . Another important observation from our serological study is the detection of antibodies in 17/42 (40.5%) of the cows that were in the dry period during the clinical outbreak in the farm. These findings suggest that non-lactating animals are also susceptible to H5N1 virus infection and as such should be considered as potential source of the virus. This is especially important when animals in the dry period are introduced into farms as replacement cows. Notably, when we assessed milk production in seropositive subclinical animals, we did not observe a decrease in milk production in these cows, indicating that clinically affected animals are the main group of animals contributing to decreased milk yields due to HPAI H5N1 virus infection. To gain a better understanding of the economic impact of an HPAI H5N1 virus outbreak in a dairy farm, we estimated the costs incurred at the target farm following introduction of the virus. Our analysis investigated sources of economic losses to the target farm including milk losses, replacement costs associated with animals that died due to influenza, and replacement costs associated with animals that were culled due to the influenza virus-induced mastitis and consequent decreased milk production. The overall cost per case of clinical influenza from these factors was estimated at ~ $ 950, resulting in ~ $ 737,500 loss for the farm targeted in our study during the 67-day period in which we monitored the herd for losses. The true cost is likely even higher if one were also to account for on-going reproductive adjustments, disruptions to milking time and other important labor considerations, supportive medical care for sick cows, changes in biosecurity, and other unmeasured factors. Although our study focused on a single herd it is one that is typical of a total-mixed-ration-fed, free-stall herd, and thus these results demonstrate the significance of an influenza A H5N1 virus outbreak in affected farms. It is important to note that differences in farm style, geographic region, or management practices may result in higher or lower economic losses to affected farms. Nonetheless, our findings highlight the high impact of influenza A H5N1 virus to the US dairy industry, as the virus continues to circulate and cause economic losses to dairy producers, posing an increased risk to animal and public health. Materials and Methods Characteristics of the study dairy farm. The dairy farm targeted in our study housed 3,433 lactating and 443 nonlactating cattle at the time of the HPAI influenza A H5N1 virus outbreak. The free-stall farm consists of five barns housing adult dairy cows and pregnant heifers. Each barn is separated longitudinally into two free-stall pens, with a feed alley in the middle of each pen. There are two rows of beds in each pen and the bedding substrate is recycled manure solids. Pens 1 through 8, located in barns 1 through 4, house lactating cattle ( Extended Data Fig. 1 ). Covered walkways connect the lactating barns, and cattle are moved to and from the milking parlor through gates and walkway. Pens 9 and 10, in barn 5, house nonlactating cattle, with a hospital pen for sick animals within pen 9. All pens hold approximately 500 cattle each, except for those located in barn 3. This barn is divided sagittally, with the milking parlor in the lower half and pens 5 and 6, each housing 250 cattle, in the upper half. There is a holding area between the parlor and pens 5 and 6 where cattle wait to enter the parlor. In this area, close contact and nose-to-nose interaction can occur. The 42 cows that were moved from a farm in TX, originating the outbreak in the study farm in OH 5 were placed in pen 5, adjacent to the holding area, upon arrival. The “double 40” parallel milking parlor milks 80 cows per milking cycle, with 40 cows on each side of the parlor. Employees who milk the cows work in the “pit”, a lower part of the parlor with access to each row of cattle. The cow’s udder is at arm level of the workers to make the milking process more accessible. An automatic gate that reads the individual animal identification can siphon animals into the sort pen for individualized treatments as they leave the parlor. The sort pen is adjacent to pen 5. Data collection. Data from the study farm was collected over a period of three months, from March 8, 2024, to June 7, 2024. The farm used a herd management software called Dairy Comp 305 (Valley Agricultural Software, Tulare, CA). Farm employees recorded all relevant management events daily, including animal movements across pens, clinical disease diagnoses, calving and dry dates, treatments, sales, deaths, and monthly somatic cell count test results. Dairy Comp was used to create reports exported as CSV spreadsheets, which were later used for statistical analyses. Daily milk production and rumination system was obtained from AfiCollar® monitoring system (Afimilk® Ltd., Kibbutz Afikim, Israel). Collection of samples. Milk, nasal swab, urine and fecal samples tested by real-time PCR were collected as part of the initial diagnostic investigation conducted at the farm targeted in our study. Blood samples (n = 810) were collected on June 20, 2024 and serum separated and used to estimate the seroprevalence of HAPI in the affected herd, with an attempt to sample 25% of the cows in each pen on the farm. No prior sample size calculation was performed to make this decision. Study activities were approved by the Cornell University Institutional Animal Care and Use Committee (IACUC; Protocol No. 2013-0064). Of those, 637 samples represented samples from cows that were present at the farm during the HPAI H5N1 clinical outbreak (March 21st to April 13th, 2024). Of those 595 samples were from cows that were lactating and 42 were from cows that were in the dry period during the H5N1 virus outbreak. Only those 637 serum samples were included in our serosurveillance assessment. Cells. Human kidney cells HEK293T (ATCC CRL-3216) and bovine uterine epithelial cells (Cal-1, developed in house at the Virology Laboratory at the Cornell University Animal Health Diagnostic Center, AHDC) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 1% L-glutamine and 10% Fetal Bovine Serum (FBS) and containing penicillin–streptomycin (Thermo Fisher Scientific; 10 U ml–1 and 100 µg ml–1, respectively) at 37 o C with 5% CO 2 . HEK293T and Cal-1 cells were used for recombinant virus rescue and Cal-1 cells were used on the virus neutralization assay as described below. Generation of recombinant HPAI TX2/24-miniGFP2 reporter virus. A reverse genetics system for the bovine H5N1 virus based on an isolate A/Cattle/Texas/06322424-1/2024 (TX2/24) obtained from milk from infected dairy cows 5 was established in Dr. Diel’s laboratory and used as backbone to generate a recombinant virus expressing the miniGFP2 reporter gene (rTX2/24-miniGFP2). Briefly, full length genome sequences of PB1, PB2, PA, HA, NA, NP and M gene segments of TX2/24 strain (H5N1 clade 2.3.4.4b, genotype B3.13, GISAID accession number: EPI_ISL_19155861) were synthesized commercially (Twist Bioscience) and cloned into the dual promoter influenza reverse genetics plasmid pHW2000 (kindly provided by Dr. Richard Webby at St. Jude Children’s Research Hospital) using the BsmBI (New England Biolabs) restriction sites. To generate the miniGFP2 reporter virus, the NS segment of the rTX2/24 recombinant virus was modified to encode a fusion protein (NS-miniGFP2) from a single nonoverlapping transcript. The miniGFP2 was cloned at the C-terminal of NS1. The NS1 and NEP open reading frames were separated by the porcine teschovirus 1 2A autoproteolytic cleavage site. The NS-miniGFP2 gene segment was synthesized (Twist Bioscience) and cloned into pHW2000 vector using the BsmBI sites. The pHW2000 plasmids containing seven TX2/24 gene segments (PB1, PB2, PA, HA, NA, NP and M) and the modified NS segment encoding miniGFP2 were co-transfected into a co-culture of HEK293T and Cal-1 (bovine uterine epithelial cells) using Lipofectamine 3000 reagents (ThermoFisher Scientific). Cell culture supernatant was harvested after 96 hours and used to infect newly seeded Cal-1 cells. Both cell lysate and culture supernatant were harvested after 72–96 hours to prepare the seed stock for the rTX2/24-miniGFP2 virus. The working stock of the virus was prepared after inoculating 10-day old embryonated chicken eggs via the allantoic cavity route and the infected allantoic fluid was harvested after 48 hours. Viruses from the initial rescue and from passages 1 and 2 were sequenced to confirm the integrity of the sequences and absence of unwanted mutations. The 50% tissue culture infectious dose (TCID 50 ) was determined using end-point dilutions and the Spearman and Karber's method and expressed as TCID 50 .mL – 1 . The sequenced verified stock rTX2/24-miniGFP2 virus was used in the virus neutralization assays below. Multi-species NP ELISA. A commercial multi-species NP-based ELISA kit (ID. Screen® Influenza A Antibody Competition Multi-species, Innovative Diagnostics, Grabels, France), was used to assess the presence of antibodies against the nucleocapsid protein (NP) in serum samples from 637 cows present in the farm at the time of the H5N1 virus outbreak. For this, the serum samples were diluted 1:4 and tested with the ELISA kits following the manufacturer's instructions. Results were interpreted based on the following criteria: <45% Pos (S/N % value), 45% to 50% Neg as recommended by the manufacturer. Fluorescent virus neutralization assay (FVNA). To confirm the presence of H5N1 specific antibodies on serum samples collected from the cows present at the farm during the outbreak, we tested all samples using a FVNA. All samples were screened at a 1:8 dilution and positive samples were then subjected to two-fold serial dilutions (1:8 to 1:2028) for VN antibody titrations. Briefly, each serum dilution was incubated with 200 TCID 50 of rTX2/24-miniGFP2 for 1 h at 37°C. Cal-1 cells were added to each well and plates were incubated at 37°C for 48 h. Plates were visualized using a fluorescence microscope (Hybrid microscope ECHO Revolve 3K) to determine neutralizing antibody (NA) titers, expressed as the reciprocal of the highest serum dilution capable of completely inhibiting HPAI H5N1 virus replication based on the expression of miniGFP2 by the rTX2/24-miniGFP2 virus. Statistical analysis. All statistical analyses were performed on R (version 4.3.2). Before analyses, data was wrangled and cleaned using functions implemented in the “tidyverse” package in R (version 2.0.0) 20 . Days in milk (DIM) were calculated as the number of days from the last calving date. Days from clinical diagnosis were defined as the number of days from the diagnosis of clinical influenza. Days with clinical signs were defined as the number of days from date that clinical signs receded to date of diagnosis of clinical influenza. Daily incidence was calculated as the proportion of animals diagnosed with clinical influenza divided by the total number of cows at risk on a given day (i.e., cows that have not been diagnosed with clinical influenza). Time-varying Cox proportional hazards regression was used to explore risk factors associated with the risk of new clinical influenza using the “Survival” package in R (version 3.6-4) 21 . Investigated explanatory variables included: DIM (0-100, 101–200, > 200), lactation number at given date (1, 2, 3 ≥), breed (Holstein, Jersey, Crossbreed; cows coded as Holstein or Jersey in Dairy Comp 305, including crossbreeds with a high proportion of Holstein or Jersey ancestry, were classified accordingly, and this recorded classification was used in the analysis), milk production at baseline (average daily milk production on the week from 3/8 to 3/15; Kg/day), and log-transformed somatic cell count at baseline (from the last available monthly farm’s test). Including all lactating cows present on the farm during the outbreak period (n = 3,662), mixed linear regression was used (“nlme” package in R [version 0.12] 22 ) to investigate the association between the presence of clinical influenza (Yes vs No) and daily milk production, and rumination time (Minutes/day). Models included an interaction between the presence of clinical influenza and date. Stratified analysis was used to compare the outcome within each date using the “emmeans” package (version 1.10.0) 23 . For lactating cows diagnosed with clinical influenza (n = 776), mixed linear regression was employed to investigate the association between the number of days from diagnosis (-21 to + 60), and daily milk production, and rumination time (Minutes/day). Stratified analyses were used to investigate the relationship between days from clinical diagnosis and dependent variables of interest (i.e., milk production and rumination time), within different subgroups of DIM (0-100,101–200,>200), parity (1,2,3≥), breed (Holstein, Jersey, crossbreed), and herd removal status (Cows that stayed on the farm during the follow-up period, cows that were removed, and cows that died). The clinical and serological virus neutralization data were used to classify animals as clinical negative and seronegative (CN + SN), clinical negative and seropositive (CN + SP; subclinical infection), and clinical positive and seropositive (CP + SP; clinical infection). The relationship between clinical-serological diagnosis (CN + SN vs CN + SP + CP + SP), rumination and milk production was investigated using mixed linear regression, including all lactating cows on-site during the outbreak period with serological data (n = 595). The non-independence of observations within each cow were accounted for by the inclusion of cow-id as random effect, and the use of an autoregressive correlation structure. The relationship between clinical influenza diagnosis, herd removal, and death risk was investigated using logistic regression, including all the lactating cows on-site during the outbreak period (n = 3,662). Relative risks were estimated using the odds_ratio_to_risk_ratio function implemented in the “effect size” package in R (version 0.8.7) 24 . All models accounted for DIM, lactation number and breed. Estimated marginal means (i.e., adjusted means) were estimated using the “emmeans” package. All visualizations were created using functions implemented in “ggplot2” package (version 3.5.0) 25 . Economic analysis. We combine economic data from the USDA, Agricultural Marketing Service (2024) with producer information about sales, deaths, milk production, and the prices paid for replacement cows (“springers”). This analysis complements and references the epidemiological portions of the paper where appropriate. The main text includes the nominal (not adjusted for inflation) price received for hundredweight of milk ( $ 21.50) and price paid for a springer cow ( $ 3,000). We formulate the expected losses to a producer at the time of diagnosis as the expected as probability-weighted costs from death, removal, and milk losses. Eq. 1 includes the conditional of each outcome given that a cow has received a clinical diagnosis, and the expected losses in milk or from replacement. E[L] = Pr(dies | clinical) * (milk_dies + replacement_dies) + Pr(sold | clinical) * (milk_sold + replacement_sold) + Pr(survives | clinical) * milk_survives, # (1) L represents the lost profit from HPAI, where the outcome is drawn from the set of possible outcomes, O = {dies, sold, survives}, with known probabilities. The milk losses (milk) and replacement costs (replacement) are also known for each outcome. It is important to note that the replacement costs for surviving animals are zero. Declarations Data availability All data related to the manuscript is presented in the main article body or is available online as raw data files. Source data are provided with this paper. Code availability Code for production data analyses can be found at: https://fepenamosca.github.io/hpai_impact_dairies/ Acknowledgements We thank the producer and veterinarian Dr. William Leone for sharing the data from the dairy herd targeted in our study. We would also like to thank Tyler J. Poole for his help with drawing the farm schematics. The authors also thank Dr. Dennis Summersand his staff and veterinarians and Lauren E. Meyer for their help with sample collection. The study was funded in part by the Cornell University Animal Health Diagnostic Center Virology Laboratory activity funds. Author contributions Conceptualization: F.P.-M., E.F., M.M., D.V.N., and D.G.D; methodology: F.P.-M., M.M., A.R.R., P.S.B de O., M.N., M.P.K, F.E., D.V.N., D.G.D.; resources: F.P.-M., E.F., M.M., M.P.K., M.Z., Z.R.L., D.V.N., and D.G.D; software: F.P.-M., M.M.; formal analysis: F.P.-M., M.M., P.S.B de O., D.V.N., D.G.D.; investigation: F.P.-M., E.F., M.M., D.V.N., and D.G.D.; data curation: F.P.-M., M.M., A.R.R., P.S.B de O.; writing – original draft: F.P.-M, E.F., M.M., D.G.D.; writing – review and editing: all authors; visualization: F.P.-M., E.F., P.S.B., de O.; supervision: D.V.N., D.G.D.; project administration: F.P.-M., E.F., M.M., D.V.N.; funding acquisition: D.V.N., D.G.D. Competing interests The authors declare no competing interests. Additional information Supplementary information The online version contains supplementary material at References Kwon, J.-H. et al. Diverse infectivity, transmissibility, and pathobiology of clade 2.3.4.4 H5Nx highly pathogenic avian influenza viruses in chickens. Emerging Microbes & Infections 12 , 2218945 (2023). Ramos, S. Impacts of the 2014-2015 Highly Pathogenic Avian Influenza Outbreak on the U.S. Poultry Sector. (2017). CDC. USDA Reported H5N1 Bird Flu Detections in Poultry. Avian Influenza (Bird Flu) https://www.cdc.gov/bird-flu/situation-summary/data-map-commercial.html (2025). Seeger, R. M., Hagerman, A. D., Johnson, K. K., Pendell, D. L. & Marsh, T. L. When poultry take a sick leave: Response costs for the 2014–2015 highly pathogenic avian influenza epidemic in the USA. Food Policy 102 , 102068 (2021). Caserta, L. C. et al. Spillover of highly pathogenic avian influenza H5N1 virus to dairy cattle. Nature 634 , 669–676 (2024). Burrough, E. R. et al. Highly Pathogenic Avian Influenza A(H5N1) Clade 2.3.4.4b Virus Infection in Domestic Dairy Cattle and Cats, United States, 2024. Emerg Infect Dis 30 , 1335–1343 (2024). CDC. Current Situation: Bird Flu in Dairy Cows. Avian Influenza (Bird Flu) https://www.cdc.gov/bird-flu/situation-summary/mammals.html (2025). Butt, S. L., Nooruzzaman, M., Covaleda, L. M. & Diel, D. G. Hot topic: Influenza A H5N1 virus exhibits a broad host range, including dairy cows. JDS Communications 5 , S13–S19 (2024). USDA NASS. USDA - National Agricultural Statistics Service Homepage. https://www.nass.usda.gov/ (2025). USDA APHIS. Highly Pathogenic Avian Influenza H5N1 Genotype B3.13 in Dairy Cattle: National Epidemiologic Brief. https://www.aphis.usda.gov/sites/default/files/hpai-dairy-national-epi-brief.pdf (2024). Baker, A. L. et al. Dairy cows inoculated with highly pathogenic avian influenza virus H5N1. Nature 637 , 913–920 (2025). Imai, M. et al. Highly pathogenic avian H5N1 influenza A virus replication in ex vivo cultures of bovine mammary gland and teat tissues. Emerg Microbes Infect 14 , 2450029 (2025). Ríos Carrasco, M., Gröne, A., van den Brand, J. M. A. & de Vries, R. P. The mammary glands of cows abundantly display receptors for circulating avian H5 viruses. J Virol 98 , e0105224 (2024). Kaufman, E. I., Asselstine, V. H., LeBlanc, S. J., Duffield, T. F. & DeVries, T. J. Association of rumination time and health status with milk yield and composition in early-lactation dairy cows. Journal of Dairy Science 101 , 462–471 (2018). Rajala-Schultz, P. J., Gröhn, Y. T., McCulloch, C. E. & Guard, C. L. Effects of clinical mastitis on milk yield in dairy cows. J Dairy Sci 82 , 1213–1220 (1999). Gröhn, Y. T. et al. Effect of Pathogen-Specific Clinical Mastitis on Milk Yield in Dairy Cows. Journal of Dairy Science 87 , 3358–3374 (2004). Sguizzato, A. L. L. et al. Understanding the dynamics of mastitis in milk yield: Decoding onset and recovery patterns in response to mastitis occurrence. JDS Communications 5 , 669–673 (2024). Voorhees, I. E. H. et al. Spread of Canine Influenza A(H3N2) Virus, United States. Emerg Infect Dis 23 , 1950–1957 (2017). Rolfes, M. A. et al. Household Transmission of Influenza A Viruses in 2021-2022. JAMA 329 , 482–489 (2023). Wickham, H. et al. Welcome to the Tidyverse. Journal of Open Source Software 4 , 1686 (2019). Therneau, T. A package for survival analysis in R. https://cran.r-project.org/web/packages/survival/vignettes/survival.pdf (2024). Pinheiro, J., Bates, D., DebRoy, S., Sarkar, D. & R Core Team. nlme: Linear and Nonlinear Mixed Effects Models. https://cran.r-project.org/web/packages/nlme/index.html (2025). Lenth, R. V. et al. emmeans: Estimated Marginal Means, aka Least-Squares Means. https://cran.r-project.org/web/packages/emmeans/index.html (2024). Grant, R. L. Converting an odds ratio to a range of plausible relative risks for better communication of research findings. BMJ 348 , f7450 (2014). Wickham, H. Ggplot2 . (Springer International Publishing, Cham, 2016). doi:10.1007/978-3-319-24277-4. Additional Declarations There is NO Competing Interest. Supplementary Files ExtendedData.docx Cite Share Download PDF Status: Published Journal Publication published 15 Jul, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6101018","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":422371804,"identity":"63411344-a6cb-4677-bf5d-86124d153760","order_by":0,"name":"Felipe Peña-Mosca","email":"","orcid":"https://orcid.org/0000-0002-7161-6203","institution":"Department of Public and Ecosystem Health, College of Veterinary Medicine, Cornell University, Ithaca, NY","correspondingAuthor":false,"prefix":"","firstName":"Felipe","middleName":"","lastName":"Peña-Mosca","suffix":""},{"id":422371805,"identity":"fab79416-030e-4269-8b62-4a8cc1094266","order_by":1,"name":"Elisha A. 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Diel","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzUlEQVRIiWNgGAWjYDCCAwwGBxgKbCCcBwVgEWK0GKRBOAkGRGphYDA4TIIWvvOLNx74YHBenl/67MEHQC1yfDcS8GuRvPGs4OAMg9uGM/vykg2AWowlCWkxuHHG4DCPwe0EgzM8ZhJALYkbiNLyx+AcSIv5D6CWesJazvcA/W5wAGwLyPsJBoT9wlZwsMcg2XBmD48x0GEShjPPPMCvhe/84c0fflTYyfPz8Bh++FBhI893nIAtDBKoCiQIKAcB/gNEKBoFo2AUjIKRDQBCn0q6/0lNQAAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-3237-8940","institution":"Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY; Animal Health Diagnostic Center, College of Veterinary Medicine, Cornell University, Ithaca, NY","correspondingAuthor":true,"prefix":"","firstName":"Diego","middleName":"G.","lastName":"Diel","suffix":""}],"badges":[],"createdAt":"2025-02-25 03:30:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6101018/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6101018/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-025-61553-z","type":"published","date":"2025-07-15T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":77581766,"identity":"85376b48-40d9-47b1-aa01-33a1c141a738","added_by":"auto","created_at":"2025-03-03 09:54:13","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":73826,"visible":true,"origin":"","legend":"\u003cp\u003eClinical incidence, mortality and removal of HPAI H5N1 affected cows. \u003cstrong\u003ea\u003c/strong\u003e. Daily incidence of clinical influenza during the study period. \u003cstrong\u003eb\u003c/strong\u003e. Number of days cows remained with clinical signs as coded by farm personnel (n=290). \u003cstrong\u003ec\u003c/strong\u003e. Number of days that affected cows remained in the hospital pen (n=176). \u003cstrong\u003ed\u003c/strong\u003e. Number of days from clinical diagnosis to death. \u003cstrong\u003ee\u003c/strong\u003e. Number of days from clinical diagnosis to herd removal.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6101018/v1/5f5393f8cd7f0301b1cc2862.png"},{"id":77581754,"identity":"eaa93551-4f03-421f-91a0-eb3c9a14923a","added_by":"auto","created_at":"2025-03-03 09:54:13","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":89643,"visible":true,"origin":"","legend":"\u003cp\u003eOutput from Cox proportional hazards regression models investigating selected risk factors for clinical influenzaduring the timeframe in which new cases were diagnosed in the herd (March 21, 2024 to April, 13 2024). HR (95%CI): Hazards ratios and their 95% confidence intervals. Milk production estimates are shown for every 10 kg change in the average milk production during baseline pre-outbreak period (March 8, 2024 to March 15, 2024). Log-SCC: Log somatic cell count at the last farm’s monthly test before the outbreak.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6101018/v1/6e26548d2de123463ff28257.png"},{"id":77581780,"identity":"8db9264c-5882-4341-9384-1a4fde333191","added_by":"auto","created_at":"2025-03-03 09:54:15","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":117529,"visible":true,"origin":"","legend":"\u003cp\u003eAdjusted means for rumination time (\u003cstrong\u003ea\u003c/strong\u003e; Minutes/day) and milk production (\u003cstrong\u003eb\u003c/strong\u003e; Kg/day) in cows with and without clinical influenza diagnosis estimated using mixed linear regression by date (n=311,701 records). Models accounted for days in milk, lactation number and breed. Models included cow-ID as a random effect and an autoregressive correlation structure. Error bars represent 95% confidence intervals. The shaded area represents the period (March 21 to April 13, 2024) with at least one cow having a new clinical diagnosis of influenza in the study herd.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6101018/v1/992232ab9a82862479fa7dc8.png"},{"id":77581761,"identity":"5ff07dcb-eeb2-4454-84ab-35466b458167","added_by":"auto","created_at":"2025-03-03 09:54:13","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":141197,"visible":true,"origin":"","legend":"\u003cp\u003eAdjusted means for rumination time (Minutes/day) and milk production (Kg/day) in cows with clinical influenza diagnosis by days from clinical diagnosis, estimated using mixed linear regression. \u003cstrong\u003ea \u003c/strong\u003eand\u003cstrong\u003e c\u003c/strong\u003erepresent lactating cows with clinical influenza diagnosis (n = 776) and \u003cstrong\u003eb \u003c/strong\u003eand\u003cstrong\u003e d\u003c/strong\u003e represent \u0026nbsp;cows with on-farm clinical diagnosis and RT-PCR laboratory diagnosis confirmation on milk (n = 38). Models accounted for days in milk, lactation number and breed. Models included cow-ID as a random effect and an autoregressive correlation structure. Error bars represent 95% confidence intervals. The dashed vertical line represents \u0026nbsp;the day of clinical influenza diagnosis. The horizontal line represents the average rumination time or milk production for non-affected cows on the farm (n = 2,886).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6101018/v1/b5cb785f59bad205a2d2923d.png"},{"id":77581778,"identity":"db2b892e-9cf4-43e2-bf70-9bda291506cc","added_by":"auto","created_at":"2025-03-03 09:54:14","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":113854,"visible":true,"origin":"","legend":"\u003cp\u003eSerological and production parameters for a subset of cows present at the farm during the H5N1 influenza A virus outbreak. \u003cstrong\u003ea.\u003c/strong\u003e Multi-species ELISA results showing the detection of influenza A nucleoprotein (NP)-specific antibodies (n=637). \u003cstrong\u003eb. \u003c/strong\u003eVirus neutralization results showing detection of influenza A H5N1 virus-specific neutralizing antibodies in serum from cows in the farm during the outbreak (n=637). \u003cstrong\u003ec. \u003c/strong\u003eNeutralizing antibody titers detected in serum from VN positive animals and expressed as reciprocal of the highest serum dilution to completely neutralize H5N1 virus (n=637). \u003cstrong\u003ed \u003c/strong\u003eand \u003cstrong\u003ee\u003c/strong\u003e, show the adjusted means for rumination time (Minutes/day) and milk production (Kg/day) by date and clinical-serological diagnosis, including data from 595 lactating cows that were on-site during the outbreak period. Models accounted for days in milk, lactation number and breed. Models included cow-ID as a random effect and an autoregressive correlation structure. Error bars represent 95% confidence intervals. CP: Cows with clinical influenza diagnosis. CN: Cows without clinical influenza diagnosis. SP: Seropositive cows. SN: Seronegative cows. The shaded area represents the period of the influenza outbreak in the study herd.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6101018/v1/9cc2d58f94decf538653a5c6.png"},{"id":77582139,"identity":"538f16df-b4d3-4608-88b9-0bf5b7beb8f4","added_by":"auto","created_at":"2025-03-03 10:02:14","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":87817,"visible":true,"origin":"","legend":"\u003cp\u003eAdjusted means for milk production (kg/day) in lactating cows with clinical influenza diagnosis by days from diagnosis and removal status (Stayed, n = 478; Died, n = 53; Sold, n= 245) estimated using mixed linear regression. Models accounted for days in milk, lactation number and breed. Models included cow-ID as a random effect and an autoregressive correlation structure. Error bars represent 95% confidence intervals.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6101018/v1/49e4a092fe42a3e32ad93c21.png"},{"id":86834852,"identity":"2bb14e8d-f551-41e6-8f63-15e97588eb8d","added_by":"auto","created_at":"2025-07-16 07:05:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1650730,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6101018/v1/d80367ad-649d-4463-af1c-5005a862ae89.pdf"},{"id":77581782,"identity":"2abe8e2c-b0fd-4d45-a3e8-1cf22531364b","added_by":"auto","created_at":"2025-03-03 09:54:15","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":493252,"visible":true,"origin":"","legend":"","description":"","filename":"ExtendedData.docx","url":"https://assets-eu.researchsquare.com/files/rs-6101018/v1/25338f578186dad7f3df9cfe.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"The impact of influenza A H5N1 virus infection in dairy cows","fulltext":[{"header":"Introduction","content":"\u003cp\u003e Highly pathogenic avian influenza virus (HPAI) H5Nx virus has been circulating in wild bird populations worldwide since 1996. These viruses cause severe disease and high mortality in domestic poultry \u003csup\u003e \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e \u003c/sup\u003e. In 2014, HPAI H5N2 virus was introduced through migratory wild birds in the United States of America (USA), resulting in a large outbreak that lasted approximately two years (2014\u0026ndash;2015) and caused the deaths or culling of over 41\u0026nbsp;million birds, leading to over \u003cspan\u003e$\u003c/span\u003e1.1\u0026nbsp;billion in economic losses due to direct mortality, market distortions for poultry products, and international trade restrictions \u003csup\u003e \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e \u003c/sup\u003e. In February of 2022, the HPAI H5N1 clade 2.3.4.4b virus was introduced in domestic poultry in the USA, causing an unprecedented outbreak that has now lasted more than three years. As of February 15, 2025 the outbreak had led to the deaths or culling of over 159\u0026nbsp;million birds \u003csup\u003e \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e \u003c/sup\u003e, which makes the current HPAI H5N1 outbreak the most economically costly animal disease outbreak in the history of the country \u003csup\u003e \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e \u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe current circulating HPAI H5N1 clade 2.3.4.4b virus has also spilled over and caused fatalities in more than 28 species of mammals. In March 2024, HPAI H5N1 virus clade 2.3.4.4b genotype B3.13 was detected in lactating dairy cattle in multiple farms in Texas (TX) and subsequently spread to several other states \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. As of February 15, 2025 there have been 973 confirmed herds in 17 US states in dairy cattle, including two additional documented spillover events of HPAI H5N1 virus clade 2.3.4.4b genotype D1.1 \u003csup\u003e7\u003c/sup\u003e. Clinically, HPAI H5N1 virus infection in lactating dairy cows presents with a decrease in feed intake and rumination time, and a pronounced decrease in milk production, with milk appearing abnormal and resembling colostrum or mastitic milk. Affected animals may develop fever and mild respiratory signs including clear nasal discharge. Increased mortality has been reported by some affected farms \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. The most important pathophysiological impact of HPAI H5N1 infection in dairy cows is associated with the virus tropism and replication in milk secreting epithelial cells in the mammary gland, which results in severe mastitis and degeneration and necrosis of infected cells \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Importantly, the effect of HPAI H5N1 infection in milk production appears to extend beyond the clinical phase of the disease.\u003c/p\u003e \u003cp\u003eHere we studied the impact of an HPAI H5N1 outbreak in a dairy herd with approximately 3,876 adult cows. We investigated risk factors associated with clinical disease and the consequences of infection on production parameters of the herd. Additionally, based on observed milk losses, mortality, and premature herd removal, we estimated the economic impact of the HPAI H5N1 outbreak in the target farm.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eClinico-epidemiological characteristics and outcomes of influenza infection in the dairy herd.\u003c/strong\u003e To assess the impact of HPAI on dairy cows, we analyzed production and clinical data from a free-stall dairy farm (n\u0026thinsp;=\u0026thinsp;3,876 cows; \u003cstrong\u003eExtended Data\u003c/strong\u003e Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e) from Ohio (OH) that experienced an HPAI H5N1 virus outbreak in the spring of 2024 following transportation of 42 apparently healthy lactating cows from a farm in TX \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. A summary of the OH farm herd demographics and production parameters in non-clinical and clinical animals is presented in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. An analysis of individual animal and herd level data collected for 91 days (March 8 to June 7, 2024), a period that encompasses pre- and post-outbreak data was performed. The data were obtained from the herd management software DairyComp 305 (Valley Ag Software, Tulare, CA) and through the AfiCollar\u0026reg; monitoring system (Afimilk\u0026reg; Ltd., Kibbutz Afikim, Israel). The first clinical influenza case in this herd was detected on March 21, 2024, and HPAI H5N1 virus diagnosis was confirmed by laboratory testing via real-time reverse transcriptase PCR (rRT-PCR) on March 29, 2024. At the beginning of the outbreak, there were 3,876 cows on the premises; 3,433 were lactating and 443 non-lactating. A total of 777 of 3,876 (20.0%) cows were diagnosed with clinical influenza by farm personnel based on production parameters (drop in milk production) and clinical signs (e.g. inappetence, apathy and decreased rumination time) recorded for each cow with the AfiCollar\u0026reg; monitoring system. Clinical diagnosis was followed by identification and segregation of sick animals to the hospital pen which is adjacent to pens used for housing of healthy nonlactating cows \u003cstrong\u003e(Extended Data\u003c/strong\u003e Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cstrong\u003e).\u003c/strong\u003e Of the 777 clinical influenza cows, 776 were lactating and 1 was in the dry period, with most affected cows being at mid-to-late stages of lactation (100\u0026ndash;200 [284/777, 36.6%] or \u0026gt;\u0026thinsp;200 [310/777, 39.9%] days in milk, respectively) and at the second (343/777, 44.1%) or greater (274/777, 35.3%) lactation (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eDescriptive characteristics of cows with and without a clinical influenza diagnosis on a Midwest dairy farm, including non-affected cows on the day of the first clinical diagnosis in the herd (March 21, 2024), and affected cows when they were diagnosed by farm personnel between March 21 and April 13, 2024 (n\u0026thinsp;=\u0026thinsp;3,876).\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"3\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eItem\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCows without clinical Influenza diagnosis (n\u0026thinsp;=\u0026thinsp;3,099)\u003c/p\u003e\n \u003cp\u003e% (number)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCows diagnosed with clinical Influenza (n\u0026thinsp;=\u0026thinsp;777)\u003c/p\u003e\n \u003cp\u003e% (number)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLactation stage\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0-100 days in milk\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e32.4% (1,004)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e23.4% (182)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u0026ndash;200 days in milk\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29.5% (914)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e36.6% (284)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;200 days in milk\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e23.8% (739)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e39.9% (310)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDry period\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14.3% (442)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.1% (1)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLactation number\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e34.6% (1,073)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e20.6% (160)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e34.6% (1,072)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e44.1% (343)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e \u0026nbsp; \u0026nbsp;3 \u0026ge;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30.8% (954)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e35.3% (274)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBreed\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHolstein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e39.4% (1,220)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e40.3% (313)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eJersey\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e21.9% (679)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e21.0% (163)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCrossbreed\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e38.7% (1,200)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e35.3% (301)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMilk production (Kg/ day)\u0026nbsp;a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e34.6\u0026thinsp;\u0026plusmn;\u0026thinsp;10.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e36.2\u0026thinsp;\u0026plusmn;\u0026thinsp;9.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRumination (Minutes/ day)\u0026nbsp;a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e403\u0026thinsp;\u0026plusmn;\u0026thinsp;87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e409.0\u0026thinsp;\u0026plusmn;\u0026thinsp;80.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSomatic cell count (cells/mL; log)\u0026nbsp;b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"3\"\u003ea Average week 3/8/2024-3/15/2024 through AfiCollar\u0026reg; monitoring system (Afimilk, Israel).\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"3\"\u003eb Measured on last dairy herd improvement monthly test.\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eCows were diagnosed with clinical influenza between March 21 and April 13, 2024, with the peak disease incidence being observed on March 31, 2024, when 121 new cases were identified among 3,876 cows at risk in the herd (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea). The clinical phase of the disease lasted, on average, 7.9\u0026thinsp;\u0026plusmn;\u0026thinsp;9.3 days, and cows stayed in the hospital pen for an average of 5.1\u0026thinsp;\u0026plusmn;\u0026thinsp;9.3 days (\u003cstrong\u003eExtended Data\u003c/strong\u003e Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e; Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eb, and \u003cstrong\u003ec\u003c/strong\u003e). Importantly, 53 of the 777 (6.8%) clinical influenza cows died or had to be euthanized within 13.6\u0026thinsp;\u0026plusmn;\u0026thinsp;15.1 days from clinical diagnosis, while another 245 influenza affected cows (31.6%) were removed from the herd within 20.6\u0026thinsp;\u0026plusmn;\u0026thinsp;15.4 days from clinical diagnosis (\u003cstrong\u003eExtended Data\u003c/strong\u003e Tables \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e and 3; Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ed and \u003cstrong\u003ee\u003c/strong\u003e).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eEconomic loss estimates incurred from milk production losses and replacement of animals that died or were removed prematurely from the herd.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"7\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eConditional probability\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e% above baseline\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eExpected milk loss\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eExpected replacement cost\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTotal per clinical cow\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTotal cost for the herd\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDied\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6.7%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.6%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cspan\u003e$\u003c/span\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cspan\u003e$\u003c/span\u003e166\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSold\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e32.9%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e23.9%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cspan\u003e$\u003c/span\u003e99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cspan\u003e$\u003c/span\u003e448\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eStayed\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e60.4%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cspan\u003e$\u003c/span\u003e221\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ex 776\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e100.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cspan\u003e$\u003c/span\u003e335\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cspan\u003e$\u003c/span\u003e615\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cspan\u003e$\u003c/span\u003e950\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cspan\u003e$\u003c/span\u003e737,510\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\"\u003e\u003csup\u003ea\u003c/sup\u003eTotal cost per cow due to milk losses and animal replacement cost.\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\"\u003e\u003csup\u003eb\u003c/sup\u003eTotal cost of the oubtreak to the target farm ($950 x 776 clinically affected cows).\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eRisk factors associated with clinical influenza and its impact on cow mortality and herd removal.\u003c/strong\u003e To identify potential risk factors associated with clinical influenza, we investigated the association of several parameters including days in milk (DIM), parity, breed, baseline milk production, and baseline somatic cell count (SCC) with clinical influenza cases. Importantly, we found that DIM and parity were associated with a greater risk of clinical influenza (Type III P-value\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). Cows between 100 and 200 DIM had a higher risk of presenting clinical influenza (hazard ratio [HR] [95% CI]: 1.39 [1.15, 1.68]), as did cows with \u0026gt;\u0026thinsp;200 DIM (HR: 1.79 [1.47, 2.17]), when compared to cows between 0 and 100 DIM (referent) (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cstrong\u003e)\u003c/strong\u003e. Additionally, multiparous cows had an increased risk of exhibiting signs of clinical influenza when compared to primiparous cows (2nd parity: HR: 1.81 [1.49, 2.20]; 3rd parity or greater: HR: 1.85 [1.46, 2.33]). In contrast, breed, baseline milk production, and SCC were not associated with the risk of new clinical influenza (P\u0026thinsp;\u0026gt;\u0026thinsp;0.18) (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eWe also evaluated the impact of clinical influenza on mortality and herd removal. Notably, compared to cows without clinical influenza, cows diagnosed with clinical disease presented an increased risk of death (relative risk [RR] [95% CI]: 6.0 [4.0, 9.1) and of being removed from the herd (RR [95% CI]: 3.6 [3.2, 4.2]) (\u003cstrong\u003eExtended Data Table\u0026nbsp;3\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImpact of influenza on rumination and milk production.\u003c/strong\u003e We evaluated the effect of clinical influenza on rumination and daily milk production in the affected herd. Rumination time during the pre-clinical period (March 8 to March 15, 2024) was higher (average 8 minutes per day; 409\u0026thinsp;\u0026plusmn;\u0026thinsp;80 minutes/day) for cows that were clinically affected with H5N1 virus when compared to cows that were not clinically affected (403\u0026thinsp;\u0026plusmn;\u0026thinsp;87 minutes/day; Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e; Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea). We observed a pronounced decrease in rumination time (average 160 minutes/day; range: -168, -151 minutes/day) in clinically affected animals which reached its lowest point on April 2, 2024, 12 days after the first clinical case was diagnosed in the herd (March 21, 2024) (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea). In the last 10 days of the clinical outbreak (April 3 to 13, 2024) rumination time in clinical cows increased; however, they remained slightly lower in clinically affected animals for at least another 30 days (11 minutes/day on average; range: -37, -7, on May 13, 2024) when compared to non-clinical animals (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea)\u003c/p\u003e\n\u003cp\u003eOur analysis showed that before the diagnosis of the first influenza case (March 21, 2024), cows that were clinically affected produced between 0.2 to 0.7 kg more milk per day than the cows that were not clinically affected (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eb). After the clinical diagnosis of the first case in the herd and throughout the clinical outbreak (March 21 to April 13, 2024), cows diagnosed with clinical influenza showed a pronounced reduction in milk production when compared to non-clinically affected cows (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eb). This became evident at the herd level 5 days (on March 26, 2024) after the first clinical case and reached its lowest point 15 days post first clinical case (April 6, 2024), with an average reduction in milk production of 21.9 kg (95% CI: -22.7, -21.0) in affected animals (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eb). Milk production remained lower in clinically affected cows, with a marked reduction compared to non-affected animals, which ranged between \u0026minus;\u0026thinsp;14.3 and \u0026minus;\u0026thinsp;8.3 kg/day during the entire post-clinical phase lasting for at least 77 days (until June 7, 2024) in which the herd was monitored (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eb). These results demonstrate a long-lasting impact of HPAI H5N1 virus infection on the productivity of clinically affected dairy cows.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDecrease in rumination and milk production precede clinical disease.\u003c/strong\u003e To determine how early clinically affected animals present a decrease in rumination and milk production, we estimated adjusted daily means for rumination time (minutes/day) and milk production (Kg/day) for the clinically affected cows in the herd using mixed linear regression modeling. To validate this approach, the same modeling was applied to the 38 clinically affected cows in the herd that were confirmed to be HPAI H5N1 positive by real time polymerase chain reaction (RT-PCR). Our analyses revealed that rumination time starts decreasing around 7 days prior to clinical diagnosis, returning to pre-outbreak lengths within 14 days (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ea). Importantly, analysis of the adjusted daily rumination time means in cows with confirmed laboratory diagnosis of HPAI H5N1 infection corroborated the results obtained in animals with clinical diagnosis only, with similar reduction in rumination time kinetics and magnitude being observed (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ea and \u003cstrong\u003eb\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eWhen investigating daily milk production in cows diagnosed with clinical influenza only (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ec), we observed that adjusted means for milk production in affected cows ranged from 35.5 to 36.3 kg/day during \u0026minus;\u0026thinsp;20 to -7 days of the first clinical diagnosis in the farm. Milk production started dropping considerably 5 days before diagnosis and reached its lowest point 2 days after clinical influenza diagnosis, with affected cows producing only 11.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4 kg/day (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ec). Milk production then increased in the next 14 days post-diagnosis ranging between 20.8 and 24.0, but it remained significantly lower in clinically affected animals when compared to their milk yields prior to the HPAI H5N1 virus outbreak (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ec \u003cstrong\u003eand d\u003c/strong\u003e). Notably, analysis of adjusted means for daily milk production for clinically affected cows that were confirmed to be HPAI H5N1 positive by RT-PCR testing mirrored the results observed in animals clinically diagnosed (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ec \u003cstrong\u003eand d\u003c/strong\u003e). A similar pattern for adjusted daily rumination times and milk production was observed for clinically affected cows at different DIM, parities, and breeds (\u003cstrong\u003eExtended Data\u003c/strong\u003e Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cstrong\u003e)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSero surveillance indicates a high rate of subclinical HPAI H5N1 virus infection in dairy cattle.\u003c/strong\u003e To determine the seroprevalence of HPAI H5N1 virus infection in the herd, we assessed the presence of serum antibodies using ELISA and virus neutralization (VN) assays. For this, serum samples from 637 cows including 595 lactating and 42 dry cows that were at the farm during the clinical phase of the outbreak (March 21 to April 13, 2024) were collected on June 20, 2024 (105 days after the first clinical case was reported). Serum was collected blindly from approximately 25% of the cows on each pen. Initial testing of the samples using a multi-species influenza A nucleoprotein-based ELISA (ID Screen\u0026reg; Influenza A Antibody Competition Multi-species, Innovative Diagnostics, Grabels, France) revealed the presence of antibodies to influenza A in 553 of 637 animals (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea, \u003cstrong\u003eExtended Data Table\u0026nbsp;4\u003c/strong\u003e). Testing of these samples with an HPAI H5N1 virus neutralization (VN) assay at a 1:8 dilution revealed a slightly higher number (570 of 637; 89.4% seroprevalence) antibody positive animals. These VN results were confirmed by a titration VN assay, in which all 570 samples presented neutralizing antibody titers ranging between 8 and 2,056 (\u003cstrong\u003eSupplementary Table\u0026nbsp;1\u003c/strong\u003e; Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ec). Importantly, HPAI H5N1 specific neutralizing antibodies were detected in lactating (553/595; 92.9%) and dry cows (17/42; 40.5%) with 67 of the sampled cows remaining seronegative. These results indicate broad exposure to HPAI H5N1 virus in animals that were in the farm at the time of the outbreak.\u003c/p\u003e\n\u003cp\u003eNext, we investigated the association of clinical influenza with the serological status of the animals. Of the 637 cows tested, 85 (13.3%) were clinically positive and seropositive for HPAI H5N1 (i.e., CP and SP), and 1 (0.1%) animal was clinically positive but seronegative (i.e., CP and SN). Notably, 485 (76.1%) seropositive animals were clinically negative (i.e., CN and SP), indicating a large proportion of subclinically infected animals in the herd. The remaining 67 cows (10.5%) were clinically and serologically negative (i.e., CN and SN). The associations between clinical-serological diagnosis, DIM, lactation number, and breed, are presented in \u003cstrong\u003eExtended Data Table\u0026nbsp;4\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eBecause of the pronounced impact on rumination and milk production that we observed in clinically affected animals (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea and \u003cstrong\u003eb\u003c/strong\u003e), we also investigated the association between serological status and rumination time and daily milk production. Daily average rumination time was similar between CN and SP and CN and SN cows throughout the study period (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ed). When evaluating pre-influenza milk production in relation to the animal\u0026rsquo;s serological status (SN \u003cem\u003evs.\u003c/em\u003e SP) post-outbreak, we noted that cows that seroconverted (SP) including clinically affected (CP and SP) and subclinically affected (CN and SP) cows produced on average 1.61 and 2.06 kg more milk per day when compared to non-infected cows (CN and SN), respectively (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ee). Following the detection of influenza, a rapid decrease in milk production (average reduction of 15.50 kg/day (95% CI: [-19.37, -11.64]) was observed in clinically affected cows that seroconverted to HPAI H5N1 (CP and SP). In contrast, subclinically infected cows (CN and SP) maintained pre-outbreak milk production levels throughout the study period (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ee). These results demonstrate a strong association between clinical disease and decrease in milk production.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEconomic impact of HPAI H5N1 virus in dairy cow production.\u003c/strong\u003e To improve our understanding of the economic impact of an HPAI H5N1 virus outbreak to dairy producers, we estimated the costs incurred due to milk production losses, mortality and premature animal removal from the herd targeted in our study. We focused our analysis on a 67-day time frame including 7 days prior to and 60 days after the first clinical influenza diagnosis in the herd. We found that the milk production per cow decreased by a cumulative 901.2 kg per cow during the targeted period (average 18.8 kg per day), when compared to milk production between days \u0026minus;\u0026thinsp;21 and \u0026minus;\u0026thinsp;8 of the influenza diagnosis. Milder losses of 44.1 kg (or 6.3 kg per day) were observed in the 7 days prior to influenza diagnosis, which brings the total milk production loss per cow to 945.3 kg in the 67-day study period. Based on the average nominal (not adjusted for inflation) price (\u003cspan\u003e$\u003c/span\u003e21.50) the producer received for 45.4 kg of milk (100 pounds) between March and May 2024 (outbreak period) \u003csup\u003e\u0026nbsp;\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026nbsp;\u003c/sup\u003e, and the total milk loss per cow (945.3 kg), we estimated that the economic loss due to decreased milk production alone per cow clinically affected that stayed in the herd was approximately \u003cspan\u003e$\u003c/span\u003e222.\u003c/p\u003e\n\u003cp\u003eWe also estimated the cost of milk losses incurred due to mortality and premature herd removal (selling) of lactating cows. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e clinically affected cows that stayed in the herd produced more milk than affected animals that died or were sold. In contrast, those that died or were sold did not contribute to milk losses past their removal date. We found that the probability-weighted expected cost of milk losses at the time of clinical diagnosis from cows that stayed in the herd, died, or were sold were approximately \u003cspan\u003e$\u003c/span\u003e222, \u003cspan\u003e$\u003c/span\u003e14, and \u003cspan\u003e$\u003c/span\u003e99 per affected cow, respectively (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eTo determine the losses associated with clinical influenza, we estimated the increase in the risk of mortality and herd removal using adjusted risks, removing background mortality and sales from non-clinical cows (\u003cstrong\u003eExtended Data Table\u0026nbsp;3\u003c/strong\u003e). We found that clinical influenza diagnosis (prevalence of 20.0%, n\u0026thinsp;=\u0026thinsp;777), increased the mortality risk by 5.5% and herd removal risk by 22.9% (n\u0026thinsp;=\u0026thinsp;3,662), above baseline compared to non-clinical cows (\u003cstrong\u003eExtended Data Table\u0026nbsp;3\u003c/strong\u003e). The average cost associated with influenza deaths above background mortality rates was then estimated at \u003cspan\u003e$\u003c/span\u003e166 per clinically affected cow, based on the number of deaths attributed to influenza (n\u0026thinsp;=\u0026thinsp;43) and the cost of a springer replacement (\u003cspan\u003e$\u003c/span\u003e3,000/head, based on producer\u0026rsquo;s records). The cost of replacing the cows removed early from the herd attributable to clinical influenza diagnosis (n\u0026thinsp;=\u0026thinsp;177) was partially offset by the average price (\u003cspan\u003e$\u003c/span\u003e1,123) that the producer received for each cow removed and sold to slaughter, resulting in a net replacement cost of \u003cspan\u003e$\u003c/span\u003e1,877 per cow. We estimated the cost attributable to clinical influenza due to early removal and replacement of affected cows above background sale rates to be \u003cspan\u003e$\u003c/span\u003e448 per clinically affected cow. Therefore, considering costs associated with decreased milk production, mortality and replacement of dead animals and animals removed early from the herd, we estimated the total cost of the influenza outbreak to be \u003cspan\u003e$\u003c/span\u003e932 per clinically affected cow, amounting to a total of approximately \u003cspan\u003e$\u003c/span\u003e737,500 for the 776 affected lactating cows in our study during the 67-day period in which we monitored the herd for losses.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eHere we investigated the impact of an HPAI H5N1 virus outbreak in a dairy herd and showed that introduction of the virus in the herd resulted in milk production losses in clinically affected animals lasting up to 60 days post diagnosis. We demonstrated that the economic losses from the HPAI H5N1 outbreak during this period were striking, including decreased milk production and compounded by even higher costs associated with mortality and premature replacement of clinically affected cows.\u003c/p\u003e \u003cp\u003eUsing individual animal data obtained from the farm management system, we found that 20.0% of the animals at risk in the herd were clinically affected by HPAI H5N1 virus, with the first clinical case being observed\u0026thinsp;~\u0026thinsp;2 weeks (13 days) after the introduction of apparently healthy lactating cows from an affected farm from TX into the herd \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. New clinical cases were recorded daily in the herd during a 3-week period with the peak incidence occurring about 10 days after the first clinical case was diagnosed in the herd. The two major clinical indicators of H5N1 influenza A viral clinical infection were associated with decreased rumination time and milk production. Our analysis demonstrates that at the herd level, declines in rumination time and daily milk production occur within 7 days of identifying the first clinically affected animal. However, when examining adjusted individual animal means for rumination time (minutes/day) and milk production (kg/day) in relation to when each animal was diagnosed with clinical influenza by farm personnel, we observed that both parameters begin to decline approximately 5 days before clinical diagnosis. Therefore, farms utilizing monitoring systems should closely track individual cow rumination times and milk production, as decreases in these parameters can serve as early warning indicators of influenza A H5N1 virus introduction into the herd.\u003c/p\u003e \u003cp\u003eThe risk of clinical disease differed by parity and lactation stage, with a higher risk observed in multiparous cows compared to first-lactation cows. Although reports from a USDA survey performed on affected farms suggested this outcome \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, results showed here represent the first epidemiological study using cow-level longitudinal data confirming a higher risk of infection in cows with greater number of lactations. The reasons behind these findings could be related to higher exposure and/or susceptibility; however, this requires further examination. The risk of clinical disease in dry cows was negligible (~\u0026thinsp;0.1%), while the risk of clinical influenza diagnosis increased as lactation progressed. These results suggest an association between cumulative exposure to the milking process and the risk of clinical disease. They also support the notion that transmission of HPAI H5N1 could be occurring during the milking process \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, as suggested by the high concentrations of the virus in milk \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e and the expression of viral receptors in mammary gland tissue \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. However, the presence of seropositive dry cows, including one clinical case, which were not milked during the outbreak period, suggest that other transmission routes (i.e. respiratory route) may also be involved, and nonlactating cattle may present with more subtle clinical signs, or remain subclinical.\u003c/p\u003e \u003cp\u003eThe most remarkable findings of our study were the magnitude and duration of reduced milk production in clinically affected cows. Within two weeks from the first detection of a clinical H5N1 influenza A virus case in the herd milk production in clinically affected animals decreased by nearly 73% (~\u0026thinsp;35 kg/day to 10 kg/day). These findings can be partially explained by the sharp drop in rumination time, compromising milk production \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Additionally, the abrupt and long-term drop in milk production could be a direct result of the virus replication in milk secreting epithelial cells in the mammary gland, which results in necrosis and destruction of these cells \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. The observed reduction in milk production represents a pronounced decrease in milk yield, even when compared to other common bacterial clinical mastitis, in which milk losses up to 18 kg have been reported \u003csup\u003e\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Future studies, evaluating milk production in subsequent lactations in affected cows will be critical to determine whether regeneration of the mammary gland epithelium that occurs during the dry period is sufficient to re-establish pre-infection milk yields in clinically affected H5N1 cows.\u003c/p\u003e \u003cp\u003eNotably, cows with clinical influenza A H5N1 mastitis in our study did not reach their pre-infection milk yields during the remainder of the period studied after the onset of the disease which resulted in a cumulative loss of 901.2 kg of milk per cow during the 60 days post diagnosis. This persistent milk loss could be overlooked when only examining herd-level milk production where, after the initial introduction of H5N1 poorly performing cows are replaced and the bulk tank recovers. In our investigation, we were able to prospectively follow individual cows with daily milk production recording allowing us to estimate the individual cow milk losses with some granularity. In turn, this allowed us to more accurately estimate the economic losses due to lost milk production when a cow experiences a case of influenza.\u003c/p\u003e \u003cp\u003eThe seroprevalence in the target study was estimated to be 89.4% (570/637) in animals that were on the farm during the clinical phase of the outbreak (March 19 to April 11, 2024) suggesting a high transmission efficiency of the virus among cows. Importantly, of the 570 seropositive animals 463 (83.7%) were not clinically affected by influenza A H5N1, indicating a large proportion of subclinical infections. Although the precise mechanism of transmission of HPAI H5N1 virus in dairy cattle remains unknown, this is consistent with infections with other influenza A viruses which can quickly spread through susceptible mammalian populations including in humans, dogs and swine \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Another important observation from our serological study is the detection of antibodies in 17/42 (40.5%) of the cows that were in the dry period during the clinical outbreak in the farm. These findings suggest that non-lactating animals are also susceptible to H5N1 virus infection and as such should be considered as potential source of the virus. This is especially important when animals in the dry period are introduced into farms as replacement cows. Notably, when we assessed milk production in seropositive subclinical animals, we did not observe a decrease in milk production in these cows, indicating that clinically affected animals are the main group of animals contributing to decreased milk yields due to HPAI H5N1 virus infection.\u003c/p\u003e \u003cp\u003eTo gain a better understanding of the economic impact of an HPAI H5N1 virus outbreak in a dairy farm, we estimated the costs incurred at the target farm following introduction of the virus. Our analysis investigated sources of economic losses to the target farm including milk losses, replacement costs associated with animals that died due to influenza, and replacement costs associated with animals that were culled due to the influenza virus-induced mastitis and consequent decreased milk production. The overall cost per case of clinical influenza from these factors was estimated at ~\u003cspan\u003e$\u003c/span\u003e950, resulting in ~\u003cspan\u003e$\u003c/span\u003e737,500 loss for the farm targeted in our study during the 67-day period in which we monitored the herd for losses. The true cost is likely even higher if one were also to account for on-going reproductive adjustments, disruptions to milking time and other important labor considerations, supportive medical care for sick cows, changes in biosecurity, and other unmeasured factors. Although our study focused on a single herd it is one that is typical of a total-mixed-ration-fed, free-stall herd, and thus these results demonstrate the significance of an influenza A H5N1 virus outbreak in affected farms. It is important to note that differences in farm style, geographic region, or management practices may result in higher or lower economic losses to affected farms. Nonetheless, our findings highlight the high impact of influenza A H5N1 virus to the US dairy industry, as the virus continues to circulate and cause economic losses to dairy producers, posing an increased risk to animal and public health.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e \u003cb\u003eCharacteristics of the study dairy farm.\u003c/b\u003e The dairy farm targeted in our study housed 3,433 lactating and 443 nonlactating cattle at the time of the HPAI influenza A H5N1 virus outbreak. The free-stall farm consists of five barns housing adult dairy cows and pregnant heifers. Each barn is separated longitudinally into two free-stall pens, with a feed alley in the middle of each pen. There are two rows of beds in each pen and the bedding substrate is recycled manure solids. Pens 1 through 8, located in barns 1 through 4, house lactating cattle (\u003cb\u003eExtended Data\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Covered walkways connect the lactating barns, and cattle are moved to and from the milking parlor through gates and walkway.\u003c/p\u003e \u003cp\u003ePens 9 and 10, in barn 5, house nonlactating cattle, with a hospital pen for sick animals within pen 9. All pens hold approximately 500 cattle each, except for those located in barn 3. This barn is divided sagittally, with the milking parlor in the lower half and pens 5 and 6, each housing 250 cattle, in the upper half. There is a holding area between the parlor and pens 5 and 6 where cattle wait to enter the parlor. In this area, close contact and nose-to-nose interaction can occur.\u003c/p\u003e \u003cp\u003eThe 42 cows that were moved from a farm in TX, originating the outbreak in the study farm in OH \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e were placed in pen 5, adjacent to the holding area, upon arrival. The \u0026ldquo;double 40\u0026rdquo; parallel milking parlor milks 80 cows per milking cycle, with 40 cows on each side of the parlor. Employees who milk the cows work in the \u0026ldquo;pit\u0026rdquo;, a lower part of the parlor with access to each row of cattle. The cow\u0026rsquo;s udder is at arm level of the workers to make the milking process more accessible. An automatic gate that reads the individual animal identification can siphon animals into the sort pen for individualized treatments as they leave the parlor. The sort pen is adjacent to pen 5.\u003c/p\u003e \u003cp\u003e \u003cb\u003eData collection.\u003c/b\u003e Data from the study farm was collected over a period of three months, from March 8, 2024, to June 7, 2024. The farm used a herd management software called Dairy Comp 305 (Valley Agricultural Software, Tulare, CA). Farm employees recorded all relevant management events daily, including animal movements across pens, clinical disease diagnoses, calving and dry dates, treatments, sales, deaths, and monthly somatic cell count test results. Dairy Comp was used to create reports exported as CSV spreadsheets, which were later used for statistical analyses. Daily milk production and rumination system was obtained from AfiCollar\u0026reg; monitoring system (Afimilk\u0026reg; Ltd., Kibbutz Afikim, Israel).\u003c/p\u003e \u003cp\u003e\u003cb\u003eCollection of samples.\u003c/b\u003e Milk, nasal swab, urine and fecal samples tested by real-time PCR were collected as part of the initial diagnostic investigation conducted at the farm targeted in our study. Blood samples (n\u0026thinsp;=\u0026thinsp;810) were collected on June 20, 2024 and serum separated and used to estimate the seroprevalence of HAPI in the affected herd, with an attempt to sample 25% of the cows in each pen on the farm. No prior sample size calculation was performed to make this decision. Study activities were approved by the Cornell University Institutional Animal Care and Use Committee (IACUC; Protocol No. 2013-0064). Of those, 637 samples represented samples from cows that were present at the farm during the HPAI H5N1 clinical outbreak (March 21st to April 13th, 2024). Of those 595 samples were from cows that were lactating and 42 were from cows that were in the dry period during the H5N1 virus outbreak. Only those 637 serum samples were included in our serosurveillance assessment.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCells.\u003c/b\u003e Human kidney cells HEK293T (ATCC CRL-3216) and bovine uterine epithelial cells (Cal-1, developed in house at the Virology Laboratory at the Cornell University Animal Health Diagnostic Center, AHDC) were cultured in Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM) supplemented with 1% L-glutamine and 10% Fetal Bovine Serum (FBS) and containing penicillin\u0026ndash;streptomycin (Thermo Fisher Scientific; 10 U ml\u0026ndash;1 and 100 \u0026micro;g ml\u0026ndash;1, respectively) at 37\u003csup\u003eo\u003c/sup\u003eC with 5% CO\u003csub\u003e2\u003c/sub\u003e. HEK293T and Cal-1 cells were used for recombinant virus rescue and Cal-1 cells were used on the virus neutralization assay as described below.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGeneration of recombinant HPAI TX2/24-miniGFP2 reporter virus.\u003c/b\u003e A reverse genetics system for the bovine H5N1 virus based on an isolate A/Cattle/Texas/06322424-1/2024 (TX2/24) obtained from milk from infected dairy cows \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e was established in Dr. Diel\u0026rsquo;s laboratory and used as backbone to generate a recombinant virus expressing the miniGFP2 reporter gene (rTX2/24-miniGFP2). Briefly, full length genome sequences of PB1, PB2, PA, HA, NA, NP and M gene segments of TX2/24 strain (H5N1 clade 2.3.4.4b, genotype B3.13, GISAID accession number: EPI_ISL_19155861) were synthesized commercially (Twist Bioscience) and cloned into the dual promoter influenza reverse genetics plasmid pHW2000 (kindly provided by Dr. Richard Webby at St. Jude Children\u0026rsquo;s Research Hospital) using the BsmBI (New England Biolabs) restriction sites. To generate the miniGFP2 reporter virus, the NS segment of the rTX2/24 recombinant virus was modified to encode a fusion protein (NS-miniGFP2) from a single nonoverlapping transcript. The miniGFP2 was cloned at the C-terminal of NS1. The NS1 and NEP open reading frames were separated by the porcine teschovirus 1 2A autoproteolytic cleavage site. The NS-miniGFP2 gene segment was synthesized (Twist Bioscience) and cloned into pHW2000 vector using the BsmBI sites. The pHW2000 plasmids containing seven TX2/24 gene segments (PB1, PB2, PA, HA, NA, NP and M) and the modified NS segment encoding miniGFP2 were co-transfected into a co-culture of HEK293T and Cal-1 (bovine uterine epithelial cells) using Lipofectamine 3000 reagents (ThermoFisher Scientific). Cell culture supernatant was harvested after 96 hours and used to infect newly seeded Cal-1 cells. Both cell lysate and culture supernatant were harvested after 72\u0026ndash;96 hours to prepare the seed stock for the rTX2/24-miniGFP2 virus. The working stock of the virus was prepared after inoculating 10-day old embryonated chicken eggs via the allantoic cavity route and the infected allantoic fluid was harvested after 48 hours. Viruses from the initial rescue and from passages 1 and 2 were sequenced to confirm the integrity of the sequences and absence of unwanted mutations. The 50% tissue culture infectious dose (TCID\u003csub\u003e50\u003c/sub\u003e) was determined using end-point dilutions and the Spearman and Karber's method and expressed as TCID\u003csub\u003e50\u003c/sub\u003e.mL\u003csup\u003e\u0026ndash;\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. The sequenced verified stock rTX2/24-miniGFP2 virus was used in the virus neutralization assays below.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMulti-species NP ELISA.\u003c/b\u003e A commercial multi-species NP-based ELISA kit (ID. Screen\u0026reg; Influenza A Antibody Competition Multi-species, Innovative Diagnostics, Grabels, France), was used to assess the presence of antibodies against the nucleocapsid protein (NP) in serum samples from 637 cows present in the farm at the time of the H5N1 virus outbreak. For this, the serum samples were diluted 1:4 and tested with the ELISA kits following the manufacturer's instructions. Results were interpreted based on the following criteria: \u0026lt;45% Pos (S/N % value), 45% to \u0026lt;\u0026thinsp;50% Susp, \u0026gt;\u0026thinsp;50% Neg as recommended by the manufacturer.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFluorescent virus neutralization assay (FVNA).\u003c/b\u003e To confirm the presence of H5N1 specific antibodies on serum samples collected from the cows present at the farm during the outbreak, we tested all samples using a FVNA. All samples were screened at a 1:8 dilution and positive samples were then subjected to two-fold serial dilutions (1:8 to 1:2028) for VN antibody titrations. Briefly, each serum dilution was incubated with 200 TCID\u003csub\u003e50\u003c/sub\u003e of rTX2/24-miniGFP2 for 1 h at 37\u0026deg;C. Cal-1 cells were added to each well and plates were incubated at 37\u0026deg;C for 48 h. Plates were visualized using a fluorescence microscope (Hybrid microscope ECHO Revolve 3K) to determine neutralizing antibody (NA) titers, expressed as the reciprocal of the highest serum dilution capable of completely inhibiting HPAI H5N1 virus replication based on the expression of miniGFP2 by the rTX2/24-miniGFP2 virus.\u003c/p\u003e \u003cp\u003e \u003cb\u003eStatistical analysis.\u003c/b\u003e All statistical analyses were performed on R (version 4.3.2). Before analyses, data was wrangled and cleaned using functions implemented in the \u0026ldquo;tidyverse\u0026rdquo; package in R (version 2.0.0) \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Days in milk (DIM) were calculated as the number of days from the last calving date. Days from clinical diagnosis were defined as the number of days from the diagnosis of clinical influenza. Days with clinical signs were defined as the number of days from date that clinical signs receded to date of diagnosis of clinical influenza. Daily incidence was calculated as the proportion of animals diagnosed with clinical influenza divided by the total number of cows at risk on a given day (i.e., cows that have not been diagnosed with clinical influenza). Time-varying Cox proportional hazards regression was used to explore risk factors associated with the risk of new clinical influenza using the \u0026ldquo;Survival\u0026rdquo; package in R (version 3.6-4) \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Investigated explanatory variables included: DIM (0-100, 101\u0026ndash;200, \u0026gt;\u0026thinsp;200), lactation number at given date (1, 2, 3 \u0026ge;), breed (Holstein, Jersey, Crossbreed; cows coded as Holstein or Jersey in Dairy Comp 305, including crossbreeds with a high proportion of Holstein or Jersey ancestry, were classified accordingly, and this recorded classification was used in the analysis), milk production at baseline (average daily milk production on the week from 3/8 to 3/15; Kg/day), and log-transformed somatic cell count at baseline (from the last available monthly farm\u0026rsquo;s test). Including all lactating cows present on the farm during the outbreak period (n\u0026thinsp;=\u0026thinsp;3,662), mixed linear regression was used (\u0026ldquo;nlme\u0026rdquo; package in R [version 0.12] \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e) to investigate the association between the presence of clinical influenza (Yes vs No) and daily milk production, and rumination time (Minutes/day). Models included an interaction between the presence of clinical influenza and date. Stratified analysis was used to compare the outcome within each date using the \u0026ldquo;emmeans\u0026rdquo; package (version 1.10.0) \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. For lactating cows diagnosed with clinical influenza (n\u0026thinsp;=\u0026thinsp;776), mixed linear regression was employed to investigate the association between the number of days from diagnosis (-21 to +\u0026thinsp;60), and daily milk production, and rumination time (Minutes/day). Stratified analyses were used to investigate the relationship between days from clinical diagnosis and dependent variables of interest (i.e., milk production and rumination time), within different subgroups of DIM (0-100,101\u0026ndash;200,\u0026gt;200), parity (1,2,3\u0026ge;), breed (Holstein, Jersey, crossbreed), and herd removal status (Cows that stayed on the farm during the follow-up period, cows that were removed, and cows that died). The clinical and serological virus neutralization data were used to classify animals as clinical negative and seronegative (CN\u0026thinsp;+\u0026thinsp;SN), clinical negative and seropositive (CN\u0026thinsp;+\u0026thinsp;SP; subclinical infection), and clinical positive and seropositive (CP\u0026thinsp;+\u0026thinsp;SP; clinical infection). The relationship between clinical-serological diagnosis (CN\u0026thinsp;+\u0026thinsp;SN vs CN\u0026thinsp;+\u0026thinsp;SP\u0026thinsp;+\u0026thinsp;CP\u0026thinsp;+\u0026thinsp;SP), rumination and milk production was investigated using mixed linear regression, including all lactating cows on-site during the outbreak period with serological data (n\u0026thinsp;=\u0026thinsp;595). The non-independence of observations within each cow were accounted for by the inclusion of cow-id as random effect, and the use of an autoregressive correlation structure. The relationship between clinical influenza diagnosis, herd removal, and death risk was investigated using logistic regression, including all the lactating cows on-site during the outbreak period (n\u0026thinsp;=\u0026thinsp;3,662). Relative risks were estimated using the odds_ratio_to_risk_ratio function implemented in the \u0026ldquo;effect size\u0026rdquo; package in R (version 0.8.7) \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. All models accounted for DIM, lactation number and breed. Estimated marginal means (i.e., adjusted means) were estimated using the \u0026ldquo;emmeans\u0026rdquo; package. All visualizations were created using functions implemented in \u0026ldquo;ggplot2\u0026rdquo; package (version 3.5.0) \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEconomic analysis.\u003c/b\u003e We combine economic data from the USDA, Agricultural Marketing Service (2024) with producer information about sales, deaths, milk production, and the prices paid for replacement cows (\u0026ldquo;springers\u0026rdquo;). This analysis complements and references the epidemiological portions of the paper where appropriate. The main text includes the nominal (not adjusted for inflation) price received for hundredweight of milk (\u003cspan\u003e$\u003c/span\u003e21.50) and price paid for a springer cow (\u003cspan\u003e$\u003c/span\u003e3,000).\u003c/p\u003e \u003cp\u003eWe formulate the expected losses to a producer at the time of diagnosis as the expected as probability-weighted costs from death, removal, and milk losses. Eq.\u0026nbsp;1 includes the conditional of each outcome given that a cow has received a clinical diagnosis, and the expected losses in milk or from replacement.\u003c/p\u003e \u003cp\u003eE[L]\u0026thinsp;=\u0026thinsp;Pr(dies | clinical) * (milk_dies\u0026thinsp;+\u0026thinsp;replacement_dies)\u0026thinsp;+\u0026thinsp;Pr(sold | clinical) * (milk_sold\u0026thinsp;+\u0026thinsp;replacement_sold)\u0026thinsp;+\u0026thinsp;Pr(survives | clinical) * milk_survives, # (1)\u003c/p\u003e \u003cp\u003eL represents the lost profit from HPAI, where the outcome is drawn from the set of possible outcomes, O = {dies, sold, survives}, with known probabilities. The milk losses (milk) and replacement costs (replacement) are also known for each outcome. It is important to note that the replacement costs for surviving animals are zero.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data related to the manuscript is presented in the main article body or is available online as raw data files. Source data are provided with this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCode availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCode for production data analyses can be found at:\u003c/p\u003e\n\u003cp\u003ehttps://fepenamosca.github.io/hpai_impact_dairies/\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the producer and veterinarian Dr. William Leone for sharing the data from the dairy herd targeted in our study. We would also like to thank Tyler J. Poole for his help with drawing the farm schematics. The authors also thank Dr. Dennis Summersand his staff and veterinarians and Lauren E. Meyer for their help with sample collection. The study was funded in part by the Cornell University Animal Health Diagnostic Center Virology Laboratory activity funds.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: F.P.-M., E.F., M.M., D.V.N., and D.G.D; methodology: F.P.-M., M.M., A.R.R., P.S.B de O., M.N., M.P.K, F.E., D.V.N., D.G.D.; resources: F.P.-M., E.F., M.M., M.P.K., M.Z., Z.R.L., D.V.N., and D.G.D; software: F.P.-M., M.M.; formal analysis: F.P.-M., M.M., P.S.B de O., D.V.N., D.G.D.; investigation: F.P.-M., E.F., M.M., D.V.N., and D.G.D.; data curation: F.P.-M., M.M., A.R.R., P.S.B de O.; writing – original draft: F.P.-M, E.F., M.M., D.G.D.; writing – review and editing: all authors; visualization: F.P.-M., E.F., P.S.B., de O.; supervision: D.V.N., D.G.D.; project administration: F.P.-M., E.F., M.M., D.V.N.; funding acquisition: D.V.N., D.G.D.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary information\u0026nbsp;\u003c/strong\u003eThe online version contains supplementary material at\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKwon, J.-H. \u003cem\u003eet al.\u003c/em\u003e Diverse infectivity, transmissibility, and pathobiology of clade 2.3.4.4 H5Nx highly pathogenic avian influenza viruses in chickens. \u003cem\u003eEmerging Microbes \u0026amp; Infections\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 2218945 (2023).\u003c/li\u003e\n\u003cli\u003eRamos, S. Impacts of the 2014-2015 Highly Pathogenic Avian Influenza Outbreak on the U.S. Poultry Sector. (2017).\u003c/li\u003e\n\u003cli\u003eCDC. USDA Reported H5N1 Bird Flu Detections in Poultry. \u003cem\u003eAvian Influenza (Bird Flu)\u003c/em\u003e https://www.cdc.gov/bird-flu/situation-summary/data-map-commercial.html (2025).\u003c/li\u003e\n\u003cli\u003eSeeger, R. M., Hagerman, A. D., Johnson, K. K., Pendell, D. L. \u0026amp; Marsh, T. L. When poultry take a sick leave: Response costs for the 2014\u0026ndash;2015 highly pathogenic avian influenza epidemic in the USA. \u003cem\u003eFood Policy\u003c/em\u003e \u003cstrong\u003e102\u003c/strong\u003e, 102068 (2021).\u003c/li\u003e\n\u003cli\u003eCaserta, L. C. \u003cem\u003eet al.\u003c/em\u003e Spillover of highly pathogenic avian influenza H5N1 virus to dairy cattle. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e634\u003c/strong\u003e, 669\u0026ndash;676 (2024).\u003c/li\u003e\n\u003cli\u003eBurrough, E. R. \u003cem\u003eet al.\u003c/em\u003e Highly Pathogenic Avian Influenza A(H5N1) Clade 2.3.4.4b Virus Infection in Domestic Dairy Cattle and Cats, United States, 2024. \u003cem\u003eEmerg Infect Dis\u003c/em\u003e \u003cstrong\u003e30\u003c/strong\u003e, 1335\u0026ndash;1343 (2024).\u003c/li\u003e\n\u003cli\u003eCDC. Current Situation: Bird Flu in Dairy Cows. \u003cem\u003eAvian Influenza (Bird Flu)\u003c/em\u003e https://www.cdc.gov/bird-flu/situation-summary/mammals.html (2025).\u003c/li\u003e\n\u003cli\u003eButt, S. L., Nooruzzaman, M., Covaleda, L. M. \u0026amp; Diel, D. G. \u003cem\u003eHot topic:\u003c/em\u003e Influenza A H5N1 virus exhibits a broad host range, including dairy cows. \u003cem\u003eJDS Communications\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, S13\u0026ndash;S19 (2024).\u003c/li\u003e\n\u003cli\u003eUSDA NASS. USDA - National Agricultural Statistics Service Homepage. https://www.nass.usda.gov/ (2025).\u003c/li\u003e\n\u003cli\u003eUSDA APHIS. Highly Pathogenic Avian Influenza H5N1 Genotype B3.13 in Dairy Cattle: National Epidemiologic Brief. https://www.aphis.usda.gov/sites/default/files/hpai-dairy-national-epi-brief.pdf (2024).\u003c/li\u003e\n\u003cli\u003eBaker, A. L. \u003cem\u003eet al.\u003c/em\u003e Dairy cows inoculated with highly pathogenic avian influenza virus H5N1. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e637\u003c/strong\u003e, 913\u0026ndash;920 (2025).\u003c/li\u003e\n\u003cli\u003eImai, M. \u003cem\u003eet al.\u003c/em\u003e Highly pathogenic avian H5N1 influenza A virus replication in ex vivo cultures of bovine mammary gland and teat tissues. \u003cem\u003eEmerg Microbes Infect\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 2450029 (2025).\u003c/li\u003e\n\u003cli\u003eR\u0026iacute;os Carrasco, M., Gr\u0026ouml;ne, A., van den Brand, J. M. A. \u0026amp; de Vries, R. P. The mammary glands of cows abundantly display receptors for circulating avian H5 viruses. \u003cem\u003eJ Virol\u003c/em\u003e \u003cstrong\u003e98\u003c/strong\u003e, e0105224 (2024).\u003c/li\u003e\n\u003cli\u003eKaufman, E. I., Asselstine, V. H., LeBlanc, S. J., Duffield, T. F. \u0026amp; DeVries, T. J. Association of rumination time and health status with milk yield and composition in early-lactation dairy cows. \u003cem\u003eJournal of Dairy Science\u003c/em\u003e \u003cstrong\u003e101\u003c/strong\u003e, 462\u0026ndash;471 (2018).\u003c/li\u003e\n\u003cli\u003eRajala-Schultz, P. J., Gr\u0026ouml;hn, Y. T., McCulloch, C. E. \u0026amp; Guard, C. L. Effects of clinical mastitis on milk yield in dairy cows. \u003cem\u003eJ Dairy Sci\u003c/em\u003e \u003cstrong\u003e82\u003c/strong\u003e, 1213\u0026ndash;1220 (1999).\u003c/li\u003e\n\u003cli\u003eGr\u0026ouml;hn, Y. T. \u003cem\u003eet al.\u003c/em\u003e Effect of Pathogen-Specific Clinical Mastitis on Milk Yield in Dairy Cows. \u003cem\u003eJournal of Dairy Science\u003c/em\u003e \u003cstrong\u003e87\u003c/strong\u003e, 3358\u0026ndash;3374 (2004).\u003c/li\u003e\n\u003cli\u003eSguizzato, A. L. L. \u003cem\u003eet al.\u003c/em\u003e Understanding the dynamics of mastitis in milk yield: Decoding onset and recovery patterns in response to mastitis occurrence. \u003cem\u003eJDS Communications\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, 669\u0026ndash;673 (2024).\u003c/li\u003e\n\u003cli\u003eVoorhees, I. E. H. \u003cem\u003eet al.\u003c/em\u003e Spread of Canine Influenza A(H3N2) Virus, United States. \u003cem\u003eEmerg Infect Dis\u003c/em\u003e \u003cstrong\u003e23\u003c/strong\u003e, 1950\u0026ndash;1957 (2017).\u003c/li\u003e\n\u003cli\u003eRolfes, M. A. \u003cem\u003eet al.\u003c/em\u003e Household Transmission of Influenza A Viruses in 2021-2022. \u003cem\u003eJAMA\u003c/em\u003e \u003cstrong\u003e329\u003c/strong\u003e, 482\u0026ndash;489 (2023).\u003c/li\u003e\n\u003cli\u003eWickham, H. \u003cem\u003eet al.\u003c/em\u003e Welcome to the Tidyverse. \u003cem\u003eJournal of Open Source Software\u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e, 1686 (2019).\u003c/li\u003e\n\u003cli\u003eTherneau, T. A package for survival analysis in R. https://cran.r-project.org/web/packages/survival/vignettes/survival.pdf (2024).\u003c/li\u003e\n\u003cli\u003ePinheiro, J., Bates, D., DebRoy, S., Sarkar, D. \u0026amp; R Core Team. nlme: Linear and Nonlinear Mixed Effects Models. https://cran.r-project.org/web/packages/nlme/index.html (2025).\u003c/li\u003e\n\u003cli\u003eLenth, R. V. \u003cem\u003eet al.\u003c/em\u003e emmeans: Estimated Marginal Means, aka Least-Squares Means. https://cran.r-project.org/web/packages/emmeans/index.html (2024).\u003c/li\u003e\n\u003cli\u003eGrant, R. L. Converting an odds ratio to a range of plausible relative risks for better communication of research findings. \u003cem\u003eBMJ\u003c/em\u003e \u003cstrong\u003e348\u003c/strong\u003e, f7450 (2014).\u003c/li\u003e\n\u003cli\u003eWickham, H. \u003cem\u003eGgplot2\u003c/em\u003e. (Springer International Publishing, Cham, 2016). doi:10.1007/978-3-319-24277-4.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6101018/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6101018/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eWe investigated the impact of influenza A-H5N1 virus infection in a dairy herd. Clinical disease, which lasted for about three weeks, was recorded in 20.0% (777/3,876) of the adult cows. Milk losses of ~900 kg per cow were recorded in affected cows during a 60 day-post-outbreak period. Seroprevalence was 89.4% (570/637) in the herd, with 76.1% (485/637) of seropositive animals being subclinically infected. Clinically affected cows presented an increased risk of death (6 times) and of premature herd removal (3.6 times), when compared to non-clinical cows. Economic losses due to decreased milk production, mortality and early herd removal were estimated at $950 per clinically affected cow for a total cost of approximately $737,500 for the herd during the observation period. Our results demonstrate a long-lasting production impact and significant financial consequences of HPAI H5N1 virus infection to dairy farms.\u003c/p\u003e","manuscriptTitle":"The impact of influenza A H5N1 virus infection in dairy cows","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-03 09:53:57","doi":"10.21203/rs.3.rs-6101018/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"154735ab-411e-4ba0-9db5-562deb68edb0","owner":[],"postedDate":"March 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":45023494,"name":"Biological sciences/Microbiology/Virology/Influenza virus"},{"id":45023495,"name":"Biological sciences/Microbiology/Clinical microbiology"}],"tags":[],"updatedAt":"2025-07-16T07:05:18+00:00","versionOfRecord":{"articleIdentity":"rs-6101018","link":"https://doi.org/10.1038/s41467-025-61553-z","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2025-07-15 04:00:00","publishedOnDateReadable":"July 15th, 2025"},"versionCreatedAt":"2025-03-03 09:53:57","video":"","vorDoi":"10.1038/s41467-025-61553-z","vorDoiUrl":"https://doi.org/10.1038/s41467-025-61553-z","workflowStages":[]},"version":"v1","identity":"rs-6101018","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6101018","identity":"rs-6101018","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}