#18,152
We are now more than 3 months since the initial detection of HPAI H5N1 (genotype B3.13) in American cattle, and yet there remain substantial (and frustrating) gaps in our understanding of this spillover event.
As a result - despite anecdotal reports of sick farm workers - fewer than 60 have actually been tested. Limited serology studies have only just begun (see CIDRAP Scientists expand H5N1 testing in dairy products, launch human serology study), and no one really knows how many cattle herds have been affected.The CDC and USDA have (diplomatically) indicated that they are often limited by a lack of cooperation on the part of farm owners, farm workers, and some state and local governments.
Yesterday, Helen Branswell et al at STAT News published a special report that sums up the lackluster response to H5N1 in American cattle: Three months into bird flu outbreak in U.S. dairy cows, experts see deep-rooted problems in response.
This week, in an HHS teleconference, USDA officials indicated that `unpublished' data suggests that H5N1 is primarily spread via mammary glands (see HogVet51's post) and not the respiratory route - which would be good news - assuming that data is correct.
But limited testing, the glacial release of information - and what appears to be a tentative response to the outbreak by many state, local, and federal agencies - does little to inspire confidence.
While we wait for better data from the U.S., we've a study - conducted by researchers at Wageningen Bioveterinary Research, Lelystad, Netherlands - that suggests that bovine respiratory epithelial cells (AECs) are susceptible to H5N1 infection.
Three points, going in.
- These researchers used 3 European genotypes of H5N1 clade 2.3.4.4b, not the U.S. B3.13 genotype which is currently infecting cows, yet they achieved positive results.
- The cytopathogenic effect on bovine AECs was limited and infectious virus titres dropped rapidly, meaning respiratory infection might be sub-clinical and more difficult to detect.
- This study involved a direct inoculation of bovine epithelial cells in a lab, and not a live-animal transmission study.
While there are limitations to this study, it does at least raise the possibility that respiratory transmission in cattle may be a genuine concern.
Due to its length, I've only posted some excerpts. Follow the link to read the study in its entirety. I'll have a brief postscript after the break.
Research Article Open Access
Luca Bordes1, Nora M. Gerhards1, Stan Peters1, Sophie van Oort1, Marit Roose1, Romy Dresken1, Sandra Venema1, Manouk Vrieling1, Marc Engelsma1, Wim H.M. van der Poel1 and Rik L. de Swart1
Published: 26 June 2024 https://doi.org/10.1099/jgv.0.002007
ABSTRACT
Highly pathogenic avian influenza (HPAI) H5N1 viruses are responsible for disease outbreaks in wild birds and poultry, resulting in devastating losses to the poultry sector. Since 2020, an increasing number of outbreaks of HPAI H5N1 was seen in wild birds. Infections in mammals have become more common, in most cases in carnivores after direct contact with infected birds.
Although ruminants were previously not considered a host species for HPAI viruses, in March 2024 multiple outbreaks of HPAI H5N1 were detected in goats and cattle in the United States. Here, we have used primary bronchus-derived well-differentiated bovine airway epithelial cells (WD-AECs) cultured at air-liquid interface to assess the susceptibility and permissiveness of bovine epithelial cells to infection with European H5N1 virus isolates.
We inoculated bovine WD-AECs with three low-passage HPAI clade 2.3.4.4b H5N1 virus isolates and detected rapid increases in viral genome loads and infectious virus during the first 24 h post-inoculation, without substantial cytopathogenic effects. Three days post-inoculation infected cells were still detectable by immunofluorescent staining.
These data indicate that multiple lineages of HPAI H5N1 may have the propensity to infect the respiratory tract of cattle and support extension of avian influenza surveillance efforts to ruminants. Furthermore, this study underscores the benefit of WD-AEC cultures for pandemic preparedness by providing a rapid and animal-free assessment of the host range of an emerging pathogen.
(SNIP)
DISCUSSION
Here we investigated the susceptibility and permissiveness of bovine WD-AECs to infection with three different HPAI H5N1 viruses isolated in the Netherlands from poultry or a red fox, with either the avian PB2-627E or the mammalian PB2-627K adaptation [17]. Until the recent outbreaks of HPAI H5N1 in dairy cattle in the USA, avian influenza was not considered to spread to ruminants [15]. Reports on the clinical signs in cattle indicate rather mild disease in most of the animals [10]. High viral loads were detected in milk, supporting the possibility of cow-to-cow transmission via milk or milking machines [10, 11, 13].
The present study demonstrates that infection of bovine epithelial cells is not unique to the North American HPAI H5N1 lineage, as European HPAI H5N1 viruses readily replicated in bovine WD-AECs and generated infectious virus.
The cytopathogenic effect of the three HPAI H5N1 viruses on bovine AECs was limited and infectious virus titres dropped rapidly, suggesting that viral propagation was not very efficient. Virus replication was inversely correlated with the number of influenza virus-infected cells, which may indicate death of infected cells after 72 hpi for the H5N1-2021 PB2-627E and H5N1-2021 PB2-627K virus isolates but not the H5N1-2020 virus isolate. Nevertheless, within the first 24 h after inoculation a substantial increase in viral RNA (depicted as EID50 equivalents) and infectious titres was observed. The possibility of an abortive infection was disputed for the H5N1-2021 PB2-627E and H5N1-2021 PB2-627K viruses by the fact that the sum of the total number of collected infectious virus particles exceeded the inoculum dosage. For the H5N1-2020 virus the sum of the total number of collected virus particles was similar to the inoculum dose, which may indicate an abortive infection. However, more likely most of the inoculum was washed away during the five subsequent washes at 4 hpi and the H5N1-2020 virus replicated to a limited extent in WD-AEC. In addition, infected cells were still detectable by immunostaining 72 hpi. This is in contrast with an earlier study in which WD-AECs obtained from monkeys, cats, ferrets, dogs, rabbits or pigs were readily infected with influenza virus pH1N1, but infected cells were not detected in bovine or caprine WD-AECs [27].
Interestingly, the two H5N1 viruses isolated from a red fox in 2021 replicated more efficiently in bovine WD-AECs than the poultry isolate from 2020. We can only speculate if this may be related to the fact that these viruses were derived from a mammal, or to the evolutionary trajectory of European HPAI H5N1 strains. However, the mammalian isolates had a similar genetic composition as H5N1 viruses isolated during the 2021 epizootic from poultry and wild birds [17], supporting the likelihood of the latter. The increased genome production of the fox virus isolate with the PB2-627K mutation is not unexpected, as this mutation is widely associated with increased polymerase function in mammalian cells [28, 29].
Virus shedding in the respiratory tract of infected cattle was limited during sampling in the field [10], which may suggest the respiratory tract is not the primary replication site of HPAI in cattle. We hypothesize that this may be due to transient viral shedding in the respiratory tract preceding clinical signs in ruminants. Similar observations have been described for other mammals infected with HPAI H5N1, where disease is not observed in acutely infected subjects and overt clinical signs coincide with viral infection of the central nervous system [17, 30].
Further investigation will be required to assess potential differences in the capacity of North American or European lineage H5N1 isolates to infect ruminants and if the respiratory tract can act as potential entry site for these viruses. However, the data shown in the current study demonstrate that European H5N1 viruses are able to replicate in bovine airway epithelial cells, creating an opportunity for further viral adaptation to ruminants.
At present, surveillance of avian influenza is largely focused on wild birds, poultry and wild carnivorous mammals. However, the H5N1 outbreaks in ruminants in the USA and the results of the current study support extension of serological surveillance to include ruminants. Moreover, it urges to test ruminants showing disease signs as reported in the cases in the USA for highly pathogenic avian influenza infection. Detection of high viral loads in the milk of infected cows poses a potential risk for farmers or consumers of unpasteurized milk. Further investigation into the tropism, shedding and clinical symptoms of the recent HPAI H5N1 virus is required to provide an informed risk assessment for ruminants and the possible implications this may have for the zoonotic potential of HPAI H5N1 viruses.
This study has several limitations. The results cannot predict if the European HPAI H5N1 viruses may have spread to ruminants, or if transmission between ruminants is possible. However, this study shows first indications of amplification of infectious virus particles in bovine WD-AECs, with limited tissue damage, suggesting that subclinical infections may have occurred in cattle during the 2020–2024 European H5N1 epizootics.
Further comparison of North American and European HPAI H5N1 virus isolates in cells and tissues of ruminants and more extensive biological replicates are required to assess the translational value of our results. Screening cattle populations, that may have been exposed to HPAI infected birds, for avian influenza virus antibodies is indicated to confirm if these kind of infections have also occurred in Europe.
Our study concludes that European HPAI H5N1 isolates can replicate in bovine WD-AECs, refuting the general assumption that ruminants are not susceptible to infection with influenza A viruses. Our study underscores the benefit of WD-AEC cultures for the pandemic preparedness toolkit, providing a rapid assessment of the host range of an emerging pathogen.
Currently, lactating dairy cows are the primary focus for HPAI H5N1 testing in the United States, and even then, testing (except prior to interstate transport) is largely voluntary.
While there is an assumption that non-dairy cows are immune - based primarily on a lack of clinical symptoms - our failure to test beef cattle remains a serious blind-spot.
Today's study suggests that H5N1 respiratory infection in cattle might well be sub-clinical, in which case we could be missing silent transmission of the virus.
But even if bovine respiratory transmission should prove insignificant today, that could always change as the virus evolves and better adapts to mammals.
But the only way we'll know if that risk is genuine, or if changes are occurring, is by extensive and continued testing. And so far, that doesn't seem to be a priority.
