Pasteurization effectively lowers H5N1 virus concentration in milk; however, further testing should still take place to ensure safety.

While pasteurization of milk supplies can significantly decrease H5N1 virus infection rates, the results of this research show that certain virus strains remain resilient requiring further examination in commercial milk supply chains to ensure safety and reliability of commercial supplies.

Research Letter on Influenza A(H5N1) Virus Resilience in Milk after Thermal Inactivation. Image Credits: Lik Studio/Shutterstock.

Researchers published in Emerging Infectious Diseases conducted a recent study that assessed how well H5N1 virus thrives under temperatures associated with pasteurization-like processes as well as thermal inactivation measures to neutralize it.

Background The recent detection of H5N1 virus infection among United States dairy cows has raised major public health concerns. Pasteurization, an effective strategy used for milk safety, involves heating milk to destroy harmful bacteria that might exist and protect consumers. U.S. pasteurization processes typically employ low-temperature long-time (LTLT) and high-temperature short-time (HTST) processes for pasteurizing milk products. Recent studies have produced contradicting results concerning the efficacy of pasteurization-like conditions, suggesting unpasteurized milk from H5N1-infected cows may contain sufficient virus to infect susceptible animals and be passed along via unpasteurization to humans, creating potential threats to health that require additional research in order to fully comprehend its resilience and develop appropriate safety measures.

About this research Researchers studied how different temperatures affected influenza virus viability in milk. Noteworthy is the fact that test tube conditions used in this experiment do not accurately mimic actual pasteurization processes in dairy farms; thus presenting one key limitation of this research. Four influenza virus strains were used for this study: one laboratory-adapted strain (PR8) and three H5N1 strains. Commercially available pasteurized whole milk (3.255% fat content) was spiked with virus strains at concentrations equal to 108 50% tissue culture infectious dose/mL of milk or Optimized Minimum Essential Medium (Opti-MEM). Samples then underwent three distinct heating treatments at temperatures 63degC for 30 minutes, 72degC for 20 seconds, and 91degC for 20 seconds respectively.

At various volumes (200, 20 and 2 milliliters), spiked samples (200mL, 20 milliliters and 2milliliters) were examined to understand their influence on heat treatments’ success. Furthermore, pasteurized and unpasteurized milk samples with PR8 strain infections were preheated at 37degC for 1 minute prior to exposure to HTST conditions; posttreatment, the samples were adjusted back down to 200 milliliters before being adjusted back to an initial volume of 200milliliters before being adjusted back down for viability testing purposes.

Study Results
Researchers found no discernable difference in viral titers between influenza viruses diluted in milk vs control media for PR8, VN/04 DH5N1 and Ty/IN/22 viruses tested; all three behaved similarly. However, it should be remembered that these findings come from controlled laboratory settings which may not adequately depict dairy processing realities. Heating to 63degC for 30 minutes effectively reduced viral viability below detection limits. At 72degC for 20 seconds, titer reduction was directly related to sample volume; non-significant decline was found for 200mL samples but significant reduction was noted with 20 and 2mL volumes. At 91degC for 20 seconds, smaller sample volumes showed greater viral inactivation. Preheating to 37degC for one minute prior to applying HTST conditions also hastens viral inactivation – especially with regard to smaller volumes.

This study investigated how various milk types and storage temperatures affected virus stability. Pasteurized, unpasteurized colostrum, and mature milk samples were spiked with viruses for up to four days and stored at different temperatures; unpasteurized unpasteurized milk at temperatures below 63degC hosted infections for more than four days; viral inactivation occurred within 24 hours at these lower temperatures; unpasteurized colostrum milk displayed enhanced viral inactivation possibly due to immunoglobulins as natural defense mechanisms against pathogens than its unpasteurized counterpart.

An important limitation lies in that injecting viruses into milk does not fully replicate natural infection scenarios. Enveloped viruses like influenza tend to be less stable than non-enveloped ones which have been more extensively investigated; as a result, more research must be undertaken in regards to viral inactivation under real world circumstances.

Though these findings appear alarming, it should be kept in mind that their presence could simply reflect detection methods’ high sensitivity instead of any actual risk to milk samples without viable viruses present. Previous studies investigating temperature conditions that approximate those found at an HTST laboratory have had mixed outcomes. One found complete viral inactivation only occurred at 105degC while another demonstrated total inactivation under these same HTST-like temperatures. These inconsistent findings highlight the complexity of viral resilience under variable environmental temperatures. While the current study provides valuable insight, its interpretation must take account of prior findings to fully appreciate its value; further investigation in this field remains necessary for its improvement.

Studies suggest that high temperature/high pressure pasteurization can effectively inactivate influenza viruses, thus decreasing their risks in milk supplies. Due to experimental limitations discussed earlier, caution must still be exercised; definitively ruling out live virus may necessitate multiple blind passages into eggs for testing, as is typically required during surveillance studies. The findings from this research, particularly with respect to PR8 strain, could serve as an invaluable reference when assessing commercial pasteurization processes. Yet real world testing must still occur to verify their reliability under industry conditions, with understanding factors impacting virus survival being key in improving milk safety as well as combatting emerging viruses threats.

Conclusions
In summary, this research shows that pasteurizing at standard temperatures – including 63degC for 30 minutes and 72degC for 20 seconds – can significantly lower viability of H5N1 virus in milk at smaller volumes, particularly with preheating. While virus inactivation was enhanced when preheated was applied. Viral inactivation was more pronounced among smaller sample volumes with preheating also contributing. Viral resilience highlights the importance of rigorous heat treatment processes within commercial dairy practices. Article RevisionsOct 7 2024 – Journal paper text and link were corrected due to errors introduced upon publication initially omitting them due to typographical mistakes during first draft editing stage of article content creation phase of revision 1. Article RevisionsOct 7 2024 – Text and link were revised due to errors introduced upon publication initially omitting them by mistake when initially posted due to typing error during content creation phase 1. Article RevisionsOct 7 2024 added due to original error when first submitted without referenced link or text provided within text/link in error before publication was revised due. Article revisionsocto 7 2024 Revision 1st October 7 2024 as first included incorrectly before publication of original post revision 1. Article RevisionsOcto 5 2024 Revision 3 2024 added The Journal paper reference text/link not present initially. Article revision revised, after it initially being left off earlier than anticipated due. Article. Article revision 7 2024 revision 2 17/2024 Revision 3 2024 added as initial version not published due. Article revised later amended according. Article revision 2. Article revision was incorrectly excluded when initially submitted due ommitted due ot added 7 October 7 2024 revised since first posted which added as initially missing by mistake due. – Revision 3Octo 7 2024 October o4 2024 This error corrected before 7-2024 corrected initially included for revision: Article add. 7.7 2024 Incorrect for initial. Article Correction/22. Article revision made changes made for changes initially O.