Guidance

Influenza A (H5N1) infection in mammals: suspect case definition and diagnostic testing criteria

Updated 20 December 2023

Applies to England, Scotland and Wales

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Influenza A (H5N1) infection in mammals is a notifiable event. This means the Chief Veterinary Officer has a legal requirement to report each case to international partners. It is therefore important that laboratories conduct the right quality-assured tests on the correct samples. Samples must be collected under control of the competent authority.

This guidance provides advice to veterinarians, diagnostic laboratories, wildlife rehabilitators and research establishments on how to submit samples for testing mammals for influenza A (H5N1).

This guidance includes:

• defining a case definition
• criteria for suspected infection with influenza of avian origin in companion animals, kept and wild mammals
• details on how to send samples for confirmatory testing to the national reference laboratory
• the regulatory and professional obligations for testing and reporting any positive test results

This document should be read in conjunction with the avian influenza and influenza of avian origin: diagnostic testing, controls and reporting obligations.

You should report suspicion of influenza A viruses in mammals first so that you receive the right advice for how to take and submit samples.

This work is subject to controls under both Control of Substances Hazardous to Health Regulations 2002 (as amended) (COSHH) and the Specified Animal Pathogens Orders (SAPO).

Influenza A viruses in mammals

Influenza A viruses isolated from birds are termed avian influenza viruses. While avian influenza viruses are predominantly considered a pathogen of birds, certain strains of the virus can infect mammals as spill-over events. The virus generally infects mammals of the order Carnivora.

There is emerging evidence that the virus responsible for the current panzootic (highly pathogenic avian influenza (HPAI) H5N1 clade 2.3.4.4b) is able to infect a broader range of mammals following close contact with infected poultry, wild birds, captive birds or contaminated equipment, environment or feed.

Globally, a huge number of kept and wild birds have been infected. However only a small number of findings of naturally-acquired influenza A (H5N1) infections in mammals have been reported.

UK infections

In the UK, there have been confirmed infections with influenza A (H5N1) in several mammals. Some of these mammals are known to scavenge dead or dying birds, including:

• red foxes (Vulpes vulpes)
• Eurasian otters (Lutra lutra)
• harbour seals (Phoca vitulina)
• grey seals (Halichoerus grypus)
• common dolphins (Delphinus delphis)
• harbour porpoises (Phocoena phocoena)
• one report from captive mammals at a zoological collection in South American bush dogs (Speothos venaticus)

Reports of findings of avian influenza in non-avian wildlife from 2021 onwards in Great Britain (England, Scotland and Wales) are published in Bird flu (avian influenza): findings in non-avian wildlife.

We still consider that avian-to-avian transmission is responsible for the burden of infection in birds in recent outbreaks in the UK. Infection of mammals are the result of spill-over events.

Worldwide infections

Globally, the majority of species where influenza A (H5N1) has been isolated are birds. They are mostly Galliformes and Anseriformes, particularly the Anatidae (swans, geese and ducks) but also the Charidriiform families (waders, gulls and auks) as well as Falconiformes (raptors), Passeriformes (song birds and perching birds) and Columbiformes (pigeons and doves), amongst others.

However, worldwide there have been isolated cases in carnivorous or omnivorous mammals:

Felis catus (domestic cats), Panthera tigris (tiger), Panthera pardus orientalis (amur leopard), Lynx rufus (bobcat), Canis lupus (grey wolf), Vulpes vulpes (fox), Canis latrans (coyote), Neovison vison (mink), Viverridae (civet), Mustela furo (ferret), Pekania pennanti (fisher), Mephitis Mephitis (skunk), Didelphis virginiana (Virginia opossum), Procyon lotor (racoon), Ursus americanus (American black bear), Ursus arctos (brown bear), grizzly bear (Ursus arctos horribilis), Phoca vitulina (harbour seal), Halichoerus grypus (grey seal), Phocoenidae (porpoise), Lagenorhynchus acutus (white-sided dolphin), Tursiops (bottlenose dolphin), Otaria flavescens (South American sea lion), Enhydra lutris (sea otter), Lutra lutra (European otter) (FAO, 2023).

There is also some limited evidence of serological positivity for related H5 viruses in 3 non-carnivore species:

• Sus scrofa (domestic pig) during the HPAI H5N8 epizootic in 2020 in Europe
• Equus asinus (donkey) in previous H5N1 epizootics
• Capra hircus (domestic goat) in unpublished evidence

The mammals which have tested positive for influenza A (H5N1) to date have generally either been found dead or have shown respiratory or neurological signs such as tremors and convulsions (Frymus et al., 2021).

You can find more information on cases of infection of mammals with influenza A of avian origin globally in the World Organisation for Animal Health (WOAH) dashboard and situation reports.

There have also been a small number of confirmed accounts of mass die offs in mammals where influenza A (H5N1) was detected, including:

• mink farm outbreak in Spain, 2022
• sea lions die-off in Peru, 2023

Mink farm outbreak in Spain, 2022

An outbreak of influenza A (H5N1) was reported in a mink farm in Spain in October 2022 (Montserrat et al. 2022). The farm had nearly 52,000 American mink (Neovison vison) placed in 30 barns.

Sampling of animals in areas with the highest mortality from 13 animals revealed positive PCR results. Virus sequence showed 4 samples clustered closely to sequences from European gull samples from various countries, including Spain, during 2022. Of the 12 workers on the farm, 11 had close contact with the mink but no evidence of infection was demonstrated. All 11 people tested negative for avian origin influenza A.

No source or spread pathway between mink has been confirmed. The sequences aligned closely with wild bird sequences which would be a highly probable pathway given the attraction of mink farms for wild birds that scavenge the feed. However, another source such as feed derived from infected poultry cannot be ruled out.

Aerosol transmission between the mink has been hypothesised, given the positivity of oral-pharyngeal and lung swabs and the ‘hotspots’ of increased mortality seen within the barns. However, faecal-oral or indirect transmission pathways from handling animals or exposure to another as yet unknown common source over time cannot be ruled out.

Sea lions die-off in Peru, 2023

In 2023, a large die-off of over 500 sea lions was reported in Peru (Gamarra-Toledo et al., 2023). Since November 2022, a large mortality event of over 22,000 birds over 4 weeks in several coastal areas had occurred, rising to an estimated 50,000 dead birds. Over 5 weeks (January to February 2023), 634 sea lions were found dead (both Otaria flavescens and Arctocephalus australis).

Clinical signs in live sea lions (some of which subsequently died) included tremors, convulsions, paralysis, respiratory signs including nasal and buccal secretions. Most were female, which is probably indicative of the population and behaviour at this time of year rather than dimorphic susceptibility.) Several abortions were also recorded.

The clinical signs of systemic infection, pneumonia and acute encephalitis are suggestive of HPAI infection. The high mortality in wild birds, including sea birds, is evidence of exposure. It has not been confirmed at this point whether there is mammal-to-mammal transmission through direct (such as nose-to-nose), aerosol, faecal-oral or indirect pathways.

How to report suspected avian influenza in mammals

Influenza of avian origin is defined as an infection of a mammalian species with an influenza A virus that is normally only detected in avian species (an avian influenza virus). It is notifiable in both wild and kept mammals.

In England, you must report it immediately if you have a kept mammal or kept mammal carcase in your possession, or under your charge, which either:

• may be infected with avian influenza virus
• has evidence of avian influenza virus or antibodies to avian influenza virus

In all parts of Great Britain, you must report it immediately if you examine or inspect any wild or kept mammal, or analyse samples from any wild or kept mammal or mammal carcase, which either:

• may be infected with avian influenza virus
• has evidence of avian influenza virus or antibodies to avian influenza virus

You should report it immediately if you find a dead wild carnivore or marine mammal and the cause of death is unknown, or the animal has shown signs of respiratory or neurological disease before death. Read suspect case definition 2 for more information.

APHA will triage reports and assess them against the risk the mammal may have been infected with an avian influenza virus. Find out how APHA monitor the risk of avian influenza infection in wild mammals.

Report it immediately by calling:

• 03000 200 301 if you’re in England
• 03003 038 268 if you’re in Wales
• your local Field Services Office if you’re in Scotland

If you do not report it, you are breaking the law.

You must report any suspicion (based on the criteria outlined in this guidance) to the administration in which the tested animal resides. It is an offence if you fail to do so immediately.

Defra’s international reporting obligations

The UK is a World Organisation for Animal Health (WOAH) member country. The detection of infection with influenza A (H5N1) in animals must be reported to the WOAH as an emerging infection in accordance with the WOAH Terrestrial Animal Health Code.

Defra reports information to WOAH on behalf of UK through WOAH’s World Animal Health Information System.

Testing criteria for influenza A in mammals

The government is aware that there is increasing interest amongst owners, veterinary practitioners, universities, wildlife rehabilitators and veterinary diagnostic laboratories to test for influenza A (H5N1) in animals.

Under the Veterinary Surgeons Act 1966 (as amended), testing for any pathogen including influenza A (H5N1) in live animals should only be undertaken if:

• it is in the interest of the health and welfare of the animal and in the case of dead animals
• it can contribute to understanding the management or epidemiology of infection

You must also take into account the risk to public health when handling or sampling suspect cases.

The Home Office regulates sampling in live animals to answer research investigations under The Animal (Scientific Procedures) Act 1986 (as amended). Clinical research may also be conducted under appropriate ethical review.

Specified animal pathogen controls

In addition, certain avian influenza viruses including influenza A (H5N1) are specified animal pathogens and are subject to controls in:

• England: The Specified Animal Pathogens Order 2008 (as amended)
• Wales: The Specified Animal Pathogens (Wales) Order 2008 (as amended)
• Scotland: The Specified Animal Pathogens (Scotland) Order 2009 (as amended)

These orders are collectively referred to as the Specified Animal Pathogens Order (SAPO).

Specified animal pathogens can only be held or used at a facility which has an appropriate SAPO licence. The licence contains a set of conditions and defined control measures that must be met.

Read the avian influenza and influenza of avian origin: diagnostic testing, controls and reporting obligations for more information on SAPO.

The government advises that mammals should only be tested for influenza A if they meet the criteria set out in this guidance. If you think the criteria has been met, you should submit samples to the Avian Influenza National Reference Laboratory at the APHA Weybridge Laboratory.

Suspect case definitions

The criteria for testing has been listed as ‘suspect case definitions’ based on the current scientific knowledge of influenza A (H5N1) infection in mammals. At present, there is no evidence to suggest that mammals other than those in the order Carnivora (such as pigs, equines and ruminants), would develop infection once exposed. If the evidence changes, these suspect case definitions may be expanded.

Keepers must report kept mammals which are present on an infected premises (where avian influenza has been confirmed in poultry or other captive birds) to the APHA case officer as part of the veterinary investigation at the premises. The animals may be sampled. The decision to do so is outside the scope of this guidance.

Suspect case definition 1

The animal is a domestic cat, dog or ferret presenting to a private veterinary surgeon and meets all 3 criteria:

1. It is (or had been prior to sudden death) exhibiting a combination of clinical signs which could be indicative of a viral infection as determined by a veterinary surgeon, including either:

• signs of respiratory disease
• signs of neurological disease

2. Other common differential diagnoses have been considered and discounted as determined by a veterinary surgeon.

3. The animal has either:

• had confirmed or highly probable contact with an infected dead or sick wild bird
• been in close proximity to a wild bird mass die-off area within 3 weeks of developing its clinical signs

Confirmed or highly probable contact in this case relates to consumption, killing, or attacking a bird and may include being fed raw wild bird meat.

Suspect case definition 2

The animal is a non-domestic mammal species of the Order Carnivora (pinnipeds, cetaceans, mustelids (except badgers which will be collected under other surveillance programmes), canids, felids etc).

It is reported as a wild animal found dead or sick, or captive in an establishment (zoo, wildlife park, rescue and rehabilitation centre) and meets the following criteria:

1. It has died suddenly from unexplained causes, or it is exhibiting or has exhibited before death a combination of clinical signs which could be indicative of a viral infection as determined by a veterinary or wildlife professional. Clinical signs include either:

• signs of respiratory disease
• signs of neurological disease

2. The mammal has either:

• had confirmed or highly probable contact with a confirmed or suspect case of influenza A (H5N1) kept or wild birds
• been in close proximity to a wild bird mass die-off area
• been living in an area where influenza A is present in poultry or wild birds, within 3 weeks of its death or developing its clinical signs

Contact in this case relates to consumption, killing, or attacking a bird and may include being fed raw wild bird meat, shot gamebird meat or raw pet food.

Sample types and diagnostic tests

Based on current scientific knowledge, nasal swabs, oropharyngeal swabs, rectal swabs and post-mortem tissue samples from the brain and lungs are the most suitable specimen types for the detection of influenza A (H5N1) infection in animals.

There may be health and safety issues with invasive tissue sampling. Please discuss this with APHA staff when you report the initial suspicion. Some carcases will be too large to transport directly to the laboratory.

Faecal and vomitus samples, swabs of the animal’s coat or fur and other environmental swabs are not suitable due to the potential for environmental contamination.

Nasal, oropharyngeal and rectal swabs and post-mortem tissue samples from the brain and lungs

The receiving laboratory should be capable of retaining the sample. The receiving laboratory should also store nucleic acid extraction from the specimen pending the test result.

Laboratories should store the sample in an appropriate manner to maintain its integrity and traceability, and in compliance with the requirements for specified animal pathogens, Control of Substances Hazardous to Health Regulations 2002 (as amended) (COSHH) and Schedule 5 of the Anti-terrorism, Crime and Security Act 2001 (as amended) (ATCSA). Read the avian influenza and influenza of avian origin: diagnostic testing, controls and reporting obligations for further information.

In the event of a non-negative result, the sample, nucleic acid extraction or carcase must be sent to the APHA Weybridge Laboratory. APHA will undertake secondary or confirmatory testing (in accordance with international standards). Follow the steps to report the outcome of diagnostic testing.

Any diagnostic testing for influenza A in mammals is based on the detection of subtype specific influenza A (H5N1) viral RNA through a polymerase chain reaction (PCR). This is typically a reverse transcriptase quantitative PCR assay (RT-qPCR) or an equivalent assay. Read the WOAH Manual of Diagnostic Tests and Vaccines for Terrestrial Animals for further information.

Lateral flow tests are not considered an appropriate test method for influenza A (H5N1) in mammals.

If there is a non-negative test resulting from a PCR, samples must be sent to the national reference lab for confirmation.

If you are responsible for holding the samples, you must follow the steps to report the outcome of diagnostic testing.

Serological assays

Serological assays for influenza A (H5N1) are not recommended as stand-alone tests. Such assays are used to detect antibodies which would demonstrate past infection only. Therefore it would not be of diagnostic or treatment value to the animal and would not meet testing criteria under the Veterinary Surgeons Act 1966 (as amended).

Read about the use of influenza antigen tests for detecting avian influenza.

Report the outcome of diagnostic testing

APHA will report any positive results that are confirmed by the Chief Veterinary Officer in accordance with our international reporting obligations.

Veterinarians and keepers of susceptible species must apply the criteria for suspect cases in either captive animals, companion animals or wild animals, as appropriate.

Testing undertaken in an unofficial laboratory or using non-official tests is not recommended. If this has been actioned already, you must make sure that owners of the suspect cases and the diagnostic laboratories you use are aware of the following actions when test results are known.

Negative results

If you receive negative results:

• you have no further obligation to report these results to the competent authority
• veterinarians should continue case management as appropriate to the animal’s condition

For cases relating to disease in free-living wildlife, you can discuss further investigation with the APHA Diseases of Wildlife Scheme (DoWS). Find your local Veterinary Investigation Centre (VIC) in the APHA national surveillance network.

Non-negative results

You must report any non-negative results to the competent authority where the tested animal resides.

This includes non-negative results for the presence of either antigen or antibodies to influenza of avian origin and those obtained from Great Britain samples sent to laboratories located outside of Great Britain.

Report it immediately by calling:

• 03000 200 301 if you’re in England

• 03003 038 268 if you’re in Wales

• your local Field Services Office if you’re in Scotland

If you do not report it, you are breaking the law.

You should be prepared to provide information on the animal and any diagnostic testing which has been undertaken. The information you will be asked is outlined in Annex A. All information provided will be handled with appropriate confidentiality.

If possible, you should have the information outlined in Annex A ready when you call to report non-negative findings. Where relevant, the official co-ordinating your report will discuss with you the need to contact public health officials in your area.

Submit samples for official testing

You (the private veterinary surgeon (PVS) or diagnostic laboratory) should be prepared to submit further material to the avian influenza NRL for official testing. The official co-ordinating your report will tell you how to submit the sample.

Samples requested will typically be:

• nasal, oropharyngeal and rectal swabs
• cadaveric tissue samples such as brain and lungs, if available
• 2ml of clotted blood

In addition, where possible you (the PVS) should arrange for the original sample to be sent from the laboratory at which the non-negative test result was achieved to the avian influenza NRL for official testing. The official co-ordinating your report will tell you how to submit the sample.

Sampling and postage costs of submitting additional samples will be at your (the PVS or clients’) cost. The laboratory costs of conducting any official testing at the avian influenza NRL will be paid for by government.

Share the results of the official testing

Results of the official testing conducted by avian influenza NRL, and any other relevant information, will be made available to you (the PVS). It is your responsibility to share these results with your client. When positive results are confirmed by official testing, APHA will contact the relevant public health officials to discuss following up for any potential human exposure.

Suitability for official testing

Official testing may not be required if the report does not meet the suspect case definition.

Additional samples from the animal for confirmatory testing may not be required if the initial samples were collected at post-mortem, or the animal is not available for retesting.

However, you may still need to submit the original sample from which the non-negative test result was obtained. The official who answers your report call will make this assessment. They will tell you the result either during the initial call or during a subsequent call-back. They will explain any subsequent requirements for you to submit samples for official testing.

Samples from other animals at the premises in direct contact with the reported case may also be considered as suitable for official testing. The official co-ordinating your report will inform you of any requests to also submit samples from these animals for testing at APHA Weybridge.

Annex A: Information you need to give when you report a non-negative test

To avoid unnecessary delays, collect this information before you report receipt of a non-negative influenza A (H5N1) test result from a mammalian sample.

However, collecting this information should not delay you reporting non-negative results promptly to the competent authorities (as required by law).

Ideally you should record this information when you consider testing.

When you telephone the competent authority, you will be asked:

• the species (and if appropriate, type) of animal from which the sample was taken
• the age and sex of the animal from which the sample was taken
• history of clinical signs of the animals concerned
• the sample types from which the non-negative test results were obtained, in addition to details of any samples where negative results were obtained from the same animal
• if any other animals were in contact or are displaying clinical signs (including humans)
• the address where the animal was found or samples were taken
• the name, address and phone number or email address of the owner or person in charge of the animal (or property if the animal is part of a commercial or charitable organisation)
• the date of sampling and the date the samples were analysed by the laboratory (if available)
• the name and address of the laboratory where the samples were analysed
• the organism considered to be detected (Influenza or H5 test)
• details of any other differential diagnostic testing of the animals which has been undertaken
• if known for animals – the assay platform used by the laboratory, and the number of cycles (commonly expressed as a Ct or Cq value) needed or viral copy number used to generate the positive result (these figures indicate the amount of viral RNA present in the sample)
• if any other animals in the same premises in which the animal resides were previously tested and if so, with what result (include details of any samples where negative results were obtained from these animals, the date when specimens were analysed by the laboratory (if available), and the name and address of the laboratory)
• the name, address and phone number or email address of the veterinarian making the report
• if there was possible contact between the animal and wild birds, poultry farm (confirmed case reference number (IP or AIV) if known), raw meat (from shot wild birds) or other epidemiological risk factors deemed appropriate by the CAD vet

Find out how your personal information will be used.

References

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Alkie, T.N., Cox, S., Embury-Hyatt, C., et al and Berhane, J. (2023) ‘Characterization of neurotropic HPAI H5N1 viruses with novel genome constellations and mammalian adaptive mutations in free-living mesocarnivores in Canada’ Emerging Microbes & Infections, 12:1.

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Leguia, M., Garcia-Glaessner, A., Muñoz-Saavedra, B., Juarez, D., eP., CeC., Jara, J., Silva, W., Ploog, K., Amaro, L., Colchao-Claux, P., Uhart,M.M., Nelson, M.I., & Lescano J. (2023) ‘Highly pathogenic avian influenza A (H5N1) in marine mammals and seabirds in Peru’ bioRxiv 2023.03.03.531008.

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