WHO laboratory guidelines for the collection of animal specimens for diagnosis of influenza infection

12 January 2005

General information

The success of virus diagnosis depends largely on the quality of the specimen and the conditions under which the specimen is transported and stored before it is processed in the laboratory. Specimens for isolation of respiratory viruses in cell cultures or embryonated chicken eggs and for the direct detection of viral antigen or nucleic acids should generally be taken during the first 3 days after onset of clinical symptoms of influenza. In mammals, including humans, pigs and horses, influenza is primarily a respiratory tract infection while in avian species it can be an infection of both the respiratory tract and the large intestinal tract.

Type of specimens

A variety of specimens from ammmals and birds are suitable for the diagnosis of virus infections of the upper respiratory tract:

• nasal swab
• throat swab
• tracheal swab

In addition to swabs from the upper respiratory tract, sampling of avian species for influenza infection should include:

• cloacal swab
• faecal specimen

Whenever possible, cloacal swabs should be collected from live or freshly killed birds. Faecal specimens collected from cages or from the environment are often the only specimens that are available and cannot be assigned with total certainty to the species of origin.

If dead animals are found as part of the investigation, highly pathogenic avian influenza virus should be suspected, and representative internal organs, including brain, spleen, heart, lung, pancreas, liver and kidney, should be sampled together with sampling of the respiratory and intestinal tracts.

Specimens for the laboratory diagnosis of influenza infection should be collected in the following order of priority:

• From live animals

• Tracheal
• Throat/nasal
• Cloacal
• Faecal (environmental)
• Drinking-water

• From dead animals

• Lung lavage
• Pooled tissue (including trachea and lung)
• Faecal (environmental)
• Cloacal
• Drinking-water

Procedures for specimen collection

Materials required

• 1–3-ml screw-cap plastic tubes
• Polyester fibre-tipped swabs
• Viral transport medium
• Instruments for post-mortem examination

Virus transport medium

(A) Transport medium 199

1. Tissue culture medium 199 containing 0.5% bovine serum albumin (BSA)

2. To 1 litre of 0.5% BSA add:
- benzylpenicillin (2 x 10⁶ IU/litre)
- streptomycin (200 mg/litre)
- polymyxin B (2 x 10⁶ IU/litre)
- gentamicin (250 mg/litre)
- nystatin (0.5 x 10⁶ IU/litre)
- ofloxacin hydrochloride (60 mg/litre), and
- sulfamethoxazole (0.2 g/litre)

3. Sterilize by filtration and distribute in 1.0–2.0-ml volumes in screw-capped tubes.

Note: With increasing use of antibiotics in animal husbandry it has become necessary to use high concentrations of antibacterial and antifungal agents.

- OR -

(B) Glycerol transport medium

1. Phosphate-buffered saline (PBS):

2. Autoclave PBS and mix 1:1 with sterile glycerol to make 1 litre

3. To 1 litre PBS/glycerol add:

- benzylpenicillin (2 x 10⁶IU/litre)
- streptomycin (200 mg/litre)
- polymyxin B (2 x 10⁶ IU/litre)
- gentamicin (250 mg/litre)
- nystatin (0.5 x 10⁶ IU/litre)
- ofloxacin hydrochloride (60 mg/litre), and
- sulfamethoxazole (0.2 g/litre).

For the appropriate choice of medium see “Preparing to take specimens” section below.

Preparing specimen collection vials

To sterile plastic screw-cap vials dispense 1.0–2.0 ml of transport medium. It is preferable to store these vials at –20 °C until use. However, they can be stored at 4°C for 48–96 hours (optimally less than 48 hours) or at room temperature for short periods of 1–2 days.

Preparing to collect specimens

Vials should be assigned a number that corresponds to that on the Field Data Collection Sheet. When possible, the following information should be recorded on the Field Data Collection Sheet: type of animal sampled, species, type of specimen, date of collection, and geographical location of specimen collection, etc.

Tissue culture medium (A) is widely used for collection and transport of clinical specimens from all species. The glycerol-based medium (B) provides longer-term stability of specimens where cooling is not immediately possible; it is suitable for egg inoculation but not suited for tissue culture inoculation.

Clinical specimens should be collected as described below and added to transport medium. All specimens should be kept on ice or at 4 °C.

Standard precautions should always be followed, and barrier protections applied whenever samples are obtained from patients.

Nasal swab

A dry polyester swab is inserted into the nostril, parallel to the palate, and left in place for a few seconds. It is then slowly withdrawn, with a rotating motion, down the inside of the nose. Specimens from both nostrils are obtained with the same swab. The tip of the swab is put into a vial containing 2–3 ml of transport medium and the applicator stick is broken off.

Throat swab

The posterior pharynx is swabbed vigorously, and the swab is placed in transport medium as described above.

Tracheal swab

The trachea of live birds is swabbed by inserting a polyester swab into the trachea and gently swabbing the wall. The swab is then placed in transport medium as described above.

Tracheal swabs from dead animals, including pigs at slaughterhouses, can be taken after removal of the lungs and trachea from the carcass. The trachea is held in a gloved hand and the swab inserted to its maximal length with vigorous swabbing of the wall. The swab is then placed in transport medium as above.

Cloacal swab

A cloacal swab from a live bird is taken by inserting a swab deeply into the vent and vigorously swabbing the wall. The swab should be deeply stained with faecal material. The swab is then placed in transport medium as above.

Faecal specimens

Faecal specimens from the cages of live poultry in bird markets or from wild birds in the field are collected from freshly deposited wet faeces; the swab should be heavily coated with faeces. The swab is then placed in transport medium as above.

Sera collection for influenza diagnosis and surveillance

Tissue specimens

Tissue specimens should ideally be frozen immediately, without transport medium, and later ground in transport medium before inoculation of eggs or tissue culture.

For diagnosis, an acute-phase serum specimen (3–5 ml of whole blood) should be taken soon after onset of clinical symptoms, and not later than 7 days after onset. A convalescent-phase serum specimen should be collected 2–4 weeks later. In serological surveillance studies at slaughterhouses or of free-flying wild birds that are bled and released, a single sample of serum is collected. The blood is allowed to clot then centrifuged at 2500 rpm for 15 minutes to separate red blood cells and serum. The serum should be pipetted off, and the red cells may be discarded. Serum samples are stored at –20 °C.

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