Download Guidelines for Sample collection and despatch

Collection of Clinical Samples/ Specimens for Foot and Mouth
Disease Diagnosis
Samples and sampling
1. Epithelium or vesicular fluid.
2. Where epithelial tissue is not available, for example in advanced or convalescent
cases, or where infection is suspected in the absence of clinical signs, samples of
oro-pharyngeal fluid is collected by means of a probang cup (or in pigs by
swabbing the throat)
3. Whole blood with anticoagulant
4. Semen from FMD suspected bulls engaged in semen straw production
5. Serum samples.
Method of sampling:
Epithelial samples and vesicular fluid: Ideally, at least 1 g of epithelial tissue should be
collected from an unruptured or recently ruptured vesicle, usually from the tongue, buccal
mucosa or feet. Epithelial samples and vesicular fluid should be placed in a transport
medium composed of equal amounts of glycerol and 0.04 M phosphate buffer, pH 7.2–7.6,
preferably with added antibiotics. If 0.04 M phosphate buffer is not available, tissue culture
medium or phosphate buffered saline (PBS) can be used instead, but it is important that the
final pH of the glycerol/buffer mixture be in the range pH 7.2–7.6. FMD virus is extremely
labile in low pH and buffering of the transport media is critical for successful sample
collection.
Nasal swabs: May be collected on occasion to attempt virus isolation from animals which
may be in the incubation period (e.g. following known contact with infected animals,
personnel or equipment).
Saliva from acutely infected animals: Saliva may be collected, diluted about 1:10 with
phosphate-buffered saline (to lower the pH for ruminant saliva in particular, and to dilute
antiviral factors present in the saliva), and maintained chilled (4-10 °C) for transport to the
laboratory. Useful if fresh epithelial samples are not available. If saliva is not readily
available, the mouth of an affected animal may be rinsed with phosphate buffered saline and
this sample submitted to the laboratory
Milk: May contain virus for several days before and after the onset of clinical signs. Collect
milk into a plain glass tube. Keep at 4 °C until arrival at the laboratory. Freeze if there is
likely to be a delay in testing, to avoid a fall in pH which may inactivate the virus
Post-mortem samples: Lymph nodes, thyroid, heart muscle from a freshly dead juvenile
animal can be used. Place in phosphate buffered saline/glycerol transport medium.
Oro-pharyngeal fluid: An OP sample is collected by inserting a probang over the tongue
into the oro-pharyngeal area and then passing it vigorously backwards and forwards 5–10
times between the first portion of the oesophagus and the back of the pharynx.
Sampling:
1. Prior to sampling, a 2 ml amount of 0.08 M phosphate buffer containing 0.01% bovine
serum albumin, phenol red (0.002%) and antibiotics adjusted to pH 7.2 should be added
to one bijou bottle or similar container for each of the animals to be sampled.
2. After collection, the sample should be poured from the “probang” cup into a wide-necked
bottle such as a 20 ml Universal bottle and examined visually for quality. Two ml of OP
fluid which contain some visible cellular material should then be added to the previously
prepared bijou bottle containing an equal volume of buffer and thoroughly mixed by
gentle shaking.
3. Samples taken from some animals may be heavily contaminated with ruminal contents.
These should be discarded and the animal's mouth should be flushed with water or
physiological saline solution before repeat sampling.
4. Samples from sheep tend to be small, mucoid and difficult to detach from the probang.
The easiest procedure is to insert the probang cup directly into a disposable Universal 20
ml bottle or similar container into which has been dispensed 3 ml of buffer solution. The
probang is then shaken in the buffer solution to free the sample from the cup, and the
sample together with the buffer should be poured into a previously labelled bijou bottle
for transport.
5. Between collections from each animal, probangs should be disinfected in a bucket
containing 4% Sodium Carbonate or 0.2% citric acid. After disinfection, the probang
should be thoroughly rinsed in running tap water or at least three separate buckets of
clean water placed in series.
6. Samples of OP fluid should be refrigerated or frozen immediately after collection.
Throat swab: For the collection of throat swabs from pigs, the animal should be held on its
back in a wooden cradle with the neck extended. Holding a swab in a suitable instrument,
such as an artery forceps, the swab is pushed to the back of the mouth and into the pharynx.
Blood samples: Blood samples should be collected under sterile conditions and mixed as
soon as possible with the anticoagulant heparin (0.1-0.2 mg per ml of whole blood).
Sequestrene EDTA can be used as an alternative anticoagulant (30 mg of Sequestrene
EDTA in one ml of 0.7% aqueous solution sodium chloride per 20 ml of whole blood). The
sample should be kept at 4°C until dispatched to the Laboratory.
Semen Samples: Semen should be collected under sterile conditions, frozen at -70°C or
lower as soon as possible and packaged.
Serum Samples: Serum, rather than whole blood, must be submitted. A minimum of 4 ml
is essential. It is essential that sterile containers should be used. If sterile, samples can be
submitted without refrigeration, but refrigeration or freezing is an added safeguard against
spoilage
1.
2.
3.
Collect under sterile conditions into plain sterile glass tube without anticoagulant.
Allow to clot.
Centrifuge then decant serum into sterile tube.
Collecting Medium
For Epithelial samples: 0.04 M phosphate buffer (Add 3.05 gm Na2HPO4.2 H2O , 0.39 gm
KH2PO4,to 500 ml sterile distilled water . Add 1 ml 1% phenol red. Add antibiotics). Adjust pH
to 7.2-7.6 with HCl.
For Probang samples: 0.08 M phosphate buffer (Add 6.11 gm Na2HPO4.2H2O, 0.78 gm
KH2PO4to 500 ml sterile distilled water . Add 1ml l% phenol red, Add antibiotics). Adjust pH to
7.2-7.4 with HCl.
To each 500 ml of 0.08 M or 0.04 M phosphate buffer add the following amounts of
reconstituted antibiotics: Penicillin 2.5 ml (final concentration 1000 units/ml), Mycostatin 1.0
ml (final concentration 100 units/ml), Neomycin 1.0 ml (final concentration l00 units/ml)
Polymyxin 0.5 ml (final concentration 50 units/ml)
Transport Medium: 50% Glycerol phosphate buffer saline solution (add equal amout of
glycerol in PBS). pH should be 7.2-7.6.
References
1. Brooskby, J.B., Davie, J. and Hedger, R.S. (1964). Transmission of virus samples. Bull.
Off. Int. Epiz. 61 (11-12), 1605-1615.
2. Kitching, R.P. and Donaldson, A.I. (1987). Collection and transportation of specimens
for vesicular virus investigation. Rev. Sci. Tech. Off. Int. Epiz. 6 (1), 263-272.
3. Foot and mouth disease. OIE Terrestrial manual, 2010.
SAMPLE SUBMISSION AND TRANSPORTATION OF
SPECIMENS FOR FOOT AND MOUTH DISEASE
DIAGNOSIS TO THE PROJECT DIRECTORATE ON FMD,
MUKTESWAR, INDIA
The laboratory investigation of Foot and Mouth Disease (FMD) begins with the collection
of appropriate samples for the intended purposes, such as disease diagnosis, disease surveillance,
health certification or monitoring the response to treatment or vaccination. The samples collected
should be adequate in number and amount to provide statistically valid results and should arrive
at the laboratory in good condition.
The samples should be carefully packaged, labeled, and transmitted to the laboratory by the
fastest practicable method, with the appropriate temperature control. If the material is sent to a
laboratory in another country, this laboratory should be consulted in advance to ensure that it is
willing to receive the material and to obtain the appropriate import license. All samples should
be accompanied by a letter or submission form, which includes the details mentioned in the
section below.name
PACKAGINGING, LABELLING AND SHIPPING
Special precautions are required when sending perishable suspect FMD material both within
and between countries. The international regulations for the transport of infectious materials by
any mode of transport are based upon the Recommendations of the United Nations Committee of
Experts on the Transport of Dangerous Goods (UN). The Universal Postal Union (UPU) reflects
these recommendations in its regulations, particularly for packaging. The International Civil
Aviation Organization (ICAO) and the International Air Transport Association (IATA) have also
incorporated the UN Recommendations in their respective regulations, as have other
international transport organizations. The World Health Organization serves in an advisory
capacity to these bodies( "Guidelines for the safe transport of infectious substances and
diagnostic specimens," WHO/ EMC/97.3 (1997), World Health Organization, http://
www.who.int/csr/emc97_3.pdf).Airline companies carrying pathological material and biological
products may also have special requirements and these should be consulted prior to shipment.
Packaging Requirements:
Specimens for diagnosis must be packaged according to PI650 requirement for UN3373 category
B classification unless exempt due to a minimal likelihood that pathogens are present (e.g. samples
for serology)
1. Samples should be packed in a Triple Packaged System (Figure I). Samples should be
placed in a watertight primary container with a leak proof screw cap with rubber washer.
The primary container should be individually wrapped in absorbent material and then
placed in a watertight unbreakable and leak proof secondary container. This may be
surrounded by sealed freezer packs or dry ice and must be enclosed in a strong outer
packaging which should allow the release of carbon dioxide if card ice is enclosed. It is
essential that packaging ensures that the contents of containers, which break or leak in transit,
cannot contaminate the outside layer of the parcel.
2. Sufficient information for the identification of the material should be written on a piece
of waterproof adhesive tape attached to the Primary container. The outside of the
container containing the sample should be disinfected by immersion in 4% Sodium
bicarbonate or 0.2% citric acid, dried off, labeled and then frozen immediately in solid
CO2 within an insulated box or in a -70°C refrigerator
3. Specimen data forms, letters and other types of information that identify or describe the
specimen and also identify the shipper and receiver should be taped to the outside of the
secondary receptacle.
4. If sample collection takes place at a point which is some distance from the point of
dispatch by air, the sample should be kept refrigerated. This container should also be
labelled.
5. It is important to avoid thawing and refreezing at any stage, so packing should be done
quickly and complete packages should be kept at -70°C or lower.
Labelling:
The outer packaging of the parcel must be clearly labelled with the following information as
shown in the diagram below

Import license number of the laboratory, if any,(eg: AHZ/1309/A for IAH Pirbright (a copy
of which should also be attached within an envelope to the outside of the package)

The name and address of the Institute and in the case of parcels sent by airfreight the
instruction that the package is “Airfreight package for the attention of Project Directorate on
FMD, IVRI Campus, Mukteswar-263138, Nainital, India.

The name and telephone number of the person responsible for sending the parcel.

Infectious Substance Hazard Label stating that the package contains a “Biological Substance,
Category B” Animal Diagnostic Specimen of no Commercial Value (Hazard for Animal
Health, not for people).

Flight Number

Air Waybill Number

Dry Ice Label (if necessary).

Label as “KEEP AT 4°C unless otherwise instructed”
Diagnostic specimen (UN3373)
Infectious substance label (UN2900)
Source:
Goods
Dangerous
Miscellaneous dangerous goods (Dry Ice)
Sample orientation label
Regulations (DGR) Spiral Manual – 2013 (54th edition)
http://www.iata.org/whatwedo/dangerous_goods
Shipment:
1. Dispatch should only be by air freight.
2. The flight number, date and time of arrival and airway bill number should be sent to the
Project Directorate on FMD, IVRI Campus, Mukteswar -263138, Nainital, India.
TEL: +91 5942 286004
FAX: +91 5942 286307
3. A letter should accompany the specimens giving as much history and epidemiological
information as possible.
4. Accompanying information must include the name, address and fax number of the sender
and instructions for notification of results.
REFERENCES
4. OIE Terrestrial manual, 2010. Foot and mouth disease. Chapter 2.01.05
http://www.oie.int/fileadmin/Home/eng/Health_standards/tahm/2.01.05_FMD.pdf
5. Institute of Animal Health, Pirbright, UK
http://www.iah.bbsrc.ac.uk/ref_Labs/docs/PackingInstructions.pdf
3. "Guidelines for the transport of infectious substances 2011 -12," publ
WHO/HSE/IHR/2010.8, World Health Organization,
http://www.who.int/ihr/publications/who_hse_ihr_20100801_en.pdf
Figure 1
Triple Packaging System
Source: "Guidelines for the safe transport of infectious substances and diagnostic
specimens," WHO/ EMC/97.3 (1997), World Health Organization,
http:// www.who.int/csr/emc97_3.pdf
Note: For shipping samples to World reference Laboratory:
World Reference Laboratory for
Foot and Mouth Disease
Institute for Animal Health,
Pirbright Laboratory,
Ash Road, Pirbright,
Woking, Surrey, GU24 0NF UK
Tel: +44 (0)1483 232446
******
Airfreight package for the attention of
Mr. Moonbridge,
Unit 304, Bedfont Industrial Park,
Challenge Rd, Ashford, Middlesex, TW15 1AX UK
Note: For shipping samples to Project Directorate on Foot and
Mouth Disease:
SAARC Regional Leading Disease Diagnostic
Laboratory for
Foot and Mouth Disease
Project Directorate on Foot and Mouth Disease
IVRI Campus, Mukteswar – 263138
Nainital, Uttarakhand
India
Tel: +91-5942-286004
******
Airfreight package for the attention of
Dr. B.Pattnaik
Project Directorate On Foot and Mouth Disease
IVRI Campus, Mukteswar – 263138
Nainital, Uttarakhand
India
Tel: +91-5942-286004