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Appendix 1: Foot-and-Mouth Disease
Laboratory Diagnosis

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Note that the CO₂ emanating from the dry ice can create acidic conditions which can impact FMD virus survival. Only use wet ice or ice packs for shipping FMD specimens to a diagnostic laboratory.

SUBMISSION DURING AN FMD OUTBREAK:

• If possible, vesicular fluid aspirated with a syringe (at least 2 ml) in a separate sterile tube.
• Two gm of affected epithelial tissue taken as aseptically as possible and placed in 5 ml of phosphate buffered glycerin virus transport medium (pH 7.6).
• Scrapings of foot lesions in separate buffered glycerin transport medium.
• Sera (10 ml) from clinically affected and recovered animals.
• Tissues from vesicular lesions of animals being destroyed or from animals being slaughtered in abattoirs may also be submitted for histology. If only a carcass is available, submit pre-scapular lymph node, adrenal gland, kidney, and thyroid gland for (viral) culture.

SUBMISSION DURING ROUTINE DISEASE INVESTIGATION

TO MAXIMIZE THE LIKELYHOOD OF ARRIVING AT A DIAGNOSIS, ALWAYS CONDUCT A FULL POST-MORTEM WHERE POSSIBLE and submit fresh or frozen tissues for culture and fixed tissues for histological examinations.

Tissue samples should include (teat skin; tonsil (P)* ; lymph nodes - submandibular (P), prescapular (B), pleural (O/C), mesenteric; spleen; liver; kidney; lung; heart; piece of terminal ileum and ileo-coecal valve (5cm long and tied off at both ends); half brain (sagitally cut); Mammary (O/C) and any observed gross lesion.

Blood samples should include 10 ml serum; 10 ml EDTA and 6 air dried smears fixed in 70% alcohol. Other samples include nasal and tracheal swabs in transport media (B, O/C); body cavity fluids; infected joints fluid (P)

* (B=bovine; P=porcine; O/C=ovine/caprine).

LABORATORY TESTS

Diagnosis of FMD for the index case is by demonstration of FMD viral antigen (double antibody sandwich ELISA) and/or virus isolation or nucleic acid specific to FMD in samples of tissue or fluid. Detection of specific humoral antibody can also be used for diagnosis, but results should be interpreted carefully for the index case.

For cases subsequent to the index case, the DAS-ELISA and real time or conventional PCR will be the main diagnostic methods. Virus isolation will be important for subsequent epidemiological investigation.

The laboratory tests currently available at NC-FAD are shown in Table 1 and include the double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), virus isolation, real time (kinetic) reverse transcription-polymerase chain reaction (RT-PCR). The serological tests are the solid phase competitive ELISA and the serum neutralization test.

The time needed to confirm the diagnosis of the index case will depend on the quantity, the quality and the type of sample received by the laboratory. Sample types include: vesicular fluid, epithelial tissues and oro-pharyngeal fluid (probang). Good quality fluid collected from vesicles is ideal, but from a practical point of view difficult to obtain. Epithelial tags collected from the edges of ruptured vesicles are the more likely sample the field staff will submit. Ideally these tissues should be clean and relatively fresh and the oral tissues should be separated from the feet lesions.

The preferred procedure for the detection of FMD viral antigen and identification of viral serotype is the DAS-ELISA. However, a negative test result may be obtained when a specimen is from an old lesion, or is too small, or contains too little antigen to be detected. The method used to determine whether any infectious virus is present is to attempt virus isolation by inoculating and passaging the original tissue suspension through susceptible cell cultures, and looking for a cytopathic effect.

Detection of specific sequences of the virus nucleic acid can be attempted by PCR by using generic primers targeting the 3D gene (RNA polymerase) of the virus.

FMD virus infection can be diagnosed by the detection of a specific antibody response. Virus neutralization (VN) and ELISA are used as serotype specific serological tests. VN tests depend on cell culture systems and are therefore more prone to variability than ELISA. Generally, antibodies to the whole virus appear in serum 7-10 days after infection.

Once the NC-FAD has isolated a FMDV, it will be forwarded to the FMD World Reference Laboratory at the Institute for Animal Health, Pirbright Laboratory, UK for conducting molecular typing and providing advice on the selection of the antigen in the vaccine bank or from another source. Molecular typing is not required to initiate the control measures.

Table 1 Diagnostic test currently available at NC-FAD for FMD

* Represents only the actual test time. Time in sampling processing, repeat and reporting is not included. A complete turn around time (TAT) is available in the CFIA laboratory test TAT.

Flow Chart of Diagnostic Procedure ACUTE POST OUTBREAK *

[D]

1. LK = Lamb Kidney
2. BHK-21 = Baby Hamster Kidney
3. PK-15 = Pig Kidney
4. CPE = Cytopathic Effects
5. DAS-ELISA = Double Anitbody Sandwich Enzyme-Linked Immundsorbent Assay

* Print in colour

Appendix 2: Foot-and-Mouth Disease Disinfectant

Note: To be verified by AERT C&D supervisors.

Varying the pH of the environment of the virus outside of the range pH 5-10 is a practical method of destroying the FMD virus. Virus sensitivity is marked in the acid range and for this reason weak acids can be used in many situations.

(Adapted from AUSVETPLAN - Tables 2.8 and 4.0.)

Note: Dilution rates for use against FMD relate to effectiveness when applied to a clean surface. A dirty surface must be cleaned before it can be satisfactorily disinfected because the dirt may make the disinfectant useless. Ensure that the surface or material is:

• thoroughly cleaned, ensuring that dung, litter, etc. is removed
• thoroughly washed or sprayed with a disinfectant

Notes:

• Products effective for decontamination of viruses on the hands and the skin are limited. Virkon is reported to have low toxicity and to be effective. Citric acid may be added to washing water to induce antiviral conditions by lowering the pH.
• Vinegar and household bleach are both common disinfectants and effective when used for the purposes and concentrations recommended - see table.
• A list of disinfectants and suppliers for North America is currently under development.
• * Requires a specialized generator and a lot of electricity. Usually, the formaldehyde gas emitted in a confined area will either have to be neutralized with ammonium carbonate or the area will have to be well ventilated before being used again. Bacterial indicators are required to control the effectiveness of the treatment.

Appendix 3: Foot-and-Mouth Disease Virus Inactivation Procedures

(From OIE Terrestrial Animal Health Code 2005), Appendix 3.6.2

Inactivation of the virus in meat (Article 3.6.2.1)

1. Canning - Meat is subjected to heat treatment in hermetically sealed container to reach an internal core temperature of at least 70ºC for a minimum of 30 minutes.

2. Thorough cooking - Meat previously deboned and defatted shall be subjected to heating so that an internal temperature of 70ºC or greater is maintained for a minimum of 30 minutes. After cooking, it shall be packed and handled in such a way that it cannot be exposed to a source of virus.

3. Drying after salting - When rigor mortis is complete, the meat must be deboned, salted with cooking salt (NaCl) and completely dried. It must not deteriorate at ambient temperature. "Drying" is defined in terms of ratio between water and protein which must not be greater than 2.25:1

Inactivation of the virus in animal products

Wool & hair (Article 3.6.2.2)

For inactivation of viruses present in wool and hair for industrial use, one of the following procedures should be used:

1. industrial washing, which consists in the immersion of the wool in a series of baths of water, soap and sodium hydroxide (NaOH or soda) or potassium hydroxide (KOH or potash);

2. chemical depilation by means of slaked lime or sodium sulphide;

3. fumigation in formaldehyde in a hermetically sealed chamber for 24 hours. A common practical method of accomplishing this objective is to add commercial formalin (53 ml) to potassium permanganate (35g per cubic metre). Warning: the reaction between formalin and potassium permanganate is quite violent and generates a considerable amount of heat. For that reason, plastic and polyethylene containers should not be used and protective clothing, including goggles, are essential. The operator should also ensure a rapid exit from the room is possible. Good ventilation of the room is also essential after the fumigation. A safer fumigation method is to use the automatic formalin generators (Certek) from paraformaldehyde with neutralization with ammonium carbonate.

4. industrial scouring which consists in the immersion of wool in a water-soluble detergent held at 60-70ºC.

5. storage of wool at 18ºC for 4 weeks, or 4ºC for 4 months or 37ºC for 8 days.

Bristles (Article 3.6.2.3)

For inactivation of viruses present in bristles for industrial use, one of the following procedures should be used:

1. boiling for at least 1 hour.

2. immersion for at least 24 hours in a 1% solution of formaldehyde prepared from 30 ml of commercial formalin per litre of water.

Raw hides and skins (Article 3.6.2.4)

For the inactivation of viruses present in raw hide and skins for industrialized use, the following procedure should be used: salting for at least 28 days in sea salt containing 2% sodium carbonate.

Milk or cream for human consumption (Article 3.6.2.5)

For inactivation of viruses present in milk and cream for human consumption, one of the following procedures should be used:

1. A sterilisation process applying a minimum temperature of 132ºC for at least 1 second (Ultra High Temperature [UHT]

2. If the milk has a pH less than 7.0, a sterilisation process applying a minimum temperature of 72ºC for at least 15 seconds (high temperature-short time pasteurization simple HTST) or

3. If the milk has a pH of 7.0 or over, the HTST process is applied twice.

Milk for animal consumption (Article 3.6.2.6)

For the inactivation of viruses present in milk for animal consumption, one of the following procedures should be used:

1. Double High Temperature Short Time (HTST) pasteurisation (72ºC for at least 15 seconds);

2. HTST combined with another physical treatment e.g. maintaining a pH 6 for at least one hour or additional heating to at least 72ºC combined with desiccation;

3. Ultra-high temperature (UHT) combined with another physical treatment referred to in point 2 above.

Disinfection of Skins and Trophies (Article 3.6.2.7)

For the inactivation of viruses present in skins and trophies from wild animals susceptible to FMD, one of the following procedures should be used prior to complete taxidermal treatment:

1. boiling in water for an appropriate time to ensure that any matter other than bone, horns, hooves, claws, antlers or teeth is removed

2. gamma irradiation at a dose at least 20 kilogray at room temperature (20ºC or higher);

3. soaking with agitation in 4% (weight/volume) solution of washing soda (sodium carbonate - Na₂ CO₃) maintained at pH 11.5 or above for at least 48 hours;

4. soaking with agitation, in a formic acid solution (100 kg of salt [NaCl] and 12 kg of formic acid per 1000 litres of water) maintained at below pH 3.0 for at least 48 hours; wetting and dressing agents may be added.

5. for raw hides, salting for at least 28 days with sea salt containing 2% washing soda (sodium carbonate - Na₂ CO₃).

Practical Field Inactivation (not in OIE Code)

Acid Treatment

The addition of acid to reduce the pH below 4:

• 3 parts glacial acetic acid to 97 parts milk;
• 500 gm citric acid or sulphamic acid to 240 litres milk, or;
• 1.5 litres of ortho-phosphoric acid technical grade to 500 litres milk).

Because the milk will curd, do not undertake this activity while milk is in the bulk tank. Open tanks or containers will facilitate the disposal.

Other Disinfectants

To add another disinfectant, see Appendix 2.

When acid treatment is employed, the resulting mix of milk and acid will be agitated and held for one hour. Milk to which acid has been added can be pH verified using pH assay strips.

Appendix 4: Summary FMD Movement Control

Movement control is described in the Procedures in the Control Area (4.6) and the Control Area Regulations Section 80 (under preparation). Transmission can be effected directly by animal movement or indirectly by fomites or things such as animal products, waste, animals, people etc. Means of transmission can be placed into logical groupings and assigned a risk (low, medium, or high) according to the innate ability to contain, sustain and transmit FMD virus. Movements are classified for epidemiological purposes based on the origin and destination, risk category of item, direction of movement, effectiveness of treatment, welfare and fate of risk good once moved.

Table I: Definition of risk categories

Table II: Table of permit decision rules based on the source of the movement, the conveyor type, and the direction of the intended movement.

* Note: all places in the infected zone are suspect infected places.

Appendix 5: Epidemiological sampling of herds/flocks undergoing depopulation

Sampling requirements to meet the statistical significance suggested by international standards will need to be added for transmittal to the field staff. It is recommended that this Appendix be written by the team of epidemiologists that will build the rationale for recognition of FMD freedom by the international trading partners.

Appendix 6: Surveillance sampling protocol for the Control Area and the disease-free zone

The sampling protocols for surveillance during and after the outbreak within the control area as well as in the FMD-free zone need to be added to build the argument for trade from the unaffected parts of the country. When to initiate surveillance both within the control area as well as in the FMD free zone, determine the confidence and the prevalence criteria if different from 95% and 1%. The OIE standard calls for 95% level of confidence at a 20% prevalence. The European Commission states 95% / 2%. It is recommended that this Appendix by written by the team of epidemiologists who will be building the rationale in support of a recognition of freedom from the trading partners.

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