Official Veterinarian Action for the Initial Inquiry

1. The OV needs to take to the premises, an appropriate number of the following items:

• scalpel handle and blades (appropriate size)
• plain swabs (maximum length of swab container 12cm)
• disposable gloves
• microchip reader (as required)
• microscope slides
• microscope slide mailers or storage boxes (type slide mailer into a search engine e.g. Google or Bing, for suppliers
• microscope (including light source as appropriate), with x100 oil immersion lens, immersion oil, 1% McFadyean's methylene blue dye (commercially ready to use), only where it is intended to examine the slides on the premises
• protective clothing and Defra/Devolved Authorities approved disinfectant
• clinical waste container
• cotton wool and tape.

2. The OV is to take appropriate biosecurity and Health and Safety (H&S) measures on entry, exit and whilst on the suspect premises, to minimise the risk of potential disease spread, particularly through infected blood. OVs should manage potential exposure through for example skin wounds obtained whilst collecting samples from an anthrax positive animal, appropriate to risk and consult their GP immediately in cases where they are particularly concerned.

3. At the beginning of the on-farm investigation, individual animal identification numbers (where applicable), species, age, and breed of animal are to be recorded and ear tags or microchips checked.

4. Prior to the collection of any samples, the OV should review the clinical history and inspect the carcase and surroundings. Further information may now be available meaning anthrax can be reasonably ruled out. The list below is not meant to be exhaustive (and applies to suspicion in cattle):

• the animal is young and there is a current disease problem, such as pneumonia, affecting the group involving high morbidity/mortality
• the body of the animal is emaciated and it has not been eating for two days
• the animal has gorged on ad-lib concentrates and was seen to be very bloated earlier in the day. It died before any treatment could be initiated
• there are obvious marks on the pasture indicating the animal was convulsing before it died. The farmer has lost one animal from the group already due to hypomagnesaemia and has put high magnesium content licks out
• a heifer has a calf stuck at the hips and has obviously been calving for a while
• the animals have escaped onto a golf course where there are numerous yew trees some of which show signs of being eaten. A couple of animals from the group are dead. Yew poisoning is suspected
• there are five young heifers down in a field with flaccid paralysis, one is dead. Poultry litter was heaped in a corner of the field three days ago. Botulism is suspected
• following a storm the night before the body of the beast is directly under a tree that has signs of being struck by lightning and there are scorch marks visible on the carcase. A lightning strike is suspected
• all milking cows down one side of a herring bone parlour dropped dead at the start of milking. The electric has been turned off and an electrician has been contacted. Electrocution is suspected.

5. A decision has to be made by the OV, as to whether anthrax can, with any new evidence available, be ruled out, as there is now a high likelihood of an alternative diagnosis being the cause of illness or death. If this is the case, then a decision can be made to rule out anthrax at this stage and no samples are to be collected. The Duty Vet at Animal and Plant Health Agency (APHA) is to be contacted by phone to confirm that further findings indicate that disease can be ruled out, without samples being collected. The Duty Vet will then need to confirm to the OV that the inquiry is to be closed. The OV needs to complete a Certificate of Non-existence of Disease (AN02) and in:

• England, return it to the APHA One Health Team, Customer Service Centre (CSC), Worcester
• Scotland and Wales, return it to the local APHA office
• Contact numbers are provided on the APHA website.

6. Once a decision has been made to rule out anthrax, the keeper can be informed by the OV that no further anthrax precautions need to be taken. The farmer remains responsible for sending carcases for appropriate disposal under the Animal By-Product Regulations (ABPR).

7. If the suspicion of anthrax remains, then a set of samples is to be collected from each suspect animal or carcase.

Collection of Samples from a Carcase

1. Key points include:

• do not send whole carcases or tissues to a laboratory for post mortem, if anthrax cannot be ruled out as a differential
• do not collect samples of blood or body fluids using evacuated blood tubes or disposable syringes. There are risks associated with this method of sample collection and handling
• do collect the following set of samples below:
  • for a live animal of any species, collect a blood sample (one to two drops) by nicking a superficial vein (preferably an ear vein) with a disposable fine needle or blade. Once sufficient blood has been collected, firm pressure should be applied with a cotton swab to prevent further bleeding, instruments and materials should be disinfected and disposed of into a biohazard sharp's container. Any blood leakage is to be effectively cleansed and disinfected using a disinfectant approved by Defra/Devolved Authorities
  • for a dead animal, the following tissues make up a sample set:

    Species	Tissues to be Sampled
    Cattle and Sheep	All cases: one blood smear from a superficial vessel (for preparation of anthrax slide and local examination) two blood smears from the same source (for sending by an APHA VI to Public Health England (PHE) Porton Down as air dried unstained smears, if disease cannot be ruled out from the initial local examination) two plain swabs impregnated from the same source (for sending by an APHA VI to PHE Porton Down, if disease cannot be ruled out from the initial local examination).
    Pigs	All cases: one blood smear from a superficial vessel (for preparation of anthrax slide and local examination) two blood smears from the same source (for sending by an APHA VI to PHE Porton Down as air dried unstained smears, if disease cannot be ruled out from the initial local examination) two plain swabs impregnated from the same source (for sending by an APHA VI to PHE Porton Down, if disease cannot be ruled out from the initial local examination) in addition: when there is a swelling of the throat - two plain swabs and three smears (from the cut surface of an associated lymphatic node, or the cut surface of the throat swelling - one for local staining and analysis) where there is no swelling of the throat - two plain peritoneal swabs and three smears (one for local staining and analysis).
    Horses, dogs and other carnivore	All cases: one blood smear from a superficial vessel (for preparation of anthrax slide and local examination) two blood smears from the same source (for sending by an APHA VI to PHE Porton Down as air dried unstained smears, if disease cannot be ruled out from the initial local examination) two plain swabs impregnated from the same source (for sending by an APHA VI to PHE Porton Down, if disease cannot be ruled out from the initial local examination) in addition when there is a swelling of the throat - two plain swabs and three smears (from the cut surface of an associated lymphatic node, or the cut surface of the throat swelling - one for local staining and analysis).

2. Maximum length of swab container used must be 12cm to fit into the packaging.

3. Samples must be collected in a way in which minimal invasion of the carcase is achieved. Disposable gloves must be worn. All surrounding areas where carcase fluid has spilled must be cleaned and disinfected appropriately. In the majority of inquiries only blood smears and swabs will need to be collected (as in the tables above).

4. For the collection of blood samples:

• blood must be collected only from a superficial vein. This is to be an ear vein, unless no blood can be obtained from an ear vein. If it is accessible, an ear vein nearest the ground (dependent ear) is the best ear to collect blood from. The ear may need to be cleaned of external contamination, before sample collection, with a dry/moist cotton wool swab. A small nick is made in the vein and one to two drops of blood extracted by normal flow/pressure collected onto the end of each of three glass microscope slides. Extracting drops of blood may be more difficult from animals that have been dead for a longer period of time and several nicks may be needed. Using the edge of another slide at an angle of approx 30°C, the blood droplet is drawn across the microscope slide to create a blood smear (see below):

Alternatively, but to be avoided if possible, as it is an inferior technique, a plain swab can be used impregnating it with blood and rolling it across the slide. Do not make the slide too thick and ensure the blood smear is only made on one side of the slide. If not made correctly, the material may contaminate the package or bench surfaces presenting a danger to staff handling the samples. If a smear is too thick, it may be difficult to examine and delay a diagnosis. Allow slides time to air-dry.

• two plain swabs must be used to collect blood from the cut surface for possible future culture if required
• to minimise the risk of blood leakage, seal the incision, e.g. put cotton wool over the incision site and secure in place. If an arm length glove has been used, this may be removed, everted and tied around the incision site to secure the cotton wool dressing in place. If blood is spilled, take appropriate action to clean and disinfect the area using an approved disinfectant.

5. When required, oedematous fluid or the cut surface of lymph nodes of the throat may be collected, as follows:

• a smear can be made from direct impression of the cut surface, or by drawing a drop of fluid across a slide, or a plain swab used to collect material and a smear made by rolling it across the microscope slide, following a minimal sized incision into the affected area
• two plain swabs are needed, as well as the smears, for possible future culture
• where there is no throat swelling in pigs, two plain swabs should be used to collect peritoneal fluid. Again the plain swab is used to make the smears, and two plain swabs are needed for possible future culture. Particular care must be taken in opening the abdomen of a pig, especially if it is distended.

6. The preparation of smears must take place on the premises. Parts of the carcase must not be removed in order to facilitate the taking of blood samples, and neither must they be removed to other premises for further examination.

7. Ideally the initial microscopic examination should be carried out on the premises. Alternatively the slides may be taken back to the OV practice to stain and examine. The microscope slides and swabs must be transported back to the OV practice, in an appropriate leak-proof container, where the slide surfaces are protected e.g. plastic microscope slide mailer.

8. Before leaving the premises the OV must ensure that orifices of the carcase are sealed with cotton wool or other natural i.e. cotton based cloth, material soaked in disinfectant and that the carcase is secured from predation or inadvertent access by people. The carcase should not be moved, however if it is not possible to secure the carcase where it is and it needs to be moved, then advise on disinfection of the place where the carcase was present and any equipment used to move it. Natural materials are preferred as the use of plastic based materials may have disposal cost implications i.e. creation of dioxins during burning.

Equipment Needed to Prepare and Examine Blood Smears

1. The following equipment is needed to prepare and examine blood smears:

• forceps
• bunsen (or similar flame)
• staining rack
• 1% McFadyean's methylene blue
• microscope (with x100 oil immersion lens)
• immersion oil.

2. To prepare a smear for visualising under the microscope, the following actions are to be taken on one slide, whilst observing appropriate H&S measures. The other two slides are not required for this examination but must be air dried, kept secure, uncontaminated and unstained:

• fixing the smear - pass the microscope slide (film-side up) slowly through a bunsen flame three times, holding in a pair of forceps. The aim is to fix cells and bacteria to the glass, without distorting how they look, achieved by heating to approximately 55 to 60°C (think egg white solidifying). Do not flame more than this as over-fixing destroys the McFadyean reaction
• staining the slide - holding the slide in forceps, place it on a staining rack and cover the fixed material with 1% McFadyean's methylene blue. Leave to stain for two minutes. Gently wash the slide using tap water and allow to air-dry
• Examination - add a drop of immersion oil and examine using the x100 oil immersion objective lens on the microscope. Examine the smear carefully for the presence of blue-stained, square(ish) ended bacilli surrounded by a purple capsule:

3. Dark/dark blue rods in a background of purple/pink stained capsular material is a positive test. The capsule may not be obvious and may have disintegrated so that background material in view is purple with blue rods devoid of capsular material. It may be broken if the slide was overheated or from a live animal that has been treated with antibiotics. The capsule may also be absent or poorly stained in preparations from porcine material.

4. The following pictures are of positive and negative anthrax smears from attenuated laboratory anthrax cultures (capsules not staining deeply):

Anthrax Positive 1

Anthrax Positive 2

Anthrax Negative 1

Anthrax Negative 2

5. All waste should be disposed of as clinical waste or cleansed and disinfected appropriately using a disinfectant approved for General Orders.

Disease can be Ruled out by the Initial Inquiry

1. In the majority of investigations the OV will be able to recommend to the APHA VI that, following examination of the carcase, immediate surrounding area and the taking, staining and examining of a blood smear, disease can be ruled out at this stage. The APHA VI should have already given prior authorisation for the OV to contact the keeper to confirm that the requirements in the Anthrax Order no longer apply, if the OV can find no evidence of disease. If no prior authorisation has been given, the OV will need to obtain verbal authorisation to inform the keeper from a Duty Vet, either directly or through APHA administrative staff.

2. The keeper remains responsible for sending carcases for appropriate disposal under the ABPR.

3. A Certificate of Non-existence of Disease (AN02) needs to be completed by the OV and in:

• England, returned to the APHA One Health CSC, Worcester
• Scotland and Wales, returned to the local APHA office.

4. Contact numbers are provided on the APHA website.

5. The veterinary inquiry will be officially closed by APHA VI sign off.

Disease cannot be Ruled out by the Initial Inquiry

1. However, if disease cannot be ruled out, if necessary for an inexperienced OV, following consultation with a more experienced appropriately trained veterinary colleague in the veterinary practice (also on Panel 1c), then the APHA Duty Vet must be contacted immediately. The Duty Vet will take a history from the OV and dispatch an APHA VI to provide a second opinion for the OV on the stained blood smear.

2. The APHA VI will visit the location, where the OV is waiting and examine the stained slide. They may decide that the stained smear or other smears collected are inadequate for examination, because they are too thickly smeared, or stained with the wrong stain. If this is the case the APHA VI may need to visit the farm location to collect additional samples.

3. If following the second opinion, a discussion of the clinical history and examination of the blood smear, disease can still not be ruled out, the APHA VI will take over the inquiry and take further action including:

• serving restriction notices
• advising the Local Authority (LA) on the need for carcase disposal and on the legal onus on the owner with regard to cleansing and disinfection of the premises
• packing and sending the samples off to the laboratory for analysis (GEN04).

4. If, during an outbreak of anthrax on a premises, there is material, such as animal feed, which is suspected as being the cause, then sampling of the product will be discussed by the APHA VI with the appropriate policy team through the Veterinary Exotic Notifiable Disease Unit (VENDU). Whilst the premises is under restriction, any material leaving the premises would need to leave under a licence.

5. The anthrax organism is not excreted in the milk until the terminal stage of disease and at this time milk production is likely to be scanty or absent. Pasteurisation of milk is expected to destroy the organism. If there is concern that milk from an affected animal may have entered the bulk tank at a time when the organisms may have been present in the milk, then the APHA VI will find out whether milk is being used, without heat treatment. In such cases, the keeper will be instructed to contact their dairy and the APHA VI will liaise with the appropriate public health authority.

• External OV Instructions/Anthrax/Veterinary_Inquiry