Diagnostic Sampling

30th August 2013

Diagnostic Sampling- the where when and why!


Your flock have taken a turn for the worse, egg production has dropped, egg quality is poor or mortality has increased- what do you do?


For most, the first step is to call their vet to find out what is going on. Your vet is best placed to advise on possible causes but to do so they have to gather as much data about your flock as possible. This history taking is crucial in deciding what “differential diagnoses” are possible. Differentials are a list of possible causes for the condition being presented, it is often not possible to identify the cause of a problem just from the information provided or from a site visit but this information is nonetheless very important indeed. For any condition the veterinarian will formulate a possible list of “differential diagnoses” which will then determine what samples are needed in order to confirm the cause and ultimately to advise on treatment and/or future prevention.


For a mortality investigation, the first step is usually to undertake a post mortem examination. It is very important that the right birds are submitted for post mortem examination as this will determine the accuracy and relevance of the findings.

1)       Ensure that dead birds are selected for post mortem examination, whilst this sounds obvious, often live birds can be selected at random from the flock and submitted, this could be because the dead have already been discarded but these birds are unlikely to be representative. Live are of limited benefit in the investigation a) because they may not be have the same problem the dead birds were affected with, b) they may be in the early stages of infection and thus may not show any abnormalities.

2)       Ensure that carcasses are fresh- in the summer carcasses deteriorate very quickly indeed. Autolyses (decomposing) carcasses more than 24 hours old are very difficult for the veterinarian to examine and as such diagnoses may not be possible. Ensure that they are from fresh (less than 24 hours dead) and they are kept refrigerated in hot weather.

3)       Ensure enough birds are submitted- if high mortality is present in a flock then 10 dead birds should be collected where possible. With more examined birds it is possible to establish with greater confidence the cause of mortality.

4)       Ensure that the birds are selected at random- sometimes birds will be selected out, ie only the best feathered, non vent damaged, good conditioned birds are selected for post mortem but this will give a skewed representation of the cause of mortality in the flock. For example selecting only the non vent pecked birds for post mortem examination will prevent the veterinarian from knowing that aggression is present within the flock.


Post mortem examination will then be undertaken and depending on the findings additional tissues may be taken for further analysis. The common additional tests that will be conducted on tissues from post mortem examination are as follows:

a)       Microbiology- tissues will be selected where certain bacterial conditions are suspected. The surface of the tissue will be seared to sterilise any contamination and then swabbed onto blood agar and Mackonkey agar. These media are very good at detecting most bacteria as part of a general screen. Most bacteria will grow within 24-48 hours. Antibiotic sensitivity testing may also be conducted. This is the main method of diagnosis of common bacterial conditions such as E.coli, Erysipelas and Pasteurella. Additional media may be used to detect bacteria such as Ornithobacter and Moraxella. If Brachyspira is suspected then specific Brachyspira media is used for intestinal gut swabs – this has to be incubated in a anaerobic (0%) oxygen environment and can take up to a week to grow.

b)      Histology- whilst we can see some abnormalities with the naked eye, and we can grow bacteria on plates to produce colonies that we can see with the naked eye there are some conditions that produce changes at a microscopic level only, ie they cause their damage at a cell level only. Therefore, if differential diagnoses that cause gross pathology, and others that usually detected by microbiology are ruled out then histological testing may be advised. Samples are taken at post mortem examination, fixed in formalin, mounted in wax blocks, sliced into very thin layers, and the mounted and stained on glass slides. These tissues can then be inspected down a microscope to see if there are any changes to the cells.

c)       Intestinal scrapings and microscopy. If the intestine appears normal to the naked eye it is still important to check for worms at a microscopic level- this is easily achieved by scraping the lining of the intestine and creating a wet smear on a glass slide. It is then possible to screen for small worms like the hairworm (Capillaria) or worm eggs that are not visible with the naked eye.

d)      PCR swabs- some viral and bacterial conditions are not easy to diagnose at post mortem examination, there may be no gross pathology, the virus or bacteria that caused the problem may already be dead so will not grow. PCR stands for polymerase chain reaction. This is what is known as molecular diagnostics. The method involves taking swabs which hopefully pick up any genetic material from a virus or bacteria (even if that organism is dead). Then in the lab, the PCR test is able to replicate that specific genetic material many millions of times so that it is then detectable with further tests that we can interpret with the naked eye. This form of testing is very sensitive and specific and is often used for conditions such as brachyspira, infectious bronchitis, Avian rhinotracheitis and mycoplasma, where genetic sequencing can also be used to determine strains for example.

e)       Virology- viruses are very small and as such cannot be seen with the naked eye. Some viruses in fact cause very little changes and may cause very limited changes at microscopic level too. Consequently we can attempt to try and grow the virus in cells in the laboratory.


But what if I do not have a mortality problem in my flock- my birds are showing a decrease in production or egg quality issue.


In the above case there are likely to be different differentials but post mortem examination is also important. In this case select a mixture of live birds- some sick and some healthy looking birds. A sample selection of 10 birds is again optimal. Post mortem examination can be useful in this case to diagnose enteric (roundworm, hairworm, caecal worm and tapeworm) or external parasitism (lice and mite). It can also be useful to detect enteritis in the case of brachyspira or general bacterial enteritis for example.


Blood sampling- for production issues some viral causes and mycoplasma may be suspected. Your veterinarian may advise blood sampling so that sera (the liquid fraction of the blood) can be extracted and screened for antibodies. It is important that enough bloods are taken- 15-20 is ideal, and that 1ml of blood is taken in a sterile manner from the brachial vein (under wing) into clean blood tubes. These bloods must be transported to the lab within 24 hours, kept cool but not frozen.

Timing is critical- it is usually recommended that two sets of bloods are taken, one batch at the outset of a new problem, and a further batch 14 days later. Antibodies take time to develop in response to an infection. By comparing the bloods before and after it is possible to see if there had been a significant increase which will help to diagnose the problem. Birds should be selected at random from the flock for blood sampling.


Water sampling- often water quality issues can impact on gut health and drinker lines are notorious for supplying birds with high levels of pathogenic bacteria due to inadequate water sanitising systems. It is easy to test the water in your flocks drinker system. It is important to take 3 samples- one at the point of entry- from mains or bore supply to house, one on the exit of the header tank if present, and one at the end of the drinker line. Take samples into a sterile container (obtained from the laboratory), ensuring that the water has run for at least 10 seconds before collecting the free running stream of water. 50ml will be adequate. Submit to the laboratory. The laboratory will run a TVC (Total viable count), Pseudomonas and E.coli/coliform test. They will then be able to tell you how many bacterial there are per millitre of water and advise whether this is likely to be impacting on bird health. Your veterinarian can advise on ways to resolve any water quality issues.


Faecal sampling- screening for worms in free range flocks is very important. This should be undertaken every 6-8 weeks of a routine worming programme is not in place. This can be done by faecal sampling or submission of cull birds for post mortem examination (the latter being more sensitive and reliable). To take a faecal sample for worm screening then take 20 individual fresh droppings from random locations throughout the house and bulk them. Submit to the laboratory for a result within 24 hours.


PCR swabbing and testing- as indicated above these can be taken at post mortem examination but also from live birds with disease or symptoms. They are taken from the upper respiratory tract- usually 10 birds are selected at random, the beak opened and a swab gently rotated in the back of the throat (ideally inserted into the choanal cleft (the V shaped opening in the under side of the upper beak.))


From the above it is clear to see that there are many samples that may need to be taken to investigate a problem but that your veterinarian is best placed to advise on which samples are to be taken. Taking the right samples in the correct way, the correct number of samples, and at the right time can all influence the accuracy and relevance of any laboratory findings so it is worth taking the time to get it right as ultimately delayed or inaccurate results are likely to impact on flock health and productivity for a longer time!


Philip Hammond, Claire Knott, Stephen Lister andIan Lowery

Crowshall Veterinary Services LLP