Laboratory diagnosis is essential to confirm the presence or absence of T. equigenitalis, K. pneumoniae and P. aeruginosa in swabs taken from mares and stallions.
Types of swab
There are two types of swab:
Clitoral swab: taken from two sites; the clitoral fossa and the clitoral sinuses using mini tip swabs to ensure appropriate sampling of sinus contents, at any point during the reproduction cycle to demonstrate whether these sites are free from infection.
In the case of pregnant mares who have had difficult foalings requiring veterinary attention and antibiotic treatments, additional clitoral swabs should be taken after foaling and more than 7 days after antibiotic treatment has finished, in addition to routine endometrial swabs, in order to rule out acquired K. pneumoniae and P. aeruginosa infections. Providing the prefoaling clitoral swab was certified negative for T. equigenitalis, the additional post-foaling clitoral swab may be tested by aerobic culture only, or by PCR.
Endometrial swab: taken during oestrus from the lining of the uterus via the open cervix to demonstrate whether the uterus is free from infection.
Mare swabs taken for disease prevention purposes should be tested according to the recommendations on pages 9–12.
Note: These are minimum recommendations. Mare owners should check whether the stallion stud, boarding stud or local breeders’ association (e.g. NSFA) has any additional requirements.
Swabs should be taken from three sites; the urethra, urethral fossa and penile sheath, plus pre-ejaculatory fluid when possible. Separate swabs should be used for each site and tested by aerobic and microaerophilic culture and/or by PCR test, in all circumstances.
Further information on how to collect equine genital swabs in stud practice for the prevention of venereal diseases, as recommended by the Codes of Practice, is available online here.
All swabs should be taken by a veterinary surgeon, who should:
• Pay attention to the PowerPoint presentation ‘How to Collect Equine Genital Swabs in Stud Practice’ by Professor Sidney Ricketts FRCVS https://www/rossdales.com/wp-content/uploads/2018/01/Collecting- Swabs-2017.pdf
• Clitoral swabs must be taken using a micro tip (mini tip) swab to get deep into the clitoral sinuses.
• label them clearly to show the date and time they were taken, the horse’s name and the site of swabbing;
• indicate clearly whether aerobic, microaerophilic or both cultures, and/or PCR test are required;
• submit them to a BEVA registered Laboratory for testing. Culture must commence within 48 hours of the swab being taken.
A list of laboratories in the UK, Ireland, France and Germany registered with BEVA for the purposes of testing for T.equigenitalis, K. pneumoniae and P. aeruginosa is available from the BEVA website.
Submitting swabs to BEVA Registered Laboratories
The BEVA registered laboratories must set up swabs for conventional microaerophilic culture for T. equigenitalis within 48 hours of them being taken from the horse as this organism is short lived, even in bacteriological transport medium. Veterinary surgeons submitting swabs by routine postal services are, therefore, advised not to take swabs on Fridays, Saturdays or Sundays as they may not arrive in time. If weekend or bank holiday swabbing is unavoidable, the veterinary surgeon should ensure that the laboratory is open and able to commence cultures within the 48 hours. In this event, a suitable courier service should be used to deliver the swabs. If a swab does not arrive in time, the laboratory should reject it and advise the veterinary surgeon to repeat the swabbing.
However, time constraints do not apply to swabs submitted to laboratories that are registered to run PCR tests for T. equigenitalis as specific DNA from non-viable organisms can be detected for long periods.
Experience suggests that swabs cultured aerobically for K. pneumoniae and P. aeruginosa are not so time sensitive and these organisms have a long life in bacteriological transport medium, as they do in the environment.
Laboratory culture of swabs
Laboratories can culture swabs in two ways: aerobically and microaerophilically (see Glossary, Appendix 10). The results of culture will be returned by the laboratory on an official Laboratory Certificate. When planning the timing of breeding activities, breeders and veterinary surgeons should be aware that the results of microaerophilic culture will not be available for at least seven days. Aerobic swabs require 24 hour (overnight) culture before the initial result can be reported. If these results are satisfactory, ‘low risk’ mares may then be mated. However, final aerobic culture results will not be available for 48 hours (to exclude the possibility of slow-growing P. aeruginosa organisms), so mating before these results are available is at the stallion stud’s own risk. PCR test results do not have these time delays.
Other laboratory tests
Polymerase chain reaction (PCR) testing of swabs for T. equigenitalis, K. pneumoniae and P. aeruginosa is validated for industry screening purposes. PCR testing is not recognised for import/export testing in the UK. Breeders and veterinary surgeons may find PCR test results helpful, as they may be available on the same day that a sample is received at a laboratory that is able to undertake PCR testing. Positive PCR test results will need to be further investigated by conventional culture to help determine their significance and, in the case of K. pneumoniae, for capsule typing, unless being tested, in the same laboratory, for capsule typing by PCR test. Positive PCR test results for T. equigenitalis must be reported to Defra/APHA (see Appendix 11).
The immunofluorescence test (IFT) for T. equigenitalis, which is available only in France, is not acceptable on its own, although it may be used in addition to culture.
Preparing mares for covering
It has been brought to the Codes of Practice Committee’s attention that some mares are now being presented for walk-in coverings with only PCR test certification and sometimes incomplete gynaecological preparation.
The Code of Practice recommends PCR testing of uterine swabs from mares because this is currently the best method to rule out the contagious endometritis infections caused specifically by T. equigenitalis, K. pneumoniae and P. aeruginosa infection, which is the principal objective of this Code. However, this test is not a substitute for traditional aerobic cultures and a proper full genital examination of all mares, for signs of other uterine endometritis infections or non-infectious endometritis, or other significant genital abnormality. This examination must be performed when the mare is in early oestrus, when she will have a sufficiently relaxed cervix to allow proper swabbing, and should include:
1. Visual inspection of the vulva, vestibule and vagina to confirm adequate vulval closure, correct post-partum healing of vulval tears or Caslick repairs and the absence of signs of pneumovagina, which will need correction, prior to covering.
2. Vaginascopic examination of the cervix and vagina to confirm a relaxed oestrous cervix and no signs of parturient tears or inflammation to suggest pneumovagina, which will need repair/resolution.
3. Rectal palpation and ultrasound examination to monitor ovarian follicular development to allow the optimal time for covering to be predicted and signs of ovarian and/or uterine abnormality (e.g. delayed uterine involution and/or excessive and/or turbid uterine fluid) to be ruled out. If found, these abnormalities should be treated and time allowed for resolution before the mare is sent for covering.
4. When the cervix is adequately relaxed, swabs should be taken for PCR testing (see above) and for aerobic bacterial culture and, ideally, at the same time, for a smear cytology test. This will allow endometritis, caused by any infectious organism (most commonly with equine skin or environmental contaminants) or non-infectious endometritis (indicating inflammation rather than active infection) to be ruled out. The smear test will provide a more precise interpretation of the significance of bacteria cultured from the swab and will allow the diagnosis of noninfectious
If any of these problems are detected, covering at this stage is more likely to result in failure of conception or early pregnancy failure. The mare will then require further, perhaps more prolonged, treatment to return her into a state for covering again and valuable time will have been lost out of the breeding season, for the mare to achieve a pregnancy. This is why such careful preparation of mares for covering is helpful for the mare and all concerned and is therefore recommended.
Note: The term 'at risk' relates to any horse which may have become infected as a result of direct or indirect transmission of the disease.
References to swab testing mean appropriate testing by culture and/or PCR, where a laboratory is registered with BEVA for this method.
Further information on how to collect equine genital swabs in stud practice for the prevention of venereal diseases, as recommended by the Codes of Practice, is available here.