Diagnostic Laboratory Services- Analysis of Equine Respiratory Samples
New report format and disease classification
The pathology team in conjunction with the epidemiologists and equine clinicians at AHT have developed a new equine respiratory cytology results format, in addition to a new scoring system for classification of the severity of inflammatory disease. This allows us to provide a consistent approach to the classification of inflammatory respiratory disease.
Tracheal Cytology Inflammation Scoring
Sample Handling and Analysis – General Points
Samples may be collected via the transtracheal route or via a nasal tube or fibreoptic endoscope. Endoscopic collection is less invasive, allows coincident viewing of the tracheal lumen, and is generally well tolerated.
We recommend that samples are collected after exercise to provide as much data as possible about the condition of the respiratory tract. Efforts should be made to reduce possible contamination of the sample, eg do not feed the horse prior to endoscopy; ensure the endoscope and catheter are sterile before use; minimise the procedure time, to lessen the likelihood of inhalation of extraneous particles around the endoscope. Care should be taken to avoid causing mucosal damage or bleeding. It is helpful to record the amount or mucopus and or haemorrhage present in the trachea.
Infuse 20ml saline or phosphate buffered saline (PBS) down the trachea via the endoscope catheter (using a 50ml syringe). If mucopus is present in quantity it may be aspirated neat directly into 20ml saline or PBS.
Mix the aspirate very well in the syringe and divide into two aliquots immediately, before it sediments out. A plain sterile tube is required for bacterial cultures and a clean tube to which fixative can be added for cytology.
We recommend that BAL samples are fixed in 40% ethanol at a ratio of 1:1, since formalin as been shown to destroy any mast cells that may be present in the sample. The fixative for tracheal and guttural pouch washes is not as critical so, 40% ethanol or 10% buffered formalin may be used.
Any pharyngeal swabbing of horses for virus detection should be carried out before the wash is obtained. Virus isolation can also be carried out on a plain aliquot of the wash sample if required.
A new submission form has been developed to allow us to collect all relevant clinical information. A copy is attached.
Two levels of cytology reporting – your preference
We are currently offering 2 levels of cytology for respiratory samples:
Respiratory Cytology Screen The stained smears are examined by trained laboratory technical staff.
Respiratory Cytology Screen with Veterinary Interpretation The stained smears are examined by trained laboratory technical staff. Results are reported according to the revised format. A comment is added by one of the veterinary pathologists, to include consideration of clinical signs, history and the cytology findings.
Please note that we no longer perform red blood cell and nucleated cell counts on tracheal wash samples, as we have shown that the smear cell density correlates well with these values without the problem of clumping due to excessive mucus. We will continue to perform absolute and differential cell counts on BAL samples as these are from a more discrete region of the respiratory tract and consequently have less variability and are more straightforward to interpret. Cell count and smear cell density tend to correlate as follows, for tracheal wash samples: low smear cell density is equivalent to, up to 1,000 cells; medium, 1,000 – 10,000 cells; and high to more than 10,000 cells.
Routine and specific requests for bacteriology
A number of studies from different parts of the world have now shown that anaerobic bacteria are isolated from only a small proportion of tracheal wash samples (<5%) and that the majority of lower respiratory tract disease is associated with specific aerobic bacterial species. For this reason the Animal Health Trust now routinely conducts only aerobic culture and antibiotic sensitivity were appropriate on tracheal wash samples. Antibiotic sensitivity is carried out on: all species of haemolytic Streptococcus; Actinobacillus/Pasteurella-like organisms: Bordetella: Pseudomonas isolated as well as coagulase positive Staphylococci, and Rhodococcus equi. Additional organisms grown on sub-cultures with more than 103cfu/ml will also be tested for antibiotic sensitivity. This will have the advantage of improving turn around time for final reports and reduce the overall cost of routine bacteriological investigations for the majority of tracheal wash samples. However, anaerobic culture will also be conducted if specifically requested, and may be diagnostically rewarding in individual cases, particularly for those horses with more severe signs of respiratory disease.
Additional Advice
Please remember that additional advice about outbreaks of respiratory disease can be obtained from the epidemiology group here at the Animal Health Trust.
