1. Simple tests

There are few causes of resting hyperglycaemia (normal 3.4-6.5 mmol/l) in the horse other than ECD so, if present, this finding is fairly specific. However, most horses with ECD have serum glucose concentrations within the reference range so this makes the test very insensitive. Other causes of hyperglycaemia to be aware of include acute stress, a2 agonist sedatives and a cereal-based feed within the past 2-3 hours. Hence the testing of blood glucose should preferably be done first thing in the morning before feeding and also the stressful effects of problems such as transportation or acute laminitis should also be considered. In any case it would be preferable to reconfirm the presence of resting hyperglycaemia before placing too much importance on its presence.

Resting serum cortisol (normal 50-175 nmol/l) is of little use in suspected ECD cases. Published reports are as likely to have found high, low or normal resting cortisol in ECD cases. Some reports have suggested there is loss of the normal diurnal cortisol rhythm in ECD cases and it has been suggested that 2 blood samples taken 8 hours apart can be compared and used to aid diagnosis – the theory being that in normal horses with a normal diurnal rhythm the cortisol concentration will differ in the two samples by >30%. ECD cases may however show similar cortisol levels in both samples. This is a simple test which requires no stimulation testing although must be interpreted with caution due to other extraneous effects on endogenous cortisol production. Clearly stress, pain (e.g. laminitis) and diet have a big effect on cortisol secretion and will influence the results. Results of this test would be more reliable in a pain-free case not receiving hard feed during for at least 4 hours before the test period.

Resting serum insulin (normal 5-36 miu/ml) has become more popular recently and can be useful but again should be interpreted with great care. Many horses with Cushing’s disease are indeed found to have elevated resting insulin concentrations due to cortisol-induced antagonism of insulin and also the pro-secretory effect of CLIP (one of the pituitary derived products in ECD cases). However, diet and stress also have profound effects on insulin secretion and these should be carefully considered. A hard feed will elevate insulin levels for up to 5 hours. We have seen insulin levels as high as 250 miu/ml in non-Cushingoid horses with painful conditions such as colic and it is likely the pain of laminitis could have a similar effect. Thus insulin is not a suitable test for ECD horses with active laminar pain and also is best measured first thing in the morning before feeding.

Resting serum ACTH (normal < 7 pmol/l) is a very sensitive and specific test for ECD but is greatly limited by the stability of ACTH in blood samples. ACTH is adsorbed by glass so blood must be taken in plastic tubes. The test must then be performed immediately or the sample must be kept frozen on its way to a laboratory.These problems severely limit the practical use of what might otherwise be a very useful test. Additionally, not all horses with Cushing's disease have been found to have increased ACTH.

Urinary corticoid : creatinine ratio (normal <20 x 10-6) in urine samples collected first thing in the morning has been used by some. This is a simple test with reasonable reliability although results will be affected by the same influences as discussed above with serum cortisol and also may not be reliable in dilute samples of urine which are commonly found in ECD cases (due to polydipsia/polyuria).

2. Dynamic tests

The overnight dexamethasone suppression test is the most accurate test to have been reported in the literature. The procedure involves measuring baseline cortisol at about 4-5 pm; injecting 40 mg/kg dexamethasone iv or im (10 ml of 2mg/ml solution per 500kg); followed by a post stimulation cortisol at about 11-12 am next day (approx. 19 hours later). Normal horses show marked suppression of cortisol (<25 nmol/l) in the post stimulation sample whereas ECD cases show incomplete suppression. In a large study in USA with post mortem confirmation of ECD this test was highly accurate. It may be overstating the real situation to say it is completely accurate but it does seem to be the best available test. Importantly reports of precipitating or worsening laminitis following the test are very rare although this potential problem should be discussed with the owner first.

The TRH stimulation test is primarily used when concerns over the use of dexamethasone suppression exist (i.e. laminitis). The procedure involves measuring baseline cortisol; injecting 1 mg TRH iv (NB. 1 mg TRH costs approx. £100); followed by a post stimulation sample about 30 minutes later. Normal horses usually show no real difference between the cortisol concentrations in both samples whereas ECD cases tend to show elevation of cortisol in the 2nd sample (at least 20% rise but usually much greater). The test gives reasonable reliability but there are significant numbers of false positive and false negative results.

The ACTH stimulation test is too unreliable for clinical use. It is reported that ECD cases show a post stimulation cortisol of > 420 nmol/l and/or > 3 x baseline at 2 hours following ACTH administration. A positive result could be considered supportive of the diagnosis of ECD but is not at all definitive and of questionable value.

Combination tests exist including combined ACTH stimulation-dexamethasone suppression test and combined dexamethasone suppression-TRH stimulation test. None of these tests have been properly validated with post mortem confirmation of diagnosis in tested horses and also there are important theoretical concerns in the accuracy of combination tests which may well simply magnify the inaccuracies in the individual tests. Therefore it is difficult to justify the use of such tests.

© The Liphook Equine Hospital 2005

Diagnosis: Of the bacterial pathogens Salmonella and Aeromonas sp identification is relatively straightforward and both can be grown with reasonable success on routine culture media. Intermittent shedding of salmonellae may lead to false negative results and repeat samples are always advisable. Successful culture of C. difficile and C. perfringens is difficult and not diagnostic per se as not all strains are toxigenic. Hence the preferred approach to diagnosis of Clostridial diarrhoeas is ELISA identification of clostridial toxins - eg. C. perfringens enterotoxin (CPE) and C. difficile toxins A and B (TOX A/B). Detection of both Rotavirus and Cryptosporidium in cases of foal diarrhoea also requires an immunological technique – namely immunochromatography (ICT). All of these tests (see over) can be easily performed on a small sample of faeces.

(* all tests performed on faecal samples; # based on only one publication)

Treatment: The use of antibiotics in bacterial diarrhoeas has always been a contentious issue but is less contentious when a specific agent has been identified for which a suitable antibiotic can be targeted. For example, metronidazole is effective against the vast majority of Clostridial enteropathogens and enrofloxacin is effective against the vast majority of Salmonellae and Aeromonads. Codeine phosphate is an old and reliable means of halting diarrhoea with typically 1-2 mg/kg bid per os being effective in most cases or up to 3 mg/kg tid in the more severe cases (NB. a slow withdrawal of treatment is vital to success). As an alternative loperamide (‘Imodium’) can be used at 0.1-0.2 mg/kg bid per os (2-5 capsules bid per 50kg foal). Adsorbents such as bismuth subsalicylate (‘Pepto-bismol’) at 1 ml/kg bid per os is a useful adjunct to acute diarrhoea in horses and foals and some cases benefit from therapy to counter bowel oedema including corticosteroids and hetastarch.

© The Liphook Equine Hospital 2005

Serum amyloid A - We have been using SAA at the Liphook Equine Hospital Laboratory for approximately 2½ years now. We have found the test both reliable and sensitive to acute inflammation in horses and it has become our preferred diagnostic and monitoring acute phase protein in cases such as viral/bacterial respiratory disease, peritonitis, colitis etc…. Most normal horses have SAA concentrations around 5 ?g/ml and with severe inflammatory disorders this can rise to approximately 1000 mcg/ml creating a pathophysiological range of about 200X normal. Compared with fibrinogen this allows a far greater ‘grading’ of severity of the inflammatory process and more sensitive monitoring of progress.

© The Liphook Equine Hospital 2005

4. Further laboratory work

On the basis of the above results it should be possible to confirm/rule out CRF and DM. Cushing’s Disease (if not already suspected from DM) is usually suggested by typical clinical signs (old, hairy, laminitic etc..) or ‘overnight’ dexamethasone suppression test.

a. Water deprivation test
A water deprivation test will be required to differentiate psychogenic polydipsia from DI (both of which produce hyposthenuric urine). This test must not be performed on azotaemic horses. The object of the test is to establish whether or not the horse can produce concentrated urine (psychogenic polydipsia cases can; DI cases cannot).

Weigh horse accurately (if possible).
Check BUN and creatinine are normal (if not don’t proceed).
Take urine sample and measure SG (will usually be < 1.008 in cases requiring this test).
Remove water.
Check serum BUN and creatinine and urinary SG at least every 6 hours.

End of test is when one of the following occurs:
- 24 hours water deprivation
- 5% reduction in bodyweight
- clinical signs of dehydration
- azotaemia develops
- urinary SG > 1.020

Interpretation:
- if SG rises above 1.020 this confirms renal concentrating ability and therefore psychogenic polydipsia.
- if urine SG stays low and horse becomes dehydrated or loses 5% bodyweight this suggests DI (often happens by 12 hours with DI), although could be psychogenic polydipsia associated with ‘medullary washout’.
- if urine SG is still low after 24 hours but horse shows no clinical signs of marked dehydration this implies psychogenic polydipsia associated with ‘medullary washout’, although could be DI.

b. Modified water deprivation test
Strictly speaking this test is used to differentiate DI from psychogenic polydipsia. However, most DI cases will become rapidly dehydrated within 12 hours of water deprivation (see above) and this test is really used to confirm the suspected diagnosis of psychogenic polydipsia with medullary washout.

Start immediately following standard water deprivation test above if:
- urine SG < 1.020 after 24 hours water deprivation
- < 5% reduction in bodyweight
- no azotaemia
- no clinical signs of dehydration.

Test involves allowing restricted access to water for 2-3 days or until one of the criteria above is reached. Allow water consumption equivalent to 4% bodyweight per day (20 litres per 500kg) offered in several aliquots through the day.
Measure serum BUN and creatinine and urinary SG at least every 6 hours.

If urine SG rises above 1.020 this confirms psychogenc polydipsia. Continued inability to concentrate urine confirms DI.

c. ADH response test
Used to differentiate central and renal DI. Central DI cases successfully concentrate urine following ADH administration. Renal DI cases shown no response to ADH administration.

© The Liphook Equine Hospital 2005

Clearly many other laboratory parameters and tests may be useful in individual cases and profiles are adaptable to individual situations. Testing for equine Cushing’s disease remains a common clinical dilemma with a multitude of potential choices of test available, none of which are absolutely sensitive or specific. However, the ‘overnight dexamethasone suppression test’ appears to produce highly reliable results although false positives and negatives are still occasionally found.

© The Liphook Equine Hospital 2005

Occasionally increased numbers of eosinophils are seen for example in rare cases of lungworm or eosinophilic interstitial pneumonitis. Some young horses with ‘Inflammatory Airway Disease’ (IAD) may be found to have a high percentage of mast cells. Rarely high lymphocyte percentages are seen possibly as a result of respiratory viral disease?

By far the commonest abnormal finding in cytological analysis of airway secretions is neutrophilia. The wide normal range for TW neutrophils makes this finding difficult to interpret and many normal horses in dusty environments show marked TW neutrophilias in the absence of disease. BAL is far more specific for detecting genuine airway neutrophilia due to the smaller reference range. BAL neutrophils accounting for > 10% total cells confirms lung disease. In adult horses, airway neutrophilia usually arises as a result of allergic lung disease caused by ‘recurrent airway obstruction’ (RAO - the term now preferred for the conditions previously known as chronic obstructive pulmonary disease (COPD) and summer pasture associated obstructive pulmonary disease (SPAOPD)). However, infectious lung disease (bacterial or viral) will usually result in airway neutrophilia also. These disease types cannot always be reliably differentiated simply on BAL although generally the magnitude of the neutrophilia is higher in RAO cases (eg typically 30-70% PMNs and up to 90%+) than infectious diseases (eg. rarely >30% PMNs although can be much higher in some cases) and also the PMNs appear more ‘healthy’ and less degenerate in allergic airway disease compared with infectious airway disease. The results of TW culture, clinical signs and history also add important differential diagnostic information.

© The Liphook Equine Hospital 2005

The three most common leucogram abnormalities are neutrophilia, eosinophilia and monocytosis. Neutrophilia (PMN > 6.5 x 109/l) is the commonest of these. Neutrophilia is usually the result of an acute or chronic inflammatory response. This may be caused by infectious (viral, bacterial or parasitic) or non-infectious (eg. azoturia, surgical trauma, immune-mediated disease, neoplasia) disease. Another potential cause of neutrophilia in the horse is stress associated with concurrent disease, overtraining or poor management. The typical effect of endogenous or exogenous corticosteroid on the leucogram is neutrophilia associated with a lymphopaenia and eosinopaenia. Therefore this type of leucocyte pattern in sick horses may be a non-specific result of the primary condition rather than a specific indicator of an inflammatory aetiology. Short term excitement perhaps associated with venepuncture of a nervous horse or transport immediately before sampling can also result in neutrophilia but lymphocytosis may be more likely in these cases.

Eosinophilia (Eos > 0.8 x 109/l) is very rarely found in association with intestinal parasitism in horses. Eosinophils undoubtedly play a role in host defence against parasitic infections but are found local to the parasite. Hence intra-arterial S.vulgaris larvae (although very rare) may well be associated with a peripheral eosinophilia, lungworm infection is usually associated with an eosinophilia in tracheal washes or bronchoalveolar lavage samples, encysted cyathostomes are associated with eosinophilic infiltrates detectable in caecal, colonic and sometimes rectal biopsies. Eosinophilia is unfortunately a fairly non-specific finding as the eosinophil has many general roles in host defence and eosinophilia is often seen as a non-specific component of a systemic inflammatory reaction (eg. viral infections). Eosinophils are attracted by mast cell degranulation and have therefore been associated with antigen-antibody interactions in tissues rich in mast cells such as skin, respiratory tract and intestine and peripheral eosinophilia is certainly seen fairly consistently in association with hypersensitivity reactions such as sweet itch.

Monocytosis (Monos > 0.5 x 109/l), similar to neutrophilia and eosinophilia, is a non-specific inflammatory indicator seen to rise in both acute and chronic inflammatory conditions and tissue damage.

Is it possible to differentiate bacterial and viral disease on the basis of haematology?

The most consistent haematologic finding associated with the early stages of viral infections (ie. the time when we are usually asked to examine the horse and take a diagnostic blood sample) is a neutrophilia (PMN > 6.5 x109/l). This has been demonstrated in association with many types of viral infection in adult horses and is indistinguishable from bacterial infections on the basis of haematology. However, later in the course of disease then mild neutropaenia and possibly lymphocytosis and monocytosis would typify viral disease, whereas bacterial infections more typically remain neutrophilic with, possibly, a monocytosis, unless severe whereupon neutropaenia may occur:

Is it possible to diagnose parasitism on the basis of haematology or serum proteins?

Nematode infections in the adult horse were once typified by intra-luminal adult worms and larval migration associated with Strongylus vulgaris. These were often associated with an eosinophilia detectable in blood samples in response to intra-arterial larvae and also, in some instances, a detectable increase in b1-globulins (especially IgG(T)). Parasitological surveys in more recent years have consistently confirmed the decline of S. vulgaris (almost to the point of extinction in many instances) in conjunction with a domination of parasitic burdens by members of the cyathostome genus (small strongyles) which consistently comprise almost 100% of nematode eggs detected in equine faecal samples in this country. Cyathostome infection results in encystment of larvae locally in the caecal and colonic wall but is not associated with larval parasitic migration. An eosinophilia is not associated with cyathostome infections and a raised b1-globulin fraction is a very inconsistent and non-specific finding.

Several research studies have failed to confirm any clinically useful relationship between serum protein electrophoresis and parasitism in horses. Normal concentrations of IgG(T) and b1-globulins are usually found in parasitised adult horses and ponies although changes may be more likely in young horses. In a recent investigation of horses with chronic diarrhoea, 55% of horses with parasitic diarrhoea did not have raised b1-globulins and 37% of horses with raised b1-globulins had no evidence of parasitism. Importantly, statistics would imply that serum protein electrophoresis would be even less predictive of parasitism in blood samples taken from non-diarrhoeic horses. ‘Cyathostomosis’, the acute diarrhoea and weight loss syndrome associated with en masse larval emergence, is consistently associated with a neutrophilia, hypoalbuminaemia and hyperfibrinogenaemia (all unfortunately non-specific findings), but other than this blood samples taken from parasitised horses show no consistent abnormalities in haematology or protein analyses.

Prime considerations in interpretation of the leucogram

These lists are not intended to be exhaustive and comprehensive but rather to serve as an aide memoire for the more common causes of abnormalities of the leucogram seen in the adult horse.

Neutropaenia (PMN < 2.5 x 109/l)

- Excessive demand/sequestration (eg. septicaemia, bacterial peritonitis, pleuritis)
- Endotoxaemia
- Viral infection (mid-late phase response)

Lymphocytosis (LC > 5.0 x 109/l)

- Infectious diseases (primarily mid-late phase viral)
- Catecholamines (acute excitement/fear)

Lymphopaenia (LC < 1.5 x 109/l)

- Infectious diseases (eg. early EHV)
- Endogenous/exogenous corticosteroid (stress, equine Cushing’s disease)

Eosinophilia (Eos > 0.8 x 109/l)

- Hypersensitivity diseases (eg. sweet itch, urticaria)
- Inflammatory diseases (see neutrophilia)
- Parasitic larval migration (rare)

Monocytosis (Monos > 0.5 x 109/l)

- Inflammatory diseases (see neutrophilia)

© The Liphook Equine Hospital 2005