EQUINE CLINICAL PATHOLOGY - Rossdale & Partners

the 

beaufort cottage laboratories Guide to 

equine clinical 

pathology

T h e B e a u f o r t c o t t a g e l a b o r a t o r i e s 

Welcome to the Beaufort Cottage Laboratories 

Guide to Equine Clinical Pathology. 

We hope this Guide will provide practical help to 

busy veterinary clinicians, nurses and students in 

need of a reference source in the course of their 

daily work or studies. It is not intended to be a 

textbook, nor do we pretend it is complete. 

For further advice on pathology issues raised in this 

Guide, please call Beaufort Cottage Laboratories 

on +44 (0)1638 663017 (office hours). In case of 

emergency, please call Rossdale & Partners on 

+44 (0)1638 663150 (24 hours).

G u i d e t o e q u i n e c l i n i c a l p a t h o l o g y 

Contents 

Introduction 4 

Using clinical pathological aids to diagnosis 6 

Clinical disease, preventive medicine, laboratory profiles, management aids 

Sampling requirements 8 

Labelling and request forms, dispatch, post & packaging, sampling equipment 

Reference ranges 11 

Haematology 12 

Erythrocytes 12, Leucocytes 14, Platelets 17 

Clinical chemistry 19 

Proteins 19, IGG 21, plasma fibrinogen, SAA, AST, 22, CK, LD 24, cardiac troponin 25, SDH, 

GLDH, GGT 27, SAP, IAP, bilirubin, bile acids 28, amylase, glucose, oral glucose absorption 

test, cholesterol & triglycerides, urea 29, creatinine, urine fractional clearance ratios 30, 

calcium, potassium & chloride, calcium, phosphate & magnesium 31, plasma lactate 32 

Blood gas analysis 33 

Endocrinology 33 

Pregnancy tests, progestagens 33, granulosa cell tumour 34, cryptorchidism, 

thyroid function, pituitary function, glucose, cortisol, insulin, overnight dexamethasone 

suppression test 35, TRH stimulation test, combined DXM suppression/TRH stim. test 36 

Urine collection and analysis 37 

Parasitology 38 

Faeces collection, faecal worm egg counts, faecal lungworm larval counts 

Microbiology 39 

Bacterialogy, skin scrapings, virology 40 

Cytology 42 

Fluid samples - peritoneal, pleural 43, synovial, tracheal washes, bronchoalveolar lavage 44, 

cerebrospinal fluid, bone marrow 45, semen samples, endometrial smears 46 

Histology 47 

Necropsy (postmortem) examinations 48 

Adult horses 48, CMSNG sampling, neonatal foals, foeti and placentae 50 

Biopsy sampling 52 

Skin, lump, liver, lung 52, kidney, endometrial 53, testicular, ileal 54, rectal 55 

Tables of reference ranges 56


introduction 

In all aspects of veterinary medicine, an 

accurate diagnosis is a pre-requisite for 

specific treatment and appropriate case 

management. Making a diagnosis involves 

a challenging combination of art and science and, if 

used correctly, laboratory investigations may be helpful. 

Following a definitive diagnosis, follow-up clinical 

and laboratory examinations can assess the effects of 

treatment. Where progress is unsatisfactory, more detailed 

investigations are often indicated as directed by the 

characteristics of the individual case. 

Making a diagnosis can be likened to ‘jigsaw puzzling’. 

The more pieces become available, the easier it becomes 

to solve the puzzle. Historical information and clinical 

examination results remain the first steps in the diagnostic 

pathway, sometimes allowing a definitive diagnosis to 

be made immediately, e.g. for a mid-shaft cannon bone 

fracture. More often, a differential diagnosis needs to be 

refined by clinical pathological aids and further clinical 

diagnostic procedures, e.g. recurrent laminitis and 

hepatopathy associated with equine Cushing’s syndrome. 

These aids must be applied appropriately, accurately and 

efficiently, to best effect. 

Incorrect results or incorrectly interpreted results may 

confuse the diagnostic pathway, leading to incorrect 

treatment and case management. In-house quality control


and external quality assurance are important for all 

veterinary laboratories to ensure reliability of results and 

to give confidence both to laboratory staff and to their 

clients. 

Veterinary surgeons have a duty to relieve suffering 

in animals as quickly and efficiently as possible. Ideally, 

to achieve this, they should provide their patients with 

the most accurate diagnosis possible, as early as possible, 

using whatever aids are available and appropriate so that 

early treatment is successful. This may appear costly in the 

short term, but can sometimes save expense in the longer 

term by avoiding delays in starting appropriate treatments 

or by avoiding the use of inappropriate treatments and 

thereby shortening time to recovery. In addition to welfare 

considerations, early accurate diagnoses and treatments 

bring significant benefits for performance horses and aid 

profitability to their owners. 

Clinicopathological aids may be applied in cases of 

clinical disease and for the assessment of their treatment, 

in preventive medicine programmes, and as management 

aids. The tables on the next two pages are presented as 

broad guides to the use of laboratory aids to diagnosis 

in specific clinical, preventive medicine and managerial 

indications. 

August 2006


Using Clinical Pathological aids to diagnosis 

Clinical disease 

Clinical condition 

Infection 

Intestinal parasitism 

Liver disease 

Kidney disease 

Pancreatic disease 

Skin disease 

Bone metabolic/parathyroid 

abnormality 

Weight loss/diarrhoea 

Pulmonary abnormality 

Fluid/electrolyte balance 

Stallion genital abnormality 

Mare genital abnormality 

Potentially useful tests 

Haematology, serum amyloid A, plasma fibrinogen, serum 

protein electrophoresis, bacteriology, mycology, virology, 

serology. 


albumin and protein electrophoresis, tapeworm ELISA, faecal 

worm egg count, rectal biopsy. 


protein electrophoresis, AST, LD and isoenzymes, GGT, GLDH, 

SAP, bile acids, liver scan and biopsy. 


proteins, urea, creatinine, urine analysis and electrolytes, 

fractional electrolyte clearance ratios, bacteriology, kidney scan 

and biopsy. 

Serum GGT, amylase and lipase. 

Skin scrapings, biopsy, bacteriology, mycology, feed allergen 

tests. 

SAP, calcium and phosphate, urine phosphate 

clearance ratios. 


albumin and protein electrophoresis, SAP, IAP, glucose 

absorption test, peritoneal fluid cytology, faecal Rotavirus assay, 

serum electrolytes, feed allergen tests. Faecal occult blood. 


proteins, blood gas analysis, faecal lungworm examination, 

tracheal wash/BAL cytology, bacteriology. 


albumin and proteins, electrolytes, urine analysis and fractional 

electrolyte clearances, blood gas analysis. 

Endocrinology, bacteriology, semen analysis, testicular biopsy. 

Endocrinology, bacteriology, endometrial cytology and biopsy.


Preventive medicine 

Clinical condition 

Intestinal parasitism 

Venereal disease 

Diarrhoea 

Nasal discharge 

Skin lesions 

Useful tests 


albumin and protein electrophoresis, tapeworm ELISA, faecal 

worm egg count. 

Penile and preputial, clitoral and endometrial aerobic and 

microaerophilic bacteriology. 

Rectal/faecal bacteriology for Salmonella spp. and 

Campylobacter spp. and fluid faeces for Rotavirus test. Faecal 

occult blood test. 

Nasopharyngeal, guttural pouch and tracheal washes for 

Streptococcus equi and Rhodococcus equi bacteriology. 

Scrapings for dermatophytes, skin biopsy. 

Laboratory Profiles 

Age/type/condition 

Useful tests 

12-36 hours Haematology, serum amyloid A, plasma fibrinogen, serum 

proteins, IgG. 

Weanling and yearling 


proteins, SAP, calcium, phosphate, urinary phosphate fractional 

clearance ratios. 

Inflammatory disease 


proteins and electrophoresis. 

Horses in training 


proteins, AST, CK. 

Mature horse complete profile Haematology, serum amyloid A, plasma fibrinogen, serum 

proteins and electrophoresis, AST, CK, LD and isoenzymes, GGT, 

GLDH, SAP, IAP, urea, creatinine. 

Management Aids 

Management requirement 

‘Unfitness’ (horses in training) 

Mare pregnancy tests 

Mare luteal function 

Anthelmintic programme efficacy 

Useful tests 


proteins, AST, CK. 

45-95 days – serum eCG. 

>120 days – serum oestrone sulphate. 

>150 days – urinary oestrogens. 

Plasma progesterone. 

WEC, tapeworm ELISA


sampling requirements 

Laboratory results are only as good as the samples submitted 

allow them to be. Samples should be taken by the correct 

techniques, with suitable equipment, into suitable containers 

and media/preservatives and securely dispatched to the 

laboratory in the shortest possible time. 

 

Labelling and request forms 

All specimens should be labelled legibly 

with the name of the horse, date and 

time of sampling and site of collection, 

if appropriate, to enable proper reporting 

and certification of results. A concise 

case history should always be included, 

to help the laboratory make sure that 

the appropriate tests are performed and 

reported quickly and efficiently and in order 

to allow the clinical pathologist to help the 

clinician interpret the results. 

Dispatch 

Avoid sending perishable specimens over 

the weekend, where they may be held 

up and deteriorate in the post. Where 

possible it is sensible to centrifuge or to 

allow clotted blood samples to stand until 

it is possible to pour or pipette off the 

serum, in order to avoid haemolysis. Do 

not refrigerate EDTA samples or haemolysis 

will render the results useless. Do not send 

frozen or unfixed tissues for histological 

examinations as they will undergo natural 

autolysis and arrive in a less than suitable 

or even useless condition for satisfactory 

examination. 

For urgent samples and for dead foals, 

foeti and placentae for postmortem 

examinations, we strongly recommend 

personal delivery, e.g. by owner or private 

courier service. 

Post and Packaging 

Please instruct your secretarial staff 

in the proper packaging of specimens 

so that disappointments do not occur. 

Sound packaging is essential to avoid 

the breakage of containers in the post 

and the loss of, or damage to samples. 

Internal and external packing is essential 

('Jiffy bags' alone cannot be relied upon). 

The leakage of pathological specimens 

can present public health hazards and 

the Royal Mail may refuse to handle 

soiled packages. The following notice was 

published and circulated by the Royal 

College of Veterinary Surgeons in August 

1988:- 

1. In general, the despatch of deleterious 

substances by post is banned by the 

Post Office. There are however special 

exemptions for pathological material sent 

to and from laboratories by veterinary


Sampling Equipment 

Test Sample Container/anticoagulant/preservative 

Haematology Whole blood EDTA (lilac Vacutainer or blue Monovette) 

Plasma fibrinogen Whole blood Sodium citrate (blue Vacutainer or green 

Monovette) 

Clinical chemistry, 

serology, minerals Serum Empty tube (red Vacutainer or brown Monovette) 

and electrolytes 

Plasma glucose Whole blood Fluoride/oxalate (grey Vacutainer or yellow 

Monovette) 

Plasma progesterone Plasma or serum Lithium heparin (green Vacutainer or orange 

Monovette) 

Mare pregnancy Serum or plasma Empty tube (red Vacutainer or brown Monovette) 

Urinalysis Urine Sterile, empty, leak-proof container 

Blood selenium or 

Lithium heparin (green Vacutainer or orange 

glutathione peroxidase Erythrocytes Monovette) 

Bacteriology Swabs 

Amies charcoal transport medium 

Fluids 

Blood grow medium 

Faeces 

Sterile, leak-proof container 

Blood 

Blood grow medium 

Virology Rotavirus Liquid faeces in sterile leak-proof container 

Swabs 

Viral transport medium 

Parasitology Faeces 

Sterile, leak-proof container 

Dermatology Skin scrapings Sterile, leak-proof container 

Cytology Endometrial and Rolled onto sterile gelatine-coated slides and 

other smears fixed with ‘smear fix’ or carbowax 

Peritoneal, pleural, One sample preserved in EDTA, another diluted 

CSF, synovial fluids, 50:50 in cytospin centrifuge fluid and another 

tracheal washes undiluted in a sterile leak-proof container 

and BALs 

(synovial fluid also in blood grow medium for 

bacterial culture) 

Histology General tissue Thin, representative samples fixed in an ample 

samples 

volume (at least 10 x) of 10% formol saline 

Endometrial and Bouin’s fluid 

testicular biopsies 

Semen analysis Semen One sample diluted 50:50 in formol citrate and 

another in an empty, sterile, leak-proof container.


surgeons and some others. Very highly 

infected materials such as that containing 

foot and mouth disease virus or some 

especially dangerous human pathogens are 

excluded from this exemption. 

2. Members of the public may send 

specimens through the post only at the 

express request of a registered laboratory 

or veterinary surgeon. 

3. Only first-class letter post or data post 

may be used. Parcel post must not be 

used. 

4. The Post Office requires that all samples 

be packed in a particular way. These rules 

must be followed otherwise the Post Office 

may remove and destroy the specimen. 

a. Every specimen must be enclosed in 

a primary container, which is securely 

sealed. This container must not exceed 

50ml (although special multi-specimen 

packs may be approved). 

b. The primary container must be wrapped 

in sufficient absorbent material to absorb all 

possible leakage in the event of damage. 

c. The container and absorbent material 

must be sealed in a leak proof plastic bag. 

d. This package must then be placed in 

either: 

i. A polypropylene clip down container. 

ii. A cylindrical light metal container. 

iii. A strong cardboard box with full 

depth lid. 

iv. A specially grooved two-piece 

polystyrene box. 

5. It is recommended that this completed 

package should be placed in a padded 

bag. 

6. Multi-specimen packs may be used 

provided that each primary container is 

separated from the next by absorbent 

packing. 

7. Any other packaging systems must have 

the prior approval of the Post Office. 

8. Labelling: The outer cover must be 

labelled ‘Pathological Specimen Fragile. 

With Care’. It must show the name and 

address of the sender to be contacted in 

case of leakage. 

9. Therapeutic and diagnostic substances, 

such as blood, serum, vaccines etc. are 

classified as pathological specimens. 

Ensure that anything you send by post 

complies with the regulations otherwise 

it may be removed from the mail and 

destroyed, and you will lose a valuable 

specimen. You may be prosecuted by 

the Royal Mail. You may cause injury or 

disease to someone handling the package 

either during its transit through the mails, 

or at the receiving laboratory. 

10


reference ranges 

Clinicians and clinical pathologists must rely upon so-called 

‘normal’ reference ranges to interpret laboratory results. 

Unfortunately it is not possible to produce one set of ‘normals’ 

to suit all equine animals, as horses and ponies of different 

ages, types, uses and stages of training all have significant 

variations in some parameters. 

Another problem is that individual horses 

vary considerably not only in their own 

‘normal’ reference results but also by the 

effects that variations from reference range 

and even clinical abnormality will have 

on their clinically-apparent health and 

performance. Sub-clinical disease, e.g. 

low-grade anaemia, viral ‘challenges’, 

low-grade myopathy, hepatopathy and 

nephropathy will all produce effects on 

the appropriate laboratory results although 

the horse is considered clinically ‘normal’. 

The owner, rider, trainer or manager’s 

assessment of each horse as an individual 

remains essential and laboratory results 

should never be used to 'train' performance 

horses. Experienced interpretations are 

required for different types of horses used 

for different purposes. 

The ‘classical’ method of producing 

reference ranges for laboratory tests was to 

use the ‘bell-shaped curve’ of two standard 

deviations either side of the mean of large 

numbers of clinically normal horses. This 

requires the measured parameter to have a 

mathematically normal distribution within 

the horse population to be applicable and 

this is rarely the case. Alternatives are to 

work with percentiles (the range within 

which the results of 90% of clinically 

normal horses fall). Even then clinically 

normal horses will have significant subclinical 

changes and individual horses will 

have idiosyncratic ranges different to the 

population range. Clinicians and clinical 

pathologists must use reference ranges that 

they are comfortable with. 

Reference ranges for the more commonly 

used haematological and clinical chemical 

parameters, for adult non-Thoroughbred 

horses, neonatal and older Thoroughbred 

foals, Thoroughbred yearlings and two 

and three-year-old Thoroughbred horses in 

training can be found on pages 56-71. 

11


Haematology 

In the presence of clinical signs of disease, haematological 

examinations, performed on sequestrated (EDTA) 

blood samples, may reveal abnormalities suggesting 

haemoconcentration, anaemia, bacterial or viral infections, 

parasitic or allergic conditions. 

12 

These examinations may also be valuable 

as part of a preventive medicine programme 

for groups of horses, when examined on a 

regular and routine basis, and interpreted 

carefully. Such a sampling programme may 

provide useful information that can be used 

as a basis for advice to trainers of race and 

performance horses. 

Most clinical pathology laboratories now use 

automated or semi-automated cytochemical 

or particle counting haematology analysers. 

These provide much more accurate and 

repeatable results than the older manual 

counting technologies if the analysers are 

correctly calibrated for equine samples. 

It is important to remember that results 

from analysers that provide granulocyte/ 

non-granulocyte differential leucocyte 

counts must be interpreted differently from 

those that produce cytochemically stained 

and laser scanned differential leucocyte 

counts. The latter, if calibrated correctly, 

can be indistinguishable from traditional 

manual counts performed on stained 

films. Automated differentials are highly 

repeatable and are more accurate than 

manual cell counts as some analysers 

differentiate and count 10,000 rather 

than 100 leucocytes. Beaufort Cottage 

Laboratories currently use a Bayer Advia 

120 automated cytochemical haematology 

analyser. 

Samples for haematological examinations 

should be taken from horses at rest with 

minimal excitement, otherwise splenic 

contraction can make the interpretation of 

erythrocyte parameters impossible. 

Erythrocytes 

(RBC, PCV, Hb, MCV, McHc, McH) 

Haemoconcentration/dehydration (raised 

total erythrocyte count, haematocrit and 

haemoglobin concentration) can only be 

interpreted in samples taken from resting 

horses that are relaxed at collection and 

this can be very difficult to achieve in 

some excitable individuals. Reflex splenic 

contraction occurs in horses in response to 

fright, excitement and exercise, increasing 

numbers of young macrocytic erythrocytes 

in the circulating pool. Routine sampling 

sessions in performance horse stables are 

therefore often conducted at standard times


after a period of rest and quiet, e.g. early 

morning or late afternoon, before mucking 

out and feeding, in order to avoid sampling 

excited horses and to add a degree of 

standardisation. 

Haemoconcentration is sometimes a 

feature of so-called ‘over-trained’ horses 

that perform poorly, appear stressed, and 

have dry scurfy skin coats, lose condition 

and drink inadequately. They usually 

respond well to fluid and electrolyte therapy 

administered by nasogastric tube followed 

by a period of rest. Chronic anhidrosis can 

produce a similar picture. Dehydration is a 

feature of horses who are clinically ill with 

acute enteritis or colitis, requiring intensive 

fluid, electrolyte, acid-base and supportive 

therapy to replace losses. Haemoconcentration 

and dehydration are features 

of exercise and heat exhaustion in horses 

performing in hot dry climates and over 

long distances (typically endurance races), 

requiring timely diagnosis and appropriate 

treatment/management. The interesting 

condition of acute anhidrosis, i.e. failure 

of normal sweating, occurs in some horses 

who are raised in temperate climates and 

then perform in hot, dry climates when 

they have failed to acclimatise, resulting 

in respiratory distress, laboured breathing, 

pyrexia, collapse and even death. 

Attempts have been made to interpret 

the significance of results of post-exercise 

blood samples in terms of fitness. In terms 

of haematologic tests, variable degrees 

of haemoconcentration and leucocytosis 

are always a feature and without rigid 

standard exercise test regimes, which are 

almost impossible to organise within most 

performance horse training environments, 

results are usually uninterpretable. Specific 

muscle enzyme tests (see page 16) 

performed on serum samples collected 

before and after exercise, can help with the 

diagnosis of exercise-induced myopathy. 

There has been considerable interest in 

lactate assays before, during and after 

exercise to determine aerobic/anaerobic 

metabolic capacity, with still considerable 

debate and differences of opinion. 

Anaemia (low total erythrocyte count, 

haematocrit and haemoglobin concentration) 

in horses is less commonly a primary 

condition and more often occurs secondary 

to some other primary condition, e.g. 

infection (bacterial or viral), parasitism (endo 

or ectoparasitism) or metabolic abnormality 

(e.g. hepatopathy, nephropathy), which 

require specific diagnosis and treatment. 

Malnutrition is rarely seen in performance 

horse stables but mineral and vitamin 

imbalances (involving deficiency or excess) 

may occur. 

Acute haemorrhage can cause acute primary 

blood loss anaemia following accidental 

injury involving a major blood vessel. The 

haemorrhage may sometimes be visible 

externally but more often occurs internally 

within a body cavity, i.e. intraperitoneal, 

intrapleural or intrapericardial haemorrhage. 

13


Chronic haemorrhage, e.g. following 

castration, may cause an anaemia and selfperpetuating 

thrombocytopenia. Platelet 

transfusion may result in haemostasis 

without further surgical interference. 

Guttural pouch mycosis may cause 

internal carotid artery ulceration, resulting 

in profound acute and sometimes fatal 

epistaxis and blood-loss anaemia. Gastric 

ulceration, not uncommonly seen in 

performance horses, can cause anaemia 

from chronic haemorrhage. Examination 

of faecal samples for the presence of 

occult blood can be performed but results 

are frequently positive, most commonly 

because of activity of intestinal parasites, 

even in well-managed horses, and are 

therefore not reliably diagnostic of gastric 

ulceration. Unfortunately, serum pepsinogen 

assays are not reliably diagnostic of gastric 

ulceration in horses. Where suspected, 

the diagnosis must be made and the 

significance assessed by gastroscopic 

examinations. 

Intravascular haemolysis, i.e. erythrocyte 

destruction resulting in haemolytic 

anaemia, has a variety of different 

causes, which require specific diagnostic 

testing. Equine Infectious Anaemia 

(Coggins’ agar gel immunodiffusion 

test), autoimmune haemolytic anaemia 

(Coombs’ antiglobulin test), Piroplasmosis 

(Babesiosis) (complement fixation test), 

disseminated intravascular coagulopathy 

(DIC) (prolonged prothrombin and activated 

partial thromboplastin times) and a variety 

of plant and environmental toxins are some 

causes of intravascular haemolysis. 

Equine anaemia is most commonly 

macrocytic (raised McV), reflecting splenic 

replacement with juvenile erythrocytes, 

as seen with blood loss, infections and 

parasitism. Reticulocytes are not commonly 

seen in equine blood samples and so 

their presence or absence is not a reliable 

means of determining regeneration or 

non-regeneration, as in other species. 

Normocytic (McV within normal range) 

anaemia is sometimes seen in horses who 

are being challenged by respiratory viruses 

but who show no clinical signs. Microcytic 

anaemia (low McV) is uncommon but is 

sometimes seen in immature individuals 

and has been reported in horses with iron/ 

folate deficiency. 

Leucocytes 

(WBC & Differential leucocyte count) 

Leucocytosis (raised total leucocyte count) 

and neutrophilia (raised segmented 

neutrophil count) most commonly occurs 

in performance horses in association 

with septic and non-septic inflammatory 

conditions. Septic inflammation is most 

commonly associated with bacterial 

infection. In performance horses this 

may occur following an injury, e.g. 

penetrating wounds followed by cellulitis, 

septic arthritis or tenosynovitis, upper 

respiratory infections and less commonly 

14


in systemic bacterial infections. Non-septic 

inflammation can occur following nonpenetrating 

injury, e.g. bruising of soft 

tissues, tendons, ligaments or periosteum 

and traumatic arthritis or tenosynovitis, 

sometimes associated with degenerative 

joint disease. Marked leucocytosis is seen 

in foals responding to bacterial infections, 

notably Rhodococcus equi (‘summer 

pneumonia’) and Streptococcus equi 

(‘strangles’) infections. 

Neutrophilic ‘shifts to the left’ (appearance of 

juvenile neutrophils or ‘band’ neutrophils), 

while diagnostically helpful in foal-hood 

infections, are seldom seen in adult horses 

unless severely and acutely infected 

with conditions such as acute cellulitis 

or lymphangitis. Some cases of acute 

salmonellosis, endotoxaemia, peracute 

enterocolitis, peritonitis and pleuritis 

have neutrophilic shifts to the left, more 

commonly associated with leucopenia and 

neutropenia. 

Leucopenia (low total leucocyte count) and 

neutropenia (low segmented neutrophil 

count) is most commonly seen in adult 

performance horses during the acute phase 

of a viral challenge, when there may or 

may not be clinical signs. These may 

include lethargy, pyrexia, nasal discharge, 

coughing and oedematous legs. Leucocytic 

changes seen with infection very much 

depend upon sampling time in relation 

to the stage of the disease process (see 

Fig.1). 

If the early acute infectious phase has 

passed then the leucopenic phase will be 

missed and haematologic examinations 

will reflect repair and sometimes-secondary 

bacterial involvement with leucocytosis and 

neutrophilia (see page 14). Therefore, blood 

samples should be taken from symptomless 

stablemates where viral infections are 

suspected, in order to help with diagnosis, 

epidemiology and assessment of recovery. 

Unfortunately, unless Equine Influenza, 

Fig.1: equine blood leucocyte response to viral challenge 

14 

Circulating blood leucocytes x 

10^9/l 

12 

10 

8 

6 

4 

2 

0 

1 2 3 4 5 6 7 8 

Days – Viral challenge starts on day1. Basal leucocyte count is 8 x 10 9 /l 

15


16 

Herpesvirus, Rhinovirus or Adenovirus 

infections are involved (for which specific 

serologic assays are available), virological 

‘screening’ investigations are seldom 

rewarding. 

Profound leucopenia is seen in neonatal 

foals with either bacterial (most commonly 

Escherichia coli) or viral (most commonly 

Equine Herpesvirus-1) septicaemia and 

is an indication for urgent intensive care. 

A blood culture should be performed 

immediately prior to starting broadspectrum 

antibiotic therapy in order to 

try to identify the pathogen and clarify 

antibiotic sensitivity. 

Occasionally, profound leucopenia with 

neutropenia and neutrophilic shifts to the 

left are seen in adult performance horses 

suffering from acute salmonellosis, peracute 

enterocolitis, peritonitis and pleuritis or 

pleuropneumonia and in newly foaled 

mares with toxic metritis/laminitis, most 

commonly following placental retention. 

Profound leucopenia is always a sign of 

severe illness, indicating the need for 

intensive care and suggesting a guarded 

prognosis. 

Lymphocytosis (raised lymphocyte 

count) is seen in horses in response 

to endogenous catecholamine release 

during excitement or exercise. Otherwise, 

it is most commonly seen in response to 

some chronic viral infections and in more 

rarely seen autoimmune diseases. Massive 

leucocytosis (sometimes greater than 100 

x 10 9 /l) with lymphocytosis is a feature of 

generalised lymphoma, in which neoplastic 

lymphocytes can be demonstrated in 

peripheral blood and sometimes in body 

cavity fluid samples and tissue biopsy 

samples. 

Lymphopenia (low lymphocyte count) is 

seen in horses in response to endogenous 

glucocorticoid release and in response to 

exogenous corticosteroid administration. 

Otherwise, it may be seen in acute viral 

infections, severe bacterial infections, 

septicaemia, endotoxaemia and immune 

deficiency conditions. Profound, persistent 

leucopenia always carries a poor 

prognosis. 

Monocytosis (raised monocyte count) 

reflects increased phagocytic demand 

as may occur with chronic suppurative 

conditions with tissue necrosis. In 

performance horses, monocytosis is most 

commonly seen during the post-acute or 

recovery phases following upper respiratory 

viral infections. 

Eosinophilia (raised eosinophil count) 

occurs with antigen-antibody response 

in tissues rich in mast cells, e.g. skin, 

lung, gastrointestinal tract and uterus 

and in parasitically sensitised horses. In 

performance horses, low-grade eosinophilia 

is most commonly seen in association 

with leucopenia or lymphocytosis in 

the acute-phase of responses to viral 

infections. In horses at pasture, the first 

differential diagnosis for eosinophilia is


intestinal parasitism and warrants further 

investigations with protein electrophoresis 

and faecal worm egg counts. Rare cases 

of eosinophilic leukaemia have been seen 

with eosinophil counts as high as 2.5 

x 10 9 /l (25% of differential leucocyte 

count). 

Basophilia (raised basophil count) is very 

uncommon in horses, as are the presence 

of basophils themselves. Basophils have 

been a feature in cases of hyperlipidaemia 

and in some horses that were recovering 

from colic. 

Platelets 

Thrombocytosis (raised platelet count) is 

seen uncommonly in adult horses but may 

occur in bacterial infections. It may occur 

with bacterial infections in foals and has 

been associated with Rh. equi infections. 

Thrombocytopenia (low platelet count) 

may be a reflection of decreased 

production, increased usage or from various 

spurious factors, e.g. drug administration, 

the presence of cold agglutinins in the 

sample or platelet clumping in EDTA). 

Thrombocytopenia is sometimes seen 

in horses with viral infections. Where 

pseudothrombocytopenia is suspected, 

platelet counts should be measured on two 

blood samples collected at the same time, 

one into EDTA and the other into sodium 

citrate anticoagulants. If the sodium citrate 

sample, after correction for dilution, is 

considerably higher than the EDTA sample, 

EDTA-induced pseudo-thrombocytopenia is 

the likely answer. Decreased production 

may occur with neoplasia or a toxic insult 

to the bone marrow, the latter is diagnosed 

by biopsy. Idiopathic thrombocytopenia 

is probably an immune-mediated 

condition and thrombocytopenia is seen 

in horses with disseminated intravascular 

coagulopathy (DIC) most commonly a 

serious complication of acute enterocolitis. 

Haematologic examinations are often used 

routinely to screen horses in training for 

subclinical disease and can be helpful 

when examined on a regular routine basis, 

and interpreted carefully. Haematologic 

signs of viral infection (leucopenia and 

neutropenia or relative lymphocytosis, 

depending on stage of sampling), in 

a clinically normal horse may suggest 

‘challenge’, i.e. the horse’s immune system 

is responding but not succumbing to the 

infection. In this condition, many horses 

do not perform to their best athletic 

potential, their recovery after exertion may 

be prolonged and they may be more 

likely to suffer secondary complications 

such as pneumonia, lung abscess, skeletal 

and/or cardiac myopathy and/or exerciseinduced 

pulmonary haemorrhage 

Haematologic examinations, in combination 

with inflammatory protein measurements 

(see later) are often used to help screen 

and monitor the progress of foals and 

older horses at studfarms when Rh. equi 

infections or Strep. equi epidemics occur. 

17


In cases of clinical disease, haematologic 

variations from 'normality' are often marked 

and obvious, but more sophisticated 

analytical equipment is required when 

screening for less obvious variations and 

trends. Modern automated cytochemical 

haematology analysers (e.g. Bayer Advia 

120) provide accurate differential cell 

counts that correlate closely with traditional 

manual differential counting techniques. 

Their automated differentials are highly 

repeatable and are likely to be more 

accurate than manual cell counts as they 

are performed on 10,000 rather than 100 

leucocytes. These analysers provide results 

that are more accurate, repeatable and 

therefore reliable for the routine monitoring 

of performance horses. However, when 

required by clinical history or results 

obtained, a traditional stained smear is 

still required to demonstrate erythrocyte 

or leucocyte abnormalities. Red cell 

abnormailities include Howell-Jolly bodies 

and nucleation, fragmentation, oxidative 

damage, spherocytes, basophilic stippling 

or Babesia spp. parasites in piroplasmosis. 

White cell changes include left shifts, toxic 

degeneration, hypersegmentation, neoplastic 

change or cytoplasmic inclusions as seen 

for example in ehrlichiosis. 

NOTES 

18


clinical chemistry 

Proteins 

Total protein, albumin and globulin 

estimations are useful in the assessment 

of general bodily condition and nutritional 

status and the response to infectious or 

parasitic disease. Electrophoresis helps 

determine the significance of raised total 

globulin levels. Specific tapeworm ELISA 

assays are now available commercially. Low 

serum albumin and/or rising globulin levels 

are a ‘red flag’ warning, most commonly seen 

with cyathostomiasis (hypoalbuminaemia), 

large strongylosis (raised beta 1 globulin), 

mixed helminthiasis (hypoalbuminaemia 

and raised beta 1 globulin), hepatopathy 

(hypoalbuminaemia and raised beta 2 

globulin), antibody response to infection 

(raised gamma globulin) or abscess 

formation (raised alpha 2 and gamma 

globulins). Globulins can be differentiated 

by electrophoresis (Fig.2). 

Protein Electrophoresis 

Strep. equi infections) will often show 

characteristic alpha 2 and gamma globulin 

responses. Serum samples should be 

used for protein electrophoresis, as raised 

fibrinogen levels in heparinised plasma 

samples will cause confusing rises in beta 

2 globulins (Fig.7). 

Occasionally, horses with generalised 

lymphosarcoma or plasma call myeloma 

have massively increased total protein 

and globulin levels, for which protein 

electrophoresis shows a massively raised, 

discrete, ‘skyrocket’ peak, usually in the 

beta 2 globulin range (Fig.8) suggesting 

monoclonal lymphoma protein production. 

Fig.2: serum protein electrophoresis 

normal horse serum 

Test : ELECTR Gel 1 – 8 03/01/2002 

This identifies elevations in specific globulin 

fractions:- 

1. Alpha 2 globulin - acute-phase 

inflammatory protein responses (Fig.3). 

2. Beta 1 globulin - Strongylus vulgaris 

and mixed strongyle larval activity (Fig.4). 

3. Beta 2 globulin – hepatopathy (Fig.5). 

4. Gamma globulin - antibody responses 

to bacterial or viral infections (Fig.6). 

Horses with abscesses (e.g. Rh. equi and 

Fraction Rel% G/L 

1 5.0 1.10 

2 26.1 5.74 

3 20.6 4.53 

4 24.9 5.48 

5 23.5 5.17 

Total G/L 22.00 

19



raised alpha 2 globulin 



raised alpha 2 globulin 



raised alpha 2 and beta 1 globulins 



raised alpha 2 and beta 1 globulins 



1 2.8 0.78 

2 33.7 13.14 

3 16.4 6.40 

4 24.5 9.56 

Fraction 5 Rel% 23.4 9.13 G/L 

Total 1 G/L 39.00 2.8 0.78 

2 33.7 13.14 

3 16.4 6.40 

4 24.5 9.56 

5 23.4 9.13 

Fig.5: Total G/L serum protein 39.00 electrophoresis 

raised beta 2 globulin 



raised beta 2 globulin 



1 1.3 0.62 

2 24.0 11.52 

3 48.2 23.14 

4 10.4 4.99 


Total 1 G/L 48.00 1.3 0.62 

2 24.0 11.52 

3 48.2 23.14 

4 10.4 4.99 

5 16.2 7.78 

Fig.6: Total G/L serum protein 48.00 electrophoresis 

raised gamma globulin 



raised gamma globulin 



1 0.9 0.49 

2 17.0 9.18 

3 15.2 8.21 

4 39.3 21.22 


Total 1 G/L 54.00 0.9 0.49 

2 17.0 9.18 

3 15.2 8.21 

4 39.3 21.22 

5 27.6 14.90 



1 2.1 0.76 

2 18.3 6.59 

3 17.5 6.30 

4 17.1 6.16 


Total 1 G/L 36.00 2.1 0.76 

2 18.3 6.59 

3 17.5 6.30 

4 17.1 6.16 

5 45.1 16.24 


20



fibrinogen spike in heparinised plasma 



monoclonal beta 2 globulin 'sky rocket' 



1 5.4 2.48 

2 19.8 9.11 

3 17.5 8.05 

4 36.0 16.56 

5 21.3 9.8 


Serum Immunoglobulin G (IgG) 

As there is no transplacental transfer of 

IgG before birth, foals are born essentially 

agammaglobulinaemic. During the last few 

months of gestation, mares concentrate 

IgG in their colostrum. Foals’ intestines are 

capable of absorbing IgG for their first 12 

hours of life. Providing the mare makes 

colostrum of good quality in terms of IgG 

concentration, she does not ‘run milk’ 

before foaling and the foal sucks sufficient 

colostrum within the first 12 hours, the foal 

acquires good circulating IgG levels and 

therefore adequate passive immunity. 

Foals should have their serum IgG levels 

checked as a routine preventive medicine 

policy. Serum samples collected from foals 

after 12 hours of age should have IgG 

levels of more than 4 g/l and ideally more 

than 6 g/l. Levels of less than 2 g/l indicate 

failure of transfer of colostral immunity and 

levels of 2-4 g/l indicate partial failure. 


1 2.1 1.91 

2 9.5 8.65 

3 9.5 8.65 

4 71.2 64.79 

5 7.6 6.92 


Foals with levels below 4 g/l are considered 

at risk for neonatal infections and should be 

transfused with hyperimmune plasma and 

their serum IgG levels re-checked 24 hours 

later to make sure that IgG levels have risen 

to acceptable levels. 

Our experience suggests that none of the 

currently available ‘stable-side’ foal serum 

IgG tests are reliably accurate at the 0-4 g/l 

end of the scale and we measure IgG by an 

immunospectrophotometric method run on 

our autoanalyser. 

IgG can be measured in colostrum 

immediately after parturition semiquantitatively, 

using a refractometer 

(Colostrometer) (see table on page 22). If 

readings suggest an IgG level of less than 

45 g/l, the foal should be considered for 

donor colostrum supplementation by bottle 

or stomach tube. 

21


Colostrometer reading Concentration IgG g/l Colostrum quality 

30% >80 Excellent 

Plasma Fibrinogen 

This is an acute-phase reactive protein, 

which increases in response to inflammation. 

Elevations are found in the presence of 

tissue damage and this assay may help 

with diagnosis and prognosis in cases of 

internal abscessation, chronic infectious or 

parasitic disease and in cases of exercise 

induced pulmonary haemorrhage (EIPH). 

The test can be performed on fresh, paired, 

non-haemolysed EDTA and serum samples 

by subtraction of total serum protein from 

plasma protein results, but more accurate 

results are obtained from samples collected 

into sodium citrate anticoagulant to 

measure fibrinogen by direct coagulometric 

assay. When measured serially with 

serum amyloid A (see right) the kinetics 

of the inflammatory response can often 

be determined (Fig.9) and this can be 

very helpful when monitoring response to 

treatment. Fibrinogen is a very useful test 

to help diagnose and monitor the response 

to treatment for a number of pyogenic 

conditions in foals and yearlings, e.g. 

Rh. equi and Strep. equi. 

Serum Amyloid A (SAA) 

This is a highly sensitive, rapidly reacting 

inflammatory protein, which can be very 

helpful in monitoring early responses to 

infection and their response to treatment. 

Most normal horses have zero measurable 

levels and in the face of acute, particularly 

septic inflammation, levels increase quickly 

(within 24 hours) to over 20 mg/l and 

often more than 100 mg/l. Levels peak 

and fall similarly quickly with subsidence 

of inflammation when the infection is 

controlled (Fig.9). SAA is a very useful 

addition to routine neonatal foal ‘profiles’ 

to help identify those who may have or 

may be developing septicaemia and require 

antibiotic therapy. 

Aspartate Aminotransferase 

(AST, AAT, SGOT) 

Elevations are seen in the presence of acute 

myopathy or hepatopathy. After myopathy, 

levels peak at 24-48 hours and return to 

baseline by 10-21 days, assuming that no 

further damage occurs. This test, taken 

with CK at first visit and then 10-14 days 

later, can therefore be a useful guide to 

recovery from acute myopathy (Fig.10). 

22


Fig.9: schematic diagram showing equine inflammatory protein dynamics 

following an inflammatory challenge 

Magnitude Magnitude of response of (%) response (%) 

100 

80 

100 

60 

80 

40 

60 

20 

40 

20 

Fig.9: schematic diagram showing equine inflammatory protein dynamics 

following an inflammatory challenge 

SAA 

Pfib 

SAA 

Pfib 

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 

Days 

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 

Days 

Fig.10: schematic diagram showing equine serum muscle enzyme levels during 

a relatively mild episode of exertional rhabdomyolysis 

iu/i 

iu/i 

3500 

3500 

2500 3500 

2000 3500 

1500 2500 

1000 2000 

1500 

Fig.10: schematic diagram showing equine serum muscle enzyme levels during 

a relatively mild episode of exertional rhabdomyolysis 

CK 

AST 

CK 

AST 

1000 0 

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 

500 

Days 

0 

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 

Days 

23


Creatine Kinase (CK, CPK) 

Elevations are specifically seen in the 

presence of acute myopathy. CK isoenzyme 

analysis was used in human medicine 

to help differentiate cardiac and skeletal 

myopathy from brain pathology, but has 

never become routinely established in 

equine sports medicine. Cardiac troponin 

(see page 25) assays are now used to 

differentiate skeletal from cardiac myopathy 

in horses. 

CK levels peak at 6-12 hours and return 

to baseline by 3-4 days, assuming that no 

further myopathy occurs. When measured 

alongside AST (see page 22), which takes 

longer to rise, peak and return to normal, 

the timing and response to treatment 

of myopathy in horses can be usefully 

monitored (Fig.10). Paired CK assays 

taken before and 2-3 hours after strenuous 

exercise, can form a useful diagnostic test 

for exercise-induced myopathy, in horses 

where the diagnosis may be in doubt. Some 

respiratory virus infections, notably Equine 

Herpesvirus-1, appear to increase muscle 

cell membrane fragility and predispose 

to exercise-induced myopathy in horses 

in training. This condition is sometimes 

associated with clinical signs of fatigue and 

stiffness in performance horses. 

Significant myopathy, demonstrable by 

higher serum CK and AST levels, occurs 

more often after exercise in unfit rather 

than in fit horses. The conditioning process 

protects equine muscle cells from exerciseinduced 

injury. 

Very high CK levels are seen in young foals 

with nutritionally associated myopathy 

(‘white muscle disease’) associated with 

selenium deficiency. 

Lactate Dehydrogenase (LD) 

A variety of disease conditions can cause 

elevations in total LD and more useful 

differentiation can sometimes be provided 

by isoenzyme analysis (Fig.11):- 

■ LD isoenzyme 1 - most dramatically 

increased by intravascular haemolysis 

(Fig.12). 

Fig.11: serum ld isoenzymes 

normal horse 

Test : LD Gel 2 – 5 16/01/2002 

Fraction Rel% IU/L 

LD 1 9.2 46 

LD 2 26.6 133 

LD 3 41.6 208 

LD 4 18.4 92 

LD 5 4.2 21 

Total IU/L 500 LD1/LD2: 0.35 

24


■ LD isoenzyme 2 - elevated in some 

cases of cardiac pathology, an indication 

for cardiac troponin assay (see below) 

(Fig.13). 

■ LD isoenzyme 3 – no known disease 

association in the horse. 

■ LD isoenzyme 4 - most commonly 

elevated by intestinal pathology (Fig.14). 

■ LD isoenzyme 5 - rises seen with 

skeletal myopathy and hepatopathy, 

requiring further differentiation with CK 

(see page 24) and liver enzyme (see 

pages 24-28) assays (Fig.15). 

Total LD levels are age-dependent to 

maturity and reference ranges must be 

consulted when interpreting results for 

young horses (see pages 56-69). 

Cardiac Troponin (cTnI) 

Two proteins (tropomyosin and troponin) 

working in concert with calcium, regulate 

muscle contraction. Troponin is a globular 

protein complex composed of three 

single chain polypeptide subunits: TnI 

(troponin inhibitory component), which 

prevents muscle contractions in the 

absence of calcium; TnT (tropomyosinbinding 

component), which connects 

the troponin complex with tropomyosin; 

and TnC (calcium binding component), 

which binds calcium. The cardiac musclespecific 

isoform cTnI (24 kDa) exhibits 

approximately 60% homology with the 

skeletal isoforms (sTnI) and has a unique 

31 amino acid extension of the N-terminus. 

Experience in human medicine has shown 

that after acute myocardial infarction 

(AMI), elevated cTnI levels appear in the 

circulation within 3-6 hours. Serum levels 

peak within 14-20 hours and return to 

normal after 5-7 days. The measurement 

of cTnI can therefore be a useful diagnostic 

aid for AMI and an aid for the monitoring 

of recovery. 

Myocardial infarction is rarely diagnosed in 

the horse but myocardial necrosis is seen in 

conditions such as atypical myoglobinuria. 

Myocarditis is sometimes suspected on the 

basis of arrhythmias, echocardiographic 

abnormalities and/or raised serum lactate 

dehydrogenase isoenzyme 2 levels. This 

can follow upper respiratory viral infections. 

Horses who have suffered in this way will 

need more rest and supportive treatment 

followed by follow-up to normality before 

return to strenuous exercise if potentially 

serious complications are to be avoided. 

Raised cTnI levels are an indication for 

cardiac ultrasound examinations and 

ambulatory ECG monitoring. 

25



raised ld1 

Test : LD Gel 2 – 5 16/01/2002 


raised ld1 

Test : LD Gel 2 – 5 16/01/2002 


raised ld2 

Test : LD Gel 2 – 10 29/06/2002 


raised ld2 

Test : LD Gel 2 – 10 29/06/2002 


LD 1 44.4 316 

LD 2 34.1 243 

LD 3 16.7 119 

Fraction LD 4 Rel% 3.3 IU/L 23 

LD 51 44.4 1.5 316 11 

Total LD IU/L 2 712 34.1 LD1/LD2: 1.3 243 

LD 3 16.7 119 

LD 4 3.3 23 

LD 5 1.5 11 



raised ld 

Test : LD Gel 1 – 3 31/07/2002 


raised ld 

Test : LD Gel 1 – 3 31/07/2002 


LD 1 22.0 180 

LD 2 39.6 324 

LD 3 27.1 221 

Fraction LD 4 Rel% 7.6 IU/L 62 

LD 51 22.0 3.6 180 29 


LD 3 27.1 221 

LD 4 7.6 62 

LD 5 3.6 29 



raised ld 

Test : LD Gel 1 – 3 10/05/2002 


raised ld 

Test : LD Gel 1 – 3 10/05/2002 


LD 1 4.9 39 

LD 2 12.1 96 

LD 3 29.4 233 

Fraction LD 4 41.4 Rel% 328 IU/L 

LD 51 12.2 4.9 97 39 


LD 3 29.4 233 

LD 4 41.4 328 

LD 5 12.2 97 



LD 1 9.3 205 

LD 2 12.7 280 

LD 3 15.8 348 

Fraction LD 4 Rel% 7.3 161 IU/L 

LD 51 54.8 9.3 1207 205 

Total LD IU/L 2 220312.7 LD1/LD2: 0.73 280 

LD 3 15.8 348 

LD 4 7.3 161 

LD 5 54.8 1207 


26


Cardiac troponin (cTnI) levels are measured 

in serum samples (lower results may be 

found in plasma samples). Clinically 

normal horses have serum cTnI levels 

of less than 0.2 ng/ml. Experience so 

far suggests that greater than 0.3 ng/ 

ml is abnormal, i.e. suggests myocardial 

pathology and 0.2-0.3 ng/ml is currently 

a ‘grey’ zone. Levels of 0.9-5.4 ng/ml 

have been measured in horses with 

ultrasound-confirmed cardiomyopathy. 

Nevertheless, results greater than 0.2 ng/ 

ml are considered ‘red flags’ for expert 

cardiological appraisals. 

Sorbitol Dehydrogenase (SDH) 

This is an enzyme found in the cytoplasm 

of hepatocytes and is therefore virtually 

liver-specific, although rises are sometimes 

seen in horses with skin conditions and 

enteropathy. It is useful for the identification 

of acute hepatocellular damage for in-house 

laboratory conditions, but the enzyme is 

highly labile. Therefore, samples must be 

handled with care and assays must be 

performed within 8-12 hours of sampling 

thus SDH assays are unsatisfactory for 

transported samples. SDH is not assayed at 

our laboratories - the more stable GLDH assay 

is now used more frequently than SDH. 

Glutamate Dehydrogenase (GLDH) 

Elevations are seen in the presence of 

acute hepatocellular damage. This is a 

mitochondrial enzyme found mainly in 

liver, heart muscle and kidney. It is a 

relatively stable enzyme and is a suitable 

replacement for sorbitol dehydrogenase 

(SDH) (see left) in transported samples. 

GLDH rises are sometimes seen in horses 

with skin conditions and enteropathy. 

L-Gamma 

Glutamyltransferase (GGT) 

GGT is found in cell membranes of 

hepatocytes and biliary epithelial cells, 

but the enzyme is also found in the 

pancreas and kidney. Elevations in serum 

levels are seen in the presence of acute 

hepatitis, chronic liver cirrhosis and in very 

rare cases of pancreatitis. Nephropathy 

does not usually result in significantly 

raised serum GGT levels so high levels 

measured in the horse are usually a sign 

of biliary or cholestatic disease. Chronic 

pyrrolizidine alkaloid toxicity (ragwort, i.e. 

Senecio jacobea poisoning) causes bile 

duct hyperplasia and biliary stasis and 

therefore typically results in raised serum 

GGT and SAP (see below) levels. This 

remains an important cause of hepatopathy 

in horses and ponies in UK, who ingest 

the plant unknowingly in poor-quality hay. 

Toxicity is uncommon in well-managed 

horses. 

Idiopathic GGT elevations are sometimes 

seen in horses in training that appear 

otherwise healthy, but perform poorly. The 

cause of these GGT rises has not yet been 

satisfactorily defined although plant and 

fungal hepatotoxins have been suspected. 

In most cases, other liver enzymes are 

27


28 

within normal range as are urea and 

creatinine levels, and liver biopsy reveals 

insignificant histopathological findings. 

It is therefore not certain that primary 

hepatopathy or nephropathy is involved. 

Some cases have raised muscle enzyme 

levels suggesting an association with 

myopathy, either directly or secondarily, 

again perhaps via respiratory virus-induced 

increased muscle cell membrane fragility. 

Most cases respond (GGT levels return 

to normal) to a period of reduction in the 

training programme. 

Urine GGT:creatinine ratios are elevated 

(>4.0) in renal tubular pathology. 

Alkaline Phosphatase (SAP) 

Elevations in this brush border enzyme are 

most commonly seen in the presence of 

chronic biliary obstructive liver pathology 

(e.g. chronic pyrrolizidine alkaloid toxicity). 

High levels are also seen with abnormalities 

of bone metabolism and intestinal 

malfunction, a useful assay in growing 

foals and yearlings with clinical signs of 

developmental orthopaedic disease, where 

SAP results may be very high and will 

respond to restricted exercise and mineral, 

vitamin and trace element supplementation. 

SAP levels are age-dependent to maturity 

and appropriate reference ranges must 

be consulted when interpreting results for 

young horses (see pages 56-69). 

Intestinal Phosphatase (IAP) 

Elevations in IAP relative to total SAP 

are seen in the presence of intestinal 

pathology. 

References ranges for serum SAP and IAP 

levels are very age-related and apparently 

high results must be interpreted carefully in 

foals, yearlings and immature performance 

horses. 

Bilirubin 

The analysis of bilirubin levels is seldom 

useful in the horse but may aid the 

classification of anaemia and jaundice 

in some cases. Owing to the horse's 

unusual biliary excretion system, indirect 

(unconjugated) bilirubin levels, may be 

higher than those in other species, without 

clinical disease, and the significance of 

elevations without other abnormalities may 

therefore be difficult to interpret. A period of 

anorexia, inanition or intestinal malfunction 

typically increases indirect bilirubin levels 

spuriously. 

Bile acids 

This is a much better guide to hepatobiliary 

status than bilirubin assays. High bile acid 

levels occur with embarrassed hepatic 

function and are a useful diagnostic and 

prognostic liver function and prognostic 

guide in horses. 

It is important to remember that none 

of the liver enzymes, measured singly 

or in a profile, give useful information


about liver function. The liver has a large 

functional reserve and compensatory 

capacity. Liver enzyme rises suggest 

hepatopathy which can be differentiated 

to a degree into acute, chronic, biliary 

obstructive or mixed pathology, but bile 

acid assays and bromsulphalein clearance 

test results reveal either adequate (normal 

levels) or impaired (high levels) functional 

compensation. Impaired hepatic function 

suggests a guarded prognosis. Liver biopsy 

and ultrasound examinations are required 

to confirm an etiologic diagnosis and to 

refine prognosis. 

Amylase 

Elevations occur in the presence of 

pancreatitis, but this condition is rarely 

diagnosed in the horse. 

Glucose 

Other than for oral glucose and xylose 

absorption tests, useful for the diagnosis 

and evaluation of intestinal malabsorption 

cases, the value of this assay in adult 

horses is limited to cases of pituitary 

pars intermedia dysfunction (equine 

Cushing’s syndrome) which are frequently 

hyperglycaemic. Measurement of blood 

glucose is invaluable in the management of 

critically ill foals as profound hypoglycaemia 

is often seen in neonatal septicaemia. 

Samples for glucose assay must be taken 

into fluoride anticoagulant or must reach 

the laboratory within hours of collection. 

Oral Glucose Absorption Test 

The horse to be tested should be fasted 

for 18-24 hours. 1 g glucose per kg body 

weight is administered by stomach tube. 

Blood samples are collected into fluoride 

anticoagulant at times 0, 30, 60, 90, 120, 

150, 180, 210 and 240 mins. Glucose 

levels should peak at double resting (0 

mins) levels at 60-120 mins and return to 

resting levels by 240 mins. 

Cholesterol and Triglycerides 

Elevations are seen in the presence 

of abnormal lipid metabolism and 

hyperlipidaemia. These conditions are 

typically seen in the Shetland and other 

small ponies, donkeys and occasionally as 

a secondary complication in horses that 

are anorexic or unable to eat. Pregnant 

Shetland pony mares appear particularly 

susceptible following a managemental and 

nutritional change/challenge. 

Urea 

Urea is produced in the liver from the 

metabolism of ammonia. Elevations are 

seen in the presence of abnormal renal 

function. Urea levels may also rise in 

the haemoconcentrated and ‘over-trained’ 

horse, associated with fluid balance shifts 

rather than renal disease. Many cases 

of equine dysautonomia (‘grass sickness’) 

have a degree of uraemia but this is 

usually caused by catabolism. A period 

of anorexia can have a similar result. 

29


30 

Nephrosis and nephritis are important 

conditions in neonatal and older foals and 

are occasionally seen in other age groups. 

Creatinine 

Creatinine is formed in muscles from 

creatine breakdown and is excreted via 

the kidneys. In normal horses, daily 

production and excretion are remarkably 

constant, leading to its use as an arithmetic 

constant for use with urinary fractional 

excretion rates (see below). Therefore 

serum elevations reflect renal malfunction 

(reduced glomerular filtration), levels being 

controlled by excretion rate. This may occur 

in horses with pre-renal (e.g. circulatory 

disturbances, dehydration or shock), renal 

(insufficiency) or post-renal conditions. 

Measurement of urine specific gravity and 

fractional excretion of electrolytes may 

help to differentiate pre-renal and renal 

azotaemia. When uroperitoneum (postrenal 

azotaemia) is suspected it can be 

useful to compare peritoneal fluid and 

serum creatinine concentrations as a ratio 

of greater than 3:1 confirms uroperitoneum. 

For both renal and post-renal azotaemia, 

ultrasonography can be very helpful. 

Urinary Fractional Electrolyte 

and Mineral Clearance Ratios 

In horses with normal renal tubular 

function, urinary excretion rate of creatinine 

is almost constant. It can therefore be 

used as an arithmetic constant to produce 

a measure of the fractional excretion of 

electrolytes and minerals in equine urine. 

Concentrations of electrolytes or minerals 

are measured in serum and urine samples 

collected at the same time or at least within 

1 hour of one another. Diuretics must not 

be used to stimulate urination or spurious 

results will be obtained. 

The percentage excretion of an electrolyte 

is calculated from the following equation:- 

Where: 

(E)u = Concentration of electrolyte in urine 

(E)u (Cr)s 

Fractional urinary 

X X 100 = 

(E)s (Cr)u 

excretion (%) 

(E)s = Concentration of electrolyte in serum 

(Cr)u = Concentration of creatinine in urine 

(Cr)s = Concentration of creatinine in serum 

Fractional excretion rates for sodium, 

potassium, chloride, magnesium, calcium 

and phosphate are most commonly 

measured. The result provides an 

assessment of the horse’s homeostatic 

regulatory status for that electrolyte or 

mineral, e.g. sodium and chloride may be 

selectively excreted at an increased rate 

because of excessive dietary salt intake. 

Phosphate may be excreted at an increased 

rate to try to maintain a normal serum 

calcium:phosphate ratio in the face of 

inadequate calcium intake. The use of this 

method applies only to horses with normal 

renal function. Impaired renal tubular 

function results in high urine excretion 

rates for all the electrolytes and minerals,


from failure of retention - in such cases, 

creatinine is not a valid arithmetic constant. 

Electrolyte imbalance may predispose 

exercise-induced myopathy in horses 

in training and so fractional clearance 

ratios may sometimes be helpful in the 

investigation and management of recurrent 

cases. Dietary deficiencies, excesses or 

imbalances can be corrected and return 

to normal excretion rates monitored, 

sometimes with useful results in terms of 

resolution of myopathy. 

In secondary nutritional hyperparathyroidism, 

which occurs in horses on a 

high phosphate diet, phosphate excretion 

rate is high, indicating the need for oral 

calcium supplementation and phosphate 

reduction, to restore balance. Experience 

has shown that in UK, many healthy, 

fit, stabled and well performing horses, 

receiving high cereal training rations, have 

urinary fractional phosphate excretion rates 

in excess of 9%, emphasising the need for 

calcium supplementation. Higher excretion 

rates are seen in horses with clinical 

manifestations of secondary nutritional 

hyperparathyroidism, which include shifting 

lameness, periosteal thickening, facial and 

mandibular swelling. 

Urinary phosphate clearance ratios are useful 

measures of calcium:phosphate balance in 

weanlings and yearlings, important for 

bone growth and development. Calcium: 

phosphate imbalance can predispose to 

physitis. 

Calcium, Potassium and Chloride 

Electrolyte imbalance and fluid loss may 

occur with diarrhoea, endotoxaemia, 

intestinal crises and exertional exhaustion. 

The latter is of particular importance for 

endurance horses and for other horses 

performing in hot and humid weather 

conditions. Serial assays are helpful with 

intensive care cases. In other cases, 

fractional urinary electrolyte excretion 

rates (see page 30) are a much better 

assessment than single serum assays. 

Electrolyte assays are very important to 

the assessment of neonatal foals under 

critical care, with specific supplementation, 

where indicated and monitoring of return 

to normality. 

Calcium, Phosphate and Magnesium 

Mineral analysis may be helpful in young 

horses, i.e. yearlings and two-year-olds 

coming into training, with signs suggesting 

abnormalities of bone metabolism. As 

homeostatic mechanisms are efficient, 

serum levels are often normal even in the 

face of whole body abnormality, and thus 

urinary fractional excretion rates (see page 

30) are of greater value. 

Hypocalcaemia is a cause of synchronous 

diaphragmatic flutter in performance horses 

and of uterine inertia in pregnant mares 

at full term, requiring cautious corrective 

therapy. 

31


32 

Plasma Lactate 

A number of hand-held and bench top blood 

lactate analysers have been used in horses. 

Most have been designed to help assess 

the human response to exercise. Results 

have been used to describe and predict 

aerobic endurance capacity in horses and 

it has been suggested that the rate of 

decline in blood lactate concentration, 

which occurs after exercise, may be a 

useful index of fitness. Persistent high 

blood lactate concentrations may suggest 

metabolic fatigue. Anaerobic capacity is 

essential for sprinters and so increases in 

blood lactate concentrations for fast, short 

distance Thoroughbred flat racehorses may 

be an advantage. Successful long distance 

endurance racehorses need to be able to 

maintain mainly aerobic metabolism (low 

blood lactate levels) rather than switching 

to increased dependence on anaerobic 

metabolism (high blood lactate levels) for 

prolonged periods of time. 

In horses, high circulating erythrocyte 

counts, particularly in response to exercise, 

makes measurement of whole blood 

lactate levels less accurate, repeatable 

and meaningful than plasma lactate levels. 

This necessitates deproteinisation and 

centrifugation before analysis of plasma 

samples, which is much less convenient 

than whole blood analyses, which can be 

performed for human athletes with instantreading 

hand-held machines. 

For racehorses, results are uninterpretable 

without carefully controlled standard exercise 

testing conditions, which are often difficult 

to organise, especially under Thoroughbred 

racehorse training conditions. 

Plasma lactate is also used for monitoring 

cardiovascular status in horses receiving 

critical care. It has been shown to be a useful 

prognostic tool in surgical colic patients 

and is particularly helpful in monitoring 

circulatory support in compromised foals. 

If you wish to assay plasma lactate, please 

contact Beaufort Cottage Laboratories for 

sample handling guidance. 

Therapeutic drug monitoring 

Therapeutic drug monitoring (TDM) is 

increasingly used in equine medicine and 

cardiology and is an invaluable guide to 

treatment regimens. The aminoglycosides, 

gentimicin and amikacin, have the potential 

to cause nephrotoxicity in horses while 

under-dosing can lead to treatment failure. 

Serum samples are drawn 30 minutes and 

8, 12 or 24 (amikacin only) hours later to 

identify the peak and trough levels. Peak 

levels should be at least 8 times the MIC of 

the organism in question (up to 4 for some 

gentimicin-sensitive equine pathogens) 

whereas a delay in excretion is indicative 

of renal tubular dysfunction and should 

prompt discontinuation of aminoglycoside 

therapy. Several of the drugs that are useful 

in equine cardiac patients have a low 

therapeutic index. Assays for phenytoin and 

digoxin are available for close monitoring for 

potential drug toxicity in patients receiving 

these drugs.


blood gas analysis 

Analyses are essential for monitoring 

respiratory function under anaesthesia 

in cases of neonatal septicaemia or 

maladjustment under critical care and 

for older horses with respiratory and 

intestinal abnormality, where acidosis or 

alkalosis are suspected, prior to therapeutic 

correction. Analyses are helpful, alongside 

fluid and electrolyte balance assessments, 

for enterotoxaemia and other criticallyill 

horses under intensive care and for 

endurance horses and others performing in 

hot, humid conditions, suffering exertional 

exhaustion and/or heat stress. 

Samples must be taken in heparinised, 

airtight syringes (preferably glass), and 

transported on ice to the laboratory 

within an hour or two of collection, so are 

practically limited to immediate in-house 

testing. 

Endocrinology 

Pregnancy Tests 

Serum gonadotrophins (eCG) may 

be detected in mares where functional 

endometrial cups are present. For accurate 

results, serum samples should be collected 

between 45 and 95 days since the last 

date of mating. False negative results are 

unusual inside this period, but can occur 

in rare cases where eCG levels are below 

test 'threshold'. False positive tests are more 

common and may occur when early foetal 

death has left residual functional cups. In 

such cases the mare’s serum may remain 

eCG positive for the functional life of the 

cups, sometimes up to 100 days. 

Oestrone sulphate may be detected in the 

serum/plasma of mares over 120 days 

pregnant. At that time, levels of >100 

ng/ml are usually found (0-25 ng/ml in 

non-pregnant mares). Most of the oestrone 

sulphate peak originates from the foetal 

gonads so this may be a useful test of 

foetal viability as well as a pregnancy test. 

Levels fall during the last few weeks of 

pregnancy. 

Urinary oestrogens, of placental origin, 

may be detected in mares after 150 days 

of gestation. Urine samples are required. 

In some mares the required fluorescent 

response may be unreliable to detect and 

the serum oestrone sulphate test is now a 

much more reliable test. 

Progestagens 

The analysis of plasma progesterone levels 

is a useful guide to diagnosis and treatment 

in the acyclic or irregularly cyclic mare. In 

the non-pregnant mare, levels >2 ng/l (6.3 

nmol/l) indicate functional luteal tissue 

33


Oestrone sulphate rig test 

Normal male Cryptorchid ‘False rig’ 

Oestrone sulphate 10-50 ng/ml 0.1-10 ng/ml 0-0.02 ng/ml 

hCG/testosterone rig test 

Testosterone stimulation test should be used for all donkeys and in horses under 3 years old. 

Testosterone levels are measured in serum (clotted) or heparinised plasma samples taken before 

and then 30-120 minutes after the intravenous injection of 6000 iu hCG. 

Normal male Cryptorchid ‘False rig’ 

Testosterone (1) 5-30 nmol/l 0.3-4.3 nmol/l 0.03-0.15 nmol/l 

Testosterone (2) 5-30 nmol/l 1.0-12.9 nmol/l 0.05-0.19 nmol/l 

and suggest that prostaglandin treatment 

should induce luteolysis, providing that the 

corpus luteum is more than 4 days old. 

In the pregnant mare, there is no proven 

relationship between progesterone levels 

and the integrity of pregnancy. Mares with 

levels 4 ng/ml but there is considerable daily 

and individual variation. The progesterone 

assay is in no way an accurate pregnancy 

test but may be helpful, or reassuring to 

managers, in monitoring individual mares 

with histories of repeated pregnancy failure. 

Granulosa Cell Tumour (GCT) 

The most common ovarian tumour in the 

mare is the granulosa cell tumour (GCT). 

GCT’s produce steroid hormones which 

can be detected in elevated quantities 

in the peripheral blood. In cases where 

the tumour has a significant theca cell 

component (approximately 54% of cases), 

serum testosterone is elevated. These 

mares are more likely to be aggressive 

and stallion like. GCT mares showing 

anoestrus or persistent oestrus may have 

normal testosterone concentrations. Inhibin 

concentrations have been found to be 

elevated above normal in approximately 

87% of mares with GCT’s, and therefore 

inhibin appears to be a more accurate 

indicator of the presence of a GCT than 

testosterone alone. We recommend 

measurement of serum testosterone and 

inhibin to support an ultrasonographic 

diagnosis of GCT, or in cases in which 

rectal palpation is undesirable. 

34


Cryptorchidism 

Oestrone sulphate assay may be used as 

a cryptorchid or 'rig' test for horses (not 

for donkeys) that are over 3 years old (see 

table on left). 

Thyroid function 

Thyroid hormones, i.e. thyroxine (T4) and 

tri-iodothyronine (T3) are measured in 

equine serum samples. As diurnal rhythms 

are involved, two samples should be 

collected ideally early in the morning and 

late in the afternoon. Low levels (T4 240 nmol/l) in classical cases 

(aged, hirsute, polydipsic, polyuric, 

glucosuric). However, basal levels are 

often within normal range for younger, 

developing cases and so TRH stimulation 

test, overnight dexamethasone suppression 

test or combinations of such tests are 

needed to help define pituitary normality 

or abnormality. 

Insulin 

Serum insulin is sometimes a useful 

‘screening’ test for equine Cushing’s 

syndrome. Results 1000 miu/ml. 

Overnight dexamethasone 

suppression test 

This test is based upon the fact that 

dexamethasone administration suppresses 

plasma cortisol in normal horses because of 

feedback on ACTH release. A resting serum 

sample is collected at 5 pm and 40 µg/kg 

DXM (Azium, Gist-Brocades, 20 mg/500 

kg horse) is injected im. A follow-up serum 

sample is collected at 12 pm the following 

day. Normal horses show a suppression of 

serum cortisol levels to


TRH stimulation test 

This is considered the safest of the dynamic 

cortisol tests with the least risk of inducing 

laminitis. Protocol is to collect a baseline 

serum sample, inject 1 mg Thyroid Releasing 

Hormone (TRH; Roche) iv and then collect 

further serum samples at 15 and 60 

minutes thereafter. Typical Cushing’s cases 

show an increase in cortisol of greater 

than 20% (up to 90%) above baseline 

at 15 minutes, compared to a rise of 

approximately 17% in the normal horse. 

Cortisol concentrations return to baseline 

values at 60 minutes in the normal horse, 

but may remain at around 55% above 

baseline in Cushing’s cases. 

Combined DXM suppression, 

TRH stimulation test 

This test has been recently recommended, 

although there is dispute about whether 

it is more helpful than the overnight 

DXM suppression described above. It is 

indicated when the results of overnight 

DXM suppression or TRH stimulation tests 

are inconclusive. A protocol is to collect 

a baseline serum sample and then inject 

40g/kg DXM iv. A follow-up serum sample 

is collected 3 hours later and then 1.0 

mg TRH is injected iv. A follow-up serum 

sample is collected 30 minutes later and 

a final serum sample is collected 24 hours 

after the DXM injection. Cushingoid horses 

show suppression of serum cortisol level by 

3 hours after DXM injection and return to 

baseline by 30 minutes after TRH injection 

and by 24 hours after DXM injection. 

Conclusions are that while typical aged cases 

of equine Cushing’s syndrome are easy to 

diagnose, often on grounds of clinical signs, 

hyperglycaemia and glucosuria, younger, 

early and atypical cases are often extremely 

difficult to confirm, with marginal clinical 

abnormality and often conflicting insulin 

and dynamic cortisol testing results. Basal 

cortisol and insulin is a useful screening 

test in horses grazing at pasture and in 

stabled horses providing their samples 

are not collected after a feed. The most 

practical and probably reliable dynamic test 

is currently considered to be the overnight 

dexamethasone suppression test. 

ACTH stimulation test 

This test is primarily used to identify 

adrenal insufficiency. In human critical care 

it is recognised that many patients with 

Systemic Inflammatory Response Syndrome 

(SIRS) have adrenal insufficiency and may 

benefit from appropriate treatment. Adrenal 

insufficiency occurs commonly in premature 

foals but its prevalence in adult horses is 

unknown. The ACTH stimulation test is NOT 

recommended for the diagnosis of equine 

pituitary pars intermedia dysfunction where 

the combined dexamethasone suppression- 

TRH stimulation test is currently considered 

the gold standard. 

36


urine collection & analysis 

Urine analysis is useful to help detect renal 

or bladder pathology and to investigate 

cases of septic nephritis, cystitis or urethritis. 

Mid-stream samples should be collected 

without the use of diuretics (which alter 

urine composition) into a sterile, empty 

universal container. Beware of owners 

collecting samples into used jam jars or 

milk bottles before pouring the urine into 

the provided universal container, resulting 

in spurious glucosuria and bacterial culture 

results. For fractional urinary electrolyte 

and mineral clearance ratio measurements 

(see page 30), paired urine (not following 

diuretic administration) and serum samples 

should be collected simultaneously (ideally) 

or within one hour of each other. 

Urine samples should be examined grossly 

for colour and consistency, the presence 

of blood (either fresh or changed), pus 

or excessive crystalline material. Horse 

urine is highly variable in colour from 

near colourless to golden or brownish 

and in its density, turbidity and mucinous 

content. Specific gravity (1.008-1.040 

in adult horses, 1.001-1.025 in foals) 

should be measured with a refractometer. 

Dipsticks are commonly used to measure 

pH (normally 7.5-8.5 in adult horses, 

5.5-8.0 in foals) and to detect other 

abnormalities. Urine pH reflects diet and 

horses grazing pasture will normally have 

alkaline urine whereas those on a cerealbased 

performance-type diet will normally 

have slightly acidic urine. Proteinuria 

may occur with renal tubular pathology. 

Glucosuria may be seen in classical 

cushingoid horses and ponies. Haematuria 

and sometimes haemoglobinuria may 

occur following traumatic injury, or 

with renal or cystic calculus formation. 

Haemoglobinuria may occur with 

haemolytic conditions.Myoglobinuria is 

seen with myopathies. Bilirubinuria may 

occur with choleliths or other causes of 

bile duct obstruction. Ketonuria is rarely 

seen in horses. Microscopic examinations 

should be used to detect casts (protein 

and cellular masses), which suggest renal 

tubular pathology, leucocytes, which 

suggest inflammation/infection, bacteria, 

which if seen following Gram’s stain in 

association with leucocytes may indicate 

infection and erythrocytes, which indicate 

haemorrhage and crystals. Horse urine is 

fundamentally a supersaturated solution of 

calcium carbonate and will normally contain 

variable amounts of predominantly calcium 

carbonate crystals. Urolithiasis cases usually 

have a degree of proteinuria and haematuria 

and may exhibit dysuria. Large amounts 

of sabulous material are not necessarily 

an indication of abnormality. Further 

investigations include bladder and kidney 

palpation, ultrasound scan and cystoscopic 

examinations, looking for sabulous (bladder) 

or discrete calculus formation. 

Fractional urinary electrolyte and mineral 

clearance ratios (see p30) are significantly 

increased in horses with nephropathy, in 

particular with renal tubular malfunction. 

37


parasitology 

38 

Faeces Collection 

Faecal analysis is helpful in providing worm 

egg counts to help monitor parasite control 

programmes and to investigate cases or 

diarrhoea and septic enterocolitis. Freshly 

produced or rectal faecal samples should 

be collected into an inverted clean rectal 

sleeve so that environmental contamination 

and alteration is minimised and there is 

no doubt about the identity of the horse 

that produced the sample. Fluid diarrhoea 

samples should be submitted in sterile 

universal containers and on sterile swabs 

immersed in Amies’ charcoal transport 

medium. It is often difficult to collect 

diarrhoea samples from foals but digital 

stimulation of the rectum sometimes 

precipitates production of a sample. 

Faecal Worm Egg Counts 

These remain the basis of equine intestinal 

parasitic surveillance and monitoring of 

worm control programmes. They are not 

always a reliable means of assessing an 

individual horse, for which haematological 

and serum protein (albumin and protein 

electrophoresis) investigations are more 

reliable (see page 19). 

Testing methods vary between laboratories. 

Our worm egg counts are performed using 

the ‘Ovassay’ method, a flotation method 

which is particularly suitable for use in 

horse samples as it is extremely sensitive 

at detecting low numbers of strongyle 

and related species eggs. Ascarid and 

Strongyloides spp. eggs are also detected. 

We believe that other methods (Stoll and 

McMaster) are more suitable for counting 

worm eggs in samples where high counts 

are expected (e.g. farm animals) as these 

methods involve dilution rather than 

concentration of the eggs into the counting 

area. Our experience is that well-managed 

horses under good endoparasite control 

regimes consistently have zero strongyle 

eggs/g of faeces, using the Ovassay 

technique. For example, the presence, of 

egg laying strongyles in the intestinal lumen 

represents the final stage of the protracted 

migration of the developing larvae through 

the horse’s tissues. Using this method, a 

positive worm egg count of any magnitude 

in the faeces is a significant finding and 

indicates that the horse needs anthelmintic 

treatment, the parasite control programme 

requires review and the horse would benefit 

from a more in-depth haematological and 

serum protein appraisal. 

Tapeworm segments may sometimes be 

seen in the faeces by gross examination 

and are often seen in the caecal content 

of tapeworm-related intussusception cases 

at surgical correction. A serological (ELISA) 

test is available for detecting tapeworm 

infestations in horses. Titres of 0.6 are considered negative or 

low-intensity, moderate and high intensity 

infestations, respectively. 

Faecal Lungworm Larval Counts 

Dictyocaulus arnfieldi larvae may be 

detected in fresh rectal-collected faeces 

of infested horses using the modified 

Baermann funnel gravitation method. A 

severe eosinophilic bronchitis may be 

detected by cytological examination of 

tracheal washes (see page 44).


microbiology 

Bacteriology 

Swabs from any site should be taken into 

Amies charcoal transport medium before 

transport to the laboratory where they may 

be cultured under aerobic, microaerophilic 

and/or anaerobic conditions. 

Where potential pathogens are isolated, 

antibiotic sensitivity tests should be 

performed. In acute septicaemias, before 

antibiotic treatment has been started, 

jugular venous blood samples may be 

taken, using sterile techniques, and 

inoculated into blood culture media, before 

transport to the laboratory. 

All genital swabs should be cultured, 

unless specifically requested otherwise, 

aerobically and microaerophilically. 

Aerobic results are available after 24/48 

hours culture. Contagious Equine Metritis 

(Taylorella equigenitalis) microaerophilic 

culture results are available after 7 

days incubation, but most specifically 

experienced laboratories are able to 

culture positive isolates by 3/4 days. 

Klebsiella pneumoniae and Pseudomonas 

aeruginosa isolates should be confirmed 

by biochemical tests and K. pneumoniae 

isolates capsule typed for the recognised 

K1, K2 or K5 types using the 'Quellung' 

or counter current immunoelectrophoretic 

methods. 

Anaerobic culture may be performed where 

indicated, using standard techniques. 

Endometrial swabs should be accompanied 

by a concurrently sampled endometrial 

smear to aid the interpretation of culture 

results (see page 46). 

In addition to routine aerobic culture, 

special methods should be used routinely 

to screen rectal swabs and biopsies (in 

transport media) and faeces for Salmonella 

spp. and Campylobacter spp. 

Nasal and pharyngeal swabs, 

submandibular and parotid abscess 

swabs should be routinely screened 

for Streptococcus equi and, in foals, 

Rhodococcus equi. 

Tracheal wash and bronchoalveolar 

lavage (BAL) samples, when submitted 

for bacteriological examinations, should 

be routinely screened for Streptococcus 

pneumoniae, Bordetella bronchiseptica 

and Pasteurella spp. 

Blood culture medium should be used for, 

in addition to blood samples, other body 

fluid samples, e.g. peritoneal and pleural 

fluids and especially for synovial fluid and 

cerebrospinal fluid (CSF) from which it is 

often difficult to isolate pathogens. 

Skin scrapings 

Skin scraping samples are collected from 

horses and ponies for the confirmation 

of suspected dermatophyte infections or 

ectoparasite infestations. Samples should 

be collected from fresh skin lesions, from 

the edges of the lesions if extensive, using 

39


a fresh sterile scalpel blade. The hairs 

and the scraped skin layers, down to 

haemorrhage, should be collected into a 

sterile universal container and submitted 

for laboratory examination without delay. 

The hairs and scraped skin layers should 

be incubated for 15 minutes in warm 

40% potassium hydroxide (KOH) and then 

examined for clear ringworm spores in 

broken hair shafts and free whole or 

fragmented ectoparasitic mites, including 

Sarcoptes, Psoroptes, Chorioptes and 

Demodex spp. (differentiated on the basis 

of their anatomical appearance). 

If negative for dermatophyte spores after 

KOH incubation, skin scrapings are 

incubated overnight in blue/black ink 

and then examined for blue/black stained 

spores in clear broken hairs. 

Skin scraping samples should be incubated 

for bacterial and fungal growth. The latter 

(taking up to 3 weeks) will differentiate the 

different ringworm-causing dermatophytes, 

including Trichophyton and Microsporum 

spp. 

Microscopic and fungal culture results for 

dermatophytes do not always correlate. 

Positive microscopic findings but negative 

culture results may suggest that the spores 

were not viable at the time of scraping. 

Negative microscopic findings but positive 

culture results may suggest that too few 

spores were present in the sample to be 

detected in spite of careful examination. 

If Dermatophilus congolensis (‘rain scald’ 

or ‘mud fever’) infection is suspected on 

clinical grounds, moist lesions should be 

collected and submitted for impression 

smear stained with methylene blue to look 

for characteristic chains (‘piles of pennies’) 

of flattened spores, sometimes in branched 

conformation. 

clostridial toxin tests 

ELISAs to detect the toxins produced 

by pathogenic forms of Clostridium 

perfringens and Clostridium difficile are 

indicated in horses and foals with acute 

diarrhoea, particularly where the problem 

may be associated with antimicrobial 

administration. Faecal samples should be 

taken before the drug metronidazole is 

given and should be chilled if delivery to 

the laboratory is likely to take more than 

an hour or two. 

Virology 

Fluid diarrhoea samples may be examined 

by latex particle agglutination for the 

presence of Rotavirus antigen. 

Specialist virology laboratory facilities and 

expertise are required for testing for the 

following equine viruses:- 

Equine Influenza: paired serum samples 

(acute and convalescent phases, collected 

14 days apart) are examined for signs 

of seroconversion (a four-fold or greater 

rise in titres between the acute and 

convalescent samples). Nasal discharge or 

40


nasopharyngeal swabs collected during the 

acute phase into viral transport medium are 

cultured specifically for the virus. A direct 

ELISA test is available for the rapid detection 

of the virus in acute-phase nasal discharge 

or nasopharyngeal swab samples. 

Equine Herpesviruses (EHV-1, EHV-3, 

EHV-4): paired serum samples (acute 

and convalescent phases, collected 14 

days apart) are examined for signs of 

seroconversion (significant rise in titres 

between the acute and convalescent 

samples). Nasal discharge or 


acute phase into viral transport medium are 

cultured specifically for EHV-1 and EHV-4. 

Aborted foetal, placental and neurological 

tissues may be examined for EHV-1 and 

EHV-4 DNA by specific polymerase chain 

reaction (PCR) test. 

Equine Viral Arteritis (EVA): paired serum 

samples (acute and convalescent phases, 

collected 14 days apart) are examined 

for signs of seroconversion (significant 

rise in titres between the acute and 

convalescent samples). Nasal discharge or 


acute phase into viral transport medium 

are cultured specifically for the virus. 

Aborted foetal and placental tissues may 

be examined for the virus DNA by specific 

PCR test. 

For the screening of equine aborted foeti 

for EHV and EVA infections, please see 

postmortem examinations (see page 48). 

NOTES 

41


cytology 

Diagnostic cytological examinations are valuable diagnostic 

tools following the collection of appropriate and good quality 

samples. Safe, reliable and appropriate techniques should be 

used to collect either fluid or smear samples. 

Fluid samples 

These should, in general, be submitted in 

sequestrene (EDTA) for a nucleated cell 

count, and fixed with a suitable fixative 

(e.g. cytospin fixation fluid) for specific 

cytological processing. Another undiluted 

and unfixed sample should be submitted 

in a sterile container or on a sterile swab 

in transport medium or ideally in blood 

culture medium (particularly for synovial 

fluid samples) for concurrent bacterial 

culture. 

Fluid samples with low total nucleated 

cell counts (


Peritoneal fluid 

Analysis of peritoneal fluid is particularly 

useful as a diagnostic aid in cases of colic, 

weight loss and other suspected abdominal 

disease. It may be of particular value in 

helping to make the decision for surgical 

intervention. 

To perform a peritoneal tap, the skin over 

the site of puncture should be clipped 

and prepared as for surgical intervention. 

With the horse restrained in the standing 

position, a 19 gauge, 1.5 or 2.0 inch 

needle or following local anaesthesia and a 

stab incision, a 7.5 cm. blunt teat cannula 

(operator preference) is carefully advanced 

through the skin at the lowest part of the 

abdomen and then through the linea alba. 

If fluid is not immediately forthcoming, the 

needle may be rotated or the tap may be 

repeated at other sites. 

In foals, prior ultrasound scan examination 

of the abdomen is recommended to 

help visualise abnormalities and prevent 

inadvertent penetration of the intestine. 

Examination of the ventral midline will help 

to locate the spleen and to find a ‘pocket’ of 

peritoneal fluid to guide productive needle 

puncture. Similarly, if fluid is not obtained 

in adult horses using the blind technique 

described above, ultrasonic guidance may 

be helpful. 

A turbid and homogeneously blood stained 

sample may indicate abdominal vascular 

embarrassment. A white, turbid fluid may 

suggest peritonitis. A brown, foul smelling 

fluid may indicate intestinal rupture or an 

intestinal tap. A thick and heavily blood 

stained sample suggests a splenic tap. 

Total nucleated cell counts


Synovial fluid 

Analysis of Synovial Fluid is useful for the 

diagnosis of reactive or septic arthritis in 

horses after injury, when a joint becomes 

distended, with or without lameness, and to 

differentiate septic ('joint-ill') from traumatic 

arthritis in foals. 

To perform a joint tap, septic techniques 

for needle puncture should be used. The 

joint to be sampled and its anatomical 

relationships govern the site of puncture 

and to a degree the techniques used. Total 

nucleated cell counts 10 x 10 9 /l, with degenerative and toxic 

cytopathological changes. 

Tracheal washes or aspirations 

These samples can be extremely useful 

in confirming and characterising airway 

inflammation (leucocytes) in cases of 

septic tracheobronchitis and in reactive and 

obstructive small airway disease. 

Samples can be collected via a suitablelength 

sterile endoscope. The endoscope 

is passed through the pharynx, larynx and 

into the trachea. A long sterile polyethylene 

tube is passed down the instrument 

channel and accumulated secretions may 

be aspirated directly. Alternatively, and 

more usually, 50 ml sterile saline may be 

injected as quickly as possible and then 

aspirated while withdrawing the tube. If 

a suitable endoscope is not available, a 

sterile polyethylene tube may be passed 

down a trochar inserted surgically between 

two tracheal rings at the mid lower third 

of the cervical trachea. This is clearly a 

more invasive technique that may lead 

to local wound complications but has the 

advantage that there is less likelihood 

of contamination of the sample with 

commensal bacteria from the oropharynx. 

Trans-tracheal aspirates, collected by this 

method, are particularly useful in cases 

of pleuropneumonia where causative 

organisms must be specifically identified to 

direct antimicrobial therapy. 

Bronchoalveolar lavage 

(BAL) samples 

BAL samples can be helpful in the 

diagnosis of small airway disease. One 

must remember that the technique samples 

a focal area only and does not help with 

the diagnosis of tracheobronchitis. It is 

therefore more commonly used in older 

horses for the characterisation of reactive 

airway obstruction. 

Cytological examinations of tracheal 

wash or BAL samples may help in the 

characterisation of mucous production and 

acute and chronic, septic and reactive 

(non-septic) inflammatory responses. The 

demonstration of significant numbers of 

eosinophils in association with signs of septic 

bronchitis/bronchiolitis may be a useful 

44


diagnostic aid for lungworm (Dictyocaulus 

arnfieldi) infestation, in horses and ponies 

coughing at pasture. Bacterial culture can 

be useful in identifying specific pathogens 

and guiding antimicrobial therapy. 

Cerebrospinal (CSF) samples 

CSF samples are most commonly collected 

in horses showing neurological signs 

for the diagnosis and differentiation of 

meningitis or traumatic injury. In horses 

imported from countries where equine 

protozoal myeloencephalitis occurs and 

who develop neurological signs, CSF 

analysis is indicated. 

For a CSF tap, samples are collected from 

the atlanto-occipital space with the horse 

restrained in lateral recumbency with the 

poll flexed. In foals it may be possible to 

perform this under heavy sedation whereas 

in adults, general anaesthesia is mandatory. 

The skin is clipped and prepared as if for 

surgical intervention and a bleb of local 

anaesthetic is placed in the midline of the 

dorsal neck at a level defined by a line 

joining the cranial borders or the atlas, 

which may be clearly palpated. A sterile 

18 gauge 2 inch needle is then inserted 

through the skin at 90° and advanced 

until it ‘pops’ through the meninges and 

CSF drips or pours from the needle into a 

sterile container or is aspirated into a sterile 

syringe. 

Alternatively, in adult horses, CSF may 

be collected via the lumbosacral (LS) 

space, by lumbosacral tap. The horse is 

restrained, sedated, in stocks. The skin 

between the tuber coxae is clipped and 

prepared as if for surgical intervention. A 

sizeable (3-4 ml) bleb of local anaesthetic 

is placed in and under the skin in the 

midline at a line bisecting the caudal 

borders of the tuber coxae. With the 

horse standing ’square’ with weight evenly 

distributed on both hind legs, a sterile 18 

gauge 6 inch spinal needle with stylette in 

place is then inserted though the skin in 

the midline at the line bisecting the tuber 

coxae and down through the palpable 

depression just caudal to the sixth lumbar 

spinous process. The horse will often flinch 

when the subarachnoid space is penetrated 

and this is an indication to start aspiration 

into a sterile syringe for a fluid sample. 

Gross examination of CSF reveals a clear 

almost colourless fluid in normality and 

a turbid and/or bloodstained fluid with 

meningitis or following traumatic injury. 

Laboratory examinations of CSF need to be 

able to measure very low total nucleated 

cell counts (


46 

Bone marrow aspirates 

These are not commonly collected from 

horses - only in cases that may have severe, 

undefined anaemia, severe persistent 

leucopenia or thrombo-cytopenia and in 

some cases of leukaemia. 

Samples are most commonly collected 

from the wing of the ilium, the ribs or the 

sternum. Sternal tap is recommended 

because it is most reliably productive. 

The ventral midline, under the sternum 

is clipped and prepared as if for surgical 

intervention. Local anaesthetic is infused 

into and under the skin of the ventral 

midline, just caudal to the olecranon, with 

the horse standing normally, restrained, 

sedated, in stocks. A bone marrow 

collection needle or an 18 gauge 3.5 inch 

spinal needle is introduced through a stab 

incision in the skin upwards to contact 

the sternum. The needle is then rotated 

with upward pressure until it enters the 

sternum, the stylette is removed and firm 

suction is used to aspirate a sample into a 

sterile 10 or 20 ml syringe, pre-treated with 

a few drops of 15% tripotassium EDTA to 

prevent clotting. 

The sample is transferred to EDTA tubes 

and labelled for laboratory examination, 

without delay. If delay is inevitable, smears 

should be made and fixed, to be sent 

alongside the wet samples, in case they 

are needed. 

The cytopathological examination of bone 

marrow smears requires specific experience. 

Normal and abnormal cell types are classified 

and their relative proportions assessed. The 

normal equine myeloid:erythroid (M:E) ratio 

should be 0.5-1.5. 

Semen samples 

Semen samples should be collected into 

an artificial vagina to allow a meaningful 

interpretation. In addition to a full case 

history, details of methods of collection, the 

colour, consistency, volume and motility of 

the ejaculate should be submitted with the 

sample. An undiluted semen sample should 

be sent in a sterile container for sperm 

density and bacteriological examinations, 

and a sample diluted immediately 1:1 with 

formol citrate solution for sperm live:dead 

and morphology examinations. 

Smear Samples 

Smear samples should, in general, be 

submitted already rolled onto a gelatincoated 

slide and fixed (‘Smear-fix’, carbowax 

or even hair spray) for specific cytological 

processing. Another undiluted and unfixed 

sample should be submitted in a sterile 

container or on a sterile swab in transport 

medium for concurrent bacterial culture. 

Endometrial smears 

Endometrial Smears are simple and quick to 

perform and provide a much more accurate 

and direct test for the diagnosis of acute 

endometritis in mares, pre-coitus than with 

swab examinations alone. When used in 

conjunction with endometrial swabs for 

bacteriological examinations, results are 

infinitely more meaningful, interpretable 

and therefore valuable. The presence of


endometrial epithelial cells is used as a 

test of smear quality and the presence or 

absence of polymorphonuclear leucocytes 

is used as the diagnostic test for acute 

endometritis. 

Smears may be collected during oestrus 

by a variety of methods, but we favour 

a simple non-guarded technique. An 

extended, sterile, large tipped swab is 

passed via a sterile speculum, through 

the relaxed cervix and rotated onto the 

endometrial lining. The swab is withdrawn 

and should be rolled onto gelatine-coated 

slides immediately and fixed with 'Smearfix', 

carbowax or even hair spray prior to 

transport to the laboratory for staining. 

Excellent results have been obtained by 

rolling smears onto 'Testsimplets' (Boehringer 

Mannheim UK Ltd.) pre-stained slides, 

then after two to three minutes at room 

temperature, washing off the background 

blue colour, drying and cover slipping. This 

technique provides an excellent permanent 

smear sample for immediate reading and/or 

for sending for a second opinion. 

Histology 

Using our state-of-the-art microwave 

processing system, same day results can be 

provided for most fixed tissue specimens. 

In general terms, equine lesions are best 

sampled, if possible and appropriate, 

by total removal and submission 

for histopathological processing and 

examination. If total removal is not possible, 

samples of representative size and location 

should be collected by wedge resection. 

Fine needle aspirates should only be 

collected if it is not possible to collect 

larger samples. Experience suggests that 

they often result in a traumatised lesion 

without a conclusive diagnosis and with 

a recommendation for the collection of a 

larger and more representative sample, 

delaying resolution. Total lesion removal is 

always preferable, if possible. 

Removed lesions or biopsies should be 

placed immediately into an adequate 

volume (10 x sample volume) of 10% 

formol saline. Reproductive tissues are 

best fixed in Bouin’s fluid because of their 

higher water content. Large lesions should 

be sectioned before submersion to allow 

adequate penetration of fixative. Sectioning 

should be performed with a thought to how 

the sample will need to be trimmed and 

orientated for histopathological processing 

to allow the pathologist to make a complete 

and meaningful appraisal. When sampling 

organs and tissues, we recommend that 

samples are taken from adjacent, grossly 

normal sites as well as from lesional 

sites. Notes of your own postmortem 

examination findings should be sent to aid 

histopathological interpretations. Samples 

should be carefully packed for transport to 

avoid leakages and breakages. 

47


necropsy (postmortem) examinations 

48 

Full necropsy examinations should be 

performed on all adult and younger horses 

who die unexpectedly, those who may pose 

a risk to in-contact horses, or for whom 

confirmation of pathology is required, 

and those who are the subject of an 

insurance claim. Ideally, the examination 

should be performed, without delay, under 

conditions of adequate facilities, by suitably 

qualified and experienced pathologists, 

so that the end result is satisfactory in 

terms of answering the questions posed. 

Nevertheless, for a variety of reasons, it 

may not always be possible for dead horses 

to be transported to a specialised equine 

pathology facility and examinations may 

need to be performed ‘in the field’. Faced 

with this need, those responsible should 

consider what conditions are required 

to enable a productive examination to 

be performed. Also, waste and carcase 

disposal, prevention of environmental 

contamination and spread of infectious 

agents to humans and other animals must 

be considered. Occasionally, it may be 

possible to refer part of a carcase for 

specific necropsy investigations, e.g. a foot 

or a leg only for orthopaedic examinations 

or the head and neck to thoracic vertebra 

3 in cases of ataxia for cervical cord 

compression examination. 

Adult horses 

Where full post-mortem facilities, including 

hoists and tables are not available, 

experience suggests that the horse should 

be positioned on the ground in right lateral 

recumbency. If an incline is available, the 

horse’s back should be positioned up the 

hill. The horse should be identified and 

an external examination performed and 

findings recorded. 

The first cut is made deeply under the left 

axilla to lay the left front leg upward and 

flat back over the horse’s withers. The 

second cut is made deeply under the left 

groin, aiming caudally towards the anus, 

through the left hip joint, to lay the left hind 

leg upwards and flat back over the horse’s 

pelvis. The abdomen is then opened along 

the midline, from xiphisternum to pubis and 

the left abdominal wall is laid back over the 

horse’s back by cutting up adjacent to the 

last rib and up into the groin. The caecum, 

large colon and small intestines are then 

removed from the abdomen while looking 

for signs of displacement or gross pathology. 

The small intestines are removed carefully, 

examining the cranial mesenteric root for 

verminous pathology and the pancreas, 

and taking care not to remove the kidneys 

and adrenal glands from their attachments 

below the spine. The spleen, stomach and 

liver are then removed and examined. The 

kidneys are then carefully removed for 

examination to leave the adrenal glands 

attached either side of the aorta. A block


of tissue is then removed to include both 

adrenal glands and the adjacent aorta, 

for overnight fixation, prior to dissecting 

out the coeliaco-mesenteric sympathetic 

nerve ganglia, which lie between the 

adrenal glands and the aorta, but which 

are less easy to find in fresh condition. 

The bladder and internal genitalia are then 

removed and examined. The diaphragm 

is then examined and opened to reveal the 

thoracic viscera. The ribs are separated 

through their costochondral junctions, 

the intercostal muscles are cut and the 

ribs are broken back over the horse’s 

back. The ‘pluck’ including the trachea, 

heart and lungs are then removed for 

examination. The thymus is examined and 

removed in immature horses. The head 

is removed through the atlanto-occipital 

joint and ideally sectioned longitudinally 

with a band saw. The brain, meninges, 

guttural pouches, paranasal sinuses, teeth, 

pharynx and larynx are then examined 

thoroughly. The limb joints are opened 

for examination of the synovial fluid and 

surfaces. If indicated by the clinical signs, 

the neck and back vertebrae are dissected 

out and opened for examination of their 

articulations and the spinal cord. It is clear 

that a satisfactory examination of the head 

and spine of an adult horse is difficult to 

achieve under field conditions. 

Samples for bacterial or viral examination 

should be collected from abscesses, areas 

of inflammation, body cavities or seared 

viscera, using sterile swabs and placed 

without delay into Amies’ charcoal or 

specific viral transport media. 

Fluid samples from abscesses, cysts or 

body cavity fluids should be aspirated 

with a sterile syringe and then submitted 

in sequestrene (EDTA) for a nucleated cell 

count, and fixed with a suitable fixative 

(e.g. cytospin fixation fluid) for specific 

cytological processing. Another undiluted 

and unfixed sample should be submitted 

in a sterile container or on a sterile swab 

in transport medium or ideally into blood 

culture medium for concurrent bacterial 

culture. 

Samples for histopathological processing 

and examination should be carefully 

selected to provide thin and small, but 

representative tissue wedges, and totally 

immersed in 10% formol saline. Large 

thick samples fail to fix adequately and 

histopathological examinations are then 

unnecessarily complicated by autolytic 

changes, abused tissues may be ruined by 

artefactual damage and unrepresentative 

samples may not include the primary 

pathological changes. 

It is often wise to retain appropriate samples, 

e.g. gastric and intestinal content, urine and 

cubes of liver and kidney, carefully labelled, 

frozen, in case toxicological studies are 

required at a later date. 

49


50 

Coelioacomesenteric sympathetic 

nerve ganglion (CMSNG) sampling 

These tissues are specifically required for 

the diagnosis of equine dysautonomia 

(‘grass sickness’). 

In addition to performing a full necropsy 

examination (see above) to investigate 

other pathological conditions, the CMSNG 

require a specific approach to sampling. 

With the horse in right lateral recumbency 

and the left front and left hind legs cut 

back, the abdomen is opened along the 

midline, from xiphisternum to pubis and 

the left abdominal wall is laid back over the 

horse’s back. The caecum, large colon and 

small intestines are then removed from the 

abdomen. The small intestines are removed 

carefully, sectioning the cranial mesenteric 

root and taking care not to remove the 

kidneys and adrenal glands from their 

attachments below the spine. The spleen, 

stomach and liver are then removed. The 

kidneys are then removed carefully for 

examination to leave the adrenal glands 

attached either side of the aorta. A block 

of tissue is then removed to include both 

adrenal glands and the adjacent aorta, for 

overnight fixation in an adequate volume of 

10% formol saline. 

The following day, the tissues are examined 

and oriented to identify the two adrenal 

glands on the section of aorta. The adrenal 

glands are then carefully dissected off 

the aorta and the CMSNG are identified, 

removed and sectioned transversely and 

longitudinally for further fixation prior to 

processing. When fixed they appear greycoloured, 

solid and clearly neurological, 

rather than vascular (tubular on transverse 

section) or lymph node-like. 

They are 

much less easy to find and identify in fresh 

unfixed condition. 

After further fixation and processing, the 

ganglia are examined for the specific 

neuronal histopathological degenerative 

changes that appear characteristic of 

equine dysautonomia. 

Neonatal Foals, Foeti and 

Placentae 

These should be examined to determine 

the cause of foetal death and abortion, 

specifically looking for signs of EHV or EVA 

infections, important infectious causes of 

potential epidemic abortion in mares, as 

specifically recommended by the Horserace 

Betting Levy Board’s Code of Practice. 

The carcase and placental membranes 

should be examined together to find signs 

of foetal/foal, placental and maternal 

pathology. The foetus or foal may be 

opened in right lateral recumbency in a 

manner similar to that described above 

for adult horses. Although they may not 

be clearly seen in all cases and although 

combinations differ in different cases, the 

important gross pathological abnormalities 

of EHV infection to look for are:-


1. A freshly aborted foetus enclosed in 

its placental membranes, suggesting 

‘explosive’ abortion or a septicaemic 

neonate. 

2. Jaundiced hooves, mucous membranes 

and/or subcutaneous tissues. 

3. Excess, jaundiced peritoneal, pleural 

and/or pericardial fluids. 

4. Jaundiced visceral serosae and gelatinous 

jaundiced peri-renal fat. 

5. Pleural oedema and pneumonia. 

6. Multiple, pale, pinpoint foci visible on 

the liver capsule. 

The foetus should be weighed, the 

crown rump length and the umbilical 

cord length measured and recorded. The 

placenta should be spread out, checked for 

completeness and abnormalities recorded. 

Samples for laboratory investigation should 

include:- 

1. Small but representative slices of liver, 

lung, thymus, spleen, adrenal gland and 

chorioallantois (horns and posterior pole), 

plus any other interesting abnormality, 

in carefully packed leak-proof sealed 

containers, in an adequate volume 

of 10% formol saline for histological 

examinations. 

3. Swab samples of peritoneal fluid, liver, 

lung, heart blood, gastric contents, 

the cervical pole of the chorioallantois 

and areas of possible placentitis in 

Amies charcoal transport medium for 

bacteriological examinations. 

4. We recommend that small fresh foetal 

liver and lung samples are stored at – 

20°C (deep-freeze), in case viral isolation 

studies are required at a later stage. 

In addition, the carcase should be thoroughly 

examined for other abnormalities in case an 

infectious cause is not involved. Maternal 

uterine and placental incompetence often 

results in a grossly impoverished foetus. 

Umbilical cord vascular embarrassment (an 

important cause of equine foetal death and 

abortion, often associated with excessive, 

i.e. more than 90 cm, cord length) may 

be grossly obvious and, if significant, will 

be accompanied by signs of foetal vascular 

engorgement, haemorrhagic peritoneal and 

pleural fluids, congested and autolysed 

viscera and meconium staining/foetal 

diarrhoea. 

Whole carcases and whole placentae to be 

referred for necropsy investigations should 

be sent to a laboratory that is specifically 

experienced with equine neonatal 

pathology. 

2. Small (0.5 cm) cubes of liver, 

lung, thymus, spleen and pieces of 

chorioallantois in viral transport medium 

for EHV and EVA DNA (PCR) tests. 

51


biopsy sampling 

Skin Biopsy 

These are best obtained by full-thickness 

wedge incision. While skin punch biopsy 

techniques may be simpler to obtain, 

they are seldom as rewarding to examine. 

Tissues obtained should be fixed 

immediately in 10% formol saline. 

Lump biopsy 

Biopsy is recommended for masses that 

appear in or under the skin and sometimes 

for deeper masses that may be palpated 

or imaged by ultrasound or laparoscopic 

examination. If a total lesion resection is 

not possible, as is the case for many skin 

and subcutaneous masses, representative 

biopsies are best obtained by full-thickness 

wedge incision, and fixed immediately in 

10% formol saline. 

Liver Biopsy 

Liver biopsy is recommended where serum 

biochemical examinations have suggested 

hepatic pathology. With the horse restrained 

standing, ideally in stocks, the liver on the 

right side of the horse is examined with 

ultrasound to determine the ideal site 

for puncture and to detect signs of gross 

pathology, e.g. choleliths or Echinococcus 

spp. cysts. 

Biopsies are best obtained with a 6' 'Trucut' 

biopsy needle inserted between the 

14th and 15th ribs, on the right side of the 

horse, along a straight line drawn between 

the point of the hip and the shoulder, 

or elsewhere, if suggested by ultrasound 

examination. Specific lesion biopsies are 

best obtained by ultrasound guidance. The 

tissue sample should be carefully removed 

from the needle and fixed in 10% formol 

saline. The needle channel should then be 

carefully swabbed for bacterial culture. 

Lung Biopsy 

Lung biopsy may be indicated where there 

is clear ultrasonic evidence of pulmonary 

pathology. As biopsy of a lung abscess 

or focus of infection is contraindicated, 

evidence should suggest neoplasia or 

focal non-septic pathology before biopsy 

is attempted. Pulmonary neoplasia is very 

rare in horses so lung biopsy is seldom 

indicated. 

Needles specifically designed for lung 

biopsy should be used and the specific 

lesion to be biopsied should be imaged by 

ultrasound scan examination. The overlying 

intercostal skin site identified, infiltrated 

with local anaesthetic and clipped and 

prepared as for surgical intervention. With 

the transducer coupled in a sterile surgical 

glove and sterile coupling gel on its surface, 

the lung lesion is imaged and a lung biopsy 

device is introduced through the skin and 

into the lesion to collect the biopsy. 

The tissue sample is fixed in 10% formol 

saline without delay and the needle channel 

may, if required, be swabbed immediately 

for bacterial culture. 

52


Kidney Biopsy 

Renal biopsy may be indicated where 

there is clinicopathological and ultrasonic 

evidence of kidney pathology. The 

technique is not without risk of injury 

to the horse and therefore should only 

be contemplated if clearly indicated and 

should only be performed with great care, 

using an ultrasound-guided technique. 

The right kidney is more easily accessible. 

Ultrasound scan examination is used to 

identify a suitable site, free from large blood 

vessels, in the posterior poles. The horse 

should be restrained, sedated in stocks. 

The skin over the kidney is clipped for 

imaging to identify the site for penetration, 

local anaesthesia is induced and the skin is 

then prepared as for surgical intervention. 

With the transducer coupled in a sterile 

surgical glove and sterile coupling gel on 

its surface, the kidney is imaged to find the 

ideal site for puncture. The biopsy needle is 

then introduced through the skin and into 

the renal parenchyma, sampling specific 

pathological features if visible. 

If biopsy of the left kidney is to be 

attempted, it is helpful for an assistant to 

palpate the kidney per rectum in order to 

stabilise it against the body wall, facilitating 

the ultrasound-guided biopsy technique. 

The tissue sample is fixed in 10% formol 

saline without delay and the needle channel 

may be swabbed immediately for bacterial 

culture. 

The horse should be stable-rested for at 

least 48 hours after biopsy and should be 

monitored for signs of ill health, particularly 

associated with renal haemorrhage. 

Endometrial Biopsy 

Endometrial biopsies are indicated for the 

routine investigation of barren mares and 

for the investigation of specific endometrial 

pathology. Ideally, they are more easily 

obtained and interpreted when the mare 

is in mid-dioestrus, but samples may be 

safely obtained at any stage of the oestrous 

cycle from any non-pregnant mare. 

Biopsies are obtained with special forceps 

(Kruuse UK or Rocket of London Ltd.), 

via the vagina and cervix. The mare is 

restrained as for routine gynaecological 

examinations with her tail bandaged, 

rectum evacuated of faeces, tail bandaged 

and perineum hygienically prepared. The 

mare is re-confirmed not pregnant. The 

sterile biopsy forceps are introduced into 

the mare’s vagina with a gloved hand, the 

cervix is identified (making sure that the 

urethral opening has not been accidentally 

entered) and the index finger is placed 

through the mare’s cervix into the uterine 

body. The forceps are then advanced along 

the index finger, as a guide, through the 

cervix and into the uterine body. The finger 

is then ‘hooked’ in the cervix, which may 

then be retracted caudally. This straightens 

the cervix and the uterine body, allows the 

biopsy forceps to fully enter the uterine 

body in a cranial direction and prevents 

53


accidental cervical injury. With the hand 

holding the forceps held against the mare’s 

buttocks (to avoid injury to the uterus if 

the mare moves suddenly) maintaining 

the forceps in the uterus, the manipulating 

hand and arm are then withdrawn from 

the vagina and placed in the rectum. The 

forceps are then palpated and advanced 

into one or other of the uterine horns. The 

jaws of the forceps are then opened and 

a fold of endometrium is gently pushed 

into the jaws, which are then closed, the 

biopsy is completed and the forceps are 

withdrawn from the uterus and vagina. 

Tissues are carefully removed from the 

forceps with fine forceps to avoid artefactual 

damage and are fixed immediately in 

Bouin's fluid. A fine-tipped sterile swab may 

be used to sample the inside of the jaws of 

the biopsy instrument for bacterial culture. 

Sometimes more than one endometrial 

fold is removed and then the smaller one 

may be placed into transport medium for 

bacterial culture. 

Endometrial biopsy samples should be 

referred to a laboratory that is specifically 

experienced in both equine endometrial 

histopathology and equine gynaecology, 

in order to receive a meaningful 

interpretation. 

Testicular Biopsy 

Testicular biopsies are best obtained by 

conventional wedge resection with the 

horse under general anaesthesia in order 

to provide adequately interpretable tissues 

and to avoid accidental injury to the 

testicular artery, by blind needle biopsy, 

which may have fatal consequences. The 

testicle is examined by ultrasound scan for 

visible pathology and best site for sampling 

and the scrotum is prepared as for surgical 

intervention. Automated punch biopsy and 

ultrasound guided techniques are available 

but the relatively small tissue samples 

are better suited to specific research 

requirements. Great care must be taken to 

avoid injuring the testicular artery. 

Testicular tissues should be fixed 

immediately in Bouin's fluid. 

Ileal biopsy 

Using our state-of-the-art microwave 

processing system, same day results can 

be provided for fixed tissue specimens. 

Ileal biopsies may be indicated to attempt 

to confirm or deny a clinical diagnosis of 

equine dysautonomia (‘grass sickness’), 

where exploratory laparotomy is indicated. 

At exploratory laparotomy, a transmural, 

i.e. full-thickness, longitudinal ellipse of 

ileum, approximately 15mm by up to 

10mm is excised at the proximal end of 

the ileocaecal fold, midway between the 

mesenteric and antimesenteric borders. 

The tissue is placed immediately into 

10% formol saline and processed for 

histopathological examination. The ganglia 

in the myenteric plexus between the 

54


muscular layers are examined for normal 

and abnormal neurones. In ‘typical’ cases 

of equine dysautonomia, these ganglia are 

devoid of normal neurones and appear 

‘skeletal’. The occasional degenerate 

neurone, with nuclear degeneration and 

cytoplasmic vacuolar degenerative changes 

are seen in some cases. Also, clusters 

or normal neurones are seldom seen in 

the submucosal layers and again, the 

occasional degenerate neurone, with 

nuclear degeneration and cytoplasmic 

vacuolar degenerative changes are seen in 

some cases. 

Ileal biopsies should be referred to a 

laboratory that is specifically experienced 

with examining these tissues. 

Rectal biopsy 

Rectal biopsies are useful for the 

investigation of weight loss and diarrhoea 

cases or where haematological and serum 

biochemical results suggest enteropathy. 

Samples are best obtained with mare 

endometrial biopsy forceps (Yeoman's 

basket-jawed), inserted just a hand's length 

through the anus. A rectal fold (mucosa 

and submucosa only) is removed laterally 

on either side (dorsal or ventral biopsies are 

more prone to puncture blood vessels and 

result in worrying haemorrhage). 

One sample is fixed in 10% formol saline 

and the other placed into Amies’ charcoal 

transport medium for bacteriological 

examinations. 

NOTES 

55


reFerence range tables 

The data in the following tables are from Beaufort Cottage 

Laboratories (www.rossdales.com/laboratory) for: 

adult non-Thoroughbred horses 

neonatal Thoroughbred foals (24-48 hours old) 

older Thoroughbred foals (approximately three weeks old) 

yearling Thoroughbred horses 

two-year-old Thoroughbred horses in training 

three-year-old Thoroughbred horses in training and 

adult Thoroughbred horses at stud. 

Adult Non-Thoroughbred Horses 

56 

Test Abbrev. Units Mean Range 

Total erythrocytes RBC x10 12 /l 8.2 6.2-10.2 

Packed cell volume pcV l/l 0.37 0.31-0.43 

Haemoglobin Hb g/dl 13.5 11.1-15.9 

Mean cell volume MCV fl 46.0 40.0-50.0 

Mean cell haemoglobin. conc. McHc g/dl 36.1 33.5-38.7 

Mean cell haemoglobin McH pg 16.6 15.2-19.0 

Total leucocytes WBC x10 9 /l 7.5 6.0-10.0 

Segmented neutrophils Segs x10 9 /l 4.4 3.4-5.4 

Segs % 58 51-65 

Lymphocytes Lymphs x10 9 /l 2.6 2.0-3.2 

Lymphs % 35 29-41 

Monocytes Monos x10 9 /l 0.3 0.2-0.4 

Monos % 4 2-6 

Eosinophils Eos x10 9 /l 0.2 0-0.4 

Eos % 2 1-3 

Platelets plts x10 9 /l 156 100-250 

Total Protein TSP g/l 63 53-73 

Albumin alb g/l 35 29-41 

Globulin glob g/l 28 18-38 

Alpha 1 globulin α1 glob g/l 1.2 0.4-2.0 


Beta 1 globulin β1 glob g/l 7.4 4.0-10.8 


Gamma globulin γ glob g/l 9.0 4.6-13.4 

Plasma fibrinogen Fib g/l 2.1 0.3-3.9



Serum amyloid A SAA mg/l 1.3 0-20 

Aspartate amino transferase AST iu/l 263 102-350 

Creatinine kinase ck iu/l 186 110-250 

Lactate dehydrogenase LD iu/l 525 225-700 

LD isoenzyme 1 LD1 % total LD 14 10-18 





Gamma glutamyl transferase GGT iu/l 16 1-40 

Glutamate dehydrogenase gLDH iu/l 3 1-10 

Serum alkaline phosphatase SAP iu/l 204 147-261 

Intestinal alk. phosphatase IAP iu/l 47 13-87 

iap % total SAP 22.6 10-34 

Urea Urea mmol/l 5.2 2.5-10.0 

Creatinine creat mmol/l 125 85-165 

Glucose glu mmol/l 4.9 4.3-5.5 

Total bilirubin TBili mmol/l 20 13-34 

Direct bilirubin DBili mmol/l 9 4-16 

Bile acids BAcids mmol/l 5.1 1-8.5 

Cholesterol chol mmol/l 2.45 2.0-3.3 

Triglycerides Trigs mmol/l 0.7 0.2-1.2 

Lipase Lip mmol/l 30 8-50 

Amylase amyl iu/l 9 3-15 

Calcium ca mmol/l 3.1 2.9-3.3 

Fractional urinary clearance Ca % 6.2 2.6-15.5 

Phosphate pO4 mmol/l 1.4 0.9-1.9 

Fractional urinary clearance PO4 % 0.3 0.02-0.53 

Magnesium Mg mmol/l 0.8 0.6-1.0 

Fractional urinary clearance Mg % 11.7 3.8-21.9 

Copper (serum) cu mmol/l 18.2 14.0-22.0 

Copper (plasma) cu mmol/l 23.0 18.0-28.0 

Zinc Zn mmol/l 12.7 10.0-15.0 

Sodium na mmol/l 138 134-142 

Fractional urinary clearance Na % 0.09 0.02-1.0 

Potassium k mmol/l 4.0 3.0-5.0 

Fractional urinary clearance K % 32.8 15-65 

Chloride cl mmol/l 99 95-103 

Fractional urinary clearance Cl % 0.72 0.04-1.6 

Cortisol cort nmol/l 136 71-240 

Fasting Insulin ins miu/ml 25.5 8.0-47.5 

Tri-iodothyronine T3 nmol/l 1.0 0.48-1.46 

Thyroxine T4 nmol/l 22.7 7.7-42.8 

Cardiac Troponin cTnI ng/ml 0.1 0.05-0.2 

Selenium Se mmol/l 3.0 0.7-8.4 

57


Neonatal Thoroughbred Foals (24-48 hrs old) 

58 


Total erythrocytes rBC x10 12 /l 9.4 6.9-11.8 








Segs % 75 58-85 


Lymphs % 21 14-37 


Monos % 2 0.5-5 

Eosinophils Eos x10 9 /l 0.1 0.1-0.2 

Eos % 1 1-2 










Immunoglobulin g igG g/l 11.7 6.9-18.6 

Plasma fibrinogen Fib g/l 1.9 0.5-3.9 










Gamma glutamyl transferase GGT iu/l 21 10-32






iap % total SAP 23.7


Older Thoroughbred Foals (approx. 3 weeks old) 

60 










Segs % 58 42-88 


Lymphs % 38 22-54 


Monos % 3 1-7 


Eos % 1 1-3 











Serum amyloid A SAA mg/l 1.2 0-5.4 








LD isoenzyme 5 LD5 % total LD 3 2-4







iap % total SAP 20.3 18.7-22.2 

Urea Urea mmol/l 3.4 2.8-4.1 

Creatinine creat mmol/l 123 97-138 

Glucose glu mmol/l 4.2 3.2-6.2 

Total bilirubin TBili mmol/l 38 22-54 

Direct bilirubin DBili mmol/l 12 5-18 

Bile acids BAcids mmol/l 0-8.0 

Cholesterol chol mmol/l 

Triglycerides Trigs mmol/l 

Lipase Lip mmol/l 

Amylase amyl iu/l 

Calcium ca mmol/l 3.0 2.9-3.1 

Fractional urinary clearance Ca % 

Phosphate pO4 mmol/l 2.4 2.2-2.7 

Fractional urinary clearance PO4 % 0.3 0.02-0.53 

Magnesium Mg mmol/l 0.8 0.7-1.0 

Fractional urinary clearance Mg % 

Copper (serum) cu mmol/l 

Copper (plasma) cu mmol/l 

Zinc Zn mmol/l 

Sodium na mmol/l 141 135-145 

Fractional urinary clearance Na % 

Potassium k mmol/l 4.8 4.1-5.5 

Fractional urinary clearance K % 

Chloride cl mmol/l 99 96-102 

Fractional urinary clearance Cl % 

Cortisol cort nmol/l 

Fasting Insulin ins miu/ml 

Tri-iodothyronine T3 nmol/l 12.7 0.5-4.2 

Thyroxine T4 nmol/l 130 60-320 

Cardiac Troponin cTnI ng/ml 

Selenium Se mmol/l 

61


Yearling Thoroughbred Horses 

62 










Segs % 51 40-53 


Lymphs % 41 35-49 


Monos % 3 2-5 


Eos % 2 1-5 


























iap % total SAP 23


Two-Year-Old Thoroughbred Horses in Training 

64 










Segs % 53 42-63 


Lymphs % 38 28-46 


Monos % 4 2-6 


Eos % 2 1-3 




























Three-Year-Old Thoroughbred Horses in Training 

66 










Segs % 56 48-64 


Lymphs % 35 28-42 


Monos % 4 2-6 


Eos % 1 1-2 










Plasma fibrinogen Fib g/l 1.8 14-2.3 


















Adult Thoroughbred Horses at Stud 

68 










Segs % 52 36-68 


Lymphs % 45 39-61 


Monos % 4 2-6 


Eos % 3 0-5 




























NOTES 

Copies of the Reference Range Tables can be downloaded (as pdf files) from our website: 

www.rossdales.com/laboratory 

While we have made every endeavor to ensure the accuracy of information in this Guide, Rossdale & 

Partners cannot be held liable for any inaccuries which may inadvertently occur. 

70


The aim of this Guide is to help equine practitioners use 

clinical pathological aids to diagnosis to good effect, to provide 

‘normal’ reference ranges for horses of different ages and 

types, and to give advice for the safe and satisfactory collection 

and handling of samples for laboratory investigation. 

The Guide was produced by Sidney W. Ricketts, LVO, BSc,BVSc, 

DESM, DipECEIM, FRCPath, FRCVS 

with the help of 

Annalisa Barrelet, BVetMed, MS, CertESM, MRCVS 

Celia M.Marr, BVMS, MVM, PhD, DEIM, DipECEIM, MRCVS 

Sarah S.Stoneham, BVSc, CertESM, MRCVS 

Katherine E. Whitwell, BVSc, DiplECVP, FRCVS 

Robert S.G.Cash BSc, FIBiol 

Mary Ashpole. 

© 2006 Rossdale & Partners 

Sidney Ricketts

Beaufort Cottage laboratories 

High Street, Newmarket, Suffolk CB8 8JS 

Tel +44 (0)1638 663017 Fax +44 (0)1638 560780 

Email bcl@rossdales.com 

www.rossdales.com

EQUINE CLINICAL PATHOLOGY - Rossdale & Partners

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