Interpreting blood haematology/biochemistry in cattle and sheep in the field

Alastair Macrae

Senior Lecturer in Farm Animal Health and Production

BVM&S, PhD, CertSHP, DCHP, DipECBHM, DipECSRHM (Non-Practising), MRCVS

Dairy Herd Health and Productivity Service, Division of Veterinary Clinical Sciences, Royal (Dick) School of Veterinary Studies and the Roslin Institute, University of Edinburgh, EBVC, Easter Bush, Roslin, Midlothian EH25 9RG

Tel – 0131 651 7474

Email A.I.Macrae@ed.ac.uk

Word count: 2484 words

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Abstract

Clinical biochemistry and haematology can be useful in farm animal practice, not only for diagnostic purposes in individual animals, but also routine herd and flock investigations and monitoring. For example albumin and globulin concentrations can serve as a relatively cheap screen in the investigation of ill-thrift and chronic disease processes, as well as the assessment of colostrum intakes. Assessment of liver enzymes can prove helpful in screening for subclinical liver damage in the early stages of chronic copper toxicity. Assessment of the extent and severity of metabolic disease is essential for the prevention of production diseases, especially in dairy cattle.

Introduction

Costs of clinical biochemistry and haematology are often thought to be prohibitively expensive for use in farm animal practice. However if tests are targeted towards specific disease investigations rather than the use of broad-spectrum profile panels which include unnecessary tests (eg. Large Animal General Profiles), and specific cost-benefit analysis performed (eg. cost of specific diagnosis and treatment/prognosis versus repeat visits and unnecessary treatments) then they can greatly assist in the diagnosis of both clinical and production diseases. The increase in the number of smallholders requiring individual animal care has also increased the demand for specific diagnoses to be made.

The use of “in-house” laboratory facilities in many practices has enabled tests to be performed quickly and at a reduced price. However, quality control is an important part of all biochemical testing, and must be performed regularly (either by the testing of control samples, or by sending duplicate samples to other outside laboratories on a regular basis). The use of cow-side BHB testing (via blood or milk), and refractometers to measure specific gravity (and thus measure total protein content) enable quick and cheap evaluation of samples.

A description of all the potential biochemical and haematological tests available is outwith the scope of this article. Readers are directed to a recent review by Otter 2013, or textbooks such as Kaneko et al 2008 for further reading in this respect.

1. Serum protein analysis (albumin/globulin/total protein)

On a limited budget and in the absence of a diagnosis based on history and/or clinical grounds, serum albumin and globulin determinations can provide a useful screening test for the investigation of chronic weight loss and ill-thrift. Albumin concentration (normal serum reference range between 24 to 35 g/L; values 10 per cent higher if plasma samples) reflects the balance between hepatic synthesis from dietary nitrogenous intake and endogenous demands/losses. Serum globulin (usually measured as total

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protein minus albumin; normal range between 35 to 45 g/L) is a relatively crude long term indicator of the body's response to chronic immune stimulation and inflammation.

Examples

Poor nutritionLow serum albumin and normal globulin concentrations (<25 g/L and <45 g/L, respectively) indicate that significant chronic bacterial infection is unlikely. Such concentrations may be due to possible dietary effects as a result of low protein intakes, often encountered in ewes or suckler cows during late pregnancy fed poor quality rations, when the proteins used for immunoglobulin production accumulate in the udder.

Failure of adequate colostrum intakeNewborn calves and lambs can be blood sampled at 2 – 7 days of age for IgG, globulin or total protein status to determine if failure of passive transfer has occurred, as the globulin concentration in newborn animals is gained entirely from colostrum intake (Weaver at al 2000). Measurement of IgG concentrations below 10g/L (globulin below 20g/L) indicates poor colostrum intake.

A cheaper and simpler diagnostic test is to measure total serum proteins using refractometers. Calves and lambs should be greater than 24-48 hours old, and less than 7 days of age (when calves and lambs start to synthesise their own proteins in the bloodstream). Concentrations below 45g/L are diagnostic of failure of adequate colostrum intake, and concentrations above 55g/L indicate adequate colostrum intake.

Protein-losing conditionsIf serum albumin concentrations are less than 18 g/L in sheep, Johne’s disease (Figure 1 and 2) or other protein-losing conditions would be strongly suspected (Scott and others 1995). Serum albumin concentrations below 15 g/L in association with normal serum globulin concentrations are highly suggestive of Johne’s disease in sheep, but could also be chronic parasitic gastroenteritis or fasciolosis (which can be ruled out by doing a faecal egg count). In cattle, albumin concentrations in Johnes disease cases do not fall as dramatically as in sheep, and concentrations seldom drop below 20 g/L.

Chronic bacterial infectionLow serum albumin and high globulin (below 25 g/L and greater than 50 g/L respectively) indicate probable chronic suppurative disease process/focus such as chronic suppurative pneumonia, endocarditis, liver abscessation (Figure 3), mastitis, infectious polyarthritis, cellulitis etc. Clinicians should also consider chronic liver fluke infestation.

2. Inflammatory response

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A) Acute inflammation

NB Clinicians should remember that a full clinical examination will probably demonstrate clinical evidence of acute inflammation (eg. pyrexia, toxaemia in cases of mastitis, respiratory disease, metritis etc.) without resorting to further laboratory analysis.

Acute phase proteins (APP). The acute phase response is a non-specific response to infection, inflammation, injury and surgery such as Caesarean section. It is characterised by alterations in the concentrations of a number of proteins in the blood of hepatic origin. The most commonly measured APPs in ruminants are haptoglobin, fibrinogen and serum amyloid A (SAA), which increase markedly during active inflammatory disease. Haptoglobin (normal range below 0.2 g/L HbBC) and other APP are therefore useful as indicators of acute disease, with significant increases within one to five days of inflammation occurring. This elevation in APP will often precede clinical diagnosis, and so the use of APP may help in the early diagnosis of clinical conditions such as post-partum metritis in dairy cows (Huzzey at al 2009). APP concentrations can remain raised for up to 2 - 3 weeks post-infection, and will remain raised during active inflammatory processes.

Routine haematology. Measurement of total white blood cells (WBC) and differential counts are not as useful in ruminants as they are in companion animals. Differentials should always be expressed as total counts, and not as percentages. In normal ruminants, the circulating lymphocyte numbers are greater than neutrophil numbers (which is in contrast to the ratio present in dogs and cats).

In the initial stages of the inflammatory response (approximately 1-3 days), WBC and neutrophil numbers transiently decrease (termed leucopenia and neutropenia respectively) due to migration of these cells to the site of infection. Neutropenia is often marked in the initial stages of the inflammatory response in cows with peracute toxic mastitis for example, and may be a feature of disease conditions such as bracken poisoning and Bovine Neonatal Pancytopenia (BNP or “Bleeding calf syndrome”)

As the inflammatory process continues, there then follows an increase in WBC (leucocytosis), mainly due to an increase in neutrophils (neutrophilia). However overall WBC concentrations may decline to be within normal ranges even with a marked neutrophilia, especially in cases of chronic disease.

Examples

Effect of corticosteroids on WBC counts. Stressful conditions such as lameness or metabolic disease, or the administration of corticosteroid injections will result in a characteristic “stress leucogram” consisting of a leucocytosis (elevated WBC), mature

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neutrophilia (elevated neutrophils), lymphopenia (decreased lymphocytes), eosinopenia (decreased eosinphils).

Eosinophilia. Usually characteristic of parasitic infections (nematodes, protozoa), but may occur in allergies. However this is non-specific for particular diseases, and so of limited clinical use.

B) Chronic inflammation

Haematology is frequently unremarkable in animals with chronic suppurative foci, and if there are any changes, they will be manifest as an increase in WBC and neutrophils. Globulin concentrations are of more value in these cases. Note that chronic viral infections (Ovine Pulmonary Adenomatosis or persistently infected BVD animals) may not result in continuous stimulation of the immune system, and thus globulin concentrations are often normal in such cases (unless the animals develop secondary infections).

Further investigations

Once the possibility of a chronic suppurative focus has been highlighted, the animal should be re-examined. Further specific tests can then be selected based on the organ system suspected of being involved, e.g. serum GGT for chronic fascioliasis, ultrasonography/chest radiographs if chronic suppurative pneumonia is suspected, abdominal paracentesis for peritonitis etc.

3. Liver disease.

There are a number of tests of liver damage and function that can be used in ruminants with suspected liver disease. Due to economic considerations, it is cheaper to target specific tests than use commercial “Ruminant Liver Damage Profile” panels. An example would be to test for AST and/or GLDH (acute liver damage) and GGT (chronic bile duct disease) along with other tests (eg. fluke egg count).

a) Aspartate aminotransferase (AST). A good indicator of acute hepatocellular injury, but not specific as this enzyme is also raised in muscle damage (eg. white muscle disease) and heart disease. Concentrations of this enzyme are typically 5 – 10 times normal in acute hepatic damage (eg. copper poisoning).

b) Glutamic dehydrogenase (GLDH) and sorbitol dehydrogenase (SDH). Both of these enzymes are specific for the liver, and are elevated in acute liver damage. However concentrations fall rapidly and are not elevated in chronic disease. SDH is unstable in serum, and analysis is recommended within 6 hours.

c) Gamma-glutamyltranspeptidase (GGT). This is found predominantly in the cell membrane of bile duct cells, and is thus is a good indicator of hepatobiliary disease (eg. chronic fascioliasis)(Figure 4)

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d) Other liver enzymes. Other enzymes tend to be non-specific (Alkaline phosphatase; AP and lactate dehydrogenase; LDH), or present at low concentrations in ruminants and are thus not a reliable indicator of liver damage (Alanine aminotransferase; ALT).

e) Bilirubin. Elevated concentrations of bilirubin are observed clinically as jaundice, and is usually seen with RBC destruction (haemolysis). Liver damage (eg. hepatogenous photosensitization) rarely results in elevated bilirubin concentrations unless severe, and thus it is of little diagnostic value.

f) Bile acids. This is used a measure of liver function, but problems arise in ruminants due to the large fluctuations in its concentration. High concentrations are diagnostic of impaired liver function.

g) Others. As previously discussed, albumin is synthesised in the liver, and thus chronic liver damage with result in low albumin concentrations. Phylloerythrin concentrations may be determined if photosensitisation is suspected.

Examples

Copper poisoning. Subclinical liver damage occurs when hepatic copper concentrations, accumulated over a period of dietary intake or parenteral administration exceed about 8000 µmol/Kg DM. This can be detected using elevated AST or GLDH concentrations as a non-specific early indicator of subclinical hepatic damage.

Continued accumulation until hepatic copper concentration exceeds approximately 15000 µmol/kg DM results in lysosomal rupture and release of large amounts of copper into the circulation. Stressful events or metabolic disturbances caused by other diseases may result in lysosomal rupture at lower hepatic copper concentrations. Circulatory copper causes haemolysis through a series of biochemical interactions with red blood cell membranes. During the haemolytic crisis blood copper concentrations may be 38 - 300 µmol/l (normal range 9 - 23 µmol/l). Characteristic biochemical abnormalities include anaemia (low PCV), jaundice (raised bilirubin) and liver damage (elevated AST typically > 400 IU/l; reference range 45 – 134 IU/l) and GLDH). A cost-effective screening method for subclinical liver damage and impending haemolytic crisis is to measure AST or GLDH concentrations.

PhotosensitizationPhotosensitisation is caused by either ingestion of a photosensitive agent (eg. St. John’s Wort), or liver damage leading to a build-up of phylloerythrin (hepatogenous photosensitisation). Diagnosis is usually based on clinical signs and history of access, but can be backed up in some cases of liver damage by measurement of GLDH (acute) or GGT (chronic), bilirubin and phylloerythrin concentration in the serum (>10g/ml).

Liver abscessationAlthough common in cattle (associated with chronic rumenitis), these are comparatively rare in sheep. They are worthy of mention, because unless the abscesses are very large

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or active, they do not result in elevations in liver enzymes (Figure 3), bilirubin or bile acids (diagnosis is better achieved via albumin/ globulin concentrations and ultrasound examination of the liver).

4. Metabolic disease

Ketosis (dairy cattle) and pregnancy toxaemia (beef cattle and sheep). This is caused by the high metabolic demands of lactation and/or the fetus during late pregnancy leading to negative energy balance (Figure 5), with a rapid rate of protein and body fat catabolism (which can be detected by measuring Non-Esterified Fatty Acids [NEFA]). Failure to adapt to the rapid rate of fat catabolism results in hepatic lipidosis and high circulatory concentrations of ketone bodies (acetoacetate and -hydroxybutyrate [BHB]). Serum BHB concentrations over 3 mmol/L (aqueous humour >2 mmol/L post-mortem) are usually considered supportive of a diagnosis of clinical ketosis. BHB concentrations can be measured either in milk samples (using Keto-Test strips) or cow-side using portable BHB blood analysers.

In dairy cattle, recent research has focused on the prevalence of subclinical ketosis (BHB concentrations between 1.0 - 3.0 mmol/L), with approximately 30% of cows affected in the first 2-3 weeks of lactation. Subclinical ketosis and elevated NEFA concentrations in both late pregnancy and early lactation have been associated with reduced milk production, increased risks of periparturient disease such as LDAs and metritis, and poorer fertility (reviewed by LeBlanc 2010).

Hypocalcaemia (milk fever)

Calcium and phosphorous are interlinked, and calcium concentrations in the plasma are maintained by homeostatic mechanisms (via parathyroid hormone/vitamin D for calcium). Although not under direct hormonal control, the balance of dietary intake and salivary and/or urinary excretion serves to regulate phosphorous levels in the bloodstream. In cattle, milk fever is a common condition affecting animals around calving, and readily diagnosed on clinical examination and response to treatment. The onset of clinical disease in sheep is often precipitated by stress, and can occur during late pregnancy or around lambing time. If necessary serum calcium concentrations below 2.0 mmol/L support a diagnosis (Figure 6). Affected cases are often also hypophospataemic (serum inorganic phosphate concentrations <0.9mmol/L) but respond clinically to intravenous calcium borogluconate, and the clinical relevance of low phosphate concentrations in uncomplicated milk fever cases is unclear.

In dairy cows subclinical hypocalcaemia is defined as blood calcium concentrations below 2.0 mmol/L in the absence of clinical signs, measured within 48 hours of calving. Studies have shown that around 50% of mature dairy cows have low blood calcium concentrations at this stage (Reinhardtet al 2011), although the clinical significance of this and associations with disease are still to be accurately determined.

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Hypomagnesaemia (grass staggers)

Ruminants can only absorb approximately 20% of the dietary magnesium via the rumenoreticulum, and this dietary magnesium absorption is affected by high concentrations of potassium and low sodium concentrations in the diet, and high rumen pH which reduce magnesium absorption (such conditions are often found in lush spring grass). Disease is associated with high levels of milk production in either dairy cows, beef cows with calves at foot, or after lambing in sheep, often with a history of inadequate mineral supplementation. There are only limited readily accessible body reserves of magnesium, and thus plasma concentrations are indicative of current dietary intake. Evidence also suggests that low plasma magnesium concentrations do not necessarily indicate the development of clinical hypomagnesaemia (clinical disease is related to CSF magnesium concentrations which are buffered against changes in plasma magnesium). Serum magnesium and CSF concentrations <0.6mmol/L support a diagnosis.

Post mortem samples may also be taken to confirm hypomagnesaemia (eg. as a cause of sudden death). Fresh CSF and urine magnesium concentrations below 0.6mmol/L (within 12 hours of death), aqueous humour magnesium concentrations below 0.4mmol/L (within 12 hours of death), and vitreous humour magnesium concentrations below 0.6mmol/L (stable for up to 24 hours) can be useful in this regard.

Summary

Clinical biochemistry and haematology can be useful in farm animal practice, not only for the investigation of clinical disease in individual animals, but also for herd screening for poor production (for example ill-thrift) and metabolic disease. As such, they can provide useful and cost-effective information for the investigation and monitoring of herd and flock health.

Key points

Clinical biochemistry parameters can be effective diagnostic tools in farm animal practice

Haematology in ruminants tends to be less informative than in companion animal practice

Testing in farm animal practice must be streamlined to get the most amount of information from the least amount of tests (ie. be cost-effective)

Ask the question: will laboratory testing provide value to the diagnostic work-up? Albumin and globulin concentrations can be useful in the investigation of ill-thrift in

cattle and sheep

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Metabolic disease screening is useful at the herd level for the monitoring and prevention of disease.

Key words

Cattle, sheep, biochemistry, haematology, diagnosis

References

Huzzey JM, Duffield TF, LeBlanc SJ, Veira DM, Weary DM, von Keyserlingk MA (2009) Haptoglobin as an early indicator of metritis. Journal of Dairy Science 92, pp621-5 Kaneko JJ, Harvey JW and Bruss ML (2008) Clinical Biochemistry of Domestic Animals. 6th edition. Elsevier Academic Press. pp 351-378

LeBlanc S (2010) Monitoring metabolic health of dairy cattle in the transition period. Journal of Reproductive Development 56 Suppl:S29-35

Otter A (2013) Diagnostic blood biochemistry and haematology in cattle. In Practice 35, pp7-16

Reinhardt TA, Lippolis JD, McCluskey BJ, Goff JP, Horst RL (2011) Prevalence of subclinical hypocalcemia in dairy herds. Veterinary Journal 188 pp122-4.

Scott PR, Clarke CJ, King TJ (1995) Serum protein concentrations in clinical cases of ovine paratuberculosis (Johne's disease) Veterinary Record 137, p7

Weaver DM, Tyler JW, VanMetre DC, Hostetler DE, Barrington GM (2000) Passive Transfer of Colostral Immunoglobulins in Calves. Journal of Veterinary Internal Medicine 14 pp569–577

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Figure 1. Johnes disease in sheep (in this case a pigmented strain) will result in profoundly low albumin results.

Figure 2. Store lamb with “bottle jaw” due to hypoalbuminaemia associated with chronic liver fluke infestation.

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Figure 3. Post-mortem picture of a bull with liver abscessation and peritonitis. Such chronic cases can be difficult to diagnose clinically, but the characteristic low albumin and high globulin concentrations on serum biochemistry helps guide the clinician to a chronic septic focus as the cause of the ill-thrift.

Figure 4. Chronic liver fluke in cattle will result in elevated GGT values due to the bile duct damage caused by adult fluke.

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Figure 5. Insufficient feed access in late pregnancy in sheep is a major risk factor for the development of pregnancy toxaemia. Risk can be assessed (and prevented) on a flock basis by blood sampling for BHB concentrations in representative groups of sheep.

Figure 6. Diagnosis of downer cows with hypocalcaemia is frequently based on clinical signs, history and response to treatment, without having to determine blood calcium concentrations.

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MCQs1. As part of an investigation into ill-thrift in a group of adult ewes, clinical examination

is unremarkable. You take a blood sample from one ewe, and the serum albumin result is 20 g/L (reference range 24 – 35 g/L) and the globulin result is 60 g/L (reference range below 50 g/L). The most likely diagnosis in this individual case is:

a. Johnes disease (paratuberculosis)b. Chronic suppurative pneumoniac. Maedi Visna Virusd. Long-term under-nutritione. Molar dental disease

Correct answer: b

2. As part of an investigation into ill-thrift in a group of adult ewes, clinical examination is unremarkable. You take a blood sample from one ewe, and the serum albumin result is 15 g/L (reference range 24 – 35 g/L) and the globulin result is 40 g/L (reference range below 50 g/L). The most likely diagnosis in this individual case is:

a. Johnes disease (paratuberculosis)b. Chronic suppurative pneumoniac. Maedi Visna Virusd. Long-term under-nutritione. Molar dental disease

Correct answer: a

3. As part of an investigation into ill-thrift in a group of adult ewes, clinical examination is unremarkable. You take a blood sample from one ewe, and the serum albumin result is 24 g/L (reference range 24 – 35 g/L) and the globulin result is 40 g/L (reference range below 50 g/L). The most likely diagnosis in this individual case is:

a. Johnes disease (paratuberculosis)b. Chronic suppurative pneumoniac. Chronic fasciolosisd. Long-term under-nutritione. Parasitic gastroenteritis

Correct answer: d

4. As part of an investigation into diarrhoea in 2 weeks old calves in a dairy unit, you take samples from calves at 10 days of age for the assessment of total serum protein concentrations. All five calves have total serum protein concentrations greater than 70 g/L. You conclude that:

a. Colostrum intakes are adequateb. The calves that have been sampled were too oldc. Total plasma protein concentrations should have been analysedd. Colostrum intakes are inadequatee. The calves that have been sampled were too young

Correct answer: b

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5. A decrease in circulating WBC and neutrophil numbers in cattle is often seen during:a. Chronic inflammationb. Parasitic gastroenteritisc. Lymphosarcomad. Malnutritione. Acute inflammation

Correct answer: e

6. You are suspicious of chronic copper poisoning in a group of pedigree Texel lambs being fed high levels of concentrate feeding prior to sale. A possible biochemical screening test for the assessment of subclinical liver damage would be to measure blood concentrations of:

a. GGTb. APc. GLDHd. CKe. BHB

Correct answer: c

7. Subclinical ketosis in a dairy herd is diagnosed by the measurement of:a. BHBb. NEFAc. Glucosed. Cholesterole. Insulin

Correct answer: a

8. The prevalence of subclinical ketosis during early lactation in dairy herds is approximately:

a. 10%b. 20%c. 30%d. 40%e. 50%

Correct answer: c

9. For the assessment of subclinical hypocalcaemia in a dairy herd, cows must be blood sampled at which stage of the production cycle to measure blood calcium concentrations?

a. One week precalvingb. Within 48 hours of calvingc. 72 hours after calvingd. One week post-calvinge. At peak lactation

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Correct answer: b

10. For the post-mortem diagnosis of hypomagnesaemia (grass staggers) in a cow that has been dead for 24 hours, which sample should be taken for analysis of magnesium concentrations:

a. Bloodb. CSFc. Urined. Aqueous humoure. Vitreous humour

Correct answer: e

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