Increase your test range and maximise your diagnostic capability in large animal practice

by Janice Thompson, Gribbles Veterinary Pathology, Mark Gilmour, Large Animal Practitioner, Bulls, and Tony Thompson, Retired Large Animal Practitioner, Waipukura

In large animal practice, a full range of tests is often not requested and a definite diagnosis may be missed.

If routine tests include a full sick animal biochemical panel with glucose estimation, haematology and urinalysis, then a diagnosis of the less frequently or unusual conditions may be made more often. Further tests, such as bone marrow aspirates, may be carried out if indicated. Large animals may show a wide range of non-specific abnormal clinical signs with a range of illnesses, including rare conditions. However, diagnosis of unexpected or rare conditions often requires a broader range of tests than is usually requested or considered.

Some of the most useful tests are those that would be regularly carried out in companion animal practice, where it is routine to carry out a much greater range of diagnostic tests than in large animal practice. Therefore, in companion animal practice, conditions such as diabetes mellitus are frequently diagnosed along with other relatively rare and interesting conditions. Satisfaction in both large and companion animal practice comes about as a result of diagnosing those unusual and rare conditions that may sometimes be encountered.

This article details some less commonly diagnosed conditions that have been diagnosed through Gribbles Veterinary simply by requesting a greater range of tests and using samples that are rarely received from large animal practitioners. It also includes the recently diagnosed bovine pancytopenia syndrome in cattle involving bovine viral diarrhoea (BVD) vaccination. While most of the following examples are from cattle cases, this can equally apply to any sick large animal.

Examples of the value of haematology and bone marrow cytology

Anaemia is often encountered as a clinical pathological finding in large animals. It can arise from a wide range of causes, and haematology is needed to examine blood smears for a reticulocytosis in order to differentiate regenerative anaemias, caused by haemorrhage or haemolysis, from non-regenerative or poorly regenerative anaemia. Note that horses do not show a reticulocytosis with any anaemia.

Poorly regenerative anaemia may arise from several conditions including chronic disease and inflammation and also bone marrow suppression, dysfunction and neoplasia. Neutropenias may arise from peracute inflammation or bone marrow suppression, dysfunction and neoplasia. Companion animal practitioners routinely consider taking bone marrow aspirates for unexplained anaemia and other cytopenias, but large animal practitioners rarely consider this step. This article discusses examples of conditions in which interpreting blood and bone marrow together has been useful to make a diagnosis in cattle. There is also an example of a rare haematological condition diagnosed in a pet pig in which the haematology showed useful significant changes and where there was no need for a bone marrow sample, although it would have been interesting.

Example 1: Congenital dyserythropoiesis

Congenital dyserythropoiesis is a rare group of genetic disorders with several subtypes that has been well described in humans and is characterised by anaemia, often macrocytosis, and the presence of multinucleated erythroid precursors in the bone marrow and associated with ineffective erythropoiesis.¹⁵ Congenital dyserythropoiesis has also been reported in Polled Hereford calves in which multinucleated rubricytes were observed.¹⁰ This condition has now been described in Polled Hereford calves in both the United States¹⁰ and Australia (Alan Kessell, pers comm), and it is thought to show similarities to a similar condition in Australian-bred Murray Grey cattle where it was described as a “congenital haemolytic anaemia and jaundice of Murray grey calves”.⁷

Some years ago, blood samples from several Murray Grey calves aged from 6–20 weeks that were born on a stud farm were received at the former Palmerston North Animal Health Laboratory. Clinically, they showed progressive lethargy and anorexia and produced red urine. Limited clinical pathological investigation was done but the calves were very anaemic with variable numbers of reticulocytes and had increased bilirubin. There was an inappropriate metarubricytosis. In other words, nucleated red blood cells (RBCs) were disproportionately increased relative to the numbers of reticulocytes. In addition, the mature RBCs in the peripheral circulation were macrocytic, and macrocytosis has also been reported in human cases of congenital dyserythropoiesis.¹⁵ A bone marrow smear for cytological evaluation was received from one calf and this contained numerous multinucleated metarubricytes with some cells containing up to six nuclei (Figure 1). The calves were copper deficient, but copper supplementation did not alleviate the clinical signs. Urinalysis was never carried out to differentiate haematuria from haemoglobinuria.

The anaemia and jaundice of this New Zealand case resembled the cases of jaundice and anaemia described in Murray Grey calves in Australia.⁷ In addition, cytological characteristics of the erythroid precursors within the bone marrow from the New Zealand Murray Grey calf were similar to characteristics seen in congenital dyserythropoiesis in Polled Hereford calves¹⁰ and humans.¹⁵ Copper deficiency has also been associated with a form of dyserythropoiesis in humans,¹⁷ but multinucleated metarubricytes were not noted in the human cases. So the significance of the copper deficiency in these calves and relevance to the anaemia is not obvious. However, copper is needed for transport of iron, and copper deficiency may result in anaemia.¹⁷ There are also differences between the Polled Hereford calves and the Murray Grey calves because the Polled Herefords also showed clinical signs of dyskeratosis and progressive alopecia, in addition to the anaemia.¹⁰

Unfortunately, in this case, when the Murray Grey stud owner received the suggestion that this may be an inherited defect he did not want to pursue investigation of the condition and no further samples were received. However, this is an example of an extremely rare and unusual condition requiring haematology, serum biochemistry and bone marrow samples for a diagnosis. If any veterinary practitioners see Murray Grey or Polled Hereford calves that fit these clinical descriptions, then blood samples for haematology and bone marrow samples for cytology, in addition to serum samples for biochemistry, are necessary to make a diagnosis.

Example 2: Bovine neonatal pancytopenia

The usefulness of bone marrow smears has been demonstrated recently with the emergence of the bovine neonatal pancytopenia (BNP) syndrome. This syndrome initially presents as a haemorrhagic diathesis. Haemorrhagic diatheses are unusual findings in cattle but have been reported with bovine virus diarrhoea virus genotype II in susceptible cattle.⁹ Likewise, bone marrow failure leading to pancytopenia is rare, usually occurring as single cases associated with antibiotics such as sulphur drugs, use of non-steroidal anti-inflammatory drugs, exposure to trichothecenes, oestrogen and bracken fern.⁹

The syndrome has been described in Europe over the past three years⁹ and has now been recognised in New Zealand. Haematological examination of the peripheral blood shows a severe neutropenia, sometimes with no neutrophils present in the blood smear, severe thrombocytopenia with almost no platelets present, a severe lymphopenia and a poorly regenerative anaemia. Bone marrow from affected calves shows severe hypoplasia or aplasia usually involving erythroid, myeloid and megakaryocytic cell lines. Both cytologically and histologically there is severe depletion of all cell lines (Figures 2 and 3). Lymphoid tissue is also severely depleted. The severe neutropenia and lymphoid depletion leads to immunodeficiency and spontaneous bacteraemia with lesions affecting many organs. The thrombocytopenia leads to severe widespread petechial and echymotic haemorrhages.

Anaemia develops more slowly because erythroid cells have a longer lifespan than myeloid cells. As the RBCs become senescent and are removed, anaemia develops. Haemorrhage as a result of the thrombocytopenia also contributes to the anaemia.⁹ Any young calf with severe pancytopenia in the peripheral blood including anaemia, and with severe bone marrow hypoplasia, should be a suspect BNP case.

The condition is seen in some calves that are fed colostrum from some but not all cows that have been vaccinated with the PregSure BVD vaccine. Antibody within the colostrum of these cows then attacks the haematopoietic precursor cells within the bone marrow of susceptible calves, but overall the incidence of the condition is low. It has now been shown that the underlying mechanism involves the major histocompatibility complexes of the bovine kidney cells within cell cultures that are used for the production of the vaccine.⁴ Where the major histocompatibility complexes of the cell culture cells are different from those of the cow that is being vaccinated, she develops an immune response with antibody production against the complex from the cell culture. If a calf has the same major histocompatibility complex to the kidney cells used in the vaccine production (in other words different from those in the dam), the antibodies absorbed from the colostrum selectively attack that calf’s haematopoietic cells.⁴ The condition develops after birth so the calves are born normal and develop clinical signs from about a week of age onwards. Calves very occasionally recover.⁹

Bone marrow was therefore useful in the above cases to help differentiate from other causes of haemorrhage, anaemia, thrombocytopenia and neutropenia in young calves. Bone marrow needs to be taken from a freshly dead animal or immediately the animal is euthanised for optimal results. Bone marrow degenerates very quickly after death and the cells rupture when the smears are made, making examination and diagnosis difficult, and this happens quite soon after death. The sternum and ribs are the best bones for taking bone marrow. If the animal has been dead for some time then, while smears should still be made and submitted, formalin-fixed samples of bone marrow for histopathology should also be taken, in addition to a full range of other fixed and fresh tissues.

Example 3: Leukaemia in a pig

A further example of the usefulness of blood evaluation in diagnosing a rare condition was previously reported in the Companion Animal Society newsletter.¹⁶ Haematology and blood smears from a pet Kune Kune pig revealed a leukaemia involving multiple cell lines (Figure 4). In this case, bone marrow was not received or needed to make a diagnosis because the changes were obvious in the blood smear.

Value of complete clinical pathological workups

This example of the value of complete clinical pathological workups includes urinalysis and glucose estimations and the monitoring of sequential clinical pathological samples.

Hyperglycaemia and glucosuria are rarely observed in production animal practice and this may be partly because in the laboratory we rarely receive urine samples for urinalysis and fluoride oxalate tubes for glucose estimation. Hyperglycaemia may arise from either diabetes mellitus or stress. Glucosuria may arise from diabetes mellitus, stress or the Fanconi syndrome.

The Fanconi syndrome is a condition in which there are renal tubular defects resulting in the inability of the renal tubules to resorb glucose and other substances that are filtered through the glomerulus.^{1, 3, 5, 18} Routine urinary examination therefore reveals glucosuria and this, in conjunction with normal blood glucose, gives a diagnosis of the Fanconi syndrome. In addition to the loss of glucose, there is also loss of some amino acids, potassium, phosphorus, sodium, calcium and bicarbonate, but measurement of these substances is much less likely to be used for a diagnosis. The Fanconi syndrome may be inherited or acquired. In the acquired syndrome, toxins or infections may damage the renal tubules preventing resorption of glucose.^{1, 3, 5, 18} Affected animals eventually die of renal failure. It has been well described in dogs and humans,^{1, 5, 18} but there has only been one report in cattle³ in 1996, in which case it was thought to be inherited. Concurrent measurements of blood glucose and urinary glucose are required to differentiate the Fanconi syndrome from diabetes mellitus.^{2, 3, 6}

We recently diagnosed the Fanconi syndrome through Gribbles Veterinary Palmerston North in a one-year-old Jersey-cross heifer that showed very poor growth compared with her herd mates. Clinical examination showed she was bright, alert and non-pyrexic with no oral ulcers or other lesions. A serum sample and a urine sample were obtained because, although the heifer was not scouring, she was much smaller than her herd mates. The urine was initially examined in the clinic where glucose and protein were detected and the urine was found to be dilute. The serum and urine were then sent to the laboratory for further evaluation. BVD and parasitism were ruled out as causes of the ill thrift. In addition, the heifer was persistently polycythaemic; in other words, there were marked and persistent unexplained increases in all RBC parameters without other evidence for dehydration. Further tests that would have been carried out in human medicine to differentiate relative versus absolute polycythaemia include erythropoietin measurements; however, these tests are not available in veterinary laboratories. Hyperfibrinogenaemia indicating inflammation was noted in this heifer on two occasions, but blood leukocytes were normal.

Urinalysis on the first urine sample, and three subsequent urine samples, showed a persistent 3+ glucosuria, with persistently isosthenuric or hyposthenuric urine in which the urine specific gravity ranged from 1.005–1.012. Biochemical examination on three serum samples showed no significant changes, including no sign of azotaemia, and the blood glucose was always within the normal reference range. The heifer showed a paradoxical glucosuria, that is, a normoglycaemia with concurrent glucosuria. This is consistent with the Fanconi syndrome that has been well described in humans and dogs^{1, 5, 17} but rarely seen in cattle, with only one report in one bull.³

The heifer was euthanised because of the poor prognosis, and post-mortem examination revealed a much enlarged right kidney (Figure 5) and histological examination revealed pyelonephritis. Because this animal was a crossbred heifer and pyelonephritis was present, the Fanconi syndrome was more likely to be acquired rather than congenital.

The Fanconi syndrome needs to be differentiated from diabetes mellitus, which is also a rare diagnosis in cattle with cases reported in the literature in 1990⁶ and 2003.² At Gribbles Veterinary Palmerston North, we have diagnosed probable diabetes mellitus in cattle twice over many years. This was differentiated from a stress hyperglycaemia by monitoring the cattle on several occasions and demonstrating the animals were persistently hyperglycaemic with concurrent glucosuria.

The heifer with the Fanconi syndrome was persistently and significantly polycythaemic. She was monitored over several occasions to rule out dehydration and relative polycythaemia as a cause of the increased RBC parameters. Total protein concentrations were also consistently normal and not increased as would be expected with dehydration and haemoconcentration.

Polycythaemia or erythrocytosis may be either relative and result from dehydration leading to haemoconcentration, which is quite commonly seen, or it may be absolute.⁸ Absolute causes of polycythaemia include neoplasia (polycythaemia vera or primary erythrocytosis) and paraneoplastic syndromes whereby some tumours produce cytokines including erythropoietin that stimulate RBC production within the bone marrow. It may also result from chronic hypoxia resulting from some forms of chronic respiratory disease and cardiac disease, or from renal hypoxia and some forms of renal disease. Bovine polycythaemia vera or primary erythrocytosis has been reported in only one case in 2006.¹¹ Polycythaemia secondary to congenital cardiac disease causing chronic hypoxia has been reported in only one case, a calf, in 2010.¹⁴ In dogs, absolute secondary polycythaemia has been diagnosed as a response to pyelonephritis⁸ and pyelonephritis was diagnosed in this heifer so seems the most likely cause.

The diagnoses of both Fanconi syndrome and an absolute polycythaemia or erythrocytosis secondary to renal disease would not have been possible if a full range of samples had not been sent and a full range of tests requested. Monitoring the heifer over several occasions was also essential to confirm a diagnosis. A more detailed discussion of this case can be seen in the June 2011 newsletter of the NZVA Society of Sheep and Beef Cattle Veterinarians¹² and the September 2011 newsletter of the NZVA Society of Dairy Cattle Veterinarians.¹³

Sample type and management

When taking samples for the diagnosis of any disease, particularly if the disease appears rare or unusual, it is preferable to take the widest possible range of fresh and fixed samples. Ask the laboratory to hold samples pending other test results because, if needed, we can then go back to “held” samples and carry out further testing as required. It is better to submit samples and have them thrown out later than to wish you had taken more. Depending on the type of sample, Gribbles Veterinary holds samples for variable lengths of time. Fresh samples that are sent to the laboratory with no test requests are kept for three-to-four weeks pending future requests. Serum is kept for up to four weeks and EDTA samples are kept for two weeks following testing. Fresh samples sent for microbiology (for example, for culture and sensitivity) are then kept for a further two weeks so it is possible to go back to them for further tests if required. Histology blocks are kept for several months to years. If there is any doubt, please phone your local laboratory and check.

Acknowledgements

Catherine Williamson, Gribbles Veterinary Auckland, examined the smears and diagnosed the porcine leukaemia (Example 3).

Fraser Hill, Gribbles Veterinary Palmerston North, carried out the histopathological examination on tissues from the heifer with Fanconi syndrome.