Diagnosis of canine leptospirosis - Veterinary Practice
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InFocus

Diagnosis of canine leptospirosis

Dr PAUL BURR of Biobest
Laboratories, and CHRIS BALL
of the University of Liverpool,
review the tests available for
diagnosing this disease

LEPTOSPIROSIS is a significant disease of dogs and other mammals. It is also an important zoonosis, as it is increasingly recognised as a cause of human disease worldwide. The dangers of leptospirosis from cattle urine have been known to farm vets and farmers for many years. There is perhaps less consideration of the potential for human infection from dog urine despite leptospirosis being a differential in all cases of acute and chronic liver or kidney disease. Historically it has been difficult to confirm leptospira infection due to the relatively expensive, slow and technically demanding diagnostic tests. New diagnostic tests are now available that can be completed within 24 hours of sample receipt. Increased awareness, together with more routine diagnostic testing either for screening or confirmation of disease, is likely to yield a more complete picture of the importance and prevalence of leptospira- related disease in the UK.

Clinical diagnosis

In common with other infectious diseases that can affect multiple organ systems, the clinical signs seen in individual cases of leptospirosis are related to the organs infected in that specific case and hence can be variable. Leptospirosis should be considered as a differential diagnosis in any case with acute renal or hepatic disease. In acute or peracute infections, clinical signs may be relatively non-specific with pyrexia, vomiting, dehydration and muscle pain. Acute involvement of the liver may lead to cholestasis and icterus. Acute renal involvement may lead to oliguric, anuric, or polyuric acute renal failure. Patients may recover from this, or
progress to chronic renal failure. Chronic liver infection may in some cases lead to overt signs of liver failure including weight loss, inappetance, hepatic encephalopathy, ascites and icterus. Reproductive failure can also occur following leptospiral infection and should be considered as a differential in cases of abortion or perinatal mortality. Sometimes
musculoskeletal disease has also been reported, with signs of stiffness, weakness and muscle pain. Less common clinical manifestations include vasculitis, pulmonary haemorrhage, polyuria/polydipsia and uveitis.

Leptospira classification

The traditional nomenclature of leptospira classification was developed from a serological approach to diagnosis. This produced the serovar/serogroup classification which is used today, based on immunological antibody testing. Multiple leptospira species can belong to the same serogroup (e.g. L. interrogans and L. borgpetersenii) and furthermore, multiple serovars can belong to a species (e.g. icterohaemorrhagiae and canicola belonging to L. interrogans), with there being multiple strains belonging to a serovar. A single serogroup can therefore be represented across a range of species depending on the serovars within that group (Table 1). For some years, the microscopic agglutination test (MAT) has been regarded as the gold standard for leptospira identification and serogroup classification. It is widely used across the globe and is used by most laboratories involved with leptospira
research. While useful it does have some drawbacks: it is labour intensive, results are subjective and it can only identify to a serogroup and species level and cannot identify individual serovars or strains. More recently, molecular-based techniques have been developed. These include PCR assays, Multi-Locus Variable Number Tandem Repeat Analysis (MLVA) and Multi-Locus Sequence Typing (MLST). Molecular techniques like PCR and MLST are based on specific genes within the whole genome. These methods are objective and allow for a potentially more accurate and precise method for diagnosis and identification of leptospira strains when compared to the serological approach using MAT. Studies using molecular approaches have led to the discovery of new strains of leptospira. Five new species (containing 11 previously classified strains) of Leptospira spp. have been discovered using these techniques, which have been dubbed genomospecies. Genomospecies is the term used for species of bacteria which have been defined based on genetic typing. To date only one has been named, L. alexanderi, which has been shown to be pathogenic. The major difference between the genomospecies classification and the traditional serovar/serogroup classification is the diagnostic technique used. While serovars are linked to antibody testing, genomospecies are linked to PCR-based tests which are not directly in line with serology or vaccination.

Laboratory diagnosis

It is rare in infectious disease for there to be a perfect diagnostic test which
allows the clinician to reach a definitive diagnosis in isolation. Diagnosis must be based on a combination of clinical signs and the correct use of laboratory tests. In particular, with many infectious diseases some consideration must be given to the difference between infection past or present, and whether clinical disease is due to that infection. Most tests for
infectious agents detect the presence of, or exposure to that organism, but the clinician must decide whether that is related to any clinical signs observed. For leptospirosis and other zoonotic pathogens, current infection,
whether or not causing clinical disease, is always worthy of investigation and
potential treatment.

Biochemistry and haematology

Non-specific alterations in clinical pathology may include a leucocytosis, neutrophilia, lymphopaenia, thrombocytopaenia and prolonged bleeding times. Azotaemia, hyperphosphataemia, elevated ALP, bilirubin and ALT levels, and electrolyte abnormalities are typical findings in acute leptospirosis, depending on which organs are involved. Urinalysis may reveal haematuria, isosthenuria or hyposthenuria, and occasionally glucosuria due to tubular damage.

Serology

Detecting the presence of antibodies to leptospira is a useful aid to diagnosis. There are three techniques available in the UK.

1. MAT

MAT is the most widely known test and works by determining the ability of serial dilutions of a test serum to agglutinate cultures of leptospires. Individual tests need to be performed for each potential infecting serogroup as the reaction is serogroup specific, although cross reactions are commonly observed. The test is both timeconsuming and expensive to perform routinely. Interpretation of canicola and icterohaemorrhagiae MAT serology
may also be complicated by vaccination, as discrimination between antibodies due to infection and vaccination is not possible with this method.
There is always a danger in over- interpreting serology data and this is particularly true for the MAT test; a study of culture-proven leptospirosis in human patients showed that the predominant serogroup (based on MAT results) predicted less than 50% of the serogroups isolated. In canine patients it appears that the identity of the serogroup with the highest titre varies between laboratories and over time in individual patients.

2. Immunocomb

An alternative test to measure antibodies to canicola, icterohaemorrhagiae, pomona and grippotyphosa is the Immunocomb produced by Biogal. This test can be performed without specialist equipment by a practice laboratory and measures antibodies on a semi-quantitative scale. It would be expected to give broadly comparable results to MAT using the same serogroups, but of course may not detect antibodies directed against other serogroups. The sensitivity and specificity of this test according to the product literature are
80% and 60% respectively.

3. Immunofluorescent antibody (IFA)

This is a new test to the UK market but has been used in other countries
fr a number of years. The test predominantly detects antibodies directed against the sheath antigen shared by all pathogenic leptospires. Preliminary evidence has demonstrated that antibodies due to vaccination were not detected by this method but it detected all animals challenged with leptospira in a clinical trial. This test does not provide information on the infecting serogroup but can be set up and completed within a few hours of sample receipt and as multiple tests are not required for each serogroup it
is less costly to perform. As experience of the test increases it is likely that more information on the use and interpretation of this test will become available. Serology is a valuable tool, but it may take 7-10 days after infection for detectable levels of antibody to appear. Whatever the test method used, if initial titres are low or negative, convalescent titres should be performed after 2-3 weeks where there are strong grounds for suspicion
of acute leptospirosis. A four-fold rise in titre would be regarded as proof of recent infection; however, the failure to detect a rising titre should not be used to exclude leptospirosis if serology is positive. A persistent negative titre makes leptospirosis unlikely. Use of the IFA test for screening, and follow-up MAT where serogroup information is desired, may be useful in diagnostic situations.

Direct identification

In theory, identification of leptospires in urine, blood or tissue by culture, microscopy, fluorescent antibody techniques, or polymerase chain reaction (PCR) are also potentially useful diagnostic techniques.

Culture and direct identification

Culture of leptospires is difficult and time-consuming and few laboratories
are able to do this. Multiple urine samples may need to be submitted because of intermittent shedding and the organism may only be present in
blood for a short period of time. Dark field microscopy has low sensitivity and specificity. These tests do not appear to be routinely available in the UK at present. Light microscopy of tissue sections and smears stained with
Giemsa or silver impregnation may also show the presence of the organism, but may be negative in the presence of low numbers. These tests can be performed in a number of UK histopathology laboratories.

PCR

PCR tests detect DNA or RNA and have the potential for great sensitivity in detecting the presence of an organism in clinical samples and can be completed within 24 hours of sample receipt in urgent cases. It remains the role of the clinician and laboratory to decide whether the presence of leptospira in a sample is related to clinical disease. Diagnostic PCRs for leptospira do not usually give serogroup information, but this is not usually
critical for management of the condition. It can be regarded as both an
advantage and disadvantage of PCR techniques that live or infectious organisms are not required for a positive test result: identifying dead organisms may be useful for fragile pathogens, where diagnosis is being
attempted after therapy has started, or where sample transit has been less
than ideal. For a disease such as leptospirosis, a positive test result should be regarded as a significant finding in most instances as leptospires should not be present in normal dog urine. The only exception to this might be where disease has previously been confirmed and treatment is being monitored, as urine in such cases might stay positive for some time.
PCR testing is routinely available from UK and European veterinary laboratories.

Summary

Definitive diagnosis of leptospirosis can be difficult and there is no perfect
diagnostic test. Careful consideration of history, clinical signs and available laboratory data is therefore required, and laboratory test results must be
interpreted in the light of clinical observations and knowledge of the test limitations. It is worthwhile to maintain an index of suspicion for this disease, as leptospirosis is a significant zoonosis.

Further reading

Burr, P., Lunn, K. and Yam, P. (2009) Current perspectives on canine leptospirosis. In Pract 31: 98-102.

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