Clinical diagnosis (including non-specific laboratory parameters) is unreliable for the following reasons:
- More than 50% of dogs with proven established infections are apparently healthy on clinical diagnosis (asymptomatic). They are either progressing towards overt disease (pre-patent cases), remaining symptomless for prolonged periods (even for life), or healing spontaneously, the latter two groups regarded as resistant.
- When present, clinical signs can be variable and mimic those caused by other diseases.
- Atypical forms are reported in dogs.
For final diagnosis three types of tests are available:
- parasitological methods aiming at the detection of parasites
- serological methods with detection of anti-leishmanial antibodies in the blood
- molecularbiological techniques like the polymerase-chain reaction (PCR), detecting the parasite DNA in host tissue after multiplication.
No consensus on the relative diagnostic efficacies of the different techniques exists, particularly with regard to specificity and sensitivity. Parameters may vary according to materials used (e.g. culture medium, antigen, oligonucleotides) and methods employed (serological technique, PCR protocol). Multi-diagnostic approaches should be followed as no single technique identifies all infected animals from cross-sectional samples.
Comparing the performance of diverse techniques in a longitudinal study, Quinnell et al. (2001) recorded highest sensitivity of PCR shortly after the infection (88%), declining to around 50% in the following months. Conversely, the sensitivity of serology was low (41%) at the beginning of the infection, but high (93-100%) thereafter. PCR thus is better at detecting the early stages of established infections and transient and self-limiting infections, whereas a reliable serological technique is better at detecting advanced stages of infection, both in patent and asymptomatic dogs (although some positive cases may convert to seronegative during the course of infection).
Parasitological methods possess 100% specificity, but are not very sensitive.
Fine needle biopsies are taken from bone marrow or lymph nodes and ideally stained with May-Grünwald-Giemsa stain. Examination is under a x100 oil immersion lens. Parasites (here amastigotes) are small, oval bodies (1.5 to 3 x 3 to 6.5 µm) with dark nucleus and small kinetoplast in vertical position. No correlation between the clinic and the number of Leishmania found in cytological preparations could be found (Denerolle, 1996). Sometimes the parasites can be found in impression smear or fine needle aspirates from skin nodules. The sensibility of the cytology is too low for proper diagnosis: Bone marrow smears are positive in 50 to 70% of infected animals, lymph node smears in about 30%. The competence of the examiner and the time taken for examination are decisive for the success of the method. In bone marrow smears Leishmania are nearly exclusively found in macrophages while in cytological preparations from lymph nodes they are mainly extracellular. Infected macrophages often burst while being smeared.
Culturing of Leishmania should be performed at 26-28°C in suitable media, e.g. Novy-MacNeal-Nicolle (NMN)-medium. Positive cultures will have promastigotes visible within two weeks.
Seroprevalence, regarded as an intermediate measure between disease and the prevalence of infection, is influenced by the fact that on the one hand serology can reveal a high proportion of asymptomatic carriers which represent half of all seropositive animals; and on the other hand the limitation due to the long periods of serological latency. Antibodies are often detectable only several months after infection. Furthermore, a proportion of seropositive infected dogs spontaneously converts to negative, or does not develop specific antibodies at all (Acedo-Sànchez et al., 1998). Thus a maximum estimated sensitivity of the most efficient technique (IFAT) hardly reaches 80% and only during a few months after exposure to Leishmania in the previous season.
Several techniques can be used for the detection of antileishmanial antibodies. Commercially available are IFAT, Dot-ELISA and DAT (direct agglutination test). They possess high specificity and sensitivity (80-100%), but should not be used solely as diagnostic means. Further techniques are competitive ELISA, K39-ELISA, Western blot (WB) and Latex test. Ideally at least a repetition should be performed in six to eight weeks due to false positive and negative results (Noli, 1999). No correlation has been found between the degree of the clinical appearance and the serological titer. Serology is not useful for the control of a success in treatment, because antibodies may remain even after clinical recovery.
Universally recognized as the gold standard is the indirect immunofluorescent antibody test (IFAT), being the most sensitive and specific test. IFAT is considered impractical for the examination of large numbers of sera, and there is no consensus on dilutions at which serum considered to be positive. Reported threshold titers range from 1/20 to 1/160. It is the front-line technique recommended by the Office International Des Epizooties manual for diagnostic tests (OIE, 2000).
With the molecularbiological technique of PCR, a highly sensitive and specific method has been developed for the diagnosis of leishmaniosis. Parasite kinetoplast-DNA in liver, spleen, lymph node or bone marrow biopsies can be detected after multiplication. Fresh as well as formaline fixed paraffin embedded samples can be examined.
PCR is generally thought to approach a 'gold standard' status, but has not been successful for mass screening e.g. in American cutaneous leishmaniosis epidemiological studies (with 31% positive by PCR versus 81% by ELISA) (Reithinger et al., 2003).
PCRs using different primers have been developed for the detection of leishmanial DNA in dogs. Alvar et al. (2002) described a nested PCR, targeting the sSSUrRNA gene (LnPCR) with very high specificity and sensitivity, which is ideal for diagnosis, monitoring the success of treatment and for predicting relapses in man (slightly less sensitive in dogs).
It is a technique in which the natural arthropod vector is used for the detection and isolation of the pathogen. This technique cannot be used as a routine measure, but has been employed to solve epidemiological questions about the role of the clinical status and drug treatment of CanL.
- Acedo-Sànchez, C., F. Morillas-Màrquez, M.C. Sanchìz-Marìn and J. Martìn-Sànchez: Changes in antibody titres against Leishmania infantum in naturally infected dogs in southern Spain. Vet. Parasitol. 75, 1998, 1-8
- Alvar, J., I. Cruz, M.A. Morales and C. Cañavate: Molecular biology tools in leishmaniasis diagnosis and epidemiology. In: Killick-Kendrick, R. (ed.): Canine leishmaniasis: moving towards a solution. Proc. 2nd Int. Can. Leishm. Forum, Sevilla, Spain, 2002, Intervet Int., Boxmeer, The Netherlands, 2002, 25-30
- Denerolle, P.: Leishmaniose canine: difficulté du diagnostic et du traitement. Prat. Méd. Chir. Anim. Comp. 31, 1996, 137-145
- Noli, C.: Leishmaniose des Hundes. Waltham Focus 9, 1999, 16-24
- OIE: Leishmaniosis. In: Manual of Standards for Diagnostic Tests and Vaccines. 4th edn., Office International Epizooties, Paris, 2000, 803-812
- Quinnell, R.J., O. Courtenay, S. Davidson, L. Garcez, B. Lambson, P. Ramos, J.J. Shaw, M. A. Shaw and C. Dye: Detection of Leishmania infantum by PCR, serology and cellular immune response in a cohort study of Brazilian dogs. Parasitol. 122, 2001, 253-261
- Reithinger, R., J.C. Espinoza, O. Courtenay and C.R. Davies: Evaluation of PCR as a diagnostic mass-screening tool to detect Leishmania (Viannia) spp. in domestic dogs (Canis familiaris). J. Clin. Microbiol. 41, 2003, 1486-1493