Canine Leishmaniosis: Evaluation of the Immunocompromised Patient
WSAVA 2002 Congress
Lluís Ferrer Dip ECVD
Universitat Autònoma de Barcelona
Barcelona, Spain


In the last years, the way we understand the pathogenesis, clinical signs and treatment of canine leishmaniosis has changed considerably. A new paradigm, based on immunological and molecular investigations, has been constructed. A new paradigm that has numerous implications in the diagnosis, treatment and control of the disease. The main characteristic of this new paradigm is that considers that the disease is consequence of an inadequate immune response rather than merely consequence of the infection by the parasite. The purposes of this lecture will be to review shortly this new paradigm and to discuss how to evaluate the immune status and immune response of dogs affected by leishmaniosis.

The old paradigm and the change

Until now, key facts about canine leishmaniosis were:

1.  The prevalence of the disease in the Mediterranean area was 1-5% and the seroprevalence 5-15% (higher in some foci).

2.  Most infected dogs develop the disease, sooner or later.

3.  Infected animals become seropositive.

Two research lines have changed this paradigm. First, immunologists demonstrated, investigating the experimental infection of mice with L. major, that the immune response plays a key role in the evolution of the infection. In mice genetically deficient in the cellular immune response (BALB/c, for instance) the infection progresses and a severe systemic disease appears. These mice develop a humoral immune response (Thelper-2, production of antibodies) which is inefficient in controlling the infection. Contrarily, mice belonging to other lines (C3H), control the infection by means of a cellular immune response (Thelper-1). In this last case CD4+ T cells are activated and produce gamma-interferon, which activates macrophages for the elimination of the parasites. Genetics, in consequence, is a key factor in the control of the immune response, which is the key factor for the evolution of the disease.

Later on it was demonstrated that the situation in the dog was very similar to those described in mice: not all infected dogs develop the disease. Furthermore, dogs which developed the disease showed a humoral immune response, contrarily to the resistant dogs which showed a cellular immune response (similar to helper type-1).

Dogs affected by the disease are strongly immunedepressed as can be demonstrated by lymphocyte proliferation tests or by the low production of cytokines by PBMCs after stimulation. The number of circulating CD4+ cells and the CD4+/CD8+ ratio drop during the disease. The number of circulating CD4+ seems to be correlated with the severity of the clinical signs and with the infectivity.

A recent paper demonstrates that besides immune response other resistance factors are important in controlling susceptibility to leishmaniosis in the dog (Altet and co-workers, 2002). These authors have mapped and sequenced the canine RAMP1 gene (Slc11a1) and demonstrated that dogs susceptible to canine leishmaniosis have mutations in this gene which controls an ion transport protein involved in the control of intraphagosomal replication of parasites. This paper together with a previous study demonstrating that Ibizian hounds (a breed authoctonous of the Balearic islands) present a predominantly cellular and protective immune response against Leishmania infection have pointed out the major role that genetics play in the outcome of Leishmania infection in the dog.

At the same time, epidemiological studies showed that the incidence of the prevalence of the infection is much higher than the prevalence of the disease. For instance, a study performed in Mallorca using the PCR techniques on different tissues demonstrated that Leishmania infects 2 out of 3 dogs. In this study, most infected dogs showed no clinical signs. Similar studies performed in France and Portugal found similar results.

Finally, both research lines melted when it was demonstrated that infected but asymptomatic dogs had a cellular, effective cellular immune response, contrarily to symptomatic dogs which had a mainly humoral immune response (although the situation is not so polarised as it is in mice). In short, the new paradigm was born.

The new paradigm

 Prevalence of infection is much higher than traditionally thought. In endemic areas probably over 50% of dogs become infected.

 Most infected dogs do not develop the disease and remain free of clinical signs. Prevalence of the disease ranges between 3 and 10%.

 Infected animals without clinical signs show a cellular immune response against Leishmania and usually are seronegative or weakly seropositive (borderline titres).

 The infected and ill dogs show a humoral immune response but a weak cellular immune response. In general these dogs are strongly immunedepressed and show very low numbers of circulating CD4+ cells.

 A given dog can change from resistant to sensible to the disease and inversely. Drugs, infections, parasitic infestations, neoplasia, can induce the change.

Implications of the new paradigm for the diagnosis of the disease

 The diagnosis of the disease is a complex task. The results of each technique have to be interpreted adequately. For instance, a positive PCR means, only, that the animal is infected (as a positive bone marrow smear). A positive means infection and humoral immune response, which usually is linked to development of clinical signs (especially if the titres are high).

 The diagnosis, at the end, is always a clinical decision. Based on several analysis, but clinical. No one single test can establish a definitive diagnosis of leishmaniosis.

 Diagnostic tests (serology, PCR, intradermal skin test with leishmanin) should be used when a dog show clinical signs compatible with the disease. The clinical behaviour to be followed in infected but clinically healthy dogs is, at the present moment uncertain. A periodic follow-up is, in any case, mandatory.

 In most cases, several diagnostic techniques have to be combined to establish adequately the diagnosis. The techniques to be used in a given case depend on the clinical signs (for instance, a skin biopsy is usually very useful when cutaneous lesions are present).

 In many patients, the disease is associated with a hidden cause which has depressed the immune response (drug treatments, parasitism, infection, chronic diseases...). In fact, scientific literature is full of case reports of leishmaniosis associated to different diseases (haemangiosarcoma, lymphoma, pemphigus foliaceous, ehrlichiosis,...). A plausible explanation would be that these dogs were chronically infected animals that developed leishmaniosis when an event (treatment, infection, neoplasia,...) change their immune response. Especially in middle-aged and old dogs affected by leishmaniosis the presence of hidden causes has always to be investigated.

 After establishing the diagnosis, it is very important to evaluate the immune system of the patient and the type of specific immune response against Leishmania. This evaluation has to be continued during and after the treatment. Dogs immunedepressed and dogs which show a strong specific humoral immune response but a weak specific cellular immune response have worse prognosis.

 The evaluation should include-if possible-number of circulating CD4+ lymphocytes, lymphocytes CD8+ and CD4+/CD8+ ratio, specific immune response (intradermal skin test, lymphocyte blastogenesis, production of gamma-interferon) and specific humoral response (proteinogramme, titre of antibodies).

Counts circulating blood lymphocytes (CD4+, CD8+) using flow cytometry

 Flow cytometry is an everyday technique for monitoring immunologic function in human beings, for example in HIV infection, but it is still a research tool in veterinary medicine. However, the technique and the markers are already in the market and very soon standardised protocols will be developed.

 Flow cytometry permits the counting of the different subpopulations of lymphocytes: CD3+ or CD5+ (T cells), CD21+ (B cells), T helper (CD4+), T cytotoxic (CD8+).

 In the healthy dog, according to our results and to published data, the percentages and counts of the different lymphocyte subpopulations are:

Bourdoiseau et al, 1997

Byrne et al, 1999


2315 cells /uL (100%)


T (CD3+CD5+)

1800 cells/uL (78%)


Thelper (CD4+)

975 cells/uL (42%)


Tcytotoxic (CD8+)

410 cells/uL (18%)


CD4+/CD8+ ratio



B cells

415 cells/uL (18%)


 In dogs with leishmaniosis, the number of circulating CD4+ cells decreases (also the CD4+/CD8+ ratio), and also the number of B cells (CD21+) (Bourdoiseau et al, 1997; Guarga et al, 2000, own results). Most dogs in the acute phase of the disease are lymphopenic and all counts are disminished.




T (CD3+/CD5+)


Thelper (CD4+)

10-30% (decrease)

Tcytotoxic (CD8+)

5-30% (increase)

CD4+/CD8+ ratio

1,5-1,8 (decrease)

B cells

6% (decrease)

 This decrease in the number of CD4+ lymphocytes and of the CD4+/CD8+ ratio is clearly associated to the clinical signs. During therapy and clinical improvement there is a significant increase in the number and percentage of circulating CD4+ lymphocytes (Moreno et al, 1999). In consequence, CD4+ count and percentage and CD4+/CD8+ ratio are good prognostic indicators.

 Furthermore, it has been demonstrated a higher infectivity to sand flies amongst dogs with lower proportions of T helper cells (CD4+) (Guarga et al, 2000).

Evaluation of the specific cellular immune response

1.  Leishmanin skin test (Montenegro test, intradermal skin test with leishmanin) is a useful tool for the evaluation of the cell-mediated immunity in Leishmania infection in human beings and dogs. The technique is easy and inexpensive and it is well correlated with the intensity of the cell-mediated immune response. A recent paper (Solano-Gállego et al, 2001) describes the standardisation of the technique. However, the lecture is quite subjective, it needs two visits to the patient and the reagents are not in the market for diagnostic purposes (only for research).

2.  Lymphocyte blastogenesis test (lymphocyte lymphoproliferation after stimulation with Leishmania antigen has been described as useful in the evaluation of cell-mediated immunity in canine leishmaniosis. However, in our hands, the correlation between this test and the clinical signs and other indicators of cell-mediated immunity is low. Furthermore, the test is very complex and the use in every-day practice would be very difficult.

3.  Measurement of the production of gamma-interferon by peripheral blood mononuclear cells (PBMCs) after stimulation with Leishmania has been described as a useful technique in the evaluation of the cell-mediated immunity. The production of gamma-interferon seems to be correlated with the protective cell-mediated immune response. At present time, interferon levels, however, can only be measured (estimated) using a quite complex bioassay, not useful for the practice. Very probably in the future ELISA tests will be available to detect the production of gamma-interferon by PBMCs. These tests are much more adequate for routine use.

Further tests to evaluate the immune response

1.  At present time, the evaluation of the humoral immune response is based on titre of anti-Leishmania antibodies and proteinogramme. It has been well demonstrated that the titre of antibodies (mostly IgG2) is well correlated with the severity of the clinical signs. Remember that the titres, after therapy, decrease very slowly in many dogs and that in some dogs remain positive years after clinical cure (confirming that animals although clinically healthy remain infected). However, in the evaluation of the response to therapy the normalisation of the proteinogramme and the decrease of the titres are indicators of good response and favourable prognosis. The ratio between IgG1 and IgG2, which was suggested by some authors as a prognostic indicator, in our hands is not correlated with clinical evolution or with the severity of the clinical signs.

2.  Remains to be investigated if the identification of the allele of the NRAMP1 gene (Slc11a1) has value in clinical practice, especially to predict the response to treatment. Apparently, the identification of the allele susceptible in the microsatellite of the gene should be easy using the published PCR technique (Altet et al, 2002).


1.  Altet, L., Francino,O., Solano-Gallego, L., Renier, C., Sánchez, A. 2002. Mapping and sequencing of the canine NRAMP1 Gene and identification of mutations in leishmaniasis-susceptible dogs. Infect. Immun. 70: 2763-2771

2.  Berrahal, F., Mary, C., Roze, M., Berenger, A., Escoffier, K., Lamoroux, D., Dunan, S. 1996. Canine leishmaniasis: identification of asymptomatic carriers by polymerase chain reaction and immunoblotting. Am. J. Trop. Med. Hyg. 55:273-277.

3.  Bourdoiseau G, Bonnefont C, Magnol JP, Saint-André I, Chabanne L.1997. Lymphocyte subset abnormalities in canine leishmaniasis. Vet Immunol Immunopathol 56: 345-351

4.  Byrne K, Kim HW, Chew BP, Reinhardt GA, Hayek MG. 2000 A standardized gating technique for the generation of flow cytometry data for normal canine and normal feline blood lymphocytes. Vet Immunol Immunopathol 73: 167-182

5.  Cabral, M., O'Grady, J.E., Gomes, S., Sousa, J.C., Thompson, H., Alexander, J. 1998. The immunology of canine leishmaniosis: strong evidence for a developing disease spectrum from asymptomatic dogs. Vet. Parasitol. 76:173-180.

6.  Fisa, R., Gállego, M., Castillejo, S., Aisa, M.J., Serra, T., Riera, C., Carrió, J., Gállego, J., Portús, M. 1999. Epidemiology of canine leishmaniosis in Catalonia (Spain). The example of the Priorat focus. Vet. Parasitol. 83:87-97.

7.  Guarga JL, Moreno J, Lucientes J, Gracia MJ, Peribáñez MA, Alvar P, Castillo JA. 2000. Canine leishmaniasis transmission: higher infectivity amongst naturally infected dogs to sand flies is associated with lower proportions of T helper cells. Res Vet Sci 69: 249-253

8.  Moreno J, Nieto , Chamizo C, González F, Blanco F, Barker DC, Alvar J. 1999. The immune response and PBMC subsets in canine visceral leishmaniasis before, and after, chemotherapy. Vet Immunol Immunopathol 71: 181-195

9.  Pinelli, E., Killick-Kendrick, R., Wagenaar, J., Bernardina, W., del Real, G., Ruitenberg, J. 1994. Cellular and humoral immune response in dogs experimentally and naturally infected with Leishmania infantum. Infect. Immun. 62:229-235.

10. Pinelli, E., Gonzalo, R.M., Boog, C.J., Rutten, V.P., Gebhard, D., del Real, G., Ruitenberg, E.J. 1995. Leishmania infantum-specific T cell lines derived from asymptomatic dogs that lyse infected macrophages in a major histocompatibility complex-restricted manner. Eur. J. Immunol. 25:1594-1600.

11. Riera, C., Valladares, J.E., Gallego, M., Aisa, M.J., Castillejo, S., Fisa, R., Ribas, N., Carrio, J., Alberola, J., Arboix, M. 1999. Serological and parasitological follow-up in dogs experimentally infected with Leishmania infantum and treated with meglumine antimoniate. Vet. Parasitol. 84:3-47.

12. Solano-Gallego, L., Llull, J., Ramos, G., Riera, C., Arboix, M., Alberola, J., Ferrer, L. 2000. The ibizian hound presents a predominantly cellular immune response against natural Leishmania infection. Vet. Parasitol. 90: 37-45.

13. Solano-Gallego, L., Llull, Arboix, M., L.Ferrer, Alberola. 2001. Evaluation of the efficacy of two leishmanins in asymptomatic dogs. Vet. Parasitol. 102: 163-166.

14. Solano-Gallego L., Riera C., Roura X., Iniesta L., Gallego M., Valladares J.E., Fisa R., Castillejo S., Alberola J., Ferrer L., Arboix M., Portús M. 2001. Leishmania infantum-specific IgG, IgG1 and IgG2 antibody responses in healthy and ill dogs from endemic areas. Evolution in the course of infection and after treatment. Veterinary Parasitology 96, 265-276

15. Solano-Gallego L., Morell P., Arboix M., Alberola J., Ferrer L. 2001. Prevalence of Leishmania infantum infection in dogs living in an area of canine leishmaniosis endemicity using PCR on several tissues and serology. 2001. Journal of Clinical Microbiology 39, 560-563

Speaker Information
(click the speaker's name to view other papers and abstracts submitted by this speaker)

LluĂ­s Ferrer, Dip ECVD
Universitat Autònoma de Barcelona
Barcelona, Spain

MAIN : : Canine Leishmaniosis
Powered By VIN