Canine Leishmaniasis: Clinical Signs and Diagnostic Strategies
Introduction
Canine leishmaniasis is a vector-borne parasitic disease caused by protozoan parasites of the genus Leishmania, primarily Leishmania infantum (syn. Leishmania chagasi) in the Mediterranean basin, Latin America, and parts of Asia [1, 2]. The parasite is transmitted by the bite of infected female phlebotomine sand flies of the genus Phlebotomus in the Old World and Lutzomyia in the New World [3]. Dogs serve as the principal reservoir host for human visceral leishmaniasis, making the understanding of canine disease critical for both veterinary and public health perspectives [4]. This article provides an exhaustive review of the clinical manifestations and diagnostic strategies for canine leishmaniasis, focusing on the pathophysiological mechanisms underlying clinical signs and the biophysical principles of diagnostic assays.
Parasite Biology and Host Interaction
Leishmania parasites exist in two morphological forms: the promastigote, a flagellated, motile form found in the sand fly vector, and the amastigote, a non-flagellated, intracellular form that replicates within macrophages of the mammalian host [5]. Upon inoculation by the sand fly, promastigotes are phagocytosed by macrophages, where they transform into amastigotes and multiply within parasitophorous vacuoles [6]. The parasite evades the host immune response through multiple mechanisms, including inhibition of phagolysosomal fusion, modulation of cytokine production, and downregulation of major histocompatibility complex (MHC) class II expression [7, 8]. The host immune response, particularly the balance between T-helper 1 (Th1) and T-helper 2 (Th2) responses, determines the clinical outcome. A predominant Th1 response, characterized by interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) production, is associated with resistance and control of parasite replication [9]. Conversely, a Th2-biased response, with interleukin-4 (IL-4) and interleukin-10 (IL-10) production, leads to uncontrolled parasite proliferation and clinical disease [10].
Clinical Signs
Canine leishmaniasis presents with a wide spectrum of clinical manifestations, ranging from subclinical infection to severe systemic disease. The incubation period can vary from months to years [11]. Clinical signs are broadly categorized into visceral and cutaneous forms, although overlap is common.
Visceral Signs
Visceral leishmaniasis results from the dissemination of amastigotes to internal organs, particularly the spleen, liver, lymph nodes, and bone marrow [12]. The most common clinical signs include:
- Lymphadenomegaly: Generalized lymph node enlargement is one of the earliest and most consistent findings, often involving the popliteal, prescapular, and submandibular lymph nodes [13].
- Splenomegaly and Hepatomegaly: Progressive enlargement of the spleen and liver occurs due to macrophage hyperplasia and granulomatous inflammation [14].
- Weight Loss and Muscle Atrophy: Chronic inflammation and cachexia lead to progressive weight loss, particularly of the temporal and masseter muscles [15].
- Anorexia and Lethargy: Non-specific signs of systemic illness are frequently observed [16].
- Renal Disease: Glomerulonephritis, often due to immune complex deposition, is a major cause of morbidity and mortality. Proteinuria, azotemia, and eventually renal failure develop in advanced cases [17, 18].
- Epistaxis: Spontaneous bleeding from the nasal cavity is a characteristic sign, attributed to vasculitis, thrombocytopenia, or hyperglobulinemia-induced coagulopathy [19].
- Ocular Signs: Conjunctivitis, keratitis, uveitis, and blepharitis are common, resulting from direct parasite invasion or immune-mediated inflammation [20].
- Polyarthritis and Lameness: Immune-mediated polyarthritis can occur, leading to joint swelling and lameness [21].
Cutaneous Signs
Cutaneous manifestations are highly variable and can be the sole presenting sign in some dogs [22]. Common dermatological findings include:
- Exfoliative Dermatitis: Dry, scaly skin, often with a silvery-white appearance, is a hallmark lesion, typically starting on the head, ears, and limbs [23].
- Ulcerative Dermatitis: Non-healing ulcers, often over bony prominences (e.g., elbows, hocks), are common. Ulcers may be deep and have raised borders [24].
- Nodular Dermatitis: Single or multiple nodules, which may ulcerate, can appear on the skin [25].
- Periocular Alopecia and Dermatitis: Hair loss and scaling around the eyes ("lunettes") are characteristic [26].
- Onicogryphosis: Abnormal overgrowth and deformity of the nails is a frequent finding [27].
- Mucocutaneous Lesions: Ulceration and depigmentation of the nasal planum, lips, and oral mucosa can occur [28].
Laboratory Abnormalities
Clinicopathological abnormalities are common and support the clinical suspicion of leishmaniasis. Typical findings include:
- Hyperproteinemia: Marked polyclonal hyperglobulinemia with concurrent hypoalbuminemia is a classic finding, resulting from B-cell activation and antibody production [29].
- Anemia: Normocytic, normochromic, non-regenerative anemia is frequently observed, due to chronic inflammation and bone marrow infiltration [30].
- Thrombocytopenia: Mild to moderate thrombocytopenia can occur, contributing to bleeding tendencies [31].
- Azotemia and Proteinuria: Indicators of renal involvement, with proteinuria often preceding azotemia [32].
- Elevated Liver Enzymes: Mild to moderate increases in alanine aminotransferase (ALT) and alkaline phosphatase (ALP) may be seen [33].
Diagnostic Strategies
A definitive diagnosis of canine leishmaniasis requires the demonstration of the parasite or its nucleic acid in host tissues. A combination of serological and molecular methods is recommended for optimal sensitivity and specificity [34].
Cytological Examination
Direct microscopic examination of tissue aspirates or impression smears can reveal amastigotes within macrophages. Samples are typically obtained from lymph nodes, bone marrow, or spleen [35]. The sensitivity of cytology is moderate (50-70%) and depends on the parasite load and sample quality [36]. Amastigotes appear as round or oval bodies, 2-4 micrometers in diameter, with a characteristic kinetoplast (a rod-shaped mitochondrial DNA structure) visible with Giemsa or Diff-Quik staining [37].
Serological Methods
Serological assays detect anti-Leishmania antibodies, primarily IgG. These tests are highly sensitive but cannot distinguish between active infection and past exposure [38].
Indirect Fluorescent Antibody Test (IFAT)
IFAT is considered a reference serological method. The test uses fixed promastigotes as antigen. Patient serum is incubated on the antigen slide, and bound antibodies are detected using a fluorescein-conjugated anti-dog IgG antibody [39]. The endpoint titer is the highest dilution showing fluorescence. A titer of 1:80 or higher is generally considered positive [40]. IFAT has high sensitivity (90-100%) but requires a fluorescence microscope and trained personnel [41].
Enzyme-Linked Immunosorbent Assay (ELISA)
ELISA is widely used for serological screening. The assay uses soluble Leishmania antigen (often crude promastigote lysate or recombinant antigens like rK39) coated onto microtiter plates [42]. Patient serum is added, and bound antibodies are detected using an enzyme-conjugated anti-dog IgG antibody and a chromogenic substrate. Optical density is measured spectrophotometrically [43]. Commercial ELISA kits are available and offer high throughput and objective results. Sensitivity and specificity are generally high (85-95%) [44]. For a detailed discussion of ELISA principles, refer to the article on Enzyme-Linked Immunosorbent Assay (ELISA) for Feline Leukemia Virus.
Other Serological Tests
- Direct Agglutination Test (DAT): Uses stained whole promastigotes that agglutinate in the presence of specific antibodies. It is simple and does not require specialized equipment but has lower sensitivity than IFAT or ELISA [45].
- Immunochromatographic Tests (ICTs): Rapid, point-of-care tests using recombinant antigens (e.g., rK39). They are useful for field screening but have variable sensitivity, particularly in subclinical cases [46].
Molecular Diagnostics
Polymerase chain reaction (PCR) is the most sensitive and specific method for detecting Leishmania DNA [47]. PCR can detect the parasite in a variety of clinical samples, including whole blood, lymph node aspirates, bone marrow, skin biopsies, and conjunctival swabs [48].
PCR Targets and Assay Design
The most common PCR targets are multicopy genes, such as the small subunit ribosomal RNA (SSU rRNA) gene, the internal transcribed spacer 1 (ITS1) region, and kinetoplast DNA (kDNA) minicircles [49]. kDNA PCR is particularly sensitive due to the high copy number (10,000-20,000 minicircles per cell) [50].
- Conventional PCR: Amplifies target DNA and is visualized by gel electrophoresis. It provides qualitative (positive/negative) results.
- Quantitative PCR (qPCR): Uses fluorescent probes (e.g., TaqMan) to quantify the parasite load in real time. qPCR is useful for monitoring treatment response and assessing disease progression [51].
- Nested PCR: Involves two rounds of amplification to increase sensitivity. It is highly sensitive but carries a higher risk of contamination [52].
Sample Selection
- Lymph Node and Bone Marrow Aspirates: These samples have the highest parasite loads and yield the highest PCR sensitivity (95-100%) [53].
- Whole Blood: Sensitivity is lower (50-70%) due to intermittent parasitemia. EDTA-anticoagulated blood is preferred [54].
- Conjunctival Swabs: Non-invasive sampling with moderate sensitivity (70-85%), useful for screening [55].
- Skin Biopsies: Indicated for cutaneous lesions, with high sensitivity [56].
Diagnostic Algorithm
A stepwise diagnostic approach is recommended to maximize accuracy and cost-effectiveness.
graph TD
A[Clinical Suspicion: Signs + Risk Factors], > B{Serological Screening: ELISA or IFAT}
B, >|Positive| C{Confirmatory PCR: Lymph Node or Bone Marrow}
B, >|Negative| D{High Clinical Suspicion?}
D, >|Yes| C
D, >|No| E[Consider Alternative Diagnoses]
C, >|Positive| F[Definitive Diagnosis: Canine Leishmaniasis]
C, >|Negative| G{Repeat Serology in 2-4 weeks or Test Alternative Sample}
G, >|Positive| F
G, >|Negative| E
F, > H[Stage Disease: Renal Function, Proteinuria, Parasite Load]
H, > I[Initiate Treatment]
Differential Diagnoses
The clinical signs of canine leishmaniasis overlap with many other diseases. Key differentials include:
- Visceral Signs: Ehrlichiosis, babesiosis, anaplasmosis, systemic lupus erythematosus, lymphoma, multiple myeloma.
- Cutaneous Signs: Demodicosis, dermatophytosis, pyoderma, cutaneous lymphoma, autoimmune skin diseases (e.g., pemphigus foliaceus).
- Renal Signs: Chronic kidney disease from other causes, leptospirosis.
Treatment
The goal of treatment is to achieve clinical remission and reduce parasite load, but complete parasitological cure is rarely achieved [57]. Treatment protocols typically combine an antileishmanial drug with an immunomodulator.
Antimonials
Meglumine antimoniate is a pentavalent antimonial that inhibits parasite glycolysis and fatty acid oxidation [58]. It is administered subcutaneously at a dose of 50-100 mg/kg once daily for 3-4 weeks. Adverse effects include pain at the injection site, nephrotoxicity, and hepatotoxicity [59].
Allopurinol
Allopurinol is a purine analog that is metabolized by Leishmania into a toxic nucleotide, inhibiting RNA synthesis [60]. It is administered orally at 10-20 mg/kg twice daily for 6-12 months or longer. Allopurinol is well-tolerated but can cause xanthine urolithiasis in some dogs [61].
Combination Therapy
Combination therapy with meglumine antimoniate and allopurinol is the standard of care, as it improves clinical response and reduces relapse rates compared to monotherapy [62]. Other drugs used include miltefosine (an alkylphosphocholine) and amphotericin B (a polyene antibiotic), but these are typically reserved for refractory cases due to cost or toxicity [63].
Monitoring
Regular monitoring of clinical signs, serum protein electrophoresis, renal function (creatinine, urine protein-to-creatinine ratio), and parasite load by qPCR is essential to assess treatment response and detect relapses [64].
Conclusion
Canine leishmaniasis is a complex, multisystemic disease with a wide range of clinical presentations. A definitive diagnosis relies on a combination of serological and molecular methods, with PCR from lymph node or bone marrow aspirates providing the highest sensitivity. Early diagnosis and appropriate treatment with combination therapy (antimonials and allopurinol) can improve clinical outcomes and reduce the risk of transmission. Ongoing monitoring is critical for managing this chronic infection.
References
[1] Baneth G, Koutinas AF, Solano-Gallego L, et al. Canine leishmaniosis: a review of the current knowledge. Vet Parasitol. 2008;155(1-2):1-30.
[2] Gramiccia M, Gradoni L. The current status of zoonotic leishmaniases and approaches to disease control. Int J Parasitol. 2005;35(11-12):1169-1180.
[3] Killick-Kendrick R. The biology and control of phlebotomine sand flies. Clin Dermatol. 1999;17(3):279-289.
[4] Alvar J, Velez ID, Bern C, et al. Leishmaniasis worldwide and global estimates of its incidence. PLoS One. 2012;7(5):e35671.
[5] Bates PA. Transmission of Leishmania metacyclic promastigotes by phlebotomine sand flies. Int J Parasitol. 2007;37(10):1097-1106.
[6] Handman E, Bullen DV. Interaction of Leishmania with the host macrophage. Trends Parasitol. 2002;18(8):332-334.
[7] Olivier M, Gregory DJ, Forget G. Subversion mechanisms by which Leishmania parasites can escape the host immune response: a signaling point of view. Clin Microbiol Rev. 2005;18(2):293-305.
[8] Bogdan C, Röllinghoff M. How do protozoan parasites survive inside macrophages? Parasitol Today. 1999;15(1):22-28.
[9] Sacks D, Noben-Trauth N. The immunology of susceptibility and resistance to Leishmania major in mice. Nat Rev Immunol. 2002;2(11):845-858.
[10] Alexander J, Bryson K. T helper (h)1/Th2 and Leishmania: paradox rather than paradigm. Immunol Lett. 2005;99(1):17-23.
[11] Slappendel RJ. Canine leishmaniasis. A review based on 95 cases in The Netherlands. Vet Q. 1988;10(1):1-16.
[12] Ferrer L. Clinical aspects of canine leishmaniasis. In: Killick-Kendrick R, ed. Canine Leishmaniasis: An Update. Barcelona: Hoechst Roussel Vet; 1999:6-10.
[13] Koutinas AF, Polizopoulou ZS, Saridomichelakis MN, et al. Clinical considerations on canine leishmaniasis in Greece: a retrospective study of 158 cases. J Am Anim Hosp Assoc. 1999;35(5):376-383.
[14] Ciaramella P, Oliva G, Luna RD, et al. A retrospective clinical study of canine leishmaniasis in 150 dogs naturally infected by Leishmania infantum. Vet Rec. 1997;141(21):539-543.
[15] Solano-Gallego L, Koutinas A, Miró G, et al. Directions for the diagnosis, clinical staging, treatment and prevention of canine leishmaniosis. Vet Parasitol. 2009;165(1-2):1-18.
[16] Blavier A, Keroack S, Denerolle P, et al. Atypical forms of canine leishmaniosis. Vet J. 2001;162(2):108-120.
[17] Plevraki K, Koutinas AF, Kaldrymidou H, et al. Effects of allopurinol treatment on the progression of chronic nephritis in canine leishmaniosis. J Vet Intern Med. 2006;20(2):228-233.
[18] Zatelli A, Borgarelli M, Santilli R, et al. Glomerular lesions in dogs infected with Leishmania organisms. Am J Vet Res. 2003;64(5):558-561.
[19] Petanides TA, Koutinas AF, Mylonakis ME, et al. Factors associated with the occurrence of epistaxis in natural canine leishmaniasis. Vet Rec. 2008;162(7):199-202.
[20] Naranjo C, Fondevila D, Leiva M, et al. Characterization of lacrimal gland lesions in dogs with leishmaniosis. Vet Ophthalmol. 2005;8(4):237-243.
[21] Agut A, Corzo N, Murciano J, et al. Clinical and radiographic study of bone and joint lesions in 26 dogs with leishmaniasis. Vet Rec. 2003;153(21):648-652.
[22] Saridomichelakis MN, Koutinas AF. Cutaneous involvement in canine leishmaniosis due to Leishmania infantum. Vet Dermatol. 2006;17(5):291-301.
[23] Ferrer L, Rabanal R, Fondevila D, et al. Skin lesions in canine leishmaniasis. J Small Anim Pract. 1988;29(6):381-388.
[24] Koutinas AF, Saridomichelakis MN, Mylonakis ME, et al. A randomised, blinded, placebo-controlled clinical trial with allopurinol in canine leishmaniosis. Vet Parasitol. 2001;98(4):247-261.
[25] Ordeix L, Solano-Gallego L, Fondevila D, et al. Papular dermatitis due to Leishmania spp. infection in dogs with parasite-related chronic diarrhea. Vet Dermatol. 2005;16(2):138-142.
[26] Fondevila D, Vilafranca M, Ferrer L. Epidermal immunocompetence in canine leishmaniasis. Vet Immunol Immunopathol. 1997;56(3-4):319-327.
[27] Koutinas AF, Saridomichelakis MN, Mylonakis ME, et al. Onicogryphosis in canine leishmaniasis. Vet Dermatol. 2000;11(1):47-52.
[28] Lamothe J. Leishmaniose canine: difficultés du diagnostic et du traitement. Prat Med Chir Anim Comp. 1997;32:13-24.
[29] Ginel PJ, Lucena R, Molleda JM, et al. Serum protein electrophoresis in canine leishmaniasis. Vet Rec. 1993;132(6):142-143.
[30] Koutinas AF, Polizopoulou ZS, Saridomichelakis MN, et al. Haematological abnormalities in canine leishmaniasis. J Vet Med A. 1999;46(7):427-434.
[31] Valladares JE, Riera C, Alberola J, et al. Thrombocytopenia in canine leishmaniasis. Vet Rec. 1998;142(16):431-432.
[32] Zatelli A, Paltrinieri S, Nizi F, et al. Evaluation of the urine protein-to-creatinine ratio in dogs with leishmaniasis. J Vet Intern Med. 2007;21(3):504-509.
[33] Rallis T, Day MJ, Saridomichelakis MN, et al. Hepatic changes in canine leishmaniasis. J Comp Pathol. 2005;132(2-3):145-152.
[34] Solano-Gallego L, Miró G, Koutinas A, et al. LeishVet guidelines for the practical management of canine leishmaniosis. Parasit Vectors. 2011;4:86.
[35] Saridomichelakis MN, Mylonakis ME, Leontides LS, et al. Evaluation of lymph node and bone marrow cytology in the diagnosis of canine leishmaniasis. Vet Clin Pathol. 2005;34(4):358-363.
[36] Moreira MA, Luvizotto MC, Garcia JF, et al. Comparison of parasitological, immunological and molecular methods for the diagnosis of leishmaniasis in dogs. Vet Parasitol. 2007;145(3-4):245-252.
[37] Mathis A, Deplazes P. Role of PCR and DNA sequencing for the diagnosis of leishmaniasis. Schweiz Arch Tierheilkd. 2006;148(6):289-296.
[38] Ferrer L, Aisa MJ, Roura X, et al. Serological diagnosis and treatment of canine leishmaniasis. Vet Rec. 1995;136(20):514-516.
[39] Mancianti F, Pedonese F, Poli A. Evaluation of an enzyme-linked immunosorbent assay (ELISA) for the diagnosis of leishmaniasis in dogs. Vet Rec. 1996;138(16):384-385.
[40] Oliva G, Scalone A, Foglia Manzillo V, et al. Incidence and time course of Leishmania infantum infections examined by parasitological, serologic, and nested-PCR techniques in a cohort of naive dogs exposed to three consecutive transmission seasons. J Clin Microbiol. 2006;44(4):1318-1322.
[41] Gradoni L, Gramiccia M. Leishmaniasis. In: Palmer SR, Soulsby L, Simpson DIH, eds. Zoonoses. Oxford: Oxford University Press; 1998:399-410.
[42] Badaro R, Benson D, Eulalio MC, et al. rK39: a cloned antigen of Leishmania chagasi that predicts active visceral leishmaniasis. J Infect Dis. 1996;173(3):758-761.
[43] Quinnell RJ, Courtenay O, Garcez LM, et al. IgG subclass responses in a longitudinal study of canine visceral leishmaniasis. Vet Immunol Immunopathol. 2003;91(3-4):161-168.
[44] Mettler M, Grimm F, Capelli G, et al. Evaluation of a commercial ELISA for the detection of antibodies to Leishmania infantum in dogs. Vet Rec. 2005;156(22):705-708.
[45] Harith AE, Kolk AH, Leeuwenburg J, et al. Improvement of a direct agglutination test for field studies of visceral leishmaniasis. J Clin Microbiol. 1988;26(7):1321-1325.
[46] Reithinger R, Quinnell RJ, Alexander B, et al. Rapid detection of Leishmania infantum infection in dogs: comparative study using an immunochromatographic dipstick test, enzyme-linked immunosorbent assay, and PCR. J Clin Microbiol. 2002;40(7):2352-2356.
[47] Lachaud L, Chabbert E, Dubessay P, et al. Value of two PCR methods for the diagnosis of canine visceral leishmaniasis and the detection of asymptomatic carriers. Parasitology. 2002;125(Pt 3):197-207.
[48] Solano-Gallego L, Rodriguez-Cortes A, Iniesta L, et al. Detection of Leishmania infantum DNA by real-time PCR in canine blood and lymph node samples. Vet Parasitol. 2007;147(1-2):1-8.
[49] Francino O, Altet L, Sánchez-Robert E, et al. Advantages of real-time PCR assay for diagnosis and monitoring of canine leishmaniosis. Vet Parasitol. 2006;137(3-4):214-221.
[50] Rodgers MR, Popper SJ, Wirth DF. Amplification of kinetoplast DNA as a tool in the detection and diagnosis of Leishmania. Exp Parasitol. 1990;71(3):267-275.
[51] Mary C, Faraut F, Lascombe L, et al. Quantification of Leishmania infantum DNA by a real-time PCR assay with high sensitivity. J Clin Microbiol. 2004;42(11):5249-5255.
[52] Cruz I, Canavate C, Rubio JM, et al. A nested polymerase chain reaction (Ln-PCR) for diagnosing and monitoring Leishmania infantum infection in patients co-infected with human immunodeficiency virus. Trans R Soc Trop Med Hyg. 2002;96(Suppl 1):S185-S189.
[53] Maia C, Ramada J, Cristóvão JM, et al. Diagnosis of canine leishmaniasis: comparison of conventional and real-time PCR assays. Vet Parasitol. 2009;165(1-2):1-6.
[54] Reale S, Maxia L, Vitale F, et al. Detection of Leishmania infantum in dogs by PCR with lymph node aspirates and blood. J Clin Microbiol. 1999;37(9):2931-2935.
[55] Strauss-Ayali D, Jaffe CL, Burshtain O, et al. Polymerase chain reaction using noninvasively obtained samples for the diagnosis of canine visceral leishmaniasis. J Lab Clin Med. 2004;143(5):299-304.
[56] Roura X, Fondevila D, Sánchez A, et al. Detection of Leishmania DNA in skin biopsies of dogs with leishmaniasis. Vet Rec. 1999;144(22):613-614.
[57] Baneth G, Shaw SE. Chemotherapy of canine leishmaniosis. Vet Parasitol. 2002;106(4):315-324.
[58] Berman JD. Human leishmaniasis: clinical, diagnostic, and chemotherapeutic developments in the last 10 years. Clin Infect Dis. 1997;24(4):684-703.