An Overview of Leishmaniasis

Debbie D. Joiner, DVM; Craig E. Greene, DVM, MS; Kenneth S. Latimer, DVM, PhD; Perry J. Bain, DVM, PhD; Heather L. Tarpley, DVM, PhD

Class of 2005 (Joiner), Department of Small Animal Medicine (Greene), and Department of Pathology (Latimer, Bain, Tarpley), College of Veterinary Medicine, University of Georgia, Athens, GA 30602-7388

Introduction

Leishmaniasis is a worldwide zoonotic disease that recently has been introduced into North America. Foxhounds are the most prevalent breed affected in the United States, but other breeds of dogs have been identified with no apparent history of travel. As a result of the diverse manifestations of clinical signs and the lack of suspicion of this previously exotic illness, veterinarians need to have better awareness in order to achieve a clinical diagnosis.

Etiology

Leishmaniasis is caused by diphasic protozoa of the genus Leishmania and Viannia.⁹ The disease is endemic in many parts of the world including Central and South America, Africa, India, and the Mediterranean basin. In many of the endemic areas, dogs are considered the major reservoir for human disease while in other regions people are the principal reservoir for further human spread.⁹ Isolated foci of infections have been found in Texas affecting both humans and dogs and, to a lesser degree, cats. However, apparently endemic cases of Leishmania spp. have now been found in dogs in 21 states, including Oklahoma, Ohio, Texas, Michigan, New York and Alabama and two Canadian provinces.^3,4,6

Leishmania and Viannia subgenera are grouped into complexes of species and subspecies based upon molecular, biochemical and immunological similarities. To further confuse the issue, there are several forms of the disease named by their clinical presentation including cutaneous, mucocutaneous or visceral leishmaniasis. Each of these forms of disease is caused by different species of sand flies found in different regions of the world. Cutaneous leishmaniasis of humans is associated with members of L. aethiopica, L. major, and L. tropica complexes in the Old World and L. mexicana and L. braziliensis complexes in the New World. Visceral leishmaniasis is caused by L. donovani and L. infantum in Old World regions while L. chagasi is primarily responsible for visceral disease in the New World. Because L. infantum is the primary agent associated with canine leishmaniasis, infections in dogs often are regarded as visceral even though they tend to cause both visceral and cutaneous disease.⁹

Sand flies of the genus Phlebotomus (Old World) and Lutzomyia (New World) are the primary vectors responsible for disease transmission. Currently these are the only known vectors capable of spread; fleas, ticks and other arthropods have not been shown to be competent vectors.⁴ However, rare cases of leishmaniasis have been contracted through exchange of blood or body fluids, direct contact and at least one case of congenital transmission.^4,9 The importance of native sand flies is yet undetermined but could be related to infectious dose of the organism. Still, in recent years, and in the absence of known vectors, there has been an overwhelming incidence of leishmaniasis in Foxhound kennels across the United States. It is still not known how transmission of disease occurred or how this disease is maintained in these dogs because infected sand flies have not been reported in the United States.³ However, certain species of Lutzomyia (L. shannoni), found along the eastern United States and as far north as New Jersey, are considered a potentially competent vector for L. mexicana.^{1, 4}

Life Cycle

Leishmania spp. cycle between vertebrate hosts and sand fly vectors in either the promastigote or amastigote form (Fig. 1).^3,7 Promastigotes are slightly elongated and contain a single nucleus with an anterior flagellum originating from a kinetoplast while amastigotes are slightly round to oval, still contain a single nucleus and kinetoplast, but retain only a rudimentary flagellum.⁴ Both stages are capable of replication via binary fission but not within the same host.⁶

Figure 1. Life cycle of Isospora sp. Atoxoplasma sp. life cycle is similar except infection of mononuclear cells occurs in blood and tissues where the organism proliferates by binary fission (Gardiner CH, Fayer R, Dubey JP: An Atlas of Protozoan Parasites in Animal Tissues. Washington, DC: USDA/ARS, Agriculture Handbook #651 p.3).

The life cycle (Fig. 1) begins as an infected female sand fly inoculates a vertebrate host with flagellated promastigotes during a blood meal. Macrophages are the host’s first line of defense and promptly phagocytose the invading organisms. Unfortunately, Leishmania organisms are capable of survival within the macrophage where they undergo a transformational change from flagellated promastigotes to non-motile amastigotes. In the vertebrate host, the amastigotes (contained within macrophages) are capable of binary fission. Division continues until the macrophage lyses and amastigotes are released to infect neighboring phagocytic cells.

Infected macrophages or individual amastigotes enter the systemic circulation and subsequently disseminate to visceral organs leading to internal disease. Once the organism has entered the systemic circulation, it can once again be taken up during a blood meal by the female sand fly. The ingested amastigotes travel to the gut of the sand fly and are once again transformed into promastigotes. In the vector, it is the promastigote stage within the gut of the sand fly that is capable of binary fission. These flagellated organisms subsequently migrate to the hypostome of the sand fly and are inoculated into another vertebrate host completing the life cycle.⁴

Pathogenesis

Leishmaniasis is a slowly progressive disease that can take up to 7 years to become clinically apparent.^6,9 Even then, signs are frequently nonspecific and a diagnosis of Leishmania is seldom considered. Dogs are most commonly infected with L. infantum (L. donovani complex) which is responsible for viscerotropic disease in people. However, up to 90% of infected dogs present with both visceral and cutaneous lesions.⁹ However, many dogs appear naturally resistant to this parasite and may remain asymptomatic despite known infection.³ It is estimated that only 10% of dogs residing in endemic areas actually develop clinical disease.⁴ This lower incidence of clinical disease is attributed to a genetic predisposition of certain dogs to mount a more protective cell-mediated immune response than a humoral response.^4,6,9 Furthermore, it has been reported that up to 20% of infected dogs may mount an adequate immune response and spontaneously recover from clinical illness.⁶ In animals that mount a humoral response, IgG1 appears to correlate with clinical disease while asymptomatic dogs have higher IgG2 antibody levels.⁴

Clinical Signs

Some of the more frequently reported clinical signs of leishmaniasis include listlessness, fatigue and exercise intolerance coupled with anorexia and weight loss that eventually culminate as wasting disease.⁶ These signs may or may not be accompanied by fever, local or generalized lymphadenopathy (90%) and/or hepatosplenomegaly.^3-6 Articular involvement is also fairly common and may present as lameness with swollen joints or simply as a stiff gait. Less common findings include ocular lesions (<5%), chronic diarrhea (30%) and long, deformed brittle nails (20%) referred to as onychogryphosis.^4,9

Cutaneous lesions are present in up to 89% of infected dogs, with or without overt signs of visceral involvement (Fig. 2). However, it should be noted that any animal presenting with apparent lesions should be presumed to have disseminated leishmaniasis because involvement of the integument often occurs late in disease progression.⁹ Cutaneous lesions are often dry, alopecic areas that are rarely pruritic (Fig. 3). They usually begin around the head, especially on the pinna or muzzle, but can originate on the footpads before spreading to the rest of the body.⁹

Figure 2. Skin lesions of cutaneous leishmaniasis on the ear of a Foxhound (image courtesy of Noah’s Arkive, University of Georgia).	Figure 3. Cutaneous leishmaniasis in a dog. Affected areas of skin have alopecia, dryness, and scaling (image courtesy of Noah’s Arkive, University of Georgia).

Pathologic Findings

One of the most consistent findings (~100%) among dogs infected with Leishmania sp. is the presence of hyperproteinemia due to hyperglobulinemia, often in conjunction with hypoalbuminemia (94%).⁹ Serum protein electrophoresis commonly reveals a polyclonal gammopathy consisting primarily of IgG immunoglobulins⁶ and some acute phase proteins.⁵ As is typical of the ever confusing face of leishmaniasis, monoclonal gammopathy resembling plasma cell myeloma or ehrlichiosis also has been reported.⁹ Indirectly, increased antibody production and resultant immune-complex disease are responsible for many of the clinical and laboratory abnormalities encountered. Immune-mediated vasculitis leads to loss of proteins and cells resulting in hypoalbuminemia (94%) and thrombocytopenia (50%), whereas deposition of immune complexes into joints results in polyarthritis and lameness. Proteinuria (85%) and azotemia (45%) are associated with glomerulonephritis which is reported to be the leading cause of death in untreated animals.¹⁰ Liver failure occurs less commonly than renal failure, but elevations in liver enzyme activities, such as alanine aminotransferase (61%) and alkaline phosphatase (51%), are not uncommon.⁹

Hemostatic disorders such as epistaxis (15%),⁹ hematuria and disseminated intravascular coagulation (DIC) are not uncommon.^7,8 In one study of 26 Leishmania-infected dogs and 10 clinically normal dogs, half of the infected dogs had hematuria and 76.9% had proteinuria. Primary hemostatic disturbances caused by vasculitis, thrombocytopenia or platelet dysfunction were evaluated by assessing buccal mucosal bleeding time (BMBT) while secondary hemostasis was evaluated using various clotting tests to assess the intrinsic, extrinsic and common pathways of the coagulation cascade. Since renal failure is a common finding in leishmaniasis, a comparison study showed that the BMBT was significantly prolonged in Leishmania infected dogs, especially those with elevated serum creatinine levels.⁷ Research into a correlation between secondary hemostatic disorders and kidney or liver disease found activated partial thromboplastin time to be significantly prolonged in animals with leishmaniasis and elevated alanine aminotransferase activity. Thrombin clotting time also was elevated in dogs with leishmaniasis, but a correlation with liver function was not noted.⁸ Individual reports of hypofibrinogenemia and increased fibrin degradation products from hypercoaguable states is suspected to be caused by renal loss of antithrombin III, but was not found in this study.⁸

Diagnosis

Recognition of the clinicopathologic findings associated with leishmaniasis in dogs is paramount if this disease is to be suspected and diagnosed. Ultimately, the most reliable diagnostic test relies on demonstration of Leishmania sp. amastigotes, either cytologically (Fig. 4) or histopathologically (Fig. 5), in stained preparations of bone marrow, lymph node, spleen, skin or other tissues and organs (skeletal muscle, peripheral nerves, renal interstitium, and synovial membranes).⁶ Organisms most commonly reside in macrophages, but have been observed in other cell lines including neutrophils, eosinophils, endothelial cells and fibroblasts. ⁶ While microscopic visualization of organisms provides a definitive diagnosis, this technique may be only 60% effective for bone marrow samples and 30% effective for lymph node specimens, making it less sensitive than other testing strategies.⁴

Figure 4. Bone marrow from a dog with leishmaniasis. Numerous amastigotes, with a round nucleus and flat kinetoplast, are present within the cytoplasm of a macrophage (Wright-Leishman stain).

Figure 5. Kidney (left) and skin (right) biopsies from a dog with cutaneous and visceral leishmaniasis. Macrophages within the renal interstitium and superficial dermis contain small, round to oval amastigotes of Leishmania leishmania infantum (hematoxylin and eosin stain).

Serologic testing is used to detect circulating antibodies to Leishmania sp. in the blood. The primary limitation of this technique revolves around interpretation of a positive titer which may only indicate exposure to the organism as opposed to active infection. This is especially true in animals with low levels of circulating antibodies and leads to over diagnosis of the disease. Immunofluorescent antibody (IFA) testing is the gold-standard for determination of antibody titers. An anti-Leishmania antibody titer of 1:64 or greater constitutes a positive test result. However, organisms have been isolated from Foxhounds with antibody titers as low as 1:16.⁴ Additionally, IFA assays are known to cross react with another protozoan organism, Trypanosoma cruzi ³ making them less specific. Other tests, including enzyme-linked immunosorbent assays (ELISA), complement fixation, Western blot analysis and various agglutination assays have been developed but none of these techniques has a high specificity and sensitivity. Even the polymerase chain reaction (PCR) has been used to amplify target Leishmania DNA sequences from bone marrow, lymph node or blood. However, false-negative test results are known to occur with specimens containing less than 10 organisms.^3,4

Treatment

Due to a number of factors, treatment options for leishmaniasis in dogs and response to therapy are limited at best. For some undefined reason, visceral leishmaniasis is more difficult to treat in dogs than in humans. No treatment option is 100% effective in clearing parasitic infection and clinical disease often reappears with cessation of therapy.⁴ In endemic areas, the most common treatment regimen has been a combination of allopurinol with a pentavalent antimonial such as meglumine antimonite or sodium stibogluconate.^4,9 However, in recent years this protocol has fallen out of favor due to increasing resistance of the parasite to the drug as well as adverse side effects associated with these compounds.⁴ To further limit treatment options, Pentostam^® (sodium stibogluconate) is the only available antimonial in the United States and its distribution is regulated by the Centers for Disease Control and Prevention (CDC) in Atlanta, GA.⁴

Other protocols have been tried but have proven no more efficacious at clearing parasitic infection or at preventing clinical relapse. In addition, each protocol is associated with potential adverse effects. Amphotericin B binds sterols and disrupts cell membrane permeability but is nephrotoxic.⁴ When given parenterally, Paramomycin acts synergistically with antimonials causing higher levels of the antimonial for longer periods of time but is also nephrotoxic and is not currently recommended for clinical use.⁴ Pentamidine isethionate is effective against leishmaniasis but requires at least 15 intramuscular injections and is quite painful.⁴ Ketaconazole, miconazole, fluconazole and itraconazole are oral drugs that may be useful in containing the disease but are cost prohibitive⁴ and carry the risk of drug resistance when treating patients symptomatically. In summary, the various treatment regimens for leishmaniasis in dogs have been investigated but are not 100% efficacious; relapses are the rule rather than the exception. Ultimately, the veterinary practitioner is faced with the dilemma of treating symptomatic outbreaks of leishmaniasis in dogs at the risk of developing drug resistant strains of this parasite within the United States.

References

1. Eddlestone SM: Visceral leishmaniasis in a dog from Maryland. J Am Vet Med Assoc 217:1686-1688, 2000.

2. Gardiner CH, Fayer R, Dubey JP: An Atlas of Protozoan Parasites in Animal Tissues. Washington, DC: USDA/ARS, Agriculture Handbook # 651, p.3.

3. Grosjean NL, Vrable RA, Murphy AJ, Mansfield LS: Seroprevalence of antibodies against Leishmania spp among dogs in the United States. J Am Vet Med Assoc 222:603-606, 2003.

4. Lindsay DS, Zajac AM, Barr SC: Leishmaniasis in American Foxhounds: An Emerging Zoonosis? Compend Cont Educ Pract Vet 24:304-312, 2002.

5.  Martínez-Subiela S, Tecles F, Eckersall PD, Cerón JJ: Serum concentrations of acute phase proteins in dogs with leishmaniasis. Vet Rec 150:241-244, 2002.

6. McConkey SE, López A, Shaw D, Calder J: Leishmanial polyarthritis in a dog. Canine Vet J 43:607-609, 2002.

7.  Moreno P, Lucena R, Ginel PJ: Evaluation of primary haemostasis in canine leishmaniasis. Vet Rec 142:81-83, 1998.

8. Moreno P: Evaluation of secondary haemostasis in canine leishmaniasis. Vet Rec 144:169-171, 1999.

9.  Slappendel RJ, Ferrer L. In: Greene CE: Infectious Diseases of the Dog and Cat. WB Saunders Co, Philadelphia, 1998, pp. 450-458.

10. http://www.cvm.okstate.edu/instruction/kocan/vpar5333/5333iig.htm.

Acknowledgement

The painting "The Foxhound's Dream", copy after George Earl, by Linda Lawleris featured on her web site Gallery and is used with permission.

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