INTRODUCTION

Leishmaniases are parasitic infections of human beings and animals caused by protozoa of the genus Leishmania. In man three forms of leishmaniases are described related to the causative agent, including: the visceral, cutaneous and mucocutaneous forms. Human visceral leishmaniasis is caused by Leishmania of the Leishmania donovani species complex and is transmitted by sand flies. Dogs are considered the main reservoir. As in dogs, clinical features vary widely, the term of Canine Leishmaniasis (CanL) is more appropriate than the previous term of Canine Visceral Leishmaniasis (CVL).Zoonotic leishmaniasis is an increasingly important public health problem. The human disease is seen in 88 countries. 12 millions people are affected. Two millions new cases a year are diagnosed. The increasing number of human cases is due to immunosuppressive factors (HIV and drugs). Concomitant infection with HIV has changed the age distribution of the human disease from children towards adults.

EPIDEMIOLOGY

1. The Parasite

According to the classification of Levine et al.(1980), the genus Leishmania belongs to the sub-kingdom of Protozoa; order: Kinetoplastida, suborder: Trypanosomatina; family: Trypanosmatidae. New techniques of identification include iso-enzymatic patterns, DNA, monoclonal antibodies, cell membrane structure and fatty acid analysis. The current most accepted classification is based on iso-enzyme analysis which identifies different zymodemes (parasites with similar iso-enzyme patterns). In the Old World, L. infantum is the agent of human visceral leishmaniasis and CanL. L. Tropica is the agent of human cutaneous leishmaniasis and CanL. In the New World, L. chagasi is the agent of human visceral leishmanisasis and CanL. L.mexicana is the agent of human cutaneous leishmaniasis. L.braziliensis is the agent of human mucocutaneous leishm. and CanL. To complete its cycle, the organisms require two hosts (a vertebrate and an insect). In the insect vector (the sand fly), the parasite can be observed in a flagellate form called a promastigote. The organism is fusiform, measuring 10-25µm. In the vertebrate host, the parasite is found without free flagellum. The form is called an amastigote. The organism is an intracellular ovoid structure of 1,5 to 5 µm size. The organisms are easily stained by May-Gruenewald -Giemsa staining. They appear as a nucleus and a rod shaped kinetoplast.

2. The Vector/Transmission

In the Old World, L. infantum is transmitted via the bite of sand flies of the genus Phlebotomus. Nine species of this insect are vectors of CanL around the Mediterranean Basin. In the New World, L. chagasi is transmitted by an other sand fly: Lutzomyia.. Phlebotomus is a very small (2-3 mm), nocturnal and silent sand fly. It is active during the summer season. It can fly over 2 kms from its breeding places. The sand fly female injects the infectious or promastigote form of the parasite into the skin of the host. The insect's saliva contains anticoagulants and active substances able to modulate the host's immune response. On the other hand, the sand fly can ingest the amastigote forms of the parasite from an infected host. If the vector is suitable, amastigotes are transformed into promastigotes which attach to the mid-gut epithelium of the insect. Then, they move to the fore-gut, ready to complete the cycle.

3. The Hosts/ Distribution

Sand flies can bite a wide range of mammals. They do not bite reptiles or birds. In endemic areas of human leishmaniasis, the dog is the main reservoir, but many other animals can be infected e.g., the horse, goat, fox, wolf, jackal, squirrel, rat, mouse and cats. Their role in the epidemiology is often not clear. The cat was considered until now as a very unusual host but recent reports, based on PCR leishmania detection, suggest a high rate of feline infection in endemic areas. The disease has a world wide distribution. Leishmania infantum is the causative agent of the Old World CanL. The disease has been reported around the Mediterranean Basin, Southern Europe, West, East and North Africa, the Middle East, India and China. Leishmania chagasi, considered now as synonymous with L. infantum is responsible for the disease in the New World (South America). More and more foci are reported outside the conventional endemic areas and recently CanL was identified in North America.

Interaction Host-Parasite

The relation between the vector, the parasite and the host are, now, better understood.

 The Immune Responses. After the injection into the host, promastigotes of Leishmania are normally phagocytosed by macrophages and transform into amastigotes. Then, two types of immune responses can occur making the dog susceptible or resistant to the disease. Resistance is associated with the activation of Th1 cells producing interferon γ and interleukin-2. Susceptibility and progressive disease are related to the activation of Th2 cells producing interleukin 4. B cells proliferation and antibodies production are not protective. The humoral response is responsible for the high serum levels of γglobulins. It may contribute to the formation of immune complexes. Between resistance and susceptibility, there are many intermediate states.

 Pathogenesis. The primary lesion of CanL consists of a granulomatous inflammatory reaction with parasitized and non parasitized macrophages, lymphocytes and plasma cells. Inflammatory reactions may be present in any part of the body including joints and central nervous system. Deposition of immune complexes may occur resulting in vasculitis, uveitis and glomerulonephritis.

Symptomatology

The clinical signs of CanL are very variable. Lymphadenopathy, skin lesions and weakness are the most usual manifestations of the disease in the dog. Skin lesions include alopecia, scaling, ulceration (ears and limbs) and nodules. In the most severe forms renal failure and hemorrhages (mainly epistaxis) occur. They can cause the death of the animal. The different clinical signs are summarised in order of prevalence (personal observations) in table 1.

Table 1.

The most frequent ocular manifestation is uveitis (75%), the common blepharitis being related to the skin lesions. Atypical manifestations are: neuralgia, polyarthritis, polymyositis, osteolytic lesions and proliferative periostitis.

For the certain detection of the infection, symptomatology alone is not sufficiently reliable . Probably more than 50% of proven infected dogs are asymptomatic.

Diagnosis

Diagnosis is based on non specific tools (clinical signs and clinical laboratory findings) and specific tools ( parasitology, serology and more recent molecular techniques).

1. Clinical signs

They are summarised in table 1. The diagnosis of CanL can be difficult. The signs are variable. They can mimic other diseases. Concomitant infection (i.e., ehrlichiosis, demodicosis) may complicate diagnosis

2. Clinical laboratory findings

The order of prevalence of laboratory findings are summarised in table 2. Among them, evaluation of serum proteins by electrophoresis is to be considered as a very useful mean to orientate the diagnosis and control the course of the disease.

Table 2.

1.  Hyperproteinemia

2.  Hypergammaglobulinemia

3.  Hyporegenerative anaemia

4.  Leucocytosis

5.  Thrombocytopenia

6.  Azotemia

7.  Proteinuria

3. Parasitological techniques

Direct observation of the parasite has a good specificity (100%) but a very low sensitivity (60%).

 Microscopy: Leishmania amastigotes can be observed in smears obtained from fine needle aspiration of lymph nodes or of bone marrow (better). They can also be identified, in impression or biopsies from skin lesions or nodules. The cytological smears are stained with May -Gruenewald -Giemsa.

 Cultures: The organisms can be cultured from infected tissues. Cultures are not routinely used in practice. They are long time to perform and need to be done by specialised laboratories.

4. Serological tests

Detection of anti-leishmanial circulating antibodies is an essential tool for Can L diagnosis. Several tests have been developed: indirect immuno-fluorescent antibody test (IFAT), complement fixation test (CFT), enzyme-linked immuno-sorbent assay (ELISA), direct agglutination test (DAT), Latex test, Westen blot (WB). The last one (WB) is the more sensitive but requires very well equipped laboratories. IFAT is considered as the most used technique. A positive result can only be interpreted as evidence of a previous contact with the infectious agent. Nevertheless, evaluating serologic titres every 6 months is a valuable means of monitoring CanL cases. Commercial immuno-chromatographic kits are now available for practitioners. They have variable sensitivity.

5. Molecular biological techniques

Leishmanial DNA can be amplified and detected in tissue or liquid media by Polymerase Chain Reaction (PCR). This recently developed technique is used more and more nowadays. The technique is considered as very specific and sensitive. It has been improved by the development of the nested PCR technique. It allows detection of the infection even in many clinically healthy dogs. Consequently, it is very useful to evaluate the status of asymptomatic carriers.

DIFFERENTIAL DIAGNOSIS

CanL can be confused with many other diseases including demodicosis, manges, pemphigus foliaceus, lupus erythematosus, ehrlichiosis, lymphoma, glomerulonephritis and polyarthritis. The differential diagnosis is complicated by the high rate of concomitant infections in endemic areas ( demodicosis, dermatophytosis, ehrlichiosis, hepatozoonosis, sarcoptic mange and filariasis are reported to be sometimes concomitant)

TIPS: How to diagnose CanL in practice: Clinical data, history (epidemiological data), non specific haematology (do not forget protein electrophoresis) results are good tools to facilitate a presumptive diagnosis. IFAT and, if available, PCR or WB should be the preferred techniques to confirm diagnosis.

PREVENTION/CONTROL

Vaccines are difficult to obtain. The results of a trial made with a vaccine using L.infantum promastigote fraction in Southern France were disappointing. Genetic vaccination is promising.

Control measures in endemic area should focus on the vector (the sand fly) and/or the main reservoir of parasites (the dog). Elimination of suspected dogs is unacceptable. Current proposed control strategies are mass anti-leishmanial therapy and the use of insecticides. Spraying campaigns are inappropriate because of environmental considerations. Individual protection of dogs is advised. Dogs are to be kept indoors in the evening. Putting deltamethrin impregnated collars on all dogs has been demonstrated to reduce the prevalence of the infection. This could be a promising solution in the future.

TREATMENT

Whilst anti-leishmanial treatments of dogs are considered to achieve clinical cure unfortunately they do not result in the complete elimination of the parasite.

 Antimonials. Pentavalent antimoniate (Glucantime and Pentostam) are widely used. They may inhibit leishmanial glycolytic enzymes. The most common protocol for Glucantime therapy is: 100mg/kg/day-SC for 4 weeks. After several injections, the therapy can be painful. It is expensive in the long term for large dogs. It may induce drug resistance.

 Allopurinol. Allopurinol is an analogue of hypoxanthine. The product is incorporated into leishmanial RNA and alters protein synthesis. The most common protocol for allopurinol is: 20 mg/kg/day-per os. The product is easy to administer orally. The treatment is usually appreciated by owners for its low cost and relative non toxicity. It can be administered for long periods of time. It is nowadays widely used as monotherapy or in combination with pentavalent antimonials. Allopurinol is the therapy to be recommended.

 Pentamidine. Dose: 4mg/kg/48hours-IM. The injections are painful.

 Amphotericine B. Amphotericine B is primarily an antifungal drug. It alters leishmanial cell membrane permeability. The conventional product has a toxic effect on canine kidneys. The liposomal presentation (AmBisome) has reduced side effects. The treatment is expensive.

 Paromomycin Aminosidine inhibits ribosomal function. Dose : 5-10 mg/kg/twice daily-IM or SC. The product can be nephro- or oto-toxic.

 Miltefosine. Miltefosine is effective as anti-leishmanial therapy in mice and humans. A liposomal presentation is currently under evaluation in dogs.

TIPS: How to treat in practice: Before starting treatment, a complete blood count, a biochemical profile to assess renal function, and a protein electrophoresis are necessary. For treatment, allopurinol is the author's personal recommendation. As relapses are common, periodic (every six months) monitoring of the patient has to be performed.

CONCLUSION

Recent advances in techniques of diagnosis, control measures and therapy have changed the approach to CanL. Improvement of the treatment is still needed to be able to eliminate the parasite from the canine host. The disease remains too often a frustrating problem for the practitioner and a danger to public health.