MENCKE N., VOLF P., VOLFOVA V. & STANNECK D.

Bayer AG, BHC-Business Group Animal Health, D-51368 Leverkusen, Germany, Charles University, Department of Parasitology, Prague, Czech Republic

Infections in dogs with the protozoan parasite Leishmania are widespread in Mediterranean countries. Furthermore, canine leishmaniases have a worldwide distribution and can be found in Asia, Africa and America. First reports on canine leishmaniasis were recently published also from the US, it remains unclear until now whether the disease was imported from abroad, or has already established within the country. Leishmaniases are vector-borne diseases: the promastigote stage of the parasite is transmitted to the host during the blood feeding of an insect vector, the sand fly.

The causative agents of canine leishmaniasis are Leishmania irifantum in the Mediterranean Basin and the Middle East and L. chagasi in South and Central America. Initially, L. chagasi was considered to be distinct from L. infantum, however, recent molecular studies indicate that they are indistinguishable. Today we believe that the parasite reached the New World in dogs transported from Europe to the Americans by the colonists. L. infantum/chagasi is also an important human health problem and dogs serve as the main reservoir animal. Severa! studies showed that the prevalence of human leishmaniasis could be significantly decreased with control ofleishmaniasis in dogs. Prevention of dogs from sand fly bites and thus reducing the risk of infection with L. infanrum is therefore our veterinaly obligation.

Fig. 1 Life cycle of Leishmania

A Development in Man: 1 Flagellated L. donovani parasites carried by Ph/ebotomus species (promastigote stage) enter macrophages (Giemsa stain); 2-6 lntracellular development in macrophages and later in endothelial cells; 7 Macrophages in perpheral blood containing amstigote stages

B Development in the sand fly vector: 8 Amastigote stages, within host cells, in the fly's midgut; 9 Growth and multiplication of the promastigote stage; 10 Flagellate stage (metacyclic promastigote form) from fly's proboscis

C Development as in man A in animal reservoir (dogs, small rodents etc.)

Transmission by the sand fly can occur as follows:

From Man to Man A➔B➔A

From Animal to Animal C➔B➔C

From Animal to Man C➔B➔A

and vice versa A➔B➔C

Taken from: PIEKARSKI G. (1989) Medical Parasitology; Springer - Berlin, Heidelberg, New York 

Biology of Leishmania and their vectors 

While there is no morphological differentiation between the Leishmania species, molecular techniques implemented today distinguish the previously described species as the L. donovani complex (three species: L. donovani, L. infantum and l. chagasz), the L. mexicana complex (three main species: L. mexicana, L. amazonensis and L. venezuelensis), further L. tropica, L. major, L. aethiopica and the subgenus Viannia with four main species (L. braziliensis, L. guyanensis, L. panamensis and L. peruviana).

Within humans and dogs Leishmania multiply within a parasitophorous vacuole of macrophages as amastigotes (forms without flagella). Finally, these macrophages rupture and amastigotes enter other phagocytic cells, mainJy of the reticulo-endothelial systems in liver, spleen, bone marrow and lymph nodes. Sand flies feeding on infected host ingest a blood meal with infected monocytic cells.

ln the midgut of the sand fly the amastigotes transform into a flagellated promastigotes, multiply and then, during tbe second blood feeding, they are transmitted to the next vertebrate host. Development to the infectious promastigote occurs under favorable tropical conditions within 5 - 8 days. The incubation period in dogs is severa! weeks to months. 

Sand flies belong to tbe insect order Diptera, suborder Nematocera. The Old World sand flies belong to genus Phlebotomus, the New World ones to the genus Lutzomyia. Both these genera are important vectors of Leishmania parasites.

The biology of the adult sand fly is that of a typical bloodsucking Nematocera: both sexes feed on sugar solutions but females need a blood meal to produce eggs. Larvae hatching from the eggs develop through 4 larval instars. They are terrestrial and live in soil rich in organic substrates where they also pupate.

The adult females seek bosts for a blood meal in a clear circadian activity. They are nocturnal blood feeders, resting over the hot day away from sunlight in relatively cool and humid places, like cellars, stables, crevices or rodent boles. Once landed on the potential host, the dog or other mammal, they hop over the coat aiming for less hairy place; in the head region they like to bite around the muzzle, eye or mouth.

Similarly to other bloodsucking insects, the sand fly saliva contains anticoagulants, vasodilatory peptides and enzymes with antiinflammatory, antihemostatic and anaesthetic properties. These components of saliva are important for transmission and serve as enhancing factors of the parasite infection. Sand flies are fast feeders; once they created a small pool of blood in epidermis of the host they rapidJy fill their midgut with blood. Leishmania is well adapted to this feeding habit, entering the host via the proboscis with the saliva injected into the host.

Clinical Canine leishmaniasis

Clinical features of canine leislunaniasis vary widely, while skin lesions are the most usual manifestation. The incubation period is 4 weeks to several years. The pathomechanism of the infection is a combination of chronic inflammation of skin, liver, GI-tract, kidneys, eyes and bones and a immune mediated polyarthritis, glomerulonephritis, arthritis and uveitis.

ln addition, dogs presented to the clinic often have concomitant infections, due to immunosuppression thus complicating the diagnosis. The skin lesions are alopecia with intense, dry desquamation, usually on the head. In most cases weight loss is common. Circulating immunocomplexes causing glomerulonephritis, proteinuria and subsequently renal failure is a common cause of death in affected animals.

Besides the dogs with visible clinical signs of the disease, asymptomatic carriers are frequently reported. While in endemic areas most dogs have contact with the parasite, the prevalence of the disease is usually up to 20%. The reason why some dogs develop the disease and others are resistant is not fully understood yet.

Diagnosis of canine leishmaniasis is complicated as most tests available are not sensitive or specific enough. Several treatment regimes are recommended but a once dog got ili the parasites will never been completely eliminated. Prevention of sand fly bites is therefore the most important way to stop the circulation of canine leishmaniasis. 

Human Leishmaniasis

In humans the protozoan parasite of the genus Leishmania causes cutaneous (CL), mucocutaneous (MCL) and visceral (VL) leishmaniases. The WHO reported that worldwide 350 million humans in 88 countries are at risk and 12 million people are affected by leishmaniases, with about 1.5-2.0 Million new cases ofCL and 0.5 Million new cases ofVL cases annually. More than 90% of the VL cases are reported from Bangladesh, Brazil, India, Nepal and Sudan. Visceral leishmaniasis, also known as 'Kalaazar' is caused by L. infantum in the Old World, L. chagasi in the New World, and by L. donovam in Africa and Asia.

Coinfection in humans with imrnunodeficiency syndroms like AIDS is common. After incubation period (usually 2-8 months) patients develop pyrexia, wasting and hepatosplenomegaly; especially children are at risk. there is a long list of clinical findings, with fever, discomfort from an enlarged spleen, abdominal swelling, weight loss, cough and diarrhea being the most prominent once. While untreated the mortality is about 90%. After successful recovery from VL due to L. donovani infection, patients may develop so-called 'post kala azar dermal leishmaniasis'. 

The mucocutaneous form (Espundia) occurs in some cases of L. braziliensis infection in South America. Disease is fully manifested months or years after the cutaneous sores have healed. Papules and ulcera tive lesions occur on the nose, mouth and larynx and finally may destroy the whole face.

In the Old World, L. major and L. tropica are the causative pathogens of the cutaneous form (oriental sore), while in the New World this form is caused mostly by the parasites of L. mexicana and L. brasiliensis complexes. Primary skin lesions occur at the site of sand fly bite, often at unprotected body regions like arms, legs and in the face. Most common type of lesion is a chronic ulcer with an indurated margin. The majority of these lesions are self-healing in several months leaving a scar. 

Efficacy of the lmidacloprid/Permethrin combination

The objective of the blinded, negative controlled laboratory GCP-study was the evaluation of the repellent and insecticidal efficacy of a combination containing Imidacloprid 10% (w/v) and Permethrin 50% (w/v) in a spot-on formulation against the sand fly species (Phlebotomus papalasi) on dogs.

The repellency efficacy criterion was based on the feeding rate of sand fly females in the treated dogs compareci to the untreated contrai ones. The insecticidal efficacy criterion was based on the survival rate of sand fly females in the treated group compared to the untreated control group. Twelve laboratory-bred beagle dogs were allocated to two groups of six dogs each.

On day 0 the animais i.n the treated group received 0.1 ml/kg bodyweight Imidacloprid 100%/Pennethrin 50% spot-on and the untreated control dogs received a placebo treatment. Dogs were sedated and exposed to laboratory-bred Phlebotomus papatasi . This species is not a specific vector of Leishmania infantum but is a very common Old World sand fly frequently biting man and dogs in Mediterranean area.

The dogs were exposed to the sand flies in finegauze cubic net-cages (1.2 m x 1.2 m square bottom and 1.8 m height) for about 1.5 hours on a weekly base, the study duration was four weeks. About 200 females plus some males were used as infestation load. The room temperature during the exposure periods ranged from 22 - 25ºC, the humidity ranged from 50 - 60% rh. Dead flies were counted immediately after the exposure and 20 hours later.

Then, live flies were collected, frozen and counted for survival rate determination. The feeding rate was determined by microscopical examination, female with any blood in the gut was assigned as the blood-fed. The survival rate of the sand flies in the contrai group ranged between 96% and 93% during the whole study that indicates favorable climatic conditions in the experimental room.

The product showed a repellent efficacy of 94.6% (day 1), 93.3% (day 8), 80.0% (day l5), 72.8% (day 22) and 55.9% (day 29). Due to the high repellent effect, the insecticidal efficacy was rather low with 60.0% (day 1), 46.2% (day 8), 42.6% (day 15), 35.2% (day 22) and 29.3% (day 29).

The general and dermal tolerance of the product was very good and no adverse reactions were observed in any of the treated dogs during the study. The study clearly demonstrated the high repellent potential of the imidaloprid/permethrin combination, thus protecting dogs from sand fly bites. This may have important consequences in protection of dogs against Leishmania infection.

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