CANINE LEISMANIASIS

Leismaniasis is a parasitic infection of dogs, affecting most organs, potentially fatal, caused by the protozoan Leismania spp. It is also a zoonosis Cats and many other animals are rarely affected

This parasitosis has an indirect life cycle and the sandfly is the intermediate host Incubation period last 1 month to several years

Comments

Infected dogs are the primary reservoir for zoonotic visceral leishmaniasis in endemic regions and are the most significant risk factor predisposing humans to infection Dogs have a wide range of clinical presentation caused by infection with L. infantum, ranging from asymptomatic to fatal visceralizing disease. Host factors which determine clinical outcome are poorly understood.

Immune status, the general condition, reinfections by the parasite, the saliva of sandfly and other factors play an important role on the clinical status of the infected dog

Species of Leishmania

L. donovani infatum
In Mediteranean basin and Middle East: Is endemic in some European countries such as Portugal, Spain, France, Italy, Greece but occasionally is found in Switzerland, Netherland, Germany and many others L. chagasi (In South America). L. infatum (in North America), L. tropica (Europe, North Africa)

Global distribution of reported cases of leishmaniasis and Leishmania/HIV co-infection, 1990-1998

Leishmaniasis is transmitted by the bite of female phlebotomine sandflies. The sandflies inject the infective stage, metacyclic promastigotes, during blood meals (1). Metacyclic promastigotes that reach the puncture wound are phagocytized by macrophages (2) and transform into amastigotes (3). Amastigotes multiply in infected cells and affect different tissues, depending in part on which Leishmania species is involved (4). These differing tissue specificities cause the differing clinical manifestations of the various forms of leishmaniasis. Sandflies become infected during blood meals on infected hosts when they ingest macrophages infected with amastigotes (5,6). In the sandfly's midgut, the parasites differentiate into promastigotes (7), which multiply, differentiate into metacyclic promastigotes and migrate to the proboscis (8).

Forms of Leishmania spp

Promastigotes (In Sandflay)

Amastigotes (In dog)

SANDFLlIES

Europe (Phlebotomus spp)

America (Lutzomyia spp)

Sand Flies
Sand flies belong to the fly family Psychodidae, members of which are characterized by their densely hairy wings which give them a moth-like appearance. Phlebotomines are distinguished from other members of the family by the way they hold their wings above the body in a vertical V. There are about 700 species of phlebotomine sand flies of which about 70 are considered to transmit diseases to people. Sand flies (Phlebotominae) are blood suckers and their larvae inhabit places where there is high organic matter such as in animal burrows, termite hills and tree holes

Classification
New World sandflies

Genus: Lutzomyia. The only genus of phlebotomine flies that suck blood from people in the New World. Old World sandflies

Genus: Phlebotomus. The main genus of phlebotomine flies that suck blood from people in the Old World and the only genus of phlebotomine flies that transmit diseases to people in this region.

Life Cycle

It is difficult studying the life cycle of sand flies because the larvae are tiny and don't live in well defined places, like mosquito larvae. The entire life cycle takes 20-40 days except in diapausing species (i.e. those that stop developing when conditions become too cold). Eggs. The female lays 30-70 eggs by scattering them around a potential breeding site. They hatch within 1-2 weeks. Larvae. Larvae feed on dead organic matter and are found in damp places containing organic matter such as cracks in walls or rock, animal burrows and shelters, caves, or in leaf litter. In regions with cool winters, larvae diapause in the fourth (final) instar. Pupae. Pupal development takes 510 days.

Adults.

Emerge from the pupae in darkness, often just before dawn. Only the female sucks blood, the food being used for egg production. Both males and females feed on sugary secretions from plants or from honeydew produced by homopteran bugs. Mating takes place at or near hosts: the males congregate in leks on or near the host and produce sex pheromones. Females home in on hosts using both host odor and the odor produced by the males. Vibration of the wings by males can be important in encouraging females to mate. Adults are mainly active in the early morning, evening and at night although they can bite during the day if disturbed. When inactive, adult sand flies have habitat-specific resting sites that are characteristic of particular species. One of the main ways in which entomologists study sand flies is by locating and studying them at their resting sites. Resting sites are often similar or near to the larval breeding sites and are usually places that are cool, humid and dark. Sand flies are able to survive in dry environments by withdrawing to cool, humid resting sites during the day and then becoming active at night when ambient temperatures drop and humidity increases.

Seasonal activity of adults is affected mainly by temperature and rainfall. Flies are very small and their temporary feeding on animals may not be noticed. These flies, lives the summer time in temperate and Mediterranean areas.

SANDFLlIES
Size

Sand flies are smaller than mosquitoes but larger than midges, with a body length of 23mm. Colour

All sand flies are brownish in daylight but their bodies are densely covered in oily hairs which give the insects a whitish appearance when illuminated

V-shaped wings

This is perhaps the most distinctive feature of the group. Phlebotomines at rest hold their wings in a raised V The wings are never closed or laid flat across the body.

Flight

Phlebotomines have a weak, direct flight and once on the host progress by a series of small hops. They do not hover round a host and as such are often not recognised as a biting nuisance.

Noise The wingbeat frequency of phlebotomines is inaudible to the human ear. They thus do not produce a buzzing or whining noise before biting, which again reduces the perceived nuisance to man.

Nocturnal habit Phlebotomines are crepuscular or nocturnal biters, although they may bite during the day if disturbed from their resting sites or when deep shade or clouds produce low light levels.
Painful bite Phlebotomines are pool feeders or telmophages which suck blood from a small wound they make in the skin of the host. Their bite is therefore relatively painful, and has been likened to a drop of hot oil or a cigarette burn.

Life Cycle of Sandfly

Unlike in most biting Diptera, development of sand flies takes place in terrestrial rather than aquatic microhabitats. Although there have been relatively few successful attempts to identify breeding sites in nature, eggs are laid in soil rich in organic matter and the larvae pass through four instars before pupation and adult emergence. The difficulty of finding breeding sites is an important constraint on vector control measures available to leishmaniasis control programmes, application of larvicides not being a practical alternative.

The eggs are elongated oval-shaped, pale at first and darkening on exposure to air with a single black eye spot. The larvae emerge through a J-shaped fissure and are legless and whitish with a dark head capsule. Those of the first instar can be distinguished by the presence of two caudal bristles, all subsequent instars bearing four. Fourth instar larvae also have a prominent sclerite on the dorsum of the penultimate segment. The pupae are golden brown and are affixed to the surface of the substrate in which they developed by the final larval exuvium. Shortly before emergence the wings and eyes turn black. Male sand flies emerge about 24 h before females, allowing their external genitalia time to rotate 180 to the correct position for mating before females have emerged. The time from oviposition to adult emergence at ambient temperature is around 4-6 weeks.

COMMON CLINICAL AND PATHOLOGIC FINDINGS WITH VISCERAL LEISHMANIASIS

Physical examination findings may include depression, loss of condition, particularly decreased muscle mass over shoulders, hips, and spine, with a mildly distended abdomen, serosanguineous nasal discharge, dull hair coat, splenomegaly, and generalized lymphadenopathy. About one third of cases have a fever. Other clinical signs may include diarrhea, vomiting, epistaxis, melena, dry brittle hair coat, and long brittle nails.

Although officially categorized as a form of visceral leishmaniasis, cutaneous lesions including bilaterally symmetric nonpruritic alopecia, hyperkeratosis, excessive epidermal scale with thickening, depigmentation, and chapping of the muzzle and footpads, occur with some regularity.

Abnormal clinical pathologic values often include decreased hematocrit, thrombocytopenia, and signs of renal failure including azotemia, increased blood urea nitrogen and creatinine, hyperphosphatemia, hypermagnesemia, and proteinuria. Signs of hepatic compromise are also common including elevated alkaline phosphatase (ALP), elevated alanine transferase (ALT), and hypercholesterolemia. Other common clinical chemistry abnormalities include hyperproteinemia observed with hypergammaglobulinemia and hypoalbuminemia.

Gross pathologic examinationmay find emaciation with minimaladipose tissue in body cavities and subcutaneous tissues. Many lymph nodes, including peripheral,mesenteric, and mediastinal, are often moderately tomarkedly enlarged. The liver and spleen will also be diffusely enlarged. Kidneys may be moderately enlarged and diffusely pale. Impression smears obtained at necropsy from the spleen, popliteal lymph node, liver stained with Diff-Quick, often will revealwidely scatteredmacrophages with intracellular amastigotes consistent with Leishmania species. Cytologically within the liver, spleen, bone marrow, and lymphnodes there will often also be amastigotes consistent with Leishmania species. These organisms are 1 to 3 mmin diameter, and have a round, deeply basophilic nucleus and a rod shaped kinetoplast

Dogs

90% of symptomatic also have cutaneous involvement There is no sex or breed predilection

Cats Cutaneous disease is rare There is no sex or breed predilection

IMMUNE ALTERATION AND PATHOGENESIS OF VISCERAL LEISHMANIASIS

Mammalian host responses which prevent progression to clinical VL has been shown to be dependent on promoting T helper-1 IFN-g productionbased immunity and parasiticidal activity within infected macrophages. A key immunologic feature of late stage clinical VL in dogs is an inability to proliferate or to produce IFN-g in response to Leishmania antigen. Pharmacologically-cured individuals are resistant to reinfection and mount antigen-specific IFN-g responses in vitro, indicating that there is not an inherent defect in host CD41 T cell responses of clinical patients once they have reached this stage. High levels of TNF-a have been proposed to stimulate production of regulatory cytokines, specifically IL-10, as a homeostatic response to prevent further inflammation-mediated pathology. IL-10 can be produced by many cell types including T cells, B cells, and macrophages. One of the proposed mechanisms of IL-10 promotion of VL is by conditioning macrophages for parasite growth and survival versus killing of intracellular parasites.

In surveillance studies, we have observed repeated cases where dogs do not show clinical signs of VL until there is secondary immunosuppression caused by pregnancy, concomitant Lyme disease, or other tick-borne illness. This clinical shift toward disease consistently appears upon a change from being seronegative to seropositive in these dogs. Further studies are required to determine the effects of immune alterations that lead to clinical disease in these dogs. Congenital infection secondary to vertical transmission may predispose to initial immune abnormalities, although by the time clinical signs of disease and seroconversion have appeared, evidence shows that CD41 T cells from these dogs are able to respond normally to parasite antigen. In advanced disease it is not unusual to see immunosuppression including T-cell changes, in terms of reduced CD41 T cell proliferation in response to whole Le infantum antigen or routine canine vaccines and decreased ability of these cells to produceIFN-g in response to Le infantum antigen.

GENETIC FACTORS RELATED TO VISCERAL LEISHMANIASIS DISEASE SUSCEPTIBILITY

Although several genetic polymorphisms, including alterations in TNF-a and solutecarrier family 11A1 (SLC11A1, formerly NRAMP1) allelic expression, have been indicated to predispose to disease, causative factors of disease susceptibility in humans and dogs, specifically those associated with heritability, remain elusive. Breed type has also been shown to alter the response to therapy, suggesting that canine breed-related genetic factors modulate disease progression and are therefore prognostically significant

DIFFERENTIAL DIAGNOSIS

Visceral - mycoses (blastomycosis, histoplasmosis), systemic lupus erythematosus, metastatic neoplasia, distember and vasculitis Cutaneous other causes of hyperceratosis: primary idiopathic seborrhea and nutritional dermatoses (vitamin A responsive, zing responsive): idiopathic nasodigital hyperceratosis, lichenoid-psoriasiform dermatosis, mucocutaneous syndrome, pemfigus foliaccus, epidermal dysplasia Hyperglobulinemia need to differentiate from chronic Erlichiosis and multiple myeloma

DIAGNOSIS OF VISCERAL LEISHMANIASIS

Cytology is the diagnostic method of choice in order to indentify the typical intracellular organisms from Lymph tissues samples Bone marrow samples Spleen samples

Skin samples By Immunohistochemical techniques

Numerous Leishmania infantum amastigotes in a section of spleen. Notice multiple amastigotes within macrophages. (A) Multiple amastigotes(H&E stain, original magnification 40). (B) Immunohistochemistry for Leishmania infantum amastigotes (red); bar 5 20 mm.

Amastigotes are small round or oval bodies 1.5-3 X 2.5-3.5 m in size and without a free flagellum. The organisms has a relatively large nucleus and a kinetoplast

DNA INDENTIFICATION BY PCR TECHNIQUE

PCR targeting Kinetoplast DNA increace sensitivity and specificity of Leishmania detection, use Fresh and frozen bone marrow samples Lymph node Skin biopsy specimens From the above samples the sensitivity of PCR approaches 95-100% however in peripheral blood samples is higher sortly after the infection (88%) and then decreases to 50-70%

SEROLOGY

A wide variety of serological assays are available, utilizing indirect immunofluorescence (IFA), direct aglutination, conventional ELISA, dotELISA, competitive ELISA and western blotting methodology. At present most tests employ crude Leishmania antigen, although a recombinant Leishmania K39 ELISA has been recently validated. Several rapid immunochromatographic test kits have also been produced but their sensitivity and specitifity varies from 35-76%

SKIN BIOPSY, FROM HYPERKERATOTIC AND

NODULAR LESIONS

TREATMENT/PROGNOSIS

Treatment of canine visceral leishmaniasis (CVL) is rarely curative. Prognosis for emaciated chronically infected animals is very poor. It is critical to advise the owner of potential zoonotic transmission of organisms from lesions to humans before maintaining a Leishmania-infected dog in their household, particularly if there are immunosupressed people sharing the household. The owner should be informed that the organism will never be completely eliminated (ie, no sterile cure) and relapse occurs very frequently requiring retreatment. Treatment should be undertaken on an outpatient basis. Because of the chronic wasting that can occur with leishmaniasis, it is important to provide a good high-quality protein diet or a diet appropriate for renal insufficiency if this manifestation of leishmaniasis is present.

First-line treatment options for canine leishmaniasis. Allopurinol (Zilapour): 7.020.0, PO, 812 hrs, for 324 months (Is a Leishmaniostatic) Milteforan (miltefosine): 2mg/kg, PO, every 24 hours for 28 days Meglumine antimoniatee (Glucantime): 100 mg/kg SC, every 24 hours for 1 month Amphotericin B Fungizone : 0.250.5 mg/kg, IV, 2-3 times a week Sodium stibogluconate (Pentostam): 3050mg/kg, IV or SC, every 24h for 1 month

COMENTS

Milteforan the only legal drug without side effects Meglutamine antimoniate (Glucantime , Pfizer/Merial, France), which has some side effects. Sodium stibogluconate (Pentostam, Wellcome Foundation Ltd, U.K.), which requires daily injection and has severe side effects, Amphotericin B in the lipid emulsion or liposomal form is non-nephrotoxic and is effective against the organism, although it is not thought to be superior to allopurinol as it is stillmore costly and more toxic. Renal insufficiency must be treated before giving antimonial drugs or amphotericin B as prognosis is dependent on renal function at the onset of treatment.

AFTER TREATMENT

Treatment efficacy is best monitored by clinical improvement and presence of organisms in biopsy or as measured by rigorously controlled qPCR. Relapses occur a few months to a year after therapy, so dogs should be rechecked. Relapses indetified by monitoring rise in blood globulins or reapepearance of clinical signs in a dog previously in remission Prognosis for a cure is very guarded, but therapydoes provide infected dogs improved quality of life.

Second-line drug, which require further clinical studies to understand their efficacy in dogs is paromomycin (Humantin). Paromymycin has been shown to have fewer side effects than other drugs in humans. Use of this drug has been primarily targeted to the cutaneous versions of Leishmania,less is known about its ability to remove organ-based infection.

Prevention measures

Sand fly vector control measures, including deltamethrin or permethrin-impregnated collars are useful todate to prevent disease. Lemon repellents every day before walk (near head) Avoid outdoor activities, especially from dusk to dawn, when sand flies are the most active. Keep dog in good hygiene conditions Give diet high quality protein Make special vaccines

Control of Sandflies

Spraying of residual insecticides on surfaces in the home. This has been the main way used for controlling sand flies but is obviously ineffective for those species which bite away from the home such as those in South American forests. This control technique is also used for killing Anopheles mosquitoes that transmit malaria and is some regions it is effective in reducing both malaria and leishmaniasis. Killing of reservoir species. Certain species of mammals can act as important reservoirs of Leishmania and by killing the reservoir species that are living near human habitation, disease rates can be decreased. For instance, rodenticides have been used against the Great Gerbil Rhombomys opimus in Central Asia. Insecticide spraying of larval habitat. This is usually not possible because, usually, so little is known about where the larvae occur.

SUMMARY FOR ROMANIA

At present Leishmaniasis is an important disease of dogs (and humans) But probably, climatic conditions will change dramatically in few years Always you must collect information about the possibility of dog transmission in endemic areas or origin of dog, specially in this case that clinical symptoms are in differential diagnosis Also you must recommend your clients witch will travel with their dogs in endemic areas to take prevention measures

Canine leishmaniasis has clinical symptoms from many organs but the more commons are, coetaneous lesions, enlarged lymph modes and glomerulonephritis Special test kits are useful in the beginning, for the diagnosis but cytology is the diagnostic method of choice. Alopourinole is a cheap drug wich you can sugest for prevention (dose 20mg/kg daily for a week) just after travel For the treatment, combination of Miltefosine and Alopourinole is very effective (without side efects) Vaccines at this time are very effective, but dont fully protect dogs