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Review

Directions for the diagnosis, clinical staging, treatment and preventionof canine leishmaniosis

L. Solano-Gallego a,1,*, A. Koutinas b,1, G. Miro c,1, L. Cardoso d,1, M.G. Pennisi e,1,L. Ferrer f,1, P. Bourdeau g,1, G. Oliva h,1, G. Baneth i,1

a Dep. Pathology and Infectious Diseases, Royal Veterinary College, University of London, Hawkshead Lane, North Mymms, Hatfield, Herts AL9 7TA, UKb Companion Animal Clinic, Faculty of Veterinary Medicine, Aristotle University of Thessaloniki, Greecec Dpto. Sanidad Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, Spaind Dep. de Ciencias Veterinarias, Universidade de Tras-os-Montes e Alto Douro, Portugale Dip.to Sanita Pubblica Veterinaria, Facolta di Medicina Veterinaria, Polo Universitario Annuziata, Messina, Italyf Dep. de Medicina i Cirurgia Animals, Universitat Autonoma de Barcelona, Spaing Ecole Nationale Veterinaire de Nantes, Franceh Dep. of Veterinary Clinical Sciences, Faculty of Veterinary Medicine, University of Naples Federico II, Italyi School of Veterinary Medicine, Hebrew University, Israel

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22. Epidemiology and pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Veterinary Parasitology 165 (2009) 1–18

A R T I C L E I N F O

Article history:Received 18 February 2009Received in revised form 11 May 2009Accepted 26 May 2009

Keywords:Leishmania infantumDogDiagnosisPrognosisTreatmentPrevention

A B S T R A C T

Canine leishmaniosis (CanL) due to Leishmania infantum is a life threatening zoonoticdisease with a wide distribution in four continents and importance also in non-endemicregions. The purpose of this report is to present a consensus of opinions on the diagnosis,treatment, prognosis and prevention of CanL in order to standardize the management ofthis infection. CanL is a disease in which infection does not equal clinical illness due to thehigh prevalence of subclinical infection among endemic canine populations. The mostuseful diagnostic approaches include serology by quantitative techniques and PCR. Highantibody levels are associated with severe parasitism and disease and are diagnostic ofclinical leishmaniosis. However, the presence of lower antibody levels is not necessarilyindicative of disease and further work-up is necessary to confirm CanL by other diagnosticmethods such as cytology, histopathology and PCR. We propose a system of four clinicalstages, based on clinical signs, clinicopathological abnormalities and serological status.Suitable therapy and expected prognosis are presented for each of the stages. Thecombination of meglumine antimoniate and allopurinol constitutes the first linepharmaceutical protocol. However, although most dogs recover clinically after therapy,complete elimination of the parasite is usually not achieved and infected dogs mayeventually relapse. Follow-up of treated dogs with blood counts, serum biochemistry,urinalysis, serology and PCR is essential for prevention of relapses. Protection against sandfly bites by topical insecticides is effective in reducing infection, and recent developmentof vaccines has indicated that prevention by vaccination is feasible.

! 2009 Elsevier B.V. All rights reserved.

* Corresponding author. Tel.: +44 1707666326; fax: +44 1707661464.E-mail address: [email protected] (L. Solano-Gallego).

1 All authors are members of LeishVet, Dpto. Sanidad Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, Spain.E-mail: [email protected].

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2.1. Why is leishmaniosis important to human and veterinary medicine? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32.2. What species of Leishmania infect dogs? What is the geographical distribution? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32.3. How is Leishmania transmitted to the dog? How does Leishmania develop in the vector? . . . . . . . . . . . . . . . . . . . . . . 42.4. What are the geographical distribution and vectorial characteristics of sand flies? . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42.5. Is there evidence of non-sand fly transmission in dogs? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42.6. What is the importance of canine leishmaniosis in non-endemic areas? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42.7. What are the public health considerations of canine leishmaniosis? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

3. Immunology and clinicopathological findings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53.1. Is there a difference between infection and disease in canine leishmaniosis? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53.2. What is the prevalence of disease, seroprevalence and prevalence of infection in endemic regions? . . . . . . . . . . . . . . 53.3. What role does the immune response play in the different clinical manifestations of Leishmania infection?. . . . . . . . 53.4. What patterns and frequencies of immune responses are found in dogs living in endemic regions? . . . . . . . . . . . . . . 63.5. What determines if an infected dog will remain clinically healthy or become severely sick? . . . . . . . . . . . . . . . . . . . . 63.6. What are the most common clinical manifestations of leishmaniosis?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73.7. How common are cutaneous lesions in dogs with leishmaniosis? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73.8. How common are renal disease and ocular manifestations in dogs with leishmaniosis? . . . . . . . . . . . . . . . . . . . . . . . 73.9. What are the uncommon clinical forms of leishmaniosis? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83.10. What additional clinicopathological findings should alert the clinician to the possibility of canine leishmaniosis? . . 8

4. Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84.1. What are the various purposes for which L. infantum infection diagnosis is carried out? . . . . . . . . . . . . . . . . . . . . . . . 84.2. How is clinical canine leishmaniosis diagnosed? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84.3. What tests are available for the evaluation of Leishmania infection in dogs with suspected clinical leishmaniosis

and in clinically healthy infected dogs?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84.4. How should serology be used for the diagnosis of canine leishmaniosis? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94.5. What reference and commercial antibody tests for Leishmania are currently available? . . . . . . . . . . . . . . . . . . . . . . . . 94.6. How should PCR be used for the diagnosis of canine leishmaniosis? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104.7. How should PCR be used in clinically healthy dogs? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104.8. Are there other useful immunodiagnostic tests for the prognosis in sick dogs?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

5. Treatment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105.1. What is the most effective specific treatment in canine leishmaniosis? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105.2. What is the expected clinical response to treatment in canine leishmaniosis? What are the most common side

effects of different therapeutical protocols? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115.3. What clinicopathological parameters should be monitored during the treatment of canine leishmaniosis? . . . . . . . 115.4. How should serology be used for monitoring the effectiveness of treatment? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115.5. When should allopurinol be discontinued? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125.6. Is there any evidence for anti-Leishmania drug resistance in dogs? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

6. Prognosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136.1. Can treated dogs with leishmaniosis truly be cured or cleared of the infection? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136.2. Can clinically healthy but infected dogs clear the infection spontaneously? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136.3. Can infected dogs be reinfected?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136.4. How could prognosis be established on the basis of the different clinicopathological features?. . . . . . . . . . . . . . . . . 13

7. Management of clinically healthy dogs in endemic areas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137.1. Should clinically healthy dogs be screened for Leishmania antibodies? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137.2. Should clinically healthy dogs be assessed for carrying Leishmania DNA? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147.3. How should a clinically healthy but seropositive dog be managed? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147.4. How should a clinically healthy but PCR-positive and seronegative dog be managed? . . . . . . . . . . . . . . . . . . . . . . . . 14

8. Prevention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148.1. What preventive measures should be applied to decrease the risk of canine leishmaniosis? . . . . . . . . . . . . . . . . . . . 148.2. What preventive measures should be used in dogs from non-endemic areas travelling to endemic areas?. . . . . . . . 148.3. Are there vaccines available for canine leishmaniosis? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

1. Introduction

Leishmania infantum infection was first described indogs in 1908 by Nicolle and Comte in Tunisia (Nicolle andComte, 1908). Since then, knowledge on L. infantum in dogshas continually expanded improving the understanding ofthis infection. The development of new sensitive andspecific diagnostic techniques showing high prevalence ofinfection versus lower prevalence of clinical leishmaniosis,the evidence of its wide spectrum of clinical manifesta-tions, the lack of ultimately effective therapy, and the

different measures proposed for disease prevention havebeen accompanied with increased interest in canineleishmaniosis (CanL) and disparity of opinions concerningthis infection in veterinary medicine. Several consensusstatements have been published on the management ofother canine infectious diseases such as ehrlichiosis (Neeret al., 2002) and borreliosis (Littman et al., 2006). However,there is insufficient scientific agreement on the manage-ment of CanL and therefore an increasing need for debateleading to shared standardization of criteria for clinicalstudies. These include evaluation of efficacy of different

L. Solano-Gallego et al. / Veterinary Parasitology 165 (2009) 1–182

therapeutic protocols or vaccines, and assisting veterinarypractitioners in providing the best clinicodiagnosticapproach to their canine patients. LeishVet is a group ofveterinary scientists from academic institutes in Europeand the Mediterranean basin with a main clinical andscientific interest in CanL. It was formed during theWorldLeish3 congress in 2005 and officially registered asan association in May 2008. One of the first goals ofLeishVet was to develop consensus recommendations thatwould represent the most current understanding of L.infantum infection in dogs and bridge controversies in theveterinary literature. The directions presented here reflectgeneral uniformity of opinion following an in-depth debateon controversial issues. For issues without clear consensus,the pros and cons are presented. These recommendationsare based on current literature and clinical experiencecombined to provide evidence-based medicine guidelines.The questions presented primarily address the diagnosis,clinical staging, treatment, clinical monitoring, prognosisand prevention of CanL to offer a consensus opinion toscientists and veterinary practitioners on these topics.

2. Epidemiology and pathogenesis

2.1. Why is leishmaniosis important to human and veterinarymedicine?

Human leishmaniasis, caused by several species ofLeishmania, comprises a group of diseases which aremostly zoonotic. These include visceral leishmaniasis (VL),which involves internal organs and is fatal if untreated, andthe cutaneous and mucocutaneous forms (CL), which affectthe skin or mucocutaneous junctions and may healspontaneously, leaving disfiguring scars (Murray et al.,2005). This group of infections is the third most importantvector-borne disease after malaria and lymphatic filariasis.It is endemic in many tropical and sub-tropical regions ofthe Old and New World. Leishmaniasis is endemic in 88countries, with more than 350 million people at risk. The

estimated incidence is 2 million new cases per year, 0.5million of VL and l.5 million of CL (Desjeux, 2004). Visceralleishmaniasis causes an estimated 59,000 deaths annually(a rate surpassed among parasitic diseases only bymalaria), and 2,357,000 disability-adjusted life years(DALYs) lost, placing leishmaniasis 9th in a global analysisof infectious diseases (Desjeux, 2004). Leishmaniasis is oneof the major infectious diseases afflicting the world’spoorest population living mainly in rural and suburbanareas (Alvar et al., 2006).

In veterinary medicine, leishmaniosis caused by L.infantum is mostly important in dogs. Dogs are consideredthe main reservoir of this parasite for humans (Gramicciaand Gradoni, 2005). Canine leishmaniosis is one of themajor zoonoses globally causing severe fatal disease indogs. Dogs are a common companion animal found in alarge number of households in developed and developingcountries and the health of pet dogs is of great concern totheir owners. Infection in cats (Martin-Sanchez et al.,2007), wild canids (Sobrino et al., 2008) and horses(Fernandez-Bellon et al., 2006) has also been reported inareas where disease is common in dogs.

2.2. What species of Leishmania infect dogs? What is thegeographical distribution?

The Leishmania species that infect the dog and theirgeographical distribution are listed in Table 1. The mostimportant species is L. infantum in the Old World which issynonymous to L. chagasi in Central and South America. Infact, L. infantum is thought to have been imported into theAmericas by the dogs of European settlers during thecolonization of South America (Tuon et al., 2008). Dogshave been found infected by several other Leishmaniaspecies responsible for cutaneous, mucocutaneous andvisceral leishmanioses in humans (Lemrani et al., 2002;Dantas-Torres, 2007). For these species, dogs do not appearto be a significant reservoir of infection for people (Dantas-Torres, 2007).

Table 1Geographical distribution of Leishmania species infecting dogs and their vector sand fly species.

Leishmania species Geographical distribution Proven sand fly vectors Suspected sand fly vectors

L. infantum Mediterranean basinMiddle East

Phlebotomus perniciosus, P. ariasi,P. perfiliewi, P. neglectus,P. langeroni, P. tobbi

P. longicuspis, P. syriacus, etc.

Southern Asia, Iran, Armenia,Afghanistan

P. kandelakii P. brevis, P. halepensis, etc.

Central Asia, China P. chinensis, P. alexandri P. smirnovi, P. transcaucasicus,P. Longiductus

L. infantum= L. chagasi

Central and South America Lutzomyia longipalpis, Lu. evansi,Lu. olmeca olmeca

Lu. antunesi, Lu. shannoni

L. donovani East Africa P. orientalis, P. martini P. rodhainiL. tropica North Africa P. sergenti, P. arabicus P. chabaudi, P. saevusL. braziliensis Central to South America Lu. wellcomei, Lu. spinicrassa,

Lu. whitmani, Lu. yucumensis,Lu. carrerai carrerai, Lu. llanosmartinsi,Lu. ovallesi, Lu. intermedia, Lu. gomezi,Lu. trapidoi, Lu. ylephiletor, Lu. umbratilis

Lu. amazonensis, Lu. migonei,Lu. panamensis, Lu. paraensis,Lu. complexus, Lu. pessoai, etc.

L. peruviana Peruvian Andes Lu. peruensis, Lu. verrucarum, Lu. ayacuchensis Lu. noguchii, Lu. pesceiL. panamensis Central America Lu. trapidoi, Lu. ylephiletor, Lu. gomezi,

Lu. panamensis, Lu. hartmanniLu. shannoni, Lu. ovallesi, etc.

L. Solano-Gallego et al. / Veterinary Parasitology 165 (2009) 1–18 3

2.3. How is Leishmania transmitted to the dog? How doesLeishmania develop in the vector?

Leishmania is a diphasic parasite that completes its lifecycle in two hosts, a phlebotomine sand fly vector whichharbours the flagellated extracellular promastigote formand a mammal where the intracellular amastigote formdevelops. The final promastigote form, the metacyclicpromastigote is transmitted to a new vertebrate hostcompleting the Leishmania life cycle. When the parasitizedphlebotomine sand fly feeds on blood from a vertebrate, itinoculates promastigotes from its gut via the proboscis.Only females are hematophagous whereas males feed onplants (Killick-Kendrick, 1999). Sand flies feed on dogsmainly in scarcely haired skin areas such as the head, nasalbridge, ear pinnae and inguinal and perianal areas. Oncethe parasite gains access to the vertebrate’s host dermis,phagocytosis by macrophages ensues. The macrophagesurrounds the parasite in a phagosome vacuole and tries toeliminate it through a cascade of oxygen-based metabo-lites, such as nitric oxide, and liberation of lysosomalhydrolases discharged into the interior of the parasito-phorous vacuole. Leishmania evades these non-specificdefenses to survive and multiply in the macrophage. Theprogress of infection depends on the efficiency of the host’simmune response (Alvar et al., 2004). Parasites can beingested by other phlebotomine sand flies in which theamastigotes are freed from their mammalian host cells andtransformed into promastigotes. The latter develop only inthe anterior segment of the gut (Bates, 2007). There is astrong association between sand fly vector species andLeishmania species transmitted due to specific enzymeactivities and ligands present in the gut of the insect thatallow only specific Leishmania spp. to remain attached toits wall and replicate without being excreted from the gutwith the digested blood meal (Volf et al., 2008). As aconsequence, natural transmission occurs only in thoseareas where adapted (competent) species of vectors arepresent (Killick-Kendrick, 1999; Volf et al., 2008). Thesurvival of the parasite during the winter is mainlysustained on infected dogs, as no transovarial transmissionof Leishmania has been shown in the sand fly vector (Bates,2007).

2.4. What are the geographical distribution and vectorialcharacteristics of sand flies?

Female sand flies from the genera Phlebotomus (OldWorld) or Lutzomyia (New World) are the principal vectorsof Leishmania (Killick-Kendrick, 1999; Sharma and Singh,2008). The geographical distribution of sand flies species isshown in Table 1. Sand flies are primarily present intropical countries or are active during the relatively warmmonths of the year in temperate countries. The activity ofadult sand flies is crepuscular and nocturnal from earlyspring to late autumn in the Mediterranean basin and allyear round in South America (Killick-Kendrick, 1999;Sharma and Singh, 2008). Its range of activity is between15 and 28 8C, and is always associated with high relativehumidity and absence of wind or rain. Sand flies can flydistances from 200 m to 2.5 km and may enter houses at

night due to their positive phototropism (Killick-Kendrick,1999; Sharma and Singh, 2008).

2.5. Is there evidence of non-sand fly transmission in dogs?

Sand flies are the only arthropods that are adapted tobiologic transmission of Leishmania. Some species of vectorsand flies are adapted to one specific Leishmania spp. whileother ‘‘permissive’’ vector sand fly species are able totransmit several Leishmania spp (Volf et al., 2008). Ticksand fleas have been evaluated as potential vectors ofLeishmania but no evidence has been shown that they havea role in natural transmission of the protozoan (Coutinhoet al., 2005; Coutinho and Linardi, 2007). Direct dog-to-dogtransmission has been implicated as being responsible fortransmission of infection among foxhounds in the USA inthe absence of apparent vectors; however, this has notbeen confirmed yet by experimental evidence (Dupreyet al., 2006). Transplacental transmission of infection indogs appears to be rare but possible (Rosypal et al., 2005).Recently, venereal transmission has been reported in dogs(Silva et al., 2009). Transmission of infection by infectedcanine blood products has been documented and is ofspecial concern in areas where blood donors could becarriers of infection (de Freitas et al., 2006; Tabar et al.,2008). Nevertheless, non-sand fly modes of transmissionprobably play only a marginal role in the natural historyand epidemiology of leishmaniosis (Baneth et al., 2008).

2.6. What is the importance of canine leishmaniosis in non-endemic areas?

The disease can be diagnosed in non-endemic countriesin dogs that have been living or have traveled to endemicareas (Shaw et al., 2003). The increased numbers of dogstravelling to Southern Europe and imported as companionanimals from endemic areas have raised serious concernsabout the introduction of vector-borne diseases, such asCanL, into the non-endemic areas of Europe (Shaw et al.,2003). A study from the Netherlands found that about58,000 dogs travel yearly from Holland to Southern Europewith their owners for vacations and the risk for acquiringCanL is 0.027–0.23% (Teske et al., 2002). Infected dogs innon-endemic areas may also contribute to the mainte-nance of the parasite within the canine population throughrare but possible non-vector transmission modes ofinfection. In addition, L. infantum infection has spreadnorth in Europe, reaching the foothills of the Alps inNorthern Italy (Maroli et al., 2008). Transmission couldoccur in a new area if infected dogs are imported and if thepopulation of sand fly vectors is large enough. Changes inthe dynamics of sand fly populations may lead to thecreation of new permanent foci.

2.7. What are the public health considerations of canineleishmaniosis?

Dogs are considered the most important peridomesticreservoir of L. infantum infection to humans. However, onlyone study carried out in Iran has shown that the ownershipof an infected dog is a risk factor for human infection


(Gavgani et al., 2002b). The presence of infected dogs in thevicinity of humans is certainly associated with transmis-sion of infection; but, the actual presence of an infecteddog in the household does not appear to greatly increasethe risk of infection to the family when transmission isalready taking place in the region. Therefore, the danger toowners of dogs with leishmaniosis appears to be small. Theefficiency of elimination of seropositive dogs in Brazil indecreasing human infection is disputable and severalreports have claimed that it is not helpful in decreasinghuman or canine infection in the long run (Dantas-Torresand Brandao-Filho, 2006; Nunes et al., 2008).

3. Immunology and clinicopathological findings

3.1. Is there a difference between infection and disease incanine leishmaniosis?

The traditional concept that all dogs infected with L.infantum will eventually develop severe clinical leishma-niosis after a variable incubation period has beendisproved (Ferrer et al., 1988). CanL is a disease in whichinfection does not equal clinical illness due to the highprevalence of subclinical infection (Solano-Gallego et al.,2001a; Baneth et al., 2008). In the past, researchers usedthe clinical classification of asymptomatic, oligosympto-matic and polysymptomatic dogs based only on the resultsof physical examination (Mancianti et al., 1988). Thisclassification has a limited value because it does notconsider clinicopathological abnormalities and disregardsdogs that have widespread organ dysfunction withoutapparent visual manifestations (Solano-Gallego andBaneth, 2008). The authors define dogs with clinicalleishmaniosis when they present clinical signs and/orclinicopathological abnormalities and have a confirmed L.infantum infection. Dogs with subclinical infection, or

clinically healthy infected dogs, are defined as those thatpresent neither clinical signs on physical examination norclinicopathological abnormalities by routine laboratorytests (CBC, biochemical profile and urinalysis) but have aconfirmed L. infantum infection.

3.2. What is the prevalence of disease, seroprevalence andprevalence of infection in endemic regions?

The majority of dogs infected with Leishmania do notdevelop clinical signs or clinicopathological abnormalitiesand the prevalence of disease is frequently lower than 10% inendemic regions (Table 2). Although seropositivity is foundin nearly all dogs with clinical disease, it is evident only insome of the clinically healthy infected ones. Epidemiologicalstudies using molecular techniques in areas where CanL isendemic have shown that the prevalence of canineLeishmania infection may be considerably higher thanseroprevalence and prevalence of disease (Baneth et al.,2008). L. infantum infection in dogs is endemic inapproximately 50 countries in Europe, Africa, Asia and theAmericas (Alvar et al., 2004), with variable rates ofprevalence, depending on ecological and climatic conditionsthat determine the abundance of vectors (Table 2). Severalreports have revealed the emergence of infection in newlocations as well as its increase in previously establishedareas of endemicity (Maroli et al., 2008).

3.3. What role does the immune response play in the differentclinical manifestations of Leishmania infection?

A broad range of immune responses and clinicalmanifestations have been described in CanL. Leishmaniainfection in dogs may be manifested as a subclinicalinfection, a self-limiting disease (Bottero et al., 2006), or anon-self-limiting and severe illness. In dogs, the two

Table 2Examples of prevalence of disease, seroprevalence and prevalence of L. infantum infection, based on both serology and molecular techniques, in severalcountries where CanL is endemic.

Country (region) Number dogs Prevalenceof disease

Seroprevalence Prevalenceof infection

Reference

France (south) 30 (apparentlyhealthy dogs)

3% (IFAT); 67% (WB) 90% Berrahal et al. (1996)

253 26% 30% 83% Lachaud et al. (2002)Greece (Athens) 1638 (apparently

healthy dogs)22.4% ND Sideris et al. (1999)

(centre) 73 12.3% 65.8% Leontides et al. (2002)(northwest) 1200 24.4% ND Papadopoulou et al. (2005)Israel (selected areas) 122 8.2% 11.5% ND Baneth et al. (1998)(West Bank, Palestine) 148 ND 6.8% 25% (n = 60) Nasereddin et al. (2006)Italy (northwest) 4456 ND 26.4% ND Zaffaroni et al. (1999)Portugal (north) 294 3.1% 20.4% ND Cardoso et al. (2004)Spain (Mallorca island) 100 13% 26% 67% Solano-Gallego et al. (2001a)

79 (37 + 42) 8–29% 8–20% 64–73%* Fernandez-Bellon et al. (2008)Croatia (centre) 306 8.2% 15% ND Zivicnjak et al. (2005)Cyprus (selected areas) 301 !3.7% 10% ND Deplazes et al. (1998)Turkey (west) 490 1.1% 5.3% ND Ozbel et al. (2000)Morocco (north) 1013 0.4% 8.6% ND Nejjar et al. (1998)Tunisia (southern-centre) 250 (apparently

healthy dogs)6% ND Chargui et al. (2007)

Brazil (northeast) 1381 11% 24% ND Rondon et al. (2008)

N.D.: not determined.* The prevalence of infection includes cellular immunity tests.


opposite extremes of this clinical spectrum are character-ized by: (1) protective immunity that is CD4 T cellmediated by the release of g-interferon, IL-2 and TNFathat induce macrophage anti-Leishmania activity, and (2)disease susceptibility that is associated with the produc-tion of a marked humoral non-protective immuneresponse and a reduced or depressed cell mediatedimmunity with a mixed Th1 and Th2 cytokines response(Alvar et al., 2004; Baneth et al., 2008). Within thisspectrum, clinical disease can range from a mild papulardermatitis associated with specific cellular immunity andlow humoral responses (Ordeix et al., 2005) to a severedisease characterized by renal damage with glomerulone-phritis due to immune complex deposition associated witha massive humoral response and high parasite loads (Costaet al., 2003).

3.4. What patterns and frequencies of immune responses arefound in dogs living in endemic regions?

The patterns of immune responses found in dogsexposed to Leishmania infection based on publishedstudies on naturally and experimentally infected dogsare described in Fig. 1. When describing the situation in anendemic area at one point of time using several techniquesfor the detection of exposure to the parasite such asserology, evaluation of cellular immunity with leishmaninskin test or lymphocyte proliferation test and clinicalevaluation, typically 5–10% of the dogs are sick and about90–95% are clinically healthy. In a hypothethical modelbased on previous studies (Cabral et al., 1998; Solano-Gallego et al., 2000), the apparently healthy dogs group canbe divided into a subgroup of about one third which is notinfected and two thirds that are infected. Twenty-twopercent of the infected dogs will be considered susceptibleto the development of clinical disease due to depressedcellular response and presence of humoral response. Thesewill probably develop clinical signs and/or clinicopatho-

logical abnormalities later on. Forty-seven percent of theinfected dogs are considered ‘‘resistant’’ due to thepresence of a demonstrable cellular response againstLeishmania. Twenty-seven percent of ‘‘resistant’’ dogs willpresent cellular and humoral response and 18% willpresent a cellular response only (Cabral et al., 1998;Solano-Gallego et al., 2000). However, subclinical infectionis not necessarily permanent and factors such as immu-nosupression and concomitant diseases could break theequilibrium and lead to the progression of clinical diseasein dogs as has been observed in humans coinfected withhuman immunodeficiency virus and Leishmania (Alvaret al., 2008).

3.5. What determines if an infected dog will remain clinicallyhealthy or become severely sick?

It is not known for certain what mechanisms in dogs areresponsible for resistance or susceptibility, nor the wayfactors such as age, gender, nutrition, host genetics,coinfections and/or concomitant disease, immunosuppres-sive conditions, cytokine environment, parasitic burden,virulence of Leishmania strain, previous infections andmethod of transmission can affect the polarity of clinicalmanifestations of Leishmania infection. Some dog breedssuch as the Boxer, Cocker spaniel, Rottweiler and Germanshepherd seem to be more susceptible to the developmentof disease (Sideris et al., 1999; Franca-Silva et al., 2003)while others such as the Ibizian Hound rarely developclinical signs (Solano-Gallego et al., 2000). The Slc11c1(Solute carrier family 11 member a1) gene, formerly namedN-RAMPI, and certain alleles of the MHC II genes have beenassociated with susceptibility to CanL (Quinnell et al.,2003; Sanchez-Robert et al., 2008). Age seems to be animportant factor. Peaks of the highest disease prevalencehave been reported in dogs younger than 3 years and olderthan 8 years (Abranches et al., 1991; Cardoso et al., 2004).Experimentally infected male hamsters seem to be more

Fig. 1. Clinical states and immune responses of dogs living in a Leishmania infantum endemic infection area.


susceptible to developing disease than females (Travi et al.,2002). In dogs, some epidemiological studies have notreported gender predisposition (Abranches et al., 1991;Miro et al., 2007b) while other studies have reported ahigher risk for the disease in male dogs (Zaffaroni et al.,1999; Zivicnjak et al., 2005). In addition, canine experi-mental infections have demonstrated how different routesof infection and life stages of the parasite used for infectionwill lead to divergent clinical manifestations. Intradermalinfection and inoculation with promastigotes will leadmost likely to a subclinical infection while intravenous andamastigotes infections will manifest as a severe disease(Moreno and Alvar, 2002).

3.6. What are the most common clinical manifestations ofleishmaniosis?

The clinical features of leishmaniosis vary widely as aconsequence of the numerous pathogenic mechanisms ofthe disease process, the different organs affected, and thediversity of immune responses mounted by individualhosts (Baneth et al., 2008). Canine leishmaniosis is asystemic disease that may potentially involve any organ,tissue and biological fluid and is manifested by non-specific clinical signs. The main clinical findings found onphysical examination in classical CanL include skin lesions,generalized lymphadenomegaly, progressive weight loss,muscular atrophy, exercise intolerance, decreased appe-tite, lethargy, splenomegaly, polyuria and polydypsia,ocular lesions, epistaxis, onychogryphosis, lameness,vomiting and diarrhea (Ciaramella et al., 1997; Koutinaset al., 1999; Baneth et al., 2008). The variable and non-specific clinical signs make the list of differential diagnosisto CanL widely extensive.

3.7. How common are cutaneous lesions in dogs withleishmaniosis?

Skin lesions are the most common manifestation of CanLin dogs admitted for treatment due to the disease(Ciaramella et al., 1997; Koutinas et al., 1999). Cutaneouslesions may be seen along with other clinical signs and/orclinicopathological abnormalities, be the sole reportedabnormality or be absent. Several dermatological entitieshave been described (Ferrer et al., 1988; Koutinas et al.,1992): (1) non-pruritic exfoliative dermatitis with orwithout alopecia which can be generalized or localizedover the face, ears and limbs, (2) ulcerative dermatitis overbony prominences, mucocutaneous junctions, paws, earpinnae, (3) focal or multifocal nodular dermatitis, (4)mucocutaneous proliferative dermatitis and (5) papulardermatitis (Ordeix et al., 2005; Bottero et al., 2006). Atypicalcutaneous manifestations such as depigmentation, panni-culitis, digital and nasal hyperkeratosis, pustular eruption,an alopecia areata-like disease or pemphigus foliaceus-likedisease and erythema multiforme are relatively uncommon(Blavier et al., 2001; Papadogiannakis et al., 2005).Staphylococcal pyoderma, either superficial or deep, is acommon complication. The most common cutaneoushistopathological picture is a periadnexal nodular to diffusepyogranulomatous/granulomatous dermatitis, together

with orthokeratotic to parakeratotic hyperkeratosis,acanthosis, crusting and ulceration. However, other histo-pathological patterns such as subcorneal pustular derma-titis, lichenoid dermatitis, vasculitis and panniculitis, havealso been described (Ferrer et al., 1988; Koutinas et al., 1992;Blavier et al., 2001; Solano-Gallego et al., 2004; Papado-giannakis et al., 2005).

3.8. How common are renal disease and ocularmanifestations in dogs with leishmaniosis?

In sick dogs, it is essential to evaluate the renal functionand to stage possible renal disease using the InternationalRenal Interest Society (IRIS) recommendations (IRIS, 2006a)since CanL is associated with a high prevalence of chronicrenal disease (Costa et al., 2003). The early diagnosis of renaldisease is beneficial to the patient and may prolong its life.Renal disease may be the sole clinical manifestation of CanLin sick dogs and can progress from mild proteinuria tonephrotic syndrome or end stage renal disease. Chronicrenal failure (CRF) is a severe manifestation of diseaseprogression and is the principal cause of animal death inCanL. Several studies have described the presence ofhistological lesions in 100% of the dogs evaluated (Costaet al., 2003; Zatelli et al., 2003). Despite the high prevalenceof renal pathology, azotemia typical of renal failure is anuncommon laboratory finding and it is evident only whenthe majority of nephrons become dysfunctional rather lateduring disease progression. Glomerulonephritis and tubu-lointerstitial nephritis are the most common pathologicalfindings while amyloidosis is very rare (Costa et al., 2003;Zatelli et al., 2003). Glomerulonephritis is frequentlyassociated with the glomerular deposition of immunecomplexes and is mainly membranoproliferative and/ormesangioproliferative (Plevraki et al., 2006). Other histolo-gical types of glomerular disease (membranous, focalsegmental, chronic, minimal change) have also beendescribed (Costa et al., 2003; Zatelli et al., 2003; Plevrakiet al., 2006). Membranoproliferative glomerulonephritis ismore frequently associated with CRF, whereas in dogswithout clinicopathological evidence of renal disease,histopathological evaluation usually reveals mesangiopro-liferative lesions and minimal change glomerulonephritis(Plevraki et al., 2006).

The relative prevalence of ocular and periocular lesionsin CanL has been reported to range from 16% to 80%(Ciaramella et al., 1997; Koutinas et al., 1999; Pena et al.,2000). Eye lesions are the sole presenting complaint andclinical manifestation of the disease in up to 15% of clinicalcases (Pena et al., 2000). The most common manifestationsare conjunctivitis, blepharitis (exfoliative, ulcerative, ornodular), anterior uveitis and keratoconjunctivitis, eithercommon or sicca (Koutinas et al., 1999; Naranjo et al.,2005). Ocular consequences of systemic hypertension (e.g.retinal detachement and/or hemorrhages, retinal arterialtortuosity, hyphema) are quite uncommon as they wereseen in only 5.7% of the hypertensive dogs with CanL(Cortadellas et al., 2006). Ocular histopathology of 60 CanLcases has revealed a lymphoplasmacytic to granulomatousinflammation in a perivascular to nodular to diffusepattern, along with the presence of the parasite (56.2%)


in ocular tissues, especially in the conjunctiva, limbus andciliary body (Pena et al., 2008).

3.9. What are the uncommon clinical forms of leishmaniosis?

Uncommon clinical forms (Blavier et al., 2001) includemucosal lesions (oral cavity, tongue and genital organs),joint swelling with erosive or non-erosive polyarthritis,osteolytic and osteoproliferative bone lesions, chronichepatitis (Rallis et al., 2005), chronic relapsing colitis(Adamama-Moraitou et al., 2007), neurological disease dueto meningitis and masseter muscle atrophic myositis orpolymyositis (Vamvakidis et al., 2000), autoimmunedisorders and cardiovascular disorders such as pericarditis,systemic vasculitis, thromboembolism and serum hyper-viscosity syndrome.

3.10. What additional clinicopathological findings shouldalert the clinician to the possibility of canine leishmaniosis?

Clinicians should suspect the possibility of CanL whendogs present persistent renal proteinuria (urinary proteincreatinine ratio (UPC) !0.5) or renal azotemia (IRIS stagesII, III or IV of CRF) (IRIS, 2006a), non-regenerative anemia(chronic disease and/or CRF), leukocytosis or leukopenia,serum hyperproteinemia, polyclonal beta and gammahyperglobulinemia, hypoalbuminemia, decreased albu-min/globulin ratio and elevated liver enzyme activities(Ciaramella et al., 1997; Koutinas et al., 1999). Serumhyperviscosity, thrombocytopathy (Petanides et al., 2008),thrombocytopenia, impaired secondary hemostasis andfibrinolysis (Ciaramella et al., 2005) may also be detected.

The type of inflammatory infiltrate found in tissuecytology (aspirates, impression smears) or histopathologyin organs such as skin, liver, intestine, eye, spleen, lymphnodes, striated muscles, synovium, nasal mucosa, etc., iscommonly either pyogranulomatous to granulomatousand/or lymphoplasmacytic (Solano-Gallego et al., 2004;Adamama-Moraitou et al., 2007; Pena et al., 2008;Petanides et al., 2008). Typically, there is lymphoid reactivehyperplasia in lymphoid organs such as lymph nodes andspleen along with bone marrow/spleen monocytic hyper-plasia (Mylonakis et al., 2005; Barrouin-Melo et al., 2006;Giunchetti et al., 2008) and variable numbers of Leishmaniaamastigotes.

4. Diagnosis

4.1. What are the various purposes for which L. infantuminfection diagnosis is carried out?

Diagnosis is usually performed for two main reasons:(1) to confirm ‘disease’, e.g. to find out if a dog with clinicalsigns and/or clinicopathological abnormalities compatiblewith CanL indeed has the disease and (2) to investigate thepresence of ‘infection’ for epidemiological studies, forscreening clinically healthy dogs living endemic regionsusually requested by the owners, to prevent transmissionfrom subclinical carriers by blood transfusion, to avoidimportation of infected dogs to non-endemic countries,and to monitor response to treatment (Miro et al., 2008).

For these reasons, it is important to separate Leishmaniainfection from disease and to apply different diagnostictechniques accordingly.

4.2. How is clinical canine leishmaniosis diagnosed?

The diagnosis of CanL is complex as the clinicalspectrum and clinicopathological abnormalities are bothwide and not specific. Accurate diagnosis of CanL oftenrequires an integrated approach consisting of clinico-pathological diagnosis and specific laboratory tests.Pertinent clinical history, a thorough physical examinationand several routine diagnostic tests such as CBC, bio-chemical profile, urinalysis and serum electrophoresis canhelp to raise the suspicion index for this disease. Otherdiagnostic tests such as coagulation profile, radiographs,abdominal ultrasound, cytological and histological evalua-tion of tissues or evaluation of biological fluids would beperformed on an individual basis. Several specific diag-nostic techniques have been developed to facilitate thediagnosis. It is essential to understand the basis of eachdiagnostic test and its limitations and appropriate inter-pretation. Reliable and specific diagnostic tests areessential for the detection of Leishmania infection in sickdogs although they lack 100% sensitivity and specificity.

4.3. What tests are available for the evaluation of Leishmaniainfection in dogs with suspected clinical leishmaniosis and inclinically healthy infected dogs?

In dogs with clinical signs and/or clinicopathologicalabnormalities consistent with leishmaniosis, the diagnos-tic methods include the detection of amastigotes in stainedcytological smears of aspirates from cutaneous lesions,lymph nodes, bone marrow and spleen (Alvar et al., 2004;Saridomichelakis et al., 2005). Less commonly cytology iscarried out on other tissues or body fluids (Agut et al.,2003; Dantas-Torres, 2006). Search for amastigotes bycytology could be unrewarding due to the low to moderatenumbers of detectable parasites present even in dogs withfull blown clinical disease (Moreira et al., 2007). Leishmaniaparasites may also be viewed in histopathologic biopsysections from the skin or other infected organs. Leishmaniainfection should be suspected when either pyogranulo-matous, granulomatous or lymphoplasmacytic inflamma-tions in different tissues (Solano-Gallego et al., 2004;Adamama-Moraitou et al., 2007; Pena et al., 2008;Petanides et al., 2008) and/or lymph node reactivehyperplasia are observed (Mylonakis et al., 2005; Giunch-etti et al., 2008). Definite identification of parasites withintissue macrophages may be difficult and an immunohis-tochemical staining method can be employed to detect orconfirm the presence of Leishmania in the tissue. Theisolation in culture of parasites from infected tissues is notsuitable for rapid diagnosis and has a lower sensitivity thanPCR and serology. Parasite culture is currently used moreoften for research purposes (Miro et al., 2008).

The most useful diagnostic approaches for investigationof infection in sick and clinically healthy infected dogsinclude: (1) detection of specific serum anti-leishmanialantibodies by several serological techniques and (2)


demonstration of the parasite DNA in tissues by applyingmolecular techniques.

4.4. How should serology be used for the diagnosis of canineleishmaniosis?

The diagnosis of CanL can be made by the detection ofspecific serum antibodies (IgG) using quantitative serolo-gical techniques, such as the immunofluorescence anti-body test (IFAT) and enzyme-linked immunosorbent assay(ELISA). High antibody levels are associated with highparasitism and disease (Reis et al., 2006). Some dogsremain seronegative for variable periods after beinginfected with Leishmania (Strauss-Ayali et al., 2004).However, due to the relatively long incubation period,sick dogs are likely to be seropositive (Oliva et al., 2006). Itis important to submit samples to a laboratory that runsquantitative serological techniques and can provide anendpoint titer (IFAT) or optical density reading (ELISA) anda classification of the levels of antibodies (negative,doubtful, low, medium and high positive levels). A highlevel of antibodies is conclusive of a diagnosis of CanL.However, the presence of low antibody levels is notnecessarily indicative of the disease and further work-up is

necessary to confirm or exclude clinical leishmaniosis byother diagnostic methods such as cytology, histopathologyand PCR (Miro et al., 2008) (Fig. 2).

4.5. What reference and commercial antibody tests forLeishmania are currently available?

The IFAT, ELISA and immunochromatographic devicesare the most commonly used techniques for detection ofantileishmanial antibodies (Alvar et al., 2004; Maia andCampino, 2008; Miro et al., 2008). False positive resultsdue to serological cross-reactivity with other pathogenshave been described in all of the serological techniquesmentioned above, especially in areas of Trypanosoma cruziinfection in North, Central and South America or with otherspecies of Leishmania (Ferreira Ede et al., 2007; Porrozziet al., 2007) and with tests using whole-parasite crudeantigens. Cross-reactions are less likely to occur whenusing recombinant peptides such as rA2, rK9, rK26 andrK39 (Boarino et al., 2005; Porrozzi et al., 2007).

IFAT, which generally uses whole promastigotes antigenis highly specific and sensitive for the detection of clinicalCanL, but may lack sensitivity to detect clinically healthy butinfected dogs (Mettler et al., 2005). The cut-off titre to

Fig. 2. Algorithm of diagnostic approach in dogs with clinical signs and/or clinicopathological abnormalities compatible with leishmaniosis.


distinguish positive and negative results varies from 1:40 to1:160 between different laboratories (Ferroglio and Vitale,2006).

ELISA sensitivity and specificity greatly depend on theantigens employed, which mainly include soluble pro-mastigote extracts and recombinant or purified proteins(Miro et al., 2008). Whole-parasite extracts are sensitivefor the detection of subclinical or clinical canine infectionsbut provide a somewhat lower specificity (Mettler et al.,2005; Ferreira Ede et al., 2007). On the other hand, ELISAwith recombinant peptides is very specific but may lacksensitivity for the detection of clinically healthy infecteddogs, depending on the antigen employed (Mettler et al.,2005; Porrozzi et al., 2007). An ELISA based on recombi-nant antigens (K9, K26 and K39 sub epitopes), has shownhigh specificity and sensitivity in infected dogs (Boarinoet al., 2005). Studies evaluating IgG1 and IgG2 usingpolyclonal antibodies have frequently reached conflictingoutcomes and are therefore not used routinely in thediagnosis of CanL (Day, 2007).

Immunochromatography-based assays are easy to useand provide qualitative results on the spot. These kitsusually have good specificity but their sensitivity is variableand their performance is still not optimal (Mettler et al.,2005). Several rapid tests kits and antigenic preparations forIFAT and ELISA are also commercially available.

4.6. How should PCR be used for the diagnosis of canineleishmaniosis?

PCR assays have greatly improved the sensitivity ofparasitological diagnosis in CanL. Detection of parasite-specific DNA in tissues by PCR allows sensitive and specificdiagnosis. Several different assays with various targetsequences using genomic or kinetoplast DNA (kDNA) havebeen developed for CanL. Assays based on kDNA appear tobe the most sensitive for direct detection in infectedtissues (Gomes et al., 2008; Maia and Campino, 2008; Miroet al., 2008). PCR can be performed on DNA extracted fromtissues, blood, biological fluids or even from histopatho-logic specimens. PCR on bone marrow, lymph node, spleenor skin is most sensitive and specific for the diagnosis ofCanL (Maia et al., 2009; Manna et al., 2008a; Reis et al.,2009). PCR on whole blood, buffy coat, and urine is lesssensitive than the aforementioned tissues (Solano-Gallegoet al., 2007; Manna et al., 2008a). Sampling using non-invasive conjunctival swabs has proven to be very sensitiveand specific for the detection of L. infantum in groups ofseropositive dogs with clinical leishmaniosis (Strauss-Ayali et al., 2004; Ferreira Sde et al., 2008). PCR on aspiratesof lymph node and bone marrow has been shown to bemore sensitive than microscopic detection of amastigotesin stained smears or parasite culture (Moreira et al., 2007).Currently, three different PCR techniques are available:conventional PCR, nested-PCR and real-time PCR (Gomeset al., 2008; Maia and Campino, 2008; Miro et al., 2008).Quantitative real-time PCR is an advanced technique thatcan detect extremely low parasitic loads when comparedwith conventional PCR (Francino et al., 2006). Real-timePCR allows the quantification of Leishmania loads in tissuesof infected dogs which is important for diagnosis as well as

for follow-up during the treatment of CanL (Pennisi et al.,2005b; Manna et al., 2008a). It is important to highlightthat information provided by PCR should not be separatedfrom the data obtained from clinicopathological andserological evaluations. These should all be combinedtogether for a comprehensive assessment.

4.7. How should PCR be used in clinically healthy dogs?

The presence of Leishmania DNA in the blood or othertissues of clinically healthy dogs living in endemic areasindicates that these dogs harbour infection (Solano-Gallego et al., 2001a) but, they may never develop clinicaldisease (Oliva et al., 2006). The interpretation of PCRresults should be done cautiously in clinically healthy dogsand with consideration of the diagnostic procedure’spurpose. For instance, for the purpose of identifyinginfected dogs and preventing their importation to non-endemic areas where infection might spread via local sandfly vectors, or for the purpose of preventing transmission ofinfection via blood products from infected donors, PCRwould be an appropriate technique in combination withquantitative serological tests. However, the decision totreat clinically healthy dogs with anti-leishmanial medica-tion based on positive PCR alone is not recommended.

4.8. Are there other useful immunodiagnostic tests for theprognosis in sick dogs?

Dogs that have a progressive infection manifested asdisease have a depressed cellular response to Leishmaniaantigens expressed by no apparent cellular response or littleresponse in cell-stimulation assays while clinically healthyinfected dogs are considered ‘‘resistant’’ due to the presenceof a demonstrable cellular response (Baneth et al., 2008). Thein vivo leishmanin skin test, the in vitro lymphocytestimulation with Leishmania antigen and the detection ofIFN-g are tests that evaluate the cellular response toLeishmania and they might be used to predict susceptibilityor resistance to infection in the near future (Dos-Santos et al.,2008). However, these tests are available only in researchlaboratories and need further standardization (Maia andCampino, 2008). The clinical usefulness of lymphocyte sub-population size, such as the CD4 and CD8 in assessing theseverity of Leishmania infection and response of dogs totherapy is questionable. A recent study has not founddifferences between lymphocyte sub-populations in sickdogs, before and after the antileishmanial treatment whencompared to healthy dogs (Miranda et al., 2007).

5. Treatment

5.1. What is the most effective specific treatment in canineleishmaniosis?

The drugs most commonly used in the treatment ofCanL and current therapeutical protocols are listed inTable 3. Meglumine antimoniate, aminosidine and milte-fosine are, up to date, the only drugs licenced in Europespecifically for treatment of CanL. The combination ofmeglumine antimoniate with allopurinol is considered as


the most effective therapy and constitutes the first lineprotocol against the disease (Denerolle and Bourdoiseau,1999) but many different therapeutic protocols aresuggested regarding the dosage, dose interval and durationof treatment (Noli and Auxilia, 2005). The pharmacoki-netics of antimonials may vary significantly in dogs withnaturally occurring CanL and renal failure may increasetheir half life as shown in hamsters (Zaghloul and Al-Jasser,2004). Risk of toxicity may therefore be increased in dogswith reduced glomerular filtration rate. Miltefosine hasbeen recently suggested for treatment of CanL in combina-tion with allopurinol as an alternative to the combinationof meglumine antimoniate with allopurinol (Manna et al.,2008b; Miro et al., in press).

Amphotericin B has also a good efficacy demonstrated bydifferent clinical trials, but this drug has serious disadvan-tages, such as the route of administration (IV) and itsnephrotoxicity, especially since CanL has a direct harmfuleffect on the kidneys. Aminosidine also has severe sideeffects (nephrotoxicity, ototoxicity) and its use as first linetherapy of CanL is not recommended. Apart from theaforementioned drugs, several other candidate medicationsagainst CanL have been studied in vitro or in laboratoryanimals but rarely in controlled clinical trials. These includepentamidine, aminosidine, ketoconazole, metronidazoleand spiramycin and marbofloxacin (Miro et al., 2008). Moreextensive clinical studies with naturally infected dogs arenecessary to confirm the therapeutic value of these drugs.

5.2. What is the expected clinical response to treatment incanine leishmaniosis? What are the most common side effectsof different therapeutical protocols?

The expected clinical response to treatment of sick dogscan vary from poor to good depending on their overall

initial clinicopathological status (Table 4). Dogs with renalinsufficiency are expected to have a lower recovery rate incomparison to those without kidney compromise. Themajority of dogs experience clinical improvement withinthe first month of therapy (Pennisi et al., 2005b; Mannaet al., 2008a), although in others, a longer period of therapyis required before any apparent improvement. Serumantibody titres and serum protein alterations are expectedto require a longer period of time before normalization.Some dogs show side effects to therapy that must bedistinguished from deterioration of the disease and arelisted in Table 3 (Noli and Auxilia, 2005).

5.3. What clinicopathological parameters should bemonitored during the treatment of canine leishmaniosis?

The clinicopathological parameters to be monitoredduring treatment would depend on the individualabnormalities. However, in general, it is recommendedto perform complete CBC, biochemical profile and urina-lysis including urine protein/creatinine ratio in proteinuricdogs. The frequency of monitoring clinicopathologicalparameters would vary in each patient but, clinicopatho-logical parameters should, in most cases, be monitoredmore frequently initially, i.e. after the first month oftreatment and then every 3–4 months. Later on, if the dogis fully recovered clinically with treatment then a recheckwould be recommended every 6 months or once a year.

5.4. How should serology be used for monitoring theeffectiveness of treatment?

The use of antibody levels to assess clinical improve-ment after treatment is controversial. Older studiesreported that antibody titer did not decrease within the

Table 3Current treatment protocols applied in canine leishmaniosis.

Protocol Drugs and dosages Main side effects Reference

1st line N-methylglucamine antimoniatea

(75–100 mg/kg/SID) for 4–8 weeks,S.C. + allopurinol (10 mg/kg/BID)for at least 6–12 months P.O.

Potential nephrotoxicity andcutaneous abcesses/cellulitis(N-methylglucamine antimoniate)

Denerolle and Bourdoiseau (1999),Ikeda-Garcia et al. (2007)and Manna et al. (2008a)

Xantine urolithiasis (allopurinol) Cavaliero et al. (1999), Koutinas et al.(2001), Pennisi et al. (2005b)and Plevraki et al. (2006)

2nd line Miltefosinea (2 mg/kg/SID) for 4 weeksP.O. + allopurinol (10 mg/kg/BID)for at least 6–12 months P.O.

Vomiting, diarrhea (Miltefosine) Manna et al. (2008b) and Miro et al. (in press)

Xantine urolithiasisAllopurinol (10 mg/kg/BID) for atleast 6–12 months P.O.

Xantine urolithiasis Cavaliero et al. (1999), Koutinas et al. (2001),Pennisi et al. (2005b) and Plevraki et al. (2006)

3rd line Amphotericin Bb (0.5–0.8 mg/kg, I.V/SID/twiceper week) for 2 months

Nephrotoxicity Lamothe (2001) and Cortadellas (2003)

Liposomal amphotericin Bb (3 mg/kg/SID)for 5 consecutive days, I.V.

Transient nephrotoxicity Oliva et al. (1995)

Metronidazole (25 mg/kg/SID) + spiramycin(150,000 U/SID) for 3 months P.O.

Not described Pennisi et al. (2005a)

Marbofloxacin (2 mg/kg/SID for 1 month P.O.) Not described Rougier et al. (2008)

P.O.: per os; S.C.: subcutaneous; I.V.: intravenous.a Registered for veterinary use in Europe.b 1st line drug for Human Visceral Leishmaniasis in Europe; not recommended by WHO for veterinary use, to avoid drug parasite resistance.


first months after the beginning of the therapy (Ferreret al., 1995). However, recent studies have demonstrated aslow and progressive decrease in IgG and IgA antibodylevels which is associated with clinical improvement(Solano-Gallego et al., 2001b; Rodriguez et al., 2006).Therefore, we recommend repeating a quantitativeserological test in the same laboratory 6 months afterthe initial treatment due to the relatively long half life ofIgG ("3 weeks in humans) (Anderson et al., 2006) and thecommon presence of initial high levels of antibodies insick dogs. Ideally, it would be better to evaluate theantibody kinetics by running sera from the initial andfollow-up dates simultaneously in the same assay. Forexample, decrease of IFAT titer would be consideredsignificant if there is more than a twofold dilutionsdifference between the first and the following sample.Some dogs would present a significant decrease inantibody levels associated with clinical improvementwithin 6 months to 1 year of treatment while others mightnot have a decrease in antibody titers despite the clinicalimprovement. In contrast, a marked increase of antibodylevels should be interpreted as a marker of relapse,especially in dogs following the discontinuation oftreatment.

5.5. When should allopurinol be discontinued?

The clinical decision on timing of allopurinol disconti-nuation is not well defined and should be based onclinicopathological, serological and parasitological assess-ments. However, the authors consider the combination offollowing criteria sufficient to discontinue allopurinoltreatment: (1) presence of complete physical and clin-icopathological recovery evaluated by a thorough physicalexamination, CBC, full biochemistry panel and urinalysis atleast 1 year after the initial allopurinol administration, and(2) marked decrease of antibody levels (negative orborderline positive by a quantitative serological assay).

It is important to highlight that some extremelysusceptible dogs never arrive at a point that would allowthe discontinuation of allopurinol while some lesssusceptible dogs would be capable of controlling theinfection without extremely lengthy treatment.

5.6. Is there any evidence for anti-Leishmania drug resistancein dogs?

While the occurrence of Leishmania resistance topentavalent antimonials, amphotericin B, aminosidine

Table 4Clinical staging of canine leishmaniosis based on serological status, clinical signs, laboratory findings, and type of therapy and prognosis for each clinicalstage.

Clinical stages Serologya Clinical signs Laboratory findings Therapy Prognosis

Stage I:mild disease

Negative tolow positiveantibodylevels

Dogs with mild clinicalsigns such as peripherallymphadenopathy,or papular dermatitis(Ordeix et al., 2005;Bottero et al., 2006)

Usually no clinicopathologicalabnormalities observed;normal renal profile:creatinine < 1.4 mg/dl;non-proteinuric: UPC < 0.5

Scientific neglect/allopurinol alone/allopurinol + meglumineantimoniate or miltefosine

Good

Stage II:moderatedisease

Low to highb

positiveantibodylevels

Dogs, which apart fromthe signs listed in stage I,may present: diffuse orsymmetrical cutaneouslesions such as exfoliativedermatitis/onychogryphosis,ulcerations (planum nasale,footpads, bony prominences,mucocutaneous junctions),anorexia, weight loss, fever,and epistaxis (Petanideset al., 2008)

Clinicopathological abnormalitiessuch as mild non-regenerativeanemia, hypergammaglobulinemia,hypoalbuminemia, serumhyperviscosity syndrome(Petanides et al., 2008).Substage—(a) normal renalprofile: creatinine < 1.4 mg/dl;non-proteinuric: UPC < 0.5. (b)Creatinine < 1.4 mg/dl; UPC = 0.5–1

Allopurinol + meglumineantimoniate or miltefosine

Good toguarded

Stage III:severe disease

Medium tohigh positiveantibodylevels

Dogs, which apart from thesigns listed in stages I and II,may present signsoriginating fromimmune-complex lesions:vasculitis, arthritis, uveitisand glomerulonephritis

Clinicopathological abnormalitieslisted in stage II

Allopurinol + meglumineantimoniate or miltefosine

Guardedto poor

Chronic kidney disease (CKD)IRIS stage I with UPC > 1 orstage II (creatinine 1.4–2 mg/dl)(IRIS, 2006a)

Follow IRIS guidelinesfor CKD (IRIS, 2006b)

Stage IV:very severedisease

Medium tohigh positiveantibodylevels

Dogs with clinical signs listedin stage III. Pulmonarythromboembolism, or nephroticsyndrome and end stagerenal disease

Clinicopathological abnormalitieslisted in stage II

Allopurinol (alone) Poor

CKD IRIS stage III (creatinine2–5 mg/dl) and stage IV(creatinine > 5 mg/dl) (IRIS, 2006a)

Follow IRIS guidelinesfor CKD (IRIS, 2006b)

Nephrotic syndrome: markedproteinuria UPC > 5

a Dogs with negative to medium positive antibody levels should be confirmed as infected with other diagnostic techniques such as cytology, histology/immunohistochemistry and PCR.

b High levels of antibodies are conclusive of a diagnosis of CanL and are defined as three- to four fold increase of a well established laboratory referencecut-off.


and miltefosine is well known in human medicine (Croftet al., 2006), only limited information is available for thedog. A decreased sensitivity to meglumine antimoniate orantimonials of L. infantum isolated from dogs after severaltreatment courses has been reported (Gramiccia et al.,1992; Carrio and Portus, 2002). In contrast, no differencesin susceptibility to antimonials of intracellular amastigotesof L. infantum strains isolated from untreated dogs werefound, despite repeated in vitro passages and hamsterinfection (Carrio and Portus, 2002). Unfortunately, there isno data available about the occurrence of Leishmaniaresistance to other drugs in dogs.

6. Prognosis

6.1. Can treated dogs with leishmaniosis truly be cured orcleared of the infection?

Treatment with antileishmanial drugs and especiallywith the combination of meglumine antimoniate/allopur-inol or allopurinol alone often leads to clinical cure (Noliand Auxilia, 2005). However, some dogs that initiallyrespond well to therapy can experience clinical relapseafter the cessation of treatment or during it suggesting thatinfection had not been eliminated. Furthermore, para-sitological cure is rarely achieved and treated dogs, eventhose on a prolonged allopurinol regime, continue toharbour the parasite and be infectious to sand flies,although to a lesser extent (Ikeda-Garcia et al., 2007;Manna et al., 2008a; Ribeiro et al., 2008).

6.2. Can clinically healthy but infected dogs clear the infectionspontaneously?

It remains unclear if some healthy infected dogs areable to clear Leishmania infection spontaneously. Based oncurrent knowledge in rodent and human studies (Dereureet al., 2003; Soliman, 2006; Okwor and Uzonna, 2008), it islikely that persistent infection occurs in some dogs withoutever producing apparent lesions or clinical disease, andthat complete clearance of infection in dogs would be anexceptional event. Long-term experimental studies havedemonstrated that healthy infected dogs harbour theparasite in lower numbers in the blood, liver, spleen,lymph node and skin when compared to sick dogs whichhave high parasitic loads and wide tissue dissemination ofthe parasite (Rodriguez-Cortes et al., 2007). A longitudinalstudy in a cohort of dogs introduced into an endemic areahas demonstrated that some of them were PCR positive inbone marrow and converted to negative over time (Olivaet al., 2006). However, this study was limited due to thesensitivity of conventional PCR and the sampling of onlyone tissue. Further studies are required to elucidate thisquestion.

6.3. Can infected dogs be reinfected?

There is no conclusive information in the literatureabout re-infection in the dog. Experimental re-challengeinfections in dogs indicated that a previous exposure couldconfer some degree of resistance in some dogs (Santos-

Gomes et al., 2003). No data is available on reinfectionunder natural conditions. However, the authors hypothe-size that reinfection in endemic areas where dogs liveoutdoors is likely. Dogs living in endemic areas are oftencontinuously exposed to infective sand fly bites. Studieshave shown that dogs may be bitten by sand flies hundredsof times during one night. In addition, a relatively low tomoderate proportion of sand flies harbours L. infantum(0.5–3%) in endemic areas (Gomez-Saladin et al., 2005;Martin-Sanchez et al., 2006). It is most probable that dogsthat are initially able to resist the establishment ofinfection may eventually succumb when the parasite iscontinuously introduced and reintroduced. It is alsopossible that dogs can be infected with one strain of theparasite and may then be infected with a different andpossibly more virulent strain.

6.4. How could prognosis be established on the basis of thedifferent clinicopathological features?

Prognosis in CanL is difficult to establish and there islimited information available. In addition, no controlledstudies have been attempted to evaluate the prognosticfactors. Nevertheless, the prognosis for each patient willvary according to its clinicopathological status. Clinicalstaging systems are aimed to group patients in which theseverity of clinical picture and prognosis are the same.These systems are useful to evaluate the efficacy ofdifferent therapies, to decide on the therapy most suitablefor each patient and also to consider a prognosis. Thecriteria of classification have to be simple and clinical, withthe use of uncomplicated diagnostic methods. According tothe established criteria, each patient is staged at a certainmoment in time. Later on, the stage can change as thedisease deteriorates or improves. We propose a system offour clinical stages, based on clinical signs, clinicopatho-logical abnormalities and serological status, along with thetype of therapy and prognosis suitable for each stage(Table 4).

7. Management of clinically healthy dogs in endemicareas

7.1. Should clinically healthy dogs be screened for Leishmaniaantibodies?

Healthy dogs should be screened for Leishmaniaantibodies as an initial indication for the presence ofinfection if they are imported, scheduled to travel to non-endemic areas, serve as blood donors, employed inepidemiological/research studies or if their owners wishto have them monitored for early infection and thepotential to develop disease. It is well known that highantibodies titers are correlated with high parasitism anddisease (Reis et al., 2006) and for this reason a high positiveantibody titer may indicate that an infected dog is headingtoward the development of a widespread infection andfuture development of clinical disease. Consequently, earlydetection of the disease is beneficial for the patient.Therefore, it is appropiate to screen dogs living in endemicareas for Leishmania antibodies, at least every 6–12


months, for proper institution of both therapeutic andpreventative measures.

7.2. Should clinically healthy dogs be assessed for carryingLeishmania DNA?

In general, healthy dogs in endemic regions should onlybe screened for Leishmania DNA if they are to be exportedto non-endemic areas, and if they are considered to be usedas blood donors, as pets for immunosupressed people, or inepidemiological/research studies. Otherwise, we recom-mend using serology alone or the combination of serologywith PCR for screening healthy dogs. It is better to avoidscreening clinically healthy dogs only with PCR.

7.3. How should a clinically healthy but seropositive dog bemanaged?

Clinically healthy but seropositive dogs should bemanaged according to their antibody level. Dogs with highantibody levels should be treated as sick dogs with thesame medication and dosage given to clinical cases and byapplying the same type of monitoring during thetreatment. Seropositive dogs with low antibody titersshould be confirmed by retesting. Confirmed seropositivedogs with low antibody titers should be monitored withphysical examinations, routine laboratory tests andserological tests on a regular basis every 3–6 months toassess the progression of the infection. If antibody titersincrease significantly during the monitoring, irrespectiveof clinical signs and clinicopathological abnormalities,these dogs should be treated as sick dogs. Healthyseropositive dogs have been found to infect sand flies(Molina et al., 1994; Michalsky et al., 2007). Therefore,they should be protected with topical insecticide repel-lents to minimize the transmission of infection during thevector season.

7.4. How should a clinically healthy but PCR-positive andseronegative dog be managed?

Clinically healthy PCR-positive and seronegative dogscan be monitored clinically and serologically overtime(every 6 months or at least once a year) to evaluateseroconversion and possible development of illness.Treatment is not recommended. However, they shouldbe put on preventative measures against parasite trans-mission (see Section 8.1).

8. Prevention

8.1. What preventive measures should be applied to decreasethe risk of canine leishmaniosis?

Effective prevention of sand fly bites can be achievedwhen the following steps are taken together (Alexanderand Maroli, 2003): (i) keeping the dog indoors during thesand fly season from dusk to dawn; (ii) reducing themicrohabitats favorable to sand flies in the vicinity of thehouse or in locations where the dog spends time; (iii) usageof environmental insecticide treatment, and (iv) usage of

topical insecticides with proven activity against the sandflies which bite dogs.

In recent years, various insecticide formulations havebeen evaluated under laboratory and field conditions withencouraging results. Deltamethrin impregnated collarsrelease the insecticide gradually and it is distributed in thesubcutaneous adipose tissue of the animals within 1–2weeks (Halbig et al., 2000). Under optimal conditions, therepellent effect of these collars can last up to 6 months(Killick-Kendrick et al., 1997). Spray or spot-on formula-tions also offer high levels of protection, though of ashorter duration (2 and 3 weeks, respectively). Topicalapplication of permethrin provides good repellent andinsecticidal effects against P. perniciosus that last severalweeks (Molina et al., 2001). These topical formulationsrequire some days for the insecticide to spread throughoutthe stratum corneum. In contrast, powder formulationsachieve an immediate effect, but have a shorter lastingeffect. The synergizing action of imidacloprid, a nicotinoidinsecticide, enhances the properties of pyrethroids likepermethrin. In this way, a strong repellent effect against P.papatasi and P. perniciosus, which lasts for 3–4 weeks, hasbeen achieved (Mencke et al., 2003; Miro et al., 2007a).Field studies have shown that some topical insecticidesused in canine populations have been effective in reducingthe transmission of infection to both dogs and humans(Maroli et al., 2001; Gavgani et al., 2002a; Otranto et al.,2007).

Veterinarians and dog owners are advised to check thelabel recommendations of products and follow themanufacturer’s instructions, especially for the correctapplication of the product and frequency of reapplication.Client education on the maintenance of appropriateinsecticide throughout the period of sand fly activity isalso crucial for the protection of dogs.

8.2. What preventive measures should be used in dogs fromnon-endemic areas travelling to endemic areas?

The preventive measures should be the same asdescribed for healthy or sick dogs that live in endemicareas (see Section 8.1). It is recommended that collarsshould be applied 2 weeks before travelling and bechanged every 5 months. Alternatively, pyrethroids inspot-on or spray formulations should be applied 2 daysbefore travelling and repeatedly administered every 2–3weeks, depending on the type of product. As recommendedfor dogs living in the endemic areas, advice should be givenfor follow-up visits after returning for clinical andlaboratory check up (see Sections 7.3 and 7.4).

8.3. Are there vaccines available for canine leishmaniosis?

After decades of attempts to produce safe and effectiveLeishmania vaccines with very limited success, severalcanine vaccines are under experimental or field evaluation,some with promising results (Miro et al., 2008). PurifiedLeishmania fraction vaccines appear to be the mostsuccessful. The main representative of this group ofvaccine antigens is the glycoprotein GP63 enrichedfraction of L. donovani also known as the ‘‘fucose mannose


ligand’’ (FML). An FML-based vaccine has been evaluated infield studies in Brazil with a good protection rate and it waslicensed in that country as the first commercial vaccine forCanL (Leishmune1) (Borja-Cabrera et al., 2002; Nogueiraet al., 2005). The FML vaccine has also been proposed forimmune therapy of sick dogs (Borja-Cabrera et al., 2004)and as a transmission-blocking vaccine (Saraiva et al.,2006). However, separating naturally infected from FML-vaccinated dogs is difficult and has lead to reluctance touse this vaccine by some veterinarians in Brazil whereseropositive dogs are officially culled (Dantas-Torres andBrandao-Filho, 2006). A second vaccine based on anexcreted/secreted antigen purified from specific-mediumculture supernatant of L. infantum promastigotes withmuramyl dipeptide as adjuvant has been studied in Francewith good protection against L. infantum experimentalinfection (Lemesre et al., 2005) and a high efficacy rate in adouble blinded field trial (Lemesre et al., 2007).

Acknowledgments

The authors would like to acknowledge the support ofDr. Norbert Mencke (Bayer HealthCare Animal Health,Monheim am Rhein, Germany), also that of Drs. SantoCaracappa and Fabrizio Vitale from the National ReferenceCentre for Leishmaniosis (Istituto Zooprofilactico dellaSicilia, Palermo, Italy) and Dr. Ricardo Molina (Instituto deSalud Carlos III, Madrid, Spain). The authors would like tothank scientists, human physicians and veterinarians thathave advanced our understanding of canine leishmaniosis.

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