LeishVet guidelines for the practical management of canine leishmaniosis

The LeishVet group has formed recommendations designed primarily to help the veterinary clinician in the management of canine leishmaniosis. The complexity of this zoonotic infection and the wide range of its clinical manifestations, from inapparent infection to severe disease, make the management of canine leishmaniosis challenging. The recommendations were constructed by combining a comprehensive review of evidence-based studies, extensive clinical experience and critical consensus opinion discussions. The guidelines presented here in a short version with graphical topic displays suggest standardized and rational approaches to the diagnosis, treatment, follow-up, control and prevention of canine leishmaniosis. A staging system that divides the disease into four stages is aimed at assisting the clinician in determining the appropriate therapy, forecasting prognosis, and implementing follow-up steps required for the management of the leishmaniosis patient.

# Background
Canine leishmaniosis (CanL) due to Leishmania infantum is a major global zoonosis potentially fatal to humans and dogs, which comprise the main reservoir of infection to humans [bib_ref] The current status of zoonotic leishmaniases and approaches to disease control, Gramiccia [/bib_ref]. CanL is endemic in more than 70 countries in the world. It is present in regions of southern Europe, Africa, Asia, South and Central America [bib_ref] Canine leishmaniosis -new concepts and insights on an expanding zoonosis: part one, Baneth [/bib_ref] and has been reported also in the United States of America (USA) [bib_ref] Canine leishmaniasis in North America: emerging or newly recognized?, Petersen [/bib_ref]. It is also an important concern in non-endemic countries where imported sick or infected dogs constitute a veterinary and public health problem [bib_ref] Canine leishmaniosis in the United Kingdom: a zoonotic disease waiting for a..., Shaw [/bib_ref].
CanL is manifested by a broad spectrum of clinical signs and degrees of severity, and there is insufficient scientific agreement on the management of this disease [bib_ref] Canine leishmaniosis -new concepts and insights on an expanding zoonosis: part one, Baneth [/bib_ref]. LeishVet is a group of veterinary scientists from academic institutes in Europe and the Mediterranean basin with a main clinical and scientific interest in CanL. The main goal of LeishVet is to develop consensus recommendations that would represent the most current understanding of L. infantum infection in dogs based on recent evidence-based literature and clinical experience [bib_ref] Canine leishmaniosis -new concepts and insights on an expanding zoonosis: part one, Baneth [/bib_ref]. The objective of these guidelines is to help practitioners in the clinical management of CanL with emphasis on diagnosis, clinical staging, treatment, clinical monitoring, prognosis and prevention.

## Life cycle and transmission
Leishmania completes its life cycle in two hosts, a phlebotomine sand fly vector, which transmits the flagellated infective promastigote form, and a mammal, where the intracellular amastigote form develops and replicates [fig_ref] Figure 1: The life cycle of L [/fig_ref]. Sand flies are the only arthropods that are adapted for biological transmission of Leishmania. The relatively low proportion of sand flies harbouring L. infantum (0.5 -3%) is sufficient for maintaining the infection in endemic areas. Non-sand fly modes of transmission have also been described but their role in the natural history and epidemiology of leishmaniosis remains unclear [fig_ref] Figure 1: The life cycle of L [/fig_ref]. Proven modes of non-sand fly transmission include infection through transfused blood products [bib_ref] Transmission of visceral leishmaniasis through blood transfusions from infected English foxhounds to..., Owens [/bib_ref] from blood donors which are carriers of infection [bib_ref] Transmission of Leishmania infantum via blood transfusion in dogs: potential for infection..., De Freitas [/bib_ref] [bib_ref] Detection of Leishmania infantum by real-time PCR in a canine blood bank, Tabar [/bib_ref] , vertical [bib_ref] Transplacental transmission of a North American isolate of Leishmania infantum in an..., Rosypal [/bib_ref] [bib_ref] Tissue distribution of Leishmania chagasi and lesions in transplacentally infected fetuses from..., Pangrazio [/bib_ref] [bib_ref] Transplacental Transmission of Leishmania infantum as a Means for Continued Disease Incidence..., Boggiatto [/bib_ref] and venereal transmission [bib_ref] Venereal transmission of canine visceral leishmaniasis, Silva [/bib_ref]. The adequate selection of canine blood donors is of great importance for the prevention of L. infantum infection and recommendations on donor selection are graphically summarized in [fig_ref] Figure 2: Algorithm describing the selection of blood donors and exclusion of infected dogs [/fig_ref]. Suspected yet unproven modes of transmission include: 1) direct dogto-dog transmission through bites or wounds, which could explain the presence of autochthonous CanL clinical cases [bib_ref] Canine leishmaniosis in the United Kingdom: A zoonotic disease waiting for a..., Shaw [/bib_ref] in non-endemic areas in the absence of apparent vectors, as described in foxhounds in the USA [bib_ref] Canine visceral leishmaniasis, United States and Canada, Duprey [/bib_ref] or in breeding kennels in Europe [bib_ref] Environmental risk mapping of canine leishmaniasis in France, Chamaille [/bib_ref] , and 2) transmission by other hematophagous arthropods such as ticks and fleas [bib_ref] Participation of Rhipicephalus sanguineus (Acari: Ixodidae) in the epidemiology of canine visceral..., Coutinho [/bib_ref] [bib_ref] Can fleas from dogs infected with canine visceral leishmaniasis transfer the infection..., Coutinho [/bib_ref] [bib_ref] Transovarial passage of Leishmania infantum kDNA in artificially infected Rhipicephalus sanguineus, Dantas-Torres [/bib_ref] [bib_ref] Evaluation of the vectorial capacity of Rhipicephalus sanguineus (Acari: Ixodidae) in the..., Paz [/bib_ref] [bib_ref] Association between the prevalence of infestation by Rhipicephalus sanguineus and Ctenocephalides felis..., Paz [/bib_ref] [bib_ref] Quantification of Leishmania infantum DNA in females, eggs and larvae of Rhipicephalus..., Dantas-Torres [/bib_ref] [fig_ref] Figure 1: The life cycle of L [/fig_ref].

## Distribution and epidemiology
Socioeconomic and possible climate factors have led to changes in the distribution of CanL in Europe [fig_ref] Figure 3: The distribution of canine L [/fig_ref]. Leishmania infantum infection has spread northward reaching the foothills of the Alps in northern Italy [bib_ref] The northward spread of leishmaniasis in Italy: evidence from retrospective and ongoing..., Maroli [/bib_ref] and of the Pyrenees in France [bib_ref] Environmental risk mapping of canine leishmaniasis in France, Chamaille [/bib_ref] and northern Spain [bib_ref] Seroprevalence of Leishmania infantum in northwestern Spain, an area traditionally considered free..., Amusategui [/bib_ref]. The large numbers of dogs travelling to southern Europe or imported as companion animals from areas where CanL is endemic have increased the number of clinical cases reported in non endemic countries such as the United Kingdom [bib_ref] Canine leishmaniosis in the United Kingdom: A zoonotic disease waiting for a..., Shaw [/bib_ref] and Germany [bib_ref] Imported and travelling dogs as carriers of canine vector-borne pathogens in Germany, Menn [/bib_ref].
Leishmania infantum frequently follows an insidious and chronic pattern of infection [bib_ref] Immune privilege in sites of chronic infection: Leishmania and regulatory T cells, Peters [/bib_ref]. Therefore, CanL is a disease in which infection does not equal clinical illness resulting in a high prevalence of subclinical infection [bib_ref] Canine leishmaniosis -new concepts and insights on an expanding zoonosis: part one, Baneth [/bib_ref] [bib_ref] Prevalence of Leishmania infantum infection in dogs living in an area of..., Solano-Gallego [/bib_ref].
A broad range of immune responses and clinical manifestations have been described in CanL [fig_ref] Figure 4: Clinical manifestations and immunological characteristics of L [/fig_ref].
Infection in dogs may be subclinical or manifested as a self-limiting disease, or a severe, and sometimes, fatal illness [bib_ref] Directions for the diagnosis, clinical staging, treatment and prevention of canine leishmaniosis, Solano-Gallego [/bib_ref]. Subclinical infection is not necessarily permanent and factors such as immunosuppression or concomitant diseases could break the equilibrium and lead to the progression of clinical disease in dogs [bib_ref] Canine leishmaniosis -new concepts and insights on an expanding zoonosis: part one, Baneth [/bib_ref] [bib_ref] Directions for the diagnosis, clinical staging, treatment and prevention of canine leishmaniosis, Solano-Gallego [/bib_ref] as observed in humans coinfected with human immunodeficiency virus and Leishmania [bib_ref] The relationship between leishmaniasis and AIDS: the second 10 years, Alvar [/bib_ref].
Several predisposing factors for the development of disease have been described including breed, age and genetic background. Some dog breeds such as the Boxer, Cocker Spaniel, Rottweiler and German Shepherd seem to be more susceptible to the development of disease [bib_ref] Asymptomatic canine leishmaniasis in Greater Athens area, Greece, Sideris [/bib_ref] [bib_ref] Epidemiology of canine visceral leishmaniosis in the endemic area of Montes Claros..., Franca-Silva [/bib_ref] , while others such as the Ibizian Hound rarely develop clinical signs of CanL [bib_ref] The Ibizian hound presents a predominantly cellular immune response against natural Leishmania..., Solano-Gallego [/bib_ref]. The Slc11c1 (Solute carrier family 11 member a1) gene, formerly named N-RAMPI, and certain alleles of the MHC II genes have been associated with susceptibility to CanL [bib_ref] Susceptibility to visceral leishmaniasis in the domestic dog is associated with MHC..., Quinnell [/bib_ref] [bib_ref] Slc11a1 (formerly Nramp1) and susceptibility to canine visceral leishmaniasis, Sanchez-Robert [/bib_ref]. Age seems to be an important factor. The distribution of the disease is bimodal, with the highest prevalence reported in dogs younger than 3 years and older than 8 years [bib_ref] Canine leishmaniasis: pathological and ecological factors influencing transmission of infection, Abranches [/bib_ref] [bib_ref] Serological survey of Leishmania infection in dogs from the municipality of Peso..., Cardoso [/bib_ref].

## Clinical manifestations and laboratory abnormalities
CanL is a systemic disease that may potentially involve any organ, tissue or body fluid and is manifested by nonspecific clinical signs. The most common clinical manifestations and clinicopathological abnormalities found in CanL are listed in [fig_ref] 1: Classical life cycle of Leishmania infantum 1a [/fig_ref] [bib_ref] Canine leishmaniosis -new concepts and insights on an expanding zoonosis: part one, Baneth [/bib_ref] [bib_ref] A retrospective clinical study of canine leishmaniasis in 150 dogs naturally infected..., Ciaramella [/bib_ref]. Skin lesions are the most frequent manifestation among them [fig_ref] Figure 5: Different patterns of cutaneous lesions in CanL [/fig_ref] and may be seen along with other clinical signs or clinicopathological abnormalities. However, dogs can be presented with other clinical signs unrelated to cutaneous lesions as the main presenting complaint [bib_ref] A retrospective clinical study of canine leishmaniasis in 150 dogs naturally infected..., Ciaramella [/bib_ref] [fig_ref] Figure 6: Some clinical signs found in CanL [/fig_ref]. Renal disease may be the sole clinical manifestation of CanL and it can progress from mild proteinuria to the nephrotic syndrome or to an end stage renal disease. Chronic renal failure is a severe result of disease progression and the main cause of mortality due to CanL. Despite the high prevalence of renal pathology in infected dogs [bib_ref] Histopathologic patterns of nephropathy in naturally acquired canine visceral leishmaniasis, Costa [/bib_ref] [bib_ref] Glomerular lesions in dogs infected with Leishmania organisms, Zatelli [/bib_ref] , renal azotemia is relatively an uncommon laboratory finding. The common pathological findings detected by cytology [fig_ref] Figure 7: Interpretation of cytology A [/fig_ref] or histology in CanL [bib_ref] Histological and immunohistochemical study of clinically normal skin of Leishmania infantum-infected dogs, Solano-Gallego [/bib_ref] [bib_ref] Histopathological features of ocular leishmaniosis in the dog, Pena [/bib_ref] [bib_ref] Factors associated with the occurrence of epistaxis in natural canine leishmaniasis (Leishmania..., Petanides [/bib_ref] [bib_ref] Cytologic patterns of lymphadenopathy in dogs infected with Leishmania infantum, Mylonakis [/bib_ref] are listed in [fig_ref] Table 2: Cytological and histopathological patterns suggestive of canine L [/fig_ref]. However, the variable and nonspecific clinical signs make the list of differential diagnoses wide and extensive.

## Diagnosis
The purposes for which diagnosis of L. infantum infection is carried out are outlined in [fig_ref] Figure 8: The [/fig_ref]. Due to these different diagnostic indications, it is important to separate Leishmania infection from disease and to apply different diagnostic techniques for each state. The definitions of sick versus clinically healthy infected dogs are shown in [fig_ref] Figure 9: Definition of L [/fig_ref] [bib_ref] Directions for the diagnosis, clinical staging, treatment and prevention of canine leishmaniosis, Solano-Gallego [/bib_ref].
The diagnosis of CanL is complex as the clinical spectrum is broad and the range of clinicopathological abnormalities based on at least a complete blood count (CBC), biochemical profile and urinalysis can be both wide and non-specific. A thorough clinicopathological diagnostic approach needs to be adapted for each patient when assessing the suspicion of this disease. In addition, dogs with leishmaniosis might be co-infected with other vector borne diseases or suffering from other concomitant infectious or non-infectious diseases making the differential diagnoses more complicated and diverse. Therefore, based on the clinicopathological problem list, a differential diagnosis and specific diagnostic approach would be made for each patient.
Different specific diagnostic methods have been described for the detection of L. infantum infection in dogs and these are shown in [fig_ref] Figure 1: The life cycle of L [/fig_ref]. Valid diagnostic tests are essential for the detection of Leishmania infection in sick dogs although they lack 100% sensitivity and specificity [bib_ref] Directions for the diagnosis, clinical staging, treatment and prevention of canine leishmaniosis, Solano-Gallego [/bib_ref]. The advantages and disadvantages of the different diagnostic methods are summarized in .
The diagnosis of CanL can be made by the detection of specific serum antibodies (IgG) using preferably quantitative serological techniques, such as the immunofluorescence antibody test (IFAT) and enzymelinked immunosorbent assay (ELISA). Immunochromatography-based assays are easy to use and provide rapid qualitative results on the spot, but their performance is still not optimal [bib_ref] Evaluation of enzymelinked immunosorbent assays, an immunofluorescent-antibody test, and two rapid tests..., Mettler [/bib_ref] [bib_ref] Rapid detection of Leishmania infantum infection in dogs: comparative study using an..., Mohebali [/bib_ref] [bib_ref] Evaluation of an ELISA rapid device for the serological diagnosis of Leishmania..., Ferroglio [/bib_ref]. The interpretation of serological qualitative rapid tests is described in [fig_ref] Figure 1: The life cycle of L [/fig_ref]. It is important to submit samples to a laboratory that runs quantitative serological assays and can provide an endpoint titer (IFAT) or an optical density reading (ELISA) and a classification of the level of antibodies [bib_ref] Directions for the diagnosis, clinical staging, treatment and prevention of canine leishmaniosis, Solano-Gallego [/bib_ref]. Detection of Leishmania DNA in tissues by PCR allows sensitive and specific diagnosis of infection. PCR can be performed on DNA extracted from tissues, blood, body fluids or even from histopathologic specimens. The different sensitivities of tissues for the detection of L. infantum by PCR [bib_ref] Directions for the diagnosis, clinical staging, treatment and prevention of canine leishmaniosis, Solano-Gallego [/bib_ref] [bib_ref] Diagnosis of canine leishmaniasis: conventional and molecular techniques using different tissues, Maia [/bib_ref] [bib_ref] Detection of Leishmania infantum DNA by fret-based real-time PCR in urine from..., Solano-Gallego [/bib_ref] and variable sensitivities of PCR techniques are listed in [fig_ref] Figure 1: The life cycle of L [/fig_ref]. Assays based on the detection of kinetoplast DNA (kDNA) appear to be the most sensitive for direct detection in infected tissues [bib_ref] Methods for diagnosis of canine leishmaniasis and immune response to infection, Maia [/bib_ref] [bib_ref] Canine leishmaniosisnew concepts and insights on an expanding zoonosis: part two, Miro [/bib_ref]. Real-time PCR allows quantification of the Leishmania parasite load in the tissues of infected dogs, which is useful for the diagnosis and the follow-up during treatment [bib_ref] Real-time PCR in dogs treated for leishmaniasis with allopurinol, Pennisi [/bib_ref] [bib_ref] Real-time PCR assay in Leishmania-infected dogs treated with meglumine antimoniate and allopurinol, Manna [/bib_ref]. It is important to highlight that information provided by PCR should not be separated from the data obtained from clinicopathological and serological evaluations.
A high level of antibodies confirms the diagnosis of CanL in a dog with clinical signs and/or clinicopathological abnormalities compatible with leishmaniosis [bib_ref] Isotype patterns of immunoglobulins: hallmarks for clinical status and tissue parasite density..., Reis [/bib_ref]. However, the presence of a low antibody level is not necessarily indicative of the disease and further work-up is necessary to confirm or exclude clinical leishmaniosis [bib_ref] Directions for the diagnosis, clinical staging, treatment and prevention of canine leishmaniosis, Solano-Gallego [/bib_ref]. The diagnostic approach for sick dogs living in an endemic area is shown in [fig_ref] Figure 1: The life cycle of L [/fig_ref]. The diagnostic approach for sick or healthy dogs living in a non-endemic area that have travelled to an endemic area, should include quantitative serology three months after the beginning of exposure in the endemic area.

## Clinical staging, treatment and prognosis
A system of four clinical stages based on clinical signs, clinicopathological abnormalities and serological status was proposed by the LeishVet group in an effort to cover the wide spectrum of clinical manifestations and degrees of severity found in CanL [bib_ref] Directions for the diagnosis, clinical staging, treatment and prevention of canine leishmaniosis, Solano-Gallego [/bib_ref]. Different treatment protocols and prognoses are suggested for each clinical stage as described in [fig_ref] Table 4: Clinical staging of canine leishmaniosis based on serological status, clinical signs, laboratory... [/fig_ref].
The most common drugs used for treatment of CanL, including dosage, combinations and side effects, are listed in [fig_ref] Table 5: Current treatment protocols for canine leishmaniosis[27] Meglumine antimoniate* 75-100 mg/kg once a... [/fig_ref]. Several other candidate medications against CanL have been studied in vitro or in laboratory animals but rarely in controlled clinical trials and they are, therefore, currently not recommended for the routine treatment of CanL [bib_ref] Directions for the diagnosis, clinical staging, treatment and prevention of canine leishmaniosis, Solano-Gallego [/bib_ref].
The clinical response to treatment of sick dogs can vary from poor to good depending on their overall initial clinicopathological status and their specific response to therapy. Dogs with renal insufficiency are expected to have a lower recovery rate in comparison to those without kidney compromise or only mild proteinuria. Therapy with antileishmanial drugs often leads to clinical cure [bib_ref] Treatment of canine Old World visceral leishmaniasis: a systematic review, Noli [/bib_ref] although treated dogs may continue to harbour the parasite and be infectious to sand flies, but to a lesser extent than pre-treatment [bib_ref] Real-time PCR assay in Leishmania-infected dogs treated with meglumine antimoniate and allopurinol, Manna [/bib_ref] [bib_ref] Clinical and parasitological evaluation of dogs naturally infected by Leishmania (Leishmania) chagasi..., Ikeda-Garcia [/bib_ref] [bib_ref] Reduced tissue parasitic load and infectivity to sand flies in dogs naturally..., Ribeiro [/bib_ref].
The vast majority of dogs experience clinical improvement within the first month of therapy [bib_ref] Real-time PCR in dogs treated for leishmaniasis with allopurinol, Pennisi [/bib_ref] [bib_ref] Real-time PCR assay in Leishmania-infected dogs treated with meglumine antimoniate and allopurinol, Manna [/bib_ref] [bib_ref] Efficacy of the treatment of dogs with leishmaniosis with a combination of..., Pennisi [/bib_ref] ; however, a longer period of therapy may be required for others before improvement is apparent. The frequencies of monitoring and clinicopathological parameters, including serology, to be followed up during treatment of CanL are summarized in .
The length of allopurinol treatment depends on the severity of the disease, the clinical and parasitological response to treatment and the individual tolerance to this drug. Some extremely susceptible dogs never reach a point that would allow the discontinuation of allopurinol, while others are capable of controlling infection without the need for extremely lengthy treatment [bib_ref] Directions for the diagnosis, clinical staging, treatment and prevention of canine leishmaniosis, Solano-Gallego [/bib_ref]. Allopurinol can be discontinued when the combination of the following criteria is made:
(1) The presence of complete physical and clinicopathological recovery evaluated by a thorough physical examination, CBC, full biochemistry panel and urinalysis.
(2) A marked decrease of antibody levels (to negative or borderline by a quantitative serological assay).
In addition, allopurinol might be discontinued if it is not possible to control or decrease the xanthinuria with low purine diets or by reducing the drug's dosage, to avoid the risk of urolithiasis, if massive xanthine crystalluria is present [bib_ref] Long term follow-up of dogs diagnosed with leishmaniosis (clinical stage II) and..., Torres [/bib_ref].

## Management of clinically healthy infected dogs in endemic areas
The management of clinically healthy infected dogs in areas where CanL is endemic is of great importance for practitioners. The presence of Leishmania DNA in the blood or other tissues of clinically healthy dogs living in endemic areas indicates that these dogs harbour infection [bib_ref] Prevalence of Leishmania infantum infection in dogs living in an area of..., Solano-Gallego [/bib_ref] , but they may never develop clinical disease [bib_ref] Incidence and time course of Leishmania infantum infections examined by parasitological, serologic,..., Oliva [/bib_ref]. In contrast, a high positive antibody titer may indicate that an infected dog is heading towards the development of a widespread infection and future development of clinical disease [bib_ref] Isotype patterns of immunoglobulins: hallmarks for clinical status and tissue parasite density..., Reis [/bib_ref]. Therefore, we recommend using serology alone or the combination of serology with PCR for screening healthy dogs. It is recommended to avoid screening clinically healthy dogs only by PCR.
Healthy dogs should be screened for Leishmania antibodies as an initial indication for the presence of infection if [bib_ref] Directions for the diagnosis, clinical staging, treatment and prevention of canine leishmaniosis, Solano-Gallego [/bib_ref] :
1. They are scheduled to travel or be exported to non-endemic areas [fig_ref] Figure 1: The life cycle of L [/fig_ref] 2. They serve as blood donors [fig_ref] Figure 2: Algorithm describing the selection of blood donors and exclusion of infected dogs [/fig_ref] 3. Their owners wish to have them monitored at least every 12 months for early detection of infection and the potential to develop disease. PCR should be used for the second above indication and as an ancillary test for the first and third above indications.
The management of a clinically healthy seropositive dog and a clinically healthy seronegative and PCR-positive dog is summarized in [fig_ref] Figure 1: The life cycle of L [/fig_ref].
Prevention Current preventative measures are mainly based on the use of veterinary registered products containing synthetic pyrethroids, permethrin, or deltamethrin with a repellent effect against sand flies whose efficacy has been demonstrated both experimentally [bib_ref] Evaluation of the efficacy of a topically administered combination of imidacloprid and..., Miro [/bib_ref] [bib_ref] Repellent efficacy of a combination containing imidacloprid and permethrin against sand flies..., Mencke [/bib_ref] [bib_ref] Protection of dogs from bites of phlebotomine sandflies by deltamethrin collars for..., Killick-Kendrick [/bib_ref] and in field studies [bib_ref] Efficacy of a combination of 10% imidacloprid/50% permethrin for the prevention of..., Otranto [/bib_ref] [bib_ref] Evidence for an impact on the incidence of canine leishmaniasis by the..., Maroli [/bib_ref] [bib_ref] Deltamethrin-impregnated collars for the control of canine leishmaniasis: evaluation of the protective..., Manzillo [/bib_ref] [bib_ref] Prevention of endemic canine vector-borne diseases using imidacloprid 10% and permethrin 50%..., Otranto [/bib_ref] [bib_ref] Effect of insecticideimpregnated dog collars on incidence of zoonotic visceral leishmaniasis in..., Gavgani [/bib_ref]. These products are available in spot on formulation or in a collar form and they reduce the risk of new infections and the biting of sand flies on already infected dogs [bib_ref] Efficacy of a combination of 10% imidacloprid/50% permethrin for the prevention of..., Otranto [/bib_ref] [bib_ref] Evidence for an impact on the incidence of canine leishmaniasis by the..., Maroli [/bib_ref] [bib_ref] Deltamethrin-impregnated collars for the control of canine leishmaniasis: evaluation of the protective..., Manzillo [/bib_ref] [bib_ref] Prevention of endemic canine vector-borne diseases using imidacloprid 10% and permethrin 50%..., Otranto [/bib_ref] [bib_ref] Effect of insecticideimpregnated dog collars on incidence of zoonotic visceral leishmaniasis in..., Gavgani [/bib_ref]. Other measures useful in the prevention of sand fly bites include [bib_ref] Control of phlebotomine sandflies, Alexander [/bib_ref] [bib_ref] Guidelines for prevention of leishmaniasis in dogs, Maroli [/bib_ref] : 1) keeping the dog indoors during the sand fly season from dusk to dawn; 2) reducing microhabitats favourable to sand flies such as piles of wood and stones in the vicinity of the house and in other locations where dogs spend time; 3) usage of indoor insecticide treatment [bib_ref] Directions for the diagnosis, clinical staging, treatment and prevention of canine leishmaniosis, Solano-Gallego [/bib_ref].
Long-acting topical insecticides evaluated in several published field studies [bib_ref] Efficacy of a combination of 10% imidacloprid/50% permethrin for the prevention of..., Otranto [/bib_ref] [bib_ref] Evidence for an impact on the incidence of canine leishmaniasis by the..., Maroli [/bib_ref] [bib_ref] Deltamethrin-impregnated collars for the control of canine leishmaniasis: evaluation of the protective..., Manzillo [/bib_ref] [bib_ref] Prevention of endemic canine vector-borne diseases using imidacloprid 10% and permethrin 50%..., Otranto [/bib_ref] [bib_ref] Effect of insecticideimpregnated dog collars on incidence of zoonotic visceral leishmaniasis in..., Gavgani [/bib_ref] should be applied to dogs living or travelling to endemic areas as follows:
-Permethrin/Imidacloprid spot on formulation: Treatment provides repellent (anti feeding) activity against sand flies (P. perniciosus) for three weeks [bib_ref] Evaluation of the efficacy of a topically administered combination of imidacloprid and..., Miro [/bib_ref]. Repeat administration every 3 weeks. It should be applied at least 2 days before travelling -Deltamethrin collars: Control of feeding by phlebotomine sand flies (P. perniciosus) for a period of 5-6 months [bib_ref] Protection of dogs from bites of phlebotomine sandflies by deltamethrin collars for..., Killick-Kendrick [/bib_ref]. Replace collar every 5-6 months. It should be applied at least 1-2 weeks before travelling.
Veterinarians and dog owners are advised to carefully check the product's label recommendations and follow the manufacturer's instructions for the correct application and frequency of reapplication. Client education on the maintenance of an appropriate insecticide [bib_ref] Directions for the diagnosis, clinical staging, treatment and prevention of canine leishmaniosis, Solano-Gallego [/bib_ref] throughout the period of sand fly activity in the Mediterranean basin (April-November) is also crucial for the protection of dogs [bib_ref] Seasonal phenology, host-blood feeding preferences and natural Leishmania infection of Phlebotomus perniciosus..., Rossi [/bib_ref].
Purified Leishmania fraction vaccines appear currently to be the most effective and promising vaccines for dogs. These include the ''fucose mannose ligand'' (FML)-based vaccine [bib_ref] FML vaccine against canine visceral leishmaniasis: from second-generation to synthetic vaccine, Palatnik-De-Sousa [/bib_ref] and an excreted/secreted antigen purified from specific-medium culture supernatant of L. infantum based vaccine [bib_ref] Long-lasting protection against canine visceral leishmaniasis using the LiESAp-MDP vaccine in endemic..., Lemesre [/bib_ref]. The FML-based vaccine is currently available commercially in Brazil. The same vaccine has also been proposed as a transmission-blocking vaccine [bib_ref] The FML-vaccine (Leishmune) against canine visceral leishmaniasis: a transmission blocking vaccine, Saraiva [/bib_ref]. Recently, another vaccine which contains the recombinant A2 protein and saponin as adjuvant has also been approved in Brazil [bib_ref] Protective immunity against challenge with Leishmania (Leishmania) chagasi in beagle dogs vaccinated..., Fernandes [/bib_ref]. In Europe, a different vaccine based on cultured L. infantum purified excreted/secreted antigens has been approved for vaccination of dogs [bib_ref] Long-lasting protection against canine visceral leishmaniasis using the LiESAp-MDP vaccine in endemic..., Lemesre [/bib_ref].
The future for CanL control should be an integrated approach to prevention including vaccination against L. infantum with an effective canine vaccine and the use of long-acting topical insecticide applications. A vaccine would prevent the establishment of infection introduced by the bites of those sand flies that escape the insecticide effect [bib_ref] Canine leishmaniosisnew concepts and insights on an expanding zoonosis: part two, Miro [/bib_ref].

## Public health considerations
In Southern Europe, human visceral leishmaniosis caused by L. infantum is a zoonotic disease that affects young children or adults suffering from the Acquired Immune Deficiency Syndrome (AIDS) or immunosuppressive conditions [bib_ref] Leishmaniases in Northern Greece: seroprevalence of the infection and incidence of the..., Diza [/bib_ref]. Dogs are considered the most important peridomestic reservoir of L. infantum infection for humans. However, the ownership of an infected dog does not appear to greatly increase the risk Advantages and disadvantages of common diagnostic methods for the detection of L. infantum infection in dogs of disease in the family when transmission is already present in the region [bib_ref] Canine leishmaniosisnew concepts and insights on an expanding zoonosis: part two, Miro [/bib_ref].

# Conclusions
The complexity of CanL and the wide range of its clinical manifestations, from inapparent infection to severe disease, make the management of CanL challenging. Diagnosis is performed based on clinicopathological manifestations and by confirmation of infection using mainly serological and molecular techniques. A staging system that divides the disease into four stages is aimed at assisting the clinician in determining the appropriate therapy, forecasting prognosis, and implementing follow-up steps required for the management of the leishmaniosis patient. Prevention should be an integrated approach including vaccination against L. infantum with Follow IRIS guidelines for CKDPoor *Dogs with negative to medium positive antibody levels should be confirmed as infected by other diagnostic techniques such as cytology, histology, immunohistochemistry or PCR. High levels of antibodies, defined as a 3-4 fold elevation above the cut off level of a well established reference laboratory, are conclusive of a diagnosis of CanL. **Dogs in stage I (mild disease) are likely to require less prolonged treatment with one or two combined drugs or alternatively monitoring with no treatment. However, there is limited information on dogs in this stage and, therefore, treatment options remain to be defined. [fig_ref] Figure 1: The life cycle of L [/fig_ref] Management of Leishmania-seropositive but clinically healthy dogs and PCR-positive but seronegative dogs. Clinically healthy but seropositive dogs would normally present with low antibody titers and should be confirmed by retesting. Confirmed seropositive dogs should be monitored with physical examinations, routine laboratory tests and serological tests on a regular basis every 3-6 months to assess the progression of infection towards disease. Treatment of canine leishmaniosis -recommended monitoring of clinicopathological parameters and serology including frequency of follow up [bib_ref] Directions for the diagnosis, clinical staging, treatment and prevention of canine leishmaniosis, Solano-Gallego [/bib_ref] Parameters Frequency
Clinical history and complete physical examination Routine laboratory tests:
Complete CBC, biochemical profile, serum electrophoresis (optional) and complete urinalysis including UPC in proteinuric dogs.
After the first month of treatment and then every 3-4 months during the first year. Later on, if the dog is fully recovered clinically with treatment, a recheck would be recommended every 6 months or once a year. Serology* Not before 6 months after initial treatment and every 6 months or once a year thereafter.
Real time PCR Can optionally be carried out at the same time as serology. The full usefulness of this assay for follow up during treatment is currently undetermined.
*Some dogs present a significant decrease in antibody levels (more than a two-fold dilutions difference between the first and the following samples) associated with clinical improvement within 6 months to 1 year of treatment. Other dogs might not have a decrease in antibody levels despite clinical improvement. In contrast, a marked increase of antibody levels (more than two-fold elevation between monitoring samples) should be interpreted as a marker of relapse, especially in dogs following the discontinuation of treatment [bib_ref] Directions for the diagnosis, clinical staging, treatment and prevention of canine leishmaniosis, Solano-Gallego [/bib_ref].
[fig] Figure 1: The life cycle of L. infantum with indication of proven and unproven non-sandfly routes of transmission to dogs. [/fig]
[fig] Figure 2: Algorithm describing the selection of blood donors and exclusion of infected dogs. Any dog infected will be excluded. [/fig]
[fig] Figure 3: The distribution of canine L. infantum infection in Europe. [/fig]
[fig] Figure 4: Clinical manifestations and immunological characteristics of L. infantum infection in dogs. [/fig]
[fig] Figure 5: Different patterns of cutaneous lesions in CanL: A) Exfoliative periocular alopecia and blepharitis; B) Ulcerative nasal mucocutaneous lesions; C) Papular dermatitis in the inguinal region; D) Nodular crateriform lesions bordering the muzzle; E) Ulcerative erythematous lesions on the plantar surface of the paw and between pads; F) Onychogryphosis. [/fig]
[fig] Figure 6: Some clinical signs found in CanL: A) Epistaxis; B) Bilateral uveitis and corneal opacity; C) Purulent conjunctivitis and blepharitis; D) Exfoliative alopecia in the rear leg and popliteal lymphadenomegaly; E) Marked cachexia and generalized exfoliative alopecia. [/fig]
[fig] Figure 7: Interpretation of cytology A) Interpretation of cytology requires time and expertise for the detection of Leishmania amastigotes when parasites are in low numbers and freed from the cells. Note the nucleus (N) and the kinetoplast (K) of extracellular amastigotes (arrows) in a fine needle aspirate of a reactive lymph node from a dog with clinical leishmaniosis (x100, Diff-quick stain); B) High numbers of intracellular and extracellular Leishmania amastigotes in a fine needle aspirate of a reactive lymph node from a dog with clinical leishmaniosis (x100, modified Giemsa stain). [/fig]
[fig] : Macrophagic inflammation (granulomatous) Neutrophilic-macrophagic inflammation (pyogranulomatous) Lymphoplasmacytic inflammation Reactive hyperplasia in lymphoid organs No evidence or variable numbers of intracellular or extracellular Leishmania amastigotes [/fig]
[fig] Figure 8: The [/fig]
[fig] Figure 9: Definition of L. infantum-infected but healthy versus sick dogs. Dogs with clinical leishmaniosis are defined as those presenting clinical signs and/or clinicopathological abnormalities and having a confirmed L. infantum infection. Dogs with subclinical infection, or clinically healthy but infected dogs, are defined as those that present neither clinical signs on physical examination nor clinicopathological abnormalities by routine laboratory tests (CBC, biochemical profile and urinalysis) but have a confirmed L. infantum infection. [/fig]
[table] 1: Classical life cycle of Leishmania infantum 1a. Promastigote 1b. Amastigote 1c. Parasite dissemination to organs in macrophages 2. Other unusual modes of transmission 2a. Vertical 2b. Blood transfusion 2c. Venereal transmission Other (unproven): Dog to dog (bites, wounds) [/table]
[table] Table 2: Cytological and histopathological patterns suggestive of canine L. infantum infection found in organs or body fluids. [/table]
[table] Table 4: Clinical staging of canine leishmaniosis based on serological status, clinical signs, laboratory findings, and type of therapy and prognosis for each stage[27] Dogs with clinical signs listed in stage III. Pulmonary thromboembolism, or nephrotic syndrome and end stage renal disease Clinicopathological abnormalities listed in stage II CKD IRIS stage III (creatinine 2-5 mg/dl) and stage IV (creatinine > [/table]
[table] Table 5: Current treatment protocols for canine leishmaniosis[27] Meglumine antimoniate* 75-100 mg/kg once a day or 40-75 mg/kg twice a day for 4 weeks, S.C.***Registered for veterinary use in most European countries; both drugs are commonly recommended in combination with allopurinol. P.O.: per os; S.C.: subcutaneous **Treatment prolongation by 2-3 weeks may be considered if patient improvement is insufficient. an effective canine vaccine and the application of a topical insecticide. of abbreviations AIDS: Acquired Immune Deficiency Syndrome; CanL: Canine leishmaniosis; CBC: Complete blood count; CKD: Chronic kidney disease); DNA: Deoxyribonucleic acid; ELISA: Enzyme-linked immunosorbent assay; FML: Fucose mannose ligand; IFAT: Immunofluorescence antibody test; IgG: Immunoglobulin G; IRIS: International Renal Interest Society; kDNA: Kinetoplast DNA; MHC II: Major histocompatibility complex type II; N-RAMPI: Natural Resistance-associated Macrophage Protein one; PCR: Polymerase chain reaction; P.O.: per os; S.C.: subcutaneous; Slc11c1: Solute carrier family 11 member a1; UPC: Urinary protein creatinine ratio; USA: United States of America [/table]
 [bib_ref] Tissue distribution of Leishmania chagasi and lesions in transplacentally infected fetuses from..., Pangrazio [/bib_ref]