Leishmaniosis in dogs and cats

Transcription

1 Paolo Silvestrini qualified from the University of Perugia, Italy, in 2002 and spent three years working in private practice in Rome. In 2007 he gained the certificate of Specialist in small animal clinic and pathology from the University of Pisa, Italy. He completed an 18-month internship at the Universitat Autónoma de Barcelona in Spain, followed by a three-year residency in internal medicine at the same university. He is now senior lecturer in small animal internal medicine at the University of Liverpool. He is a diplomate of the European College of Veterinary Internal Medicine and holds RCVS recognised Specialist status in small animal internal medicine. In 2016 he completed a PhD focusing on novel clinicopathological aspects of canine leishmaniosis. doi: /inp.k5122 Leishmaniosis in dogs and cats Paolo Silvestrini Canine leishmaniosis is pushing northwards out of its traditional endemic regions as the distribution of its vector expands and increasing numbers of dogs travel between countries. In non-endemic areas, imported disease represents a threat to animal and human health. This article provides an understanding of the epidemiology, life cycle and transmission of Leishmania infantum, reviews its diagnosis and treatment, and briefly discusses the current understanding of feline leishmaniosis. CANINE leishmaniosis (CanL) is caused by the protozoan parasite Leishmania infantum (syn Leishmania chagasi). It is endemic in more than 70 countries throughout the world (Solano-Gallego and others 2011), including those of the Mediterranean basin, Portugal, Latin America and southern Asia. However, CanL is also an important concern in non-endemic countries where imported disease constitutes a veterinary and public health problem. Dogs are the main animal reservoir for human visceral leishmaniosis and, in people, the disease is usually fatal if not treated. Susceptible dogs commonly develop both visceral and cutaneous forms and if left untreated they generally die or are euthanased due to progressive severe renal disease. Cats have been considered as a relatively resistant host species but several studies in the past decade have confirmed that feline leishmaniosis may be relatively common in areas where CanL is endemic (Box 1). Epidemiology, life cycle and transmission CanL is endemic in the Mediterranean areas of Europe (Cyprus, Greece, Albania, Croatia, Italy, Malta, France, Spain) and Portugal, the Middle East, Latin America and southern Asia (Maia and Cardoso 2015). Canine infection rates approach 70 per cent to 90 per cent, as shown by PCR and serology, in highly endemic foci, such as the Balearic Islands of Spain (Solano-Gallego and others 2001), the Marseille area in France (Berrahal and others 1996), throughout Greece (Leontidas and others 2002) and the Naples area in Italy (Oliva and others 2006). At least 2.5 million dogs are infected in south-western Europe alone (Moreno and Alvar 2002). The infection is spreading to non-endemic areas, such as North America and northern Europe, probably due to wider distribution of its vector and especially to larger numbers of dogs travelling from/ to endemic countries. Leishmania species completes its life cycle in two hosts, a phlebotomine sandfly vector, which transmits the flagellated infective promastigote form (Fig 1a), and a mammal, where the amastigote form (Fig 1b) develops and replicates inside the host s macrophages. Female sandflies of the genus Phlebotomus in the Old World and Lutzomyia in the New World are the main vectors of CanL but other modes of transmission are possible. In utero transmission from an infected dam to its offspring and venereal transmission from infected males to healthy bitches have been documented (da Silva and others 2009). Transmission by haematophagus arthropods other than sandflies has been suspected but is not yet proven to have an epidemiological significance. Rhipicephalus sanguineous ticks have been shown to acquire Leishmania species in their gut after Box 1: Feline leishmaniosis Feline leishmaniosis (FeL) has often been underestimated since clinical illness due to Leishmania infantum in cats is rare and cats have been generally considered a relatively resistant host species. However, subclinical feline infections are common in areas endemic for canine leishmaniosis. The prevalence rates of feline infection in serological and/or molecular-based surveys range from 0 per cent to more than 60 per cent (Pennisi and others 2015). Sandflies are able to acquire the parasite from cats and, therefore, cats may act as a secondary reservoir. Cutaneous lesions such as dermal nodules, ulceration and crusts are predominant in FeL and are often localised on the head, nose and lips or on the distal parts of the limbs. Currently, there is no strong evidence regarding the best treatment options for FeL, but the majority of cases have been treated with variable success with allopurinol alone. Prognosis varies from good to poor and dissemination of Leishmania amastigotes to the spleen, lymph nodes, liver and gastrointestinal tract is often associated with clinical deterioration and death. No vaccines against Leishmania are available for cats and the single preventive strategy is topical insecticides. Unfortunately, most pyrethroids cannot be used in cats due to their toxicity in this species. feeding on infected dogs (Coutinho and others 2005). Blood transfusion is another possible route of transmission and canine blood donors living in endemic countries should be routinely tested for CanL (Owens and others 2001, Giger and others 2002, Tabar and others 2008). Direct dog-to-dog transmission has also been suspected, especially in those areas where sandflies are not present (Duprey and others 2006). Pathogenesis and clinical signs CanL is the classical example of a disease where the clinical signs and underlying pathology are intrinsically related to the interaction between the parasite (virulence), arthropod vector (repeated infectious bites) and host (genetic background, immune response, coexisting diseases). Leishmania species has three general pathogenic features: The parasite s target cell is the macrophage, which is the site for its replication; Establishment of infection and evolution of the disease both depend on the host s immunological responses; Once established, the infection usually persists in tissues. In Practice January/February 2019 Volume

2 (a) (b) (a) Fig 1: (a) Infective promastigote and (b) amastigote forms of Leishmania species Leishmania species tend to localise in all tissues in which monocytic-macrophagic cells exist in high numbers. Not all dogs infected with L infantum will eventually develop clinical leishmaniosis (Killick-Kendrick and others 1994, Pinelli and others 1995). Dogs that respond to infection with a T cell-mediated immunity (T helper 1; Th1) will remain asymptomatic (resistant dogs) while those that respond predominantly with a humoral immunity (T helper 2; Th2) will develop clinical signs (susceptible dogs). However, subclinical infection is not necessarily permanent, and factors such as immunosuppressive conditions or concomitant disease can break the equilibrium and lead to the progression of clinical disease. (c) (b) Fig 2: (a) Exfoliative and ulcerative dermatitis and (b,c) vasculitis and ischaemia on the extremities in a dog with leishmaniosis The usually prolonged incubation period, which can extend from three months to seven years after infection, may explain the inconsistent and variable transition from a resistant to a susceptible state (Baneth and Solano-Gallego 2012). There is a strong genetic basis influencing susceptibility and resistance to CanL. Boxers, cocker spaniels, rottweilers and German shepherd dogs are more susceptible to developing signs of leishmaniosis in contrast to Ibizan hounds, in which clinical disease is rather rare due to the predominant cell-mediated Th1- type immune response (Sideris and others 1999, Solano- Gallego and others 2000, Franca-Silva and others 2003). Age seems to be another important risk factor and clinical disease generally has two peaks, one in young dogs (two to four years old) and another in older dogs (more than seven years old) (Miranda and others 2008). CanL can potentially involve any organ, tissue and/or biological fluid, and can manifest with a wide range of clinical signs. The proliferation of B lymphocytes, plasma cells, histiocytes and macrophages results in generalised lymphadenomegaly, splenomegaly and hyperglobulinaemia. This last response is not protective but detrimental, with the generation of autoantibodies and circulating immune complexes, which can consequently deposit in various tissues and organs, resulting in glomerulonephritis, vasculitis, uveitis, myositis and polyarthritis. Renal disease is recognised as the main cause of death or euthanasia in dogs with leishmaniosis (Planellas and others 2009) and generally progresses from mild azotaemia and proteinuria to nephrotic syndrome and end-stage kidney disease. History and physical examination may reveal reduced appetite or anorexia, lethargy, emaciation, cachexia, peripheral lymphadenomegaly, exercise intolerance, skin lesions, temporal muscle atrophy, splenomegaly, polyuria/polydipsia, epistaxis, ocular lesions, onychogryphosis, lameness, vomiting and diarrhoea, which appear alone or, more often, in various combinations. Skin lesions occur in 80 per cent to 90 per cent of cases (Ciaramella and others 1997, Koutinas and others 1999). They are rarely pruritic and include exfoliative dermatitis with focal or multifocal alopecia, generally localised on the face, ears and limbs; ulcerative dermatitis over bony prominences and in mucocutaneous junctions, paws and the ear pinnae; and focal or multifocal nodular dermatitis (Fig 2) (Ordeix and others 2005). Ocular lesions are also common and can include uveitis, conjunctivitis, keratoconjunctivitis sicca, blepharitis, glaucoma and a combination of these (Fig 3). Dogs with leishmaniosis may show signs of a haemorrhagic diathesis, manifest primarily as epistaxis, and less commonly as haematuria and haemorrhagic diarrhoea. The leading causes of epistaxis, which may be acute or chronic/ recurrent, unilateral or bilateral, and sometimes severe enough to cause anaemia due to uncontrollable blood loss, are consistent with thrombocytopathy, increased serum viscosity due to hyperglobulinaemia, and rhinitis, ulcerative or not (Mylonakis and others 2008, Petanides and others 2008). Vasculitis may also contribute in some cases, causing bleeding ulcerations of the nasal philtrum and/ or nostrils. Anaemia usually develops as a sequel to the decreased erythropoiesis of chronic disease or chronic kidney disease but may be aggravated by blood loss. Less commonly, CanL has been recognised as the cause of myositis (Paciello and others 2009, Vamvakidis and others 2000), erosive or non-erosive mono- or polyarthritis, tongue nodules and papules or multifocal to diffuse ulcerative glossitis and stomatitis, chronic hepatitis and chronic colitis, pericarditis, myocarditis and pneumonia (Font and others 1993, Torrent and others 2005), meningoencephalomyelitis (Vinuelas and others 2001, Font and others 2004, Jose-Lopez and others 2012), orchitis, epididymitis, 6 In Practice January/February 2019 Volume

3 Fig 3: (a) Conjunctivitis and blepharitis and (b) uveitis (a) chronic prostatitis, penile granulomatous disease and balanoposthitis (Diniz and others 2006, Manna and others 2012, Mir and others 2012). Diagnosis The diagnosis of CanL is difficult and complex because of the spectrum of clinical signs and laboratory abnormalities, the high prevalence of subclinical infection and coinfections with other vectorborne diseases (Baneth and others 2008) and, more recently, the use of vaccines in endemic areas. The main purpose for which diagnosis of CanL is carried out is to confirm disease in affected dogs. However, it is also important to investigate possible subclinical infection in clinically healthy dogs living in endemic areas, including blood donors, breeding dogs and dogs before vaccination; in dogs heading towards disease progression; and in clinically healthy dogs in non-endemic areas (travelling dogs), to avoid importing infected dogs to non-endemic regions and to monitor response to treatment (Solano-Gallego and others 2009, 2011). The diagnostic investigation should always be based on an integrated approach considering signalment, history, clinical findings and the results of basic blood and urine analysis and of more specific diagnostic tests. Table 1 summarises the laboratory findings generally associated with CanL (Paltrinieri and others 2010). Diagnostic techniques Numerous techniques have been developed to help in the diagnosis of CanL. The detection of L infantum infection in dogs includes parasitological (cytology, histology, immunochemistry and culture of the organism), molecular (conventional, nested and real-time PCR) and serological methods (qualitative and quantitative antibody tests). Cutaneous lesions, bone marrow, lymph nodes, and spleen, and, less commonly, other tissues or body fluids such as joint, cerebrospinal and abdominal fluids, are good choice samples to observe Leishmania species amastigotes in both cytological and histological specimens (Solano-Gallego and others 2009). Histopathology Definitive histopathological identification of parasites within tissue macrophages may be difficult and an immunohistochemical staining method can be used. PCR on bone marrow, lymph node, spleen or skin has a high sensitivity and specificity, and quantitative real-time PCR allows the quantification of Leishmania species loads in tissues of infected dogs, which is important for diagnosis as well as for follow-up during the treatment. (b) Serology Various serological methods have been used to detect serum anti-leishmania-specific IgG antibodies, including indirect fluorescent antibody tests (IFAT), ELISA, immunochromatography with rapid in-house devices, direct agglutination assays and Western blotting. Generally, IFAT, ELISA and the rapid in-house kits are the most commonly employed methods (Bourdeau and others 2014). The IFAT is considered the reference method for anti- Leishmania serology in dogs (Gradoni and Gramiccia 2008, EFSA AHAW Panel 2015) based on its high sensitivity and specificity (near 100 per cent for both) except in areas endemic for Trypanosoma cruzi where it may give false positive results. ELISA is also very sensitive and specific (near 100 per cent for both) when a combination of immunodominant recombinant proteins is used as antigen; it has slightly lower specificity when crude parasite lysates are employed instead (Maia and Campiono 2008, Rodríguez- Cortés and others 2010, Solano-Gallego and others 2014). Immunochromatographic rapid in-house tests have a quite acceptable specificity, but their sensitivity is usually low (in the range of 30 to 70 per cent) and largely dependent on the stage of infection (Paltrinieri and others 2016). Lowest sensitivity is associated with infected dogs without clinical signs, while highest sensitivity is seen for dogs with overt disease. Various in-house serological tests are commercially available and are particularly attractive to practising veterinarians because they give immediate results (Bourdeau and others 2014). However, their major disadvantage is that they are qualitative and thus any positive result needs to be followed by a quantitative test. High antibody concentrations in a non-vaccinated dog with compatible clinical signs and clinicopathological abnormalities are almost diagnostic of the disease (Solano- Gallego and others 2016). However, the presence of lower antibody levels is not necessarily indicative of latent disease and needs to be confirmed by other diagnostic methods such as PCR, cytology or histology. Moreover, the interpretation of serological titres should take into consideration the possibility of the presence of antibodies elicited by vaccines since these have been introduced as preventive strategies against CanL in endemic areas. Serological assays based on the detection of antibodies reactive with recombinant proteins and rapid in-house tests are usually less sensitive for the recognition of antibodies elicited by vaccination than those based on wholeparasite antigens and quantitative serological techniques (Marcondes and others 2013, Moreno and others 2014, Starita and others 2016). The rapid serological test Speed Photograph: Xavier Roura In Practice January/February 2019 Volume

4 Table 1: Laboratory test results associated with canine leishmaniosis (CanL) (based on Paltrinieri and others 2010) Basic test Findings Additional tests* Haematology Poorly regenerative or non-regenerative anaemia Possible regenerative anaemia (due to an immune-mediated process) Neutrophilia and monocytosis with lymphopenia and eosinopenia Leukopenia Possible thrombocytopenia Leish K (Virbac) detects circulating antibodies against kinesins of L infantum and might be used to distinguish between vaccinated and naturally infected dogs. However, its diagnostic performance has been variable in some studies (Montoya and others 2017). It therefore remains important to develop new serological techniques that are able to discriminate between naturally occurring antibodies and antibodies elicited by vaccination; currently, if there is a recent history of vaccination then other diagnostic techniques based on observation of lesions (cytology/histology) and detection of parasites or parasite DNA (PCR) should be employed. Therapy and follow up CanL is more resistant to therapy than human leishmaniosis, and only rarely are Leishmania organisms completely eliminated with available drugs (Baneth and Shaw 2002, Noli and Auxillia 2005). Relapses necessitating retreatment are common, although dogs frequently become cured of the clinical disease. In addition, in endemic areas, reinfections occur and contribute to apparent treatment failure. In order to prevent a recurrence of CanL, parasitostatic drugs, such as allopurinol, are usually combined with leishmanicidal therapy and continued for several months or years beyond apparent clinical cure (Baneth and Shaw 2002, Noli and Auxillia 2005). Allopurinol can be stopped only when all the following conditions are met: Complete clinical recovery; Clinicopathological normalisation; Antibody levels negative or below the test s cut-off level (Solano-Gallego and others 2009, Martinez and others 2011). The most commonly prescribed dosages for allopurinol range between 5 and 20 mg/kg every 12 hours. Use of Coombs test or flow cytometry to detect antibodies against red blood cells Bone marrow cytology aptt, PT, FDPs, AT, D-dimers to rule in/out disseminated intravascular coagulation; tests for coinfection (eg, with Ehrlichia canis); flow cytometry to detect antibodies against PLTs Basic coagulation profile Hyperfibrinogenaemia and increased PT and aptt Extended coagulation profile (FDPs, AT, D-dimers) Serum biochemistry Serum protein electrophoresis Urinalysis Hyperproteinaemia, hypoalbuminaemia, hyperglobulinaemia, reduced albumin:globulin ratio Azotaemia (increased urea and/or creatinine) Increased hepatic enzymes Hypoalbuminaemia, increased a2-globulin concentration, polyclonal or oligoclonal gammapathy Isothenuria (specific gravity to 1.012) or poorly concentrated urine (<1.030) Proteinuria (determined by dipstick test and UPC) Acute phase proteins (CRP, haptoglobin, SAA) Lipid concentrations (hypocholesterolaemia) Electrolyte concentrations (hypokalaemia) Mineral concentrations (hyperphosphataemia and hypermagnesaemia) Blood gas analysis (metabolic acidosis) Liver function tests Acute phase proteins (CRP, haptoglobin, SAA) SDS-AGE of urine to detect evidence of mixed or glomerular proteinuria *To be performed for a more complete staging system, if findings of initial tests are consistent with CanL aptt Activated partial thromboplastin time, AT Antithrombin III, CRP C-reactive protein, FDPs Fibrin or fibrinogen degradation products, PLTs Platelets, PT Prothrombin time, SAA Serum amyloid A, UPC Urine protein:creatinine ratio, SDS-AGE SDS-agarose gel electrophoresis allopurinol causes hyperxanthinuria, which may produce urolithiasis in about 12 per cent of treated dogs (Torres and others 2011). N-methyl-glucamine (meglumine) antimoniate is the most used pentavalent antimony compound for treating leishmaniosis in dogs and people. The drug selectively inhibits leishmanial glycolysis and fatty acid oxidation, causing the subsequent death of the parasite. Treatment with meglumine antimoniate induces a generalised reduction of the parasite load, together with a temporary restoration of cell-mediated immunological response. Pain and swelling of the injection site are the most common adverse effects. Fever, diarrhoea and loss of appetite have been also reported (Denerolle and Bourdoiseau 1999). To date, there has been no evidence of renal damage induced by antimonials in dogs. The most commonly reported treatment regimen is 100 mg/kg subcutaneously once daily for four weeks. Miltefosine at 2 mg/kg orally once daily for four weeks combined with allopurinol is an alternative first-line protocol. Miltefosine was originally developed as an antineoplastic agent and is able to kill parasites in vitro and in vivo by disturbing signalling pathways and cell membrane synthesis, thus leading to apoptosis (Verma and Dey 2004, Farca and others 2012). The side effects usually include vomiting, seen in about 11 to 23 per cent of treated dogs (Mateo and others 2009, Woerly and others 2009, Andrade and others 2011). The combination of miltefosine and allopurinol is clinically as effective as the standard protocol based on meglumine antimoniate and allopurinol. In one study (Miró and others 2009), a significant reduction in clinical scores and parasite load was observed in both groups with no significant differences. Dogs should be re-evaluated after one, three and six 8 In Practice January/February 2019 Volume

5 months of therapy and then once every six months for life. Each evaluation should include complete physical examination, haematology, biochemistry profile, serum protein electrophoresis and urinalysis with urine protein:creatinine ratio to quantify the level of proteinuria. After one month, dogs responding to therapy and showing clinical improvement will have a significant reduction of total proteins and globulins and an increase of albumin on serum protein electrophoresis. In contrast, the serological titre will remain elevated and a significant decrease will be evident only around the third month after the start of therapy. It is therefore recommended that serology should be rechecked after three and six months of therapy (omitting serology at the one month check) to document a progressive reduction in the titre. Real-time PCR can help monitor the response to therapy and identify relapses. Table 2 summarises clinical staging of CanL based on serological status, clinical signs and laboratory findings, and the types of therapy and prognosis for each clinical stage (Solano-Gallego and others 2017). Prevention There are two predominant methods to prevent CanL: Vector control through the use of insect repellent to avoid infectious bites; Chemotherapeutic and/or immunological control using leishmanicidal/leishmaniostatic drugs, immunomodulators and vaccines. As discussed above, sandflies are the primary route of infection in endemic areas, so the first line of control is the blockade of transmission though the use of insect repellents. Multiple studies have evaluated the efficacy of various active ingredients and compounding, including collars, spot-ons and sprays. Immunotherapy is an expanding area of research in which new and old molecules have been tested. Given that CanL dramatically and negatively modulates the host s immune system, the main objective of immunotherapy is to help the host s immune system to control the infection. Domperidone is a gastric prokinetic and antiemetic drug, acting as a dopamine D2 receptor antagonist, that is able to stimulate the production of serotonin, which in turn increases prolactin secretion. Prolactin, besides its well-known role in milk production, is an important proinflammatory lymphocyte-derived cytokine that is able to stimulate the cell-mediated Th1 lymphocyte-driven immune response. Domperidone has been licensed for the veterinary market with an indication for the treatment and prevention of CanL at the dose of 0.5 mg/kg orally once daily for one month, repeated every three months (Sabaté and others 2014). Three commercial vaccines Leishmune (Fort Dodge), Leish-Tec (Hertape Calier Saúde Animal) and CaniLeish (Virbac) have been licensed for the control of CanL, the first two in Brazil, and the third in Europe. The European Medicines Agency has authorised a new vaccine product (Letifend; Laboratorios LETI) for the European market, while the Brazilian health authorities have withdrawn Leishmune. Table 3 summarises the main vaccine products for CanL in South America and Europe (Solano- Gallego and others 2017). Past and currently marketed vaccines appear to elicit a long-lasting, parasite-specific, cellular immunity Table 2: Clinical staging of canine leishmaniosis based on serological status, clinical signs and laboratory findings, and types of therapy and prognosis for each clinical stage (based on Solano-Gallego and others 2017) Clinical stage Serology* Clinical signs Laboratory findings Therapy Prognosis Stage I Mild disease Stage II Moderate disease Stage III Severe disease Stage IV Very severe disease Negative to low positive Low to high positive Medium to high positive Medium to high positive Dogs with clinical signs such as solitary lymphadenomegaly or papular dermatitis Dogs that, apart from the signs listed in Stage I, may present with diffuse or symmetrical cutaneous lesions such as exfoliative dermatitis/onychogryphosis, ulcerations, generalised lymphadenomegaly, loss of appetite, weight loss Dogs that, apart from the signs listed in Stage I and II, may present signs originating from immune complex lesions (eg, uveitis and glomerulonephritis) Dogs with clinical signs listed in Stage III. Pulmonary thromboembolism, or nephritic syndrome and end-stage renal disease Usually no clinicopathological abnormalities Mild nonregenerative anaemia, hypergammaglobulinaemia, hypoalbuminaemia, serum hyperviscosity syndrome Substage: Normal renal profile: creatinine <124 μmol/l; non-proteinuric UPC <0.5 Creatinine <124 μmol/l; UPC = 0.5 to 1 Clinicopathological abnormalities listed in Stage II CKD IRIS Stage I with UPC = 1 to 5 or Stage II (creatinine 124 to 180 μmol/l) Clinicopathological abnormalities listed in Stage II CKD IRIS Stage III (creatinine 181 to 440 μmol/l) and Stage IV (creatinine >440 μmol/l) or nephritic syndrome: marked proteinuria UPC >5 Scientific neglect or monitoring of disease progression Allopurinol + meglumine antimoniate or miltefosine Allopurinol + meglumine antimoniate or miltefosine Follow IRIS guidelines for CKD Specific treatment should be tailored to the individual Follow IRIS guidelines for CKD *Dogs with negative to medium positive antibody levels should be confirmed as infected with other diagnostic techniques such as cytology, histology/ immunohistochemistry and PCR. Good Good to guarded Guarded to poor Dogs in Stage I (mild disease) are likely to require less prolonged treatment with one or two combined drugs (allopurinol, domperidone, meglumine antimoniate or miltefosine) or alternatively monitoring with no treatment. There is limited information on dogs in this stage and, therefore, treatment options remain to be defined CKD Chronic kidney disease, IRIS International Renal Interest Society, UPC Urine protein:creatinine ratio Poor In Practice January/February 2019 Volume

6 Table 3: Main vaccines against canine leishmaniosis in South America and Europe (based on Solano-Gallego and others 2017) Vaccine, manufacturer and country/region of licensing Leish-Tec Hertape Calier Saúde Animal Brazil CaniLeish Virbac Europe Letifend Laboratorios LETI Europe Composition of vaccine Vaccine protocol Antibodies elicited by vaccine Recombinant A2 antigen of Leishmania donovani; saponin adjuvant Purified LiESP; saponinderived QA-21 adjuvant Recombinant chimeric protein Q (five antigenic fragments of four Leishmania infantum proteins [histone H2A, LiP2a, LiP2b, and LiPo]); no adjuvant (Moreno and others 2014). However, the main controversy regarding CanL vaccines is that they do not block the establishment of infection. This could potentially keep an infected dog (without clinical disease) healthy, but possibly able to spread infection to people and dogs. Moreover, all vaccines are recommended solely for clinically healthy and seronegative dogs. This can sometimes be very difficult to ensure as prescreening tests may not be sensitive enough to detect all subclinically infected dogs. It is important to highlight that vaccinated infected dogs have been shown to be infectious to sandflies (Bongiorno and others 2013). Moreover, the consequences of vaccinating seropositive dogs are currently unknown, but there is a risk that these dogs would develop clinical leishmaniosis (Solano-Gallego and others 2017). Summary Three primary vaccination doses (subcutaneously) at 21- day intervals; one annual booster Three primary vaccination doses (subcutaneously) at 21- day intervals; one annual booster One primary vaccination dose (subcutaneous); one annual booster Antibodies to A2 (and potentially to LPA) Antibodies to LiESP and LPA (and potentially to Speed Leish K [Virbac] kinesins) Antibodies to protein Q Cases of CanL are uncommon in the UK, occurring only in dogs that have travelled to endemic areas. The UK also lacks the sandfly vector for the parasite, removing the threat of onward transmission of the disease by natural means. However, with the increasing numbers of dogs that enter the UK each year from countries where the disease is endemic, practitioners need to be aware of the signs of the disease, how to diagnose it, and the action to take should they confirm a case. References ANDRADE, H. M., TOLEDO, V. P., PINHEIRO, M. B., GUIMARÃES, T. M., OLIVEIRA, N. C., CASTRO, J. A. AND OTHERS (2011) Evaluation of miltefosine for the treatment of dogs naturally infected with L infantum (=L chagasi) in Brazil. Veterinary Parasitology 181, BANETH, G., KOUTINAS, A. F., SOLANO-GALLEGO, L., BOURDEAU, P. & FERRER. L. (2008) Canine leishmaniosis: new concepts and insights on an expanding zoonosis. Part one. Trends in Parasitology 24, BANETH, G. & SHAW, S. E. (2002) Chemotherapy of canine leishmaniosis. Veterinary Parasitology 106, BANETH, G. & SOLANO-GALLEGO, L. (2012) Leishmaniases. In: Infectious Diseases of the Dog and Cat, 4th edn. Ed C. E. Green. Elsevier. pp BERRAHAL, F., MARY, C., ROZE, M., BERENGER, A., ESCOFFIER, K., LAMOUROUX, D. & DUNAN, S. (1996) Canine leishmaniasis: identification of asymptomatic carriers by polymerase chain reaction and immunoblotting. American Journal of Tropical Kinetics of antibodies at primary vaccination (peak and duration) Peak: 21 days after the second dose of primary vaccination Duration: decrease at six months Peak: two weeks after the third dose of primary vaccination Duration: variable, but may persist for four to 12 months Peak: 14 days after primary vaccination Duration: at least 60 days (not assessed beyond this) IFAT Indirect fluorescent antibody test, LiESP Leishmania infantum excreted-secreted proteins, LPA Leishmania promastigote antigen Diagnostic interference associated with vaccine Detection of vaccinal antibodies with official ELISA Detection of vaccinal antibodies with quantitative tests (ELISA and IFAT); rare detection of vaccinal antibodies with Speed Leish K No detection of vaccinal antibodies by quantitative tests (IFAT and ELISA) or rapid tests Medicine and Hygiene 55, BONGIORNO, G., PAPARCONE, R., FOGLIA MANZILLO, V., OLIVA, G., CUISINIER, A. M. & GRADONI, L. 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L., MELO, M. N. & MICHALICK, M. S. (2009) First report of vertical transmission of Leishmania (Leishmania) infantum in a naturally infected bitch from Brazil. Veterinary Parasitology 166, DENEROLLE, P. & BOURDOISEAU, G. (1999) Combination allopurinol and antimony treatment versus antimony alone and allopurinol alone in the treatment of canine leishmaniasis (96 cases). Journal of Veterinary Internal Medicine 13, DINIZ, S. A., MELO, M. S., BORGES, A. M., BUENO, R., REIS, B. P., TAFURI, W. L. AND OTHERS (2006) Genital lesions associated with visceral leishmaniasis and shedding of Leishmania sp in the semen of naturally infected dogs. Veterinary Pathology 42, DUPREY, Z. H., STUERER, F. J., ROONEY, J. A. KIRCHHOFF, L. V., JACKSON, J. E., ROWTON, E. D. & SCHANTZ, P. M. (2006) Canine visceral leishmaniasis, United States and Canada, Emerging Infectious Diseases 12, EFSA AHAW Panel (2015) Scientific opinion on canine leishmaniosis. EFSA Journal 13, 4075, efsajournal/pub/4075. Accessed December 2, 2018 FARCA, A. M., MINISCALCO, B., BADINO, P., ODORE, R., MONTICELLI, P., TRISCIUOGLIO, A. & FERROGLIO, E. (2012) Canine leishmaniosis: In vitro efficacy of miltefosine and marbofloxacin alone or in combination with allopurinol against clinical strains of Leishmania infantum. Parasitology Research 110, FONT, A., DURALL, N., DOMINGO, M., CLOSA, J. M., MASCORT, J. & FERRER, L. (1993) Cardiac tamponade in a dog with visceral 12 In Practice January/February 2019 Volume

7 leishmaniasis. Journal of the American Animal Hospital Association 29, FONT, A., MASCORT, J., ALTIMIRA, J., CLOSA, J. M. & VILAFRANCA, M. (2004) Acute paraplegia associated with vasculitis in a dog with leishmaniasis. Journal of Small Animal Practice 45, FRANCA-SILVA, J. C., DA COSTA, R. T., SIQUIERA, A. M., MACHADO-COELHO, G. L., DA COSTA, C. A., MAYRINK, W. AND OTHERS (2003) Epidemiology of canine visceral leishmaniosis in the endemic area of Montes Claros Municipality, Minas Gerais State, Brazil. Veterinary Parasitology 11, GIGER, U., OAKLEY, D. A., OWENS, S. D. & SCHANTZ, P. (2002) Leishmania donovani transmission by packed RBC transfusion to anemic dogs in the United States. Transfusion 42, GRADONI, L. & GRAMICCIA, M. (2008) Leishmaniosis. In: Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. World Organisation for Animal Health (OIE); 2008: update available at Health_standards/tahm/ _LEISHMANIOSIS.pdf. Accessed 2 December 2018 JOSE-LOPEZ, R., LA FUENTE, C. D. & AÑOR, S. (2012) Presumed brain infarctions in two dogs with systemic leishmaniasis. Journal of Small Animal Practice 53, KILLICK-KENDRICK, R., KILLICK-KENDRICK, M., PINELLI, E., DEL REAL, G., MOLINA, R., VITUTIA, M. M. AND OTHERS (1994) A laboratory model of canine leishmaniasis: the inoculation of dogs with Leishmania infantum promastigotes from midguts of experimentally infected phlebotomine sandflies. Parasite 1, KOUTINAS, A. F., POLIZOPOULOU, Z. S., SARIDOMICHELAKIS, M. N., ARGYRIADIS, D., FYTIANOU, A. & PLEVRAKI, K. G. (1999) Clinical considerations on canine visceral leishmaniasis in Greece: a retrospective study of 158 cases ( ). Journal of the American Animal Hospital Association 35, LEONTIDAS, L. S., SARIDOMICHELAKIS, M. N., BILLINIS, C., KONTOS, V., KOUTINAS, A. F., GALATOS, A. D. & MYLONAKIS, M. E. (2002) A cross-sectional study of Leishmania spp infection in clinically healthy dogs with polymerase chain reaction and serology in Greece. Veterinary Parasitology 109, MAIA, C. & CAMPIONO, L. (2008) Methods for diagnosis of canine leishmaniasis and immune response to infection. Veterinary Parasitology 158, MAIA, C. & CARDOSO, L. (2015) Spread of Leishmania infantum in Europe with dog travelling. Veterinary Parasitology 213, 2-11 MANNA, L., PACIELLO, O., MORTE, R. D. & GRAVINO, A. E. (2012) Detection of Leishmania parasites in the testis of a dog affected by orchitis: case report. Parasites & Vectors 5, 216 MARCONDES, M., LIMA, V. M., DE ARAÚJO MDE, F., HIRAMOTO, R. M., TOLEZANO, J. E., VIEIRA, R. F. & BIONDO, A. W. (2013) Longitudinal analysis of serological tests officially adopted by the Brazilian Ministry of Health for the diagnosis of canine visceral leishmaniosis in dogs vaccinated with Leishmune. Veterinary Parasitology 197, MARTÍNEZ, V., QUILEZ, J., SANCHEZ, A., ROURA, X., FRANCINO, O. & ALTET L. (2011) Canine leishmaniasis: The key points for qpcr result interpretation. Parasites & Vectors 4, 57 MATEO, M., MAYNARD, L., VISCHER, C., BIANCIARDI, P. & MIRÓ, G. (2009) Comparative study on the short term efficacy and adverse effects of miltefosine and meglumine antimoniate in dogs with natural leishmaniosis. Parasitology Research 105, MIR, F., FONTAINE, E., REYES-GOMEZ, E., CARLUS, M. & FONTBONNE, A. (2012) Subclinical leishmaniasis associated with infertility and chronic prostatitis in a dog. Journal of Small Animal Practice 53, MIRANDA, S., ROURA, X., PICADO, A., FERRER, L. & RAMIS, A. (2008) Characterization of sex, age, and breed for a population of canine leishmaniosis diseased dogs. Research in Veterinary Science 85, MIRÓ, G., OLIVA, G., CRUZ, I., CAÑAVATE, C., MORTARINO, M., VISCHER, C & BIANCIARDI, P. (2009) Multicentric, controlled clinical study to evaluate effectiveness and safety of miltefosine and allopurinol for canine leishmaniosis. Veterinary Dermatology 20, MONTOYA, A., CHECA, R., MARINO, V., GáLVEZ, R., RUPEREZ, C., DE MARIE, K. & MIR, G. (2017) Antibodies elicited by primary vaccination and annual booster with CaniLesh in dogs from Spain: a clinical field study. Abstract C th World Congress on Leishmaniasis, May 2017, Toledo, Spain. worldleish2017.org/#/abstarcts, p Accessed 26 November 2018 MORENO, J. & ALVAR, J. (2002) Canine leishmaniasis: epidemiological risk and the experimental model. Trends in Parasitology 18, MORENO, J., VOULDOUKIS, I., SCHREIBER, P., MARTIN, V., MCGAHIE, D., GUEGUEN, S. & CUISINIER, A. M. (2014) Primary vaccination with LiESP/QA-21 vaccine (CaniLeish) produces a cell-mediated immune response which is still present 1 year later. Veterinary Immunology and Immunopathology 158, MYLONAKIS, M. E., SARIDOMICHELAKIS, M. N., LAZARIDIS, V., LEONTIDES, L. S., KOSTOULAS, P. & KOUTINAS, A. F. (2008) A retrospective study of 61 cases of spontaneous canine epistaxis (1998 to 2001). Journal of Small Animal Practice 48, NOLI, C. & AUXILIA, S. (2005) Treatment of Old World visceral leishmaniasis: A systemic review. Veterinary Dermatology 16, OLIVA, G., SCALONE, A., FOGLIA MANZILLO, V., GRAMICCIA, M., PAGANO, A., DI MUCCIO, T. & GRADONI, L. (2006) Incidence and time course of Leishmania infantum infections examined by parasitological, serologic, and nested-pcr techniques in a cohort of naive dogs exposed to three consecutive transmission seasons. Journal of Clinical Microbiology 44, ORDEIX, L., SOLANO-GALLEGO, L., FONDEVILA, D., FERRER, L. & FONDATI, A. (2005) Papular dermatitis due to Leishmania spp infection in dogs with parasite-specific cellular immune responses. Veterinary Dermatology 16, OWENS, S. D., OAKLEY, D. A., MARRYOTT, K., HATCHETT, W., WALTON, R., NOLAN, T. J. AND OTHERS (2001) Transmission of visceral leishmaniasis through blood transfusions from infected English foxhounds to anemic dogs. Journal of the American Veterinary Medical Association 219, PACIELLO, O., OLIVA, G., GRADONI, L., MANNA, L., FOGLIA MANZILLO, V., WOJCIK, S. AND OTHERS (2009) Canine inflammatory myopathy associated with Leishmania infantum infection. Neuromuscular Disorders 19, PALTRINIERI, S., GRADONI, L., ROURA, X., ZATELLI, A. & ZINI, E. (2016) Laboratory tests for diagnosing and monitoring canine leishmaniasis. Veterinary Clinical Pathology 45, PALTRINIERI, S., SOLANO-GALLEGO, L., FONDATI, A., LUBAS, G., GRADONI, L., CASTAGNARO, M. AND OTHERS (2010) Guidelines for diagnosis and clinical classification of leishmaniasis in dogs. Journal of the American Veterinary Medical Association 236, PENNISI, M. G., CARDOSO, L., BANETH, G., BOURDEAU, P., KOUTINAS, A., MIRÓ, G. AND OTHERS (2015) LeishVet update and recommendations on feline leishmaniosis. Parasites & Vectors 8, 302 PETANIDES, T. A., KOUTINAS, A. F., MYLONAKIS, M. E., DAY, M. J., SARIDOMICHELAKIS, M. N., LEONTIDES, L. S. AND OTHERS (2008) Factors associated with the occurrence of epistaxis in natural canine leishmaniasis (Leishmania infantum). Journal of Veterinary Internal Medicine 22, PINELLI, E., GONZALO, R. M., BOOG, C. J. P., RUTTEN, V. P., GEBHARD, D., DEL REAL, G. & RUITENBERG, E. J. (1995) Leishmania infantum-specific T cell lines derived from asymptomatic dogs that lyse infected macrophages in a major histocompatibility complex-restricted manner. European Journal of Immunology 25, PLANNELAS, M., ROURA, X. & LLORET, A. (2009) Presence of renal disease in dogs with patent leishmaniasis. Parassitologia 51, RODRÍGUEZ-CORTÉS, A., OJEDA, A., FRANCINO, O., LÓPEZ- FUERTES, L., TIMÓN, M. & ALBEROLA, J. (2010) Leishmania infection: laboratory diagnosing in the absence of a gold standard. American Journal of Tropical Medicine and Hygiene 82, SABATÉ, D., LLINÁS, J., HOMEDES, J., SUST, M. & FERRER, L. 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8 Self-assessment quizzes In Practice has partnered with BMJ OnExamination to host the self-assessment quizzes provided with each clinical article. These can be completed on the online version of each article at www. inpractice.bmj.com SIDERIS, V., PAPADOPOULOU, G., DOTSIKA, E. & KARAGOUNI, E. (1999) Asymptomatic canine leishmaniasis in Greater Athens area, Greece. European Journal of Epidemiology 15, SOLANO-GALLEGO, L., CARDOSO, L., PENNISI, M. G., PETERSEN, C., BOURDEAU, P., OLIVA, G. AND OTHERS (2017) Diagnostic challenges in the era of canine Leishmania infantum vaccines. Trends in Parasitology 33, SOLANO-GALLEGO, L., KOUTINAS, A., MIRÓ, G., CARDOSO, L., PENNISI, M. G., FERRER, L. AND OTHERS (2009) Directions for the diagnosis, clinical staging, treatment and prevention of canine leishmaniosis. Veterinary Parasitology 165, 1-18 SOLANO-GALLEGO, L., LLULL, J., RAMOS, G., RIERA, C., ARBOIX, M., ALBEROLA, J. & FERRER, L. (2000) The Ibizian hound presents a predominantly cellular immune response against natural Leishmania infection. Veterinary Parasitology 90, SOLANO-GALLEGO, L., MIRÓ, G., KOUTINAS, A., CARDOSO, L., PENNISI, M. G., FERRER, L. AND OTHERS (2011) LeishVet guidelines for the practical management of canine leishmaniosis. Parasites & Vectors 4, SOLANO-GALLEGO, L., MONTSERRAT-SANGRA, S., ORDEIX, L. & MARTÍNEZ-ORELLANA, P. (2016) Leishmania infantum-specific production of IFN-γ and IL-10 stimulated blood from dogs with clinical leishmaniosis. Parasites & Vectors 9, 317 SOLANO-GALLEGO, L., MORELL, P., ARBOIX, M., ALBEROLA, J. & FERRER, L. (2001) Prevalence of Leishmania infantum infection in dogs living in an area of canine leishmaniasis endemicity using PCR on several tissues and serology. Journal of Clinical Microbiology 39, SOLANO-GALLEGO, L., VILLANUEVA SAZ, S., CARBONELL, M., TROTTA, M., FURLANELLO, T. & NATALE, A. (2014) Serological diagnosis of canine leishmaniosis: comparison of three commercial ELISA tests (Leiscan, ID Screen and Leishmania 96), Self assessment: Leishmaniosis in dogs and cats 1. Which of the following is the infective stage of Leishmania infantum? a. Promastigote b. Trophozoite c. Amastigote d. Oocyst 2. In which target cells can Leishmania species amastigotes replicate? a. Neutrophils b. Eosinophils c. Macrophages d. Histiocytes 3. Which of the following is the most common cause of epistaxis in dog with leishmaniosis? a. Thrombocytopathy b. Systemic hypertension c. Neoplasia d. Self-trauma secondary to intense pruritus a rapid test (Speed Leish K) and an in-house IFAT. Parasites & Vectors 7, 111 STARITA, C., GAVAZZA, A. & LUBAS, G. (2016) Hematological, biochemical, and serological findings in healthy canine blood donors after the administration of CaniLeish vaccine. Veterinary Medicine International TABAR, M. D., ROURA, X., FRANCINO, O., ALTET, L. & RUIZ DE GOPEGUI, R. (2008) Detection of Leishmania infantum by realtime PCR in a canine blood bank. Journal of Small Animal Practice 47, TORRENT, E., LEIVA, M., SEGALES, J., FRANCH, J., PEÑA, T., CABRERA, B. & PASTOR, J. (2005) Myocarditis and generalised vasculitis associated with leishmaniosis in a dog. Journal of Small Animal Practice 46, TORRES, M., BARDAGÍ, M., ROURA, X., ZANNA, G., RAVERA, I. & FERRER, L. (2011) Long term follow-up of dogs diagnosed with leishmaniosis (clinical stage II) and treated with meglumine antimoniate and allopurinol. Veterinary Journal 188, VAMVAKIDIS, C. D., KOUTINAS, A. F., KANAKOUDIS, G., GEORGIADIS, G. & SARIDOMICHELAKIS, M. (2000) Masticatory and skeletal muscle myositis in canine leishmaniasis (Leishmania infantum). Veterinary Record 146, VERMA, N. K. & DEY, C. S. (2004) Possible mechanism of miltefosine mediated death of Leishmania donovani. Antimicrobial Agents and Chemotherapy 48, VINUELAS, J., GARCIA-ALONSO, M., FERRANDO, L., NAVARRETE, I., MOLANO, I., MIRÓN, C. AND OTHERS (2001) Meningeal leishmaniosis induced by Leishmania infantum in naturally infected dogs. Veterinary Parsitology 101, WOERLY, V., MAYNARD, L., SANQUER, A. & EUN, H. M. (2009) Clinical efficacy and tolerance of miltefosine in the treatment of canine leishmaniosis. Parasitology Research 105, Which of the following is the typical immunological response of resistant dogs? a. Humoral immunity (T helper 2) b. Proliferation of B lymphocytes and plasma cells c. T cell-mediated immunity (T helper 1) d. Production of specific anti-leishmania antibodies 5. Which of the following are the most common clinical signs of feline leishmaniosis? a. Epistaxis b. Vomiting and diarrhoea c. Jaundice d. Lymph node enlargement and skin lesions mainly on the head or distal limbs Answers: a, c, a, c, d 14 In Practice January/February 2019 Volume