Canine Leishmaniasis Research Articles

Epidemiology of canine visceral leishmaniasis in a vulnerable region in Brazil.

Visceral leishmaniasis (VL) is a neglected and endemic zoonosis that occurs throughout Brazil; nevertheless, few studies have focused on the early detection of the disease. The municipality of Ourinhos is a non-receptive, silent and vulnerable area for VL, where the seroprevalence of this disease has so far not been investigated. The present study aimed to determine the seroprevalence of canine VL in Ourinhos-SP, and to identify the presence of risk factors. Blood samples were obtained from 604 dogs during a rabies vaccination campaign together with application of a socioeconomic questionnaire, environmental and animal characteristics and tutor's knowledge about the disease. The samples were subjected to indirect ELISA and new samples were collected from reactive and suspect animals, including whole blood and lymph node aspiration evaluated by parasitological method, complete blood count and PCR. No animal was diagnosed as positive based on the combination of direct and indirect tests and the tutors' answers indicated little knowledge about leishmaniasis, being often confused with other diseases transmitted by arthropods; hence, according to the proposed methods, the presence of canine leishmaniasis in the city of Ourinhos was not confirmed and health education campaigns about the disease should be carried out.

Polymerase chain reaction using conjunctival swab samples for detecting Leishmania DNA in dogs.

The dog is the main domestic reservoir of Leishmania and font of infection for the vector, constituting an important host for the transmission of the parasite to humans. Non-invasive collection of swab samples for leishmaniasis diagnosis has been a promising alternative. This study analyzed the positivity of polymerase chain reaction (PCR) for the diagnosis of canine leishmaniasis in conjunctiva samples. DNA extraction was performed using SDS 20% and PCR was performed using 13A/13B primers that amplify 120-bp of Leishmania kDNA. Of the 77 dogs analyzed, 50 (64.93%) had ocular changes: 25 (32.47%) dogs had periocular lesion, 41 (53.25%) dogs had purulent eye discharge, and 17 (22.08%) dogs had both signals. PCR was positive in 35 dogs (45.45%), and there was no significant difference between dogs with and without ocular signals (p=0.4074). PCR positivity was significant higher in dogs without periocular injury (p=0.0018). Conjunctive PCR, a less invasive, fast, and painless collection technique, is indicated to complement the diagnosis, especially in dogs without periocular injury, independent of the presence of purulent eye discharge.

The spread of visceral leishmaniasis in Brazil: the first canine cases described in Ji-Paraná, Rondônia, Brazil.

Leishmaniases are zoonotic diseases caused by protozoa of the genus Leishmania. The disease has two clinical manifestations described in humans: visceral (VL) and cutaneous (CL) leishmaniasis. In Brazil, there has been an expansion of human VL. The participation of the dog as a reservoir of Leishmania infantum, the agent of VL, is important for the epidemiology of the disease since canine cases generally precede human cases. The present study aimed to evaluate the occurrence of Leishmania spp. infection in dogs in the municipality of Ji-Paraná by PCR assays using blood samples. Leishmania DNA was detected in two of the 105 studied dogs. The PCR products were sequenced and confirmed that the two samples (1.90%) correspond to L. infantum. The dogs had allochthonous history. Therefore, the positive results found here should serve as a warning to public health agencies. This is because Ji-Paraná is the third municipality to register cases of canine leishmaniasis (CanL) in Rondônia state. Thus, reinforcing the importance of expanding studies on the epidemiology and surveillance of VL in the region.

Anatomomopathological and immunohistochemical analyses of the spleen and lymph node of dogs seropositives for leishmaniasis in serological tests

Abstract Canine leishmaniasis (CanL) is a zoonosis caused by the protozoan of the species Leishmania infantum. The spleen and lymph nodes undergo morphological changes during CanL. This research aimed to perform an anatomopathological and immunohistochemical study of these organs in dogs reactive to leishmaniasis in the Dual-path Platform chromatographic immunoassay (DPP®) and Enzyme Immunoabsorption Assay (ELISA). Twenty-seven dogs were evaluated for anatomopathological examination with 92.6% showing changes at gross evaluation, specially splenomegaly and lymphadenomegaly. All dogs showed changes in the spleen unrelated to the parasitic load, with granulomatous splenitis being the most severe change. Diffuse cortical and paracortical hyperplasia, and hyperplasia and hypertrophy of the medullary cords were observed in the lymph node. Amastigote forms of Leishmania spp. were found in the spleen and lymph node at histopathological and immunohistochemical evaluations, with good agreement between these evaluations (k = 0.55, p = 0.00124), but no difference was observed in the parasitic intensity of these organs at immunohistochemistry (p = 0.23). It was concluded that spleen and lymph node from dogs reactive to leishmaniasis on the DPP® and ELISA tests show histomorphological changes resulting from the disease, independent to the parasitic load, as well as these organs show similar parasitic load at immunohistochemical test.

Dogs as a Model for Chemotherapy of Chagas Disease and Leishmaniasis

Dogs are natural reservoir of Chagas disease (CD) and leishmaniasis and have been used for studies of these infections as they develop different clinical forms of these diseases similar to humans. This article describes publications on the dog model relative to CD and leishmaniasis chemotherapy. The search of articles was based on PubMed, Scopus and MESH using the keywords: dog, Trypanosoma cruzi, treatment (T. cruzi chemotherapy analysis), Leishmania chagasi, Leishmania infantum, canine visceral leishmaniasis, treatment (Leishmania chemotherapy evaluation). Benznidazole and nifurtimox were used as a reference in the treatment of CD and in combination with other compounds. Eleven out of the fifteen studies have authors from the same team, using similar protocols and post-treatment evaluations, which assured more reproducibility and credibility. Twenty leishmaniasis studies, especially on visceral leishmaniasis, presenting at least one parasitological analysis tested in distinct monochemotherapy and polychemotherapy approaches were accessed. Data demonstrated that polychemotherapy was more effective in improving the clinical signs and parasitism control. The benefits of treatment in terms of reducing or eliminating lesions and/or cardiac dysfunctions were demonstrated at acute and/or chronic phases relative to parasite load and/or the T. cruzi strain resistance to treatment. BZ presented better therapeutic results than the two EBI compounds evaluated. Although treatment of the canine visceral leishmaniasis was not able to induce complete parasite clearance, it can improve clinical recovery. Thus, the dog is a good model for CD and leishmaniasis studies of chemotherapy and may be indicated for pre-clinical trials of new treatments.

Allopurinol y domperidona en el tratamiento de leishmaniosis visceral canina

<p>El tratamiento de la leishmaniosis humana en Argentina está normatizado con el esquema<br />terapéutico sugerido por la OMS y es suministrado por el estado nacional en forma<br />gratuita. La mayoría de las drogas utilizadas son desaconsejadas en el tratamiento farmacológico de perros afectados por leishmaniosis visceral por la dificultad de acceder al elevado costo y al riesgo de aumentar la generación de cepas resistentes. El presente trabajo tuvo como objetivo evaluar la progresión de los signos clínicos, valores de laboratorio y carga parasitaria, al inicio y durante la administración combinada de allopurinol y domperidona por<br />vía oral, en la Facultad de Veterinaria de Corrientes (Argentina). Se estudiaron 10 pacientes<br />caninos en los cuales se evaluaron los signos clínicos, se efectuó el análisis serológico y se<br />obtuvieron muestras de médula ósea. Se inició un protocolo terapéutico mediante la combinación de ambos fármacos y se efectuó un seguimiento con controles de los parámetros<br />clínicos, laboratoriales y parasitológicos. Todos los pacientes mostraron mejoría clínica al<br />inicio, pero la evolución fue irregular y particular para cada caso, con reclasificaciones de<br />acuerdo a lo sugerido por el grupo Leishvet. Al realizar ajustes de acuerdo a los signos clínicos,<br />perfiles bioquímicos y estimación de la carga parasitaria, la combinación de allopurinol<br />y domperidona mostró resultados favorables a la dosis recomendada. Si bien es necesario una<br />mayor casuística y tiempo de ensayo, los resultados permiten inferir que este protocolo podría<br />constituir una alternativa en el tratamiento farmacológico y en el control epidemiológico<br />de la leishmaniosis visceral canina.</p>

Potential of Artesunate in the treatment of visceral leishmaniasis in dogs naturally infected by Leishmania infantum: Efficacy evidence from a randomized field trial.

Leishmaniasis is among the world’s most neglected diseases. Dogs are the main reservoirs/hosts of Leishmania infantum, causative agent of both canine and human visceral leishmaniosis. Canine leishmaniasis (CanL) represents a public health problem as one of the most prevalent zoonotic diseases worldwide. Current therapeutics present drawbacks; thus, there is a need for more effective, safer, and cheaper drugs. The aim of this study was to evaluate and to compare the efficacy of oral administration of artesunate or meglumine antimoniate/allopurinol in dogs with clinical leishmaniasis. Forty-two dogs with naturally occurring clinical leishmaniasis were included in this open-label, simple randomized positive-control clinical field trial with 6 months of follow-up. Dogs received meglumine antimoniate 100 mg/kg/day and allopurinol 30 mg/kg/day for 28 days (control group, n = 26) or artesunate 25 mg/kg/day for 6 days (test group, n = 16). The animals were evaluated for their clinical evolution, parasite load (by qPCR) and humoral response at different time points: 0, 30, 90, and 180 days after treatment. Data analyses showed a significant improvement in both groups in clinical scores, parasitemia and antibody titers after treatment. Compared to the control group, the artesunate group showed significantly lower clinical score (P = 0.0001), lower parasitemia (P = 0.0001) and antibody titers after 6 months of follow-up. Compared to baseline values, a rapid, significant reduction (P < 0.012) in antibody levels, 2.28- versus 3.04-fold for the control versus artesunate groups, respectively, was observed 30 days after treatment. Antibody levels continued to decrease further in the artesunate group, where 58% of cases became seronegative at the 6-month follow-up. All qPCR-positive dogs were negative after treatment with artesunate, while 14.3% remained positive with the appearance of two new cases in the control group. Artesunate was well tolerated, and no side effects were recorded. Treatment failures were similar in both groups with 27.27% (6/22), including 18.18% (4/22) mortality in the control group, versus 26.66% (4/15), including 13.33% (2/15) mortality in the artesunate group. This is the first report showing the potential of artesunate in the treatment of dogs with clinical leishmaniasis. Artesunate showed higher efficacy than the current first-line treatment for CanL without any adverse effects. It could be a good alternative chemotherapy for CanL, and may be considered for further studies in human leishmaniases. Further clinical trials are needed to confirm these findings, to determine if there are relapses after treatment and if dogs remain infective to sandflies, to define the ideal therapeutic dosage and duration of treatment with artesunate.

Alteraciones ecográficas y hematológicas en caninos con leishmaniosis visceral

&lt;p&gt;La leishmaniosis visceral es una enfermedad multisistémica que en los caninos ostenta signos clínicos tales como esplenomegalia, hepatomegalia, linfadenopatía y glomerulonefritis. Es frecuente la presentación de anemia asociada a hiperproteinemia. La ecografía puede revelar anomalías morfológicas y estructurales de los órganos de la cavidad abdominal. El objetivo del trabajo fue evaluar mediante eco-grafía las lesiones visibles de los órganos abdominales, así como establecer relaciones con los análisis de laboratorio. La detección del parásito se efectuó mediante diagnósticos parasitológico y serológico indirecto. Los órganos abdominales afectados se evaluaron por ecografía (forma, tamaño, estructura y ecogenicidad). En los riñones se exploró la relación corteza/médula. En el laboratorio se investigaron las variables sanguíneas, serológicas y renales. La estadificación clínica de los caninos resultó del 50% en estadio moderado, 40% grave y 10% muy grave. Las principales alteraciones fueron hepatomegalia&lt;br /&gt;(8 pacientes), esplenomegalia (1 caso) e incremento de la ecogenicidad de la corteza&lt;br /&gt;renal con disminución de la definición corticomedular y linfoadenomegalia (5 animales). El&lt;br /&gt;90% de los caninos reveló anemia acompañada de hiperproteinemia. Se concluye que el uso de&lt;br /&gt;la ecografía en conjunto con los análisis sanguíneos, pueden ayudar a establecer la severidad&lt;br /&gt;de las lesiones de los órganos abdominales en pacientes con leishmaniosis.&lt;/p&gt;

Development and validation of a stability-indicating analytical method for simultaneous determination of drugs employed in canine leishmaniasis treatment

We developed and validated a stability-indicating method for the simultaneous determination of allopurinol and ketoconazole in the pharmaceutical form of capsules. A Dionex® liquid chromatograph equipped with a diode array detector (DAD) and InertSustain® C18 column (4.6 x 100 mm x 3 μm) was used and InertSustain® C18 column (4.6 x 100 mm x 3 μm). The chromatographic separation occurred in the isocratic mode with a flow rate of 0.45 mL min-1 and mobile phase composed of acetonitrile: water (52:48 v/v) with pH adjusted to 3.0. The wavelengths used were 250 nm for allopurinol and 225 nm for ketoconazole. The method was validated by evaluating the parameters of linearity, limits of detection and quantification, precision, accuracy, robustness, and selectivity. The method presented linearity. It was precise, with coefficients of variation lower than 2.0%, and accurate, with recovery close to 100.00%. Robustness was indicated by the Plackett-Burman model, and the method was not significantly influenced by any of the variations. The selectivity was proven by the peak purities close to 1000 in both the presence of excipients and drug degradation products. Therefore the proposed method is simple and fast, with separation for both drugs less than four minutes.

3D-Hepatocyte Culture Applied to Parasitology: Immune Activation of Canine Hepatic Spheroids Exposed to Leishmania infantum.

The application of innovative three-dimensional (3D) spheroids cell culture strategy to Parasitology offers the opportunity to closely explore host–parasite interactions. Here we present a first report on the application of 3D hepatic spheroids to unravel the immune response of canine hepatocytes exposed to Leishmania infantum. The liver, usually considered a major metabolic organ, also performs several important immunological functions and constitutes a target organ for L. infantum infection, the etiological agent of canine leishmaniasis (CanL), and a parasitic disease of major veterinary and public health concern. 3D hepatic spheroids were able to sense and immunologically react to L. infantum parasites, generating an innate immune response by increasing nitric oxide (NO) production and enhancing toll-like receptor (TLR) 2 and interleukin-10 gene expression. The immune response orchestrated by canine hepatocytes also lead to the impairment of several cytochrome P450 (CYP450) with possible implications for liver natural xenobiotic metabolization capacity. The application of meglumine antimoniate (MgA) increased the inflammatory response of 3D hepatic spheroids by inducing the expression of Nucleotide oligomerization domain (NOD) -like receptors 1 and NOD2 and TLR2, TLR4, and TLR9 and enhancing gene expression of tumour necrosis factor α. It is therefore suggested that hepatocytes are key effector cells and can activate and orchestrate the immune response to L. infantum parasites.

Hematological changes in different clinical forms as a complementary diagnostic tool in canine Leishmaniasis

Hematological changes in different clinical forms as a complementary diagnostic tool in canine Leishmaniasis

Identification of immunoreactive proteins in secretions of Leishmania infantum promastigotes: an immunoproteomic approach.

In the Mediterranean region, Leishmania infantum is the main cause of visceral leishmaniasis. Dogs with canine visceral leishmaniasis are an important reservoir of visceral leishmaniasis. Control of canine visceral leishmaniasis could disrupt transmission of visceral leishmaniasis to humans. The secreted antigens of Leishmania promastigotes are potential stimuli of the host immune system. Proteomic techniques facilitate the identification of new protein markers. This study aimed to identify immunoreactive proteins in the secretions of L. infantum promastigotes which could be possible targets for the diagnosis and treatment of canine visceral leishmaniasis and the development of vaccines against the disease. Secretions of L. infantum promastigotes were obtained from the cultivation of 6 × 109 promastigotes in serum- free RPMI-1640 medium during a period of 72 h. After deionization and lyophilization, two-dimensional gel electrophoresis was used for protein separation followed by Western blotting. Thirteen common and repeatable immunoreactive spots were analysed by mass spectrometry. Nine proteins were identified by spectrometry. Most of these proteins were involved in metabolism pathways, survival and pathogenicity of Leishmania parasites. Phospholipase C, immune inhibitor A, chitin-binding protein and a single peptide match to chain A crystal structure of selenomethionine were observed in the secretions of L. infantum promastigotes. The proteins identified in metabolism pathways, survival and pathogenicity of Leishmania parasites are possible targets that could be used for the diagnosis and treatment of canine visceral leishmaniasis and the development of vaccines against the disease in the future.

Registro del primer caso de leishmaniasis visceral canina (alóctona) en Indaial - SC: Reporte de caso

Canine visceral leishmaniasis is a tropical climate disease, commonly found in countries of the Americas. In Brazil, its incidence has been increasing due to the animal migrations within from the country itself, which have been rising the rate of animals infected by the protozoan Leishmania chagasi. This report describes a female dog, 2.5 years old, from São Paulo state, which made a trip to Maranhão state and whose tutors came to establish housing in Indaial, SC. Soon after settling in this city, the animal began to show characteristic symptoms of canine visceral leishmaniasis. Biochemical tests and serological diagnoses were requested to confirm the suspected and performed treatment.

Influence of domperidone supplementation on short-term changes in C-reactive protein and paraoxonase-1 in dogs with leishmaniasis undergoing meglumine antimoniate and allopurinol therapy.

C-reactive protein (CRP) and paraoxonase 1 (PON1) might increase and decrease in canine leishmaniasis (CanL), , and both can rapidly normalize after therapy. Recently, supplementation of domperidone with conventional therapy , increasing the activity of cells involved in acute phase responses in vitro. This combined therapy has been recommended to treat mild forms of CanL; however, no studies have investigated the effects of domperidone supplementation on early CRP or PON1 changes in dogs with CanL. The aim of this study was to evaluate whether domperidone, added to conventional treatments, modifies CRP concentration and PON1 activity kinetics in CanL dogs responsive to conventional therapy. Serum CRP concentrations and PON1 activities were measured in dogs with mild CanL before (t-0) and 3 (t-1), 7 (t-2), 14 (t-3), and 21 (t-4) days after treatment with N-methylglucamine antimoniate and allopurinol alone (n=18) or combined with domperidone (n=18). C-reactive protein concentrations increased at t-1 in the domperidone group, especially when the CRP concentration at t-0 was normal. However, the concentrations normalized at t-4 in 18/18 dogs compared with 14/18 dogs not receiving domperidone. The median PON1 activity decreased at t-1 in the domperidone group, and this decrease was more significant in dogs with normal PON1 activity at t-0. Based on these results, transient increases in CRP concentrations or decreases in PON1 activities after domperidone administration should not be erroneously interpreted as signs of a worsening disease process.

Canine Leishmaniasis: Serological Results in Private and Kennel Dogs Tested over a Six-Year Period (2009-2014) in Abruzzo and Molise Regions, Italy.

This paper reports the results of serological tests for the detection of antibodies against Leishmania spp. in Abruzzo and Molise regions from 2009 to 2014, with the aim of evaluating the presence and distribution of canine leishmaniasis. Data were extracted from the Laboratory Information Management System (LIMS) of the Istituto Zooprofilattico Sperimentale of Abruzzo and Molise, and then the dog identification numbers were matched with those stored in the Canine Registries of the two regions to get information about the age of dogs at time of testing. Dogs were considered positive when having an IFAT (Indirect Fluorescent Antibody Test) titer ≥1:80. In total, 41,631 dogs were tested, 85.3% from Abruzzo and 14.7% from Molise. At the provincial level, the percentage of positive dogs ranged from 5.2% (L’Aquila, Abruzzo region) to 21.8% (Campobasso, Molise region). Findings are consistent with the hypothesis that in the coastal areas, the relationships between the host, the vector, and the agent are more favorable for the spreading of CanL, and it seems that densely populated urban internal areas have less favorable conditions. Being a dog hosted in a kennel seems not to be a factor increasing the probability that dogs show positivity, even in long-term sheltering conditions.

Mammary carcinoma in a mixed tumor with myeloid metaplasia component presenting Leishmania spp. in a dog

Here is described a 9-years-old, intact female dog with cytopathological analysis from mammary tumors containing Leishmania spp. amastigotes in macrophages. The animal was submitted to euthanasia and necropsy was performed. Histopathological diagnosis was a mammary carcinoma in a mixed tumor, grade I, with myeloid metaplasia component containing macrophages laden with a large amount of amastigotes. For confirmation, immunohistochemistry was used in neoplastic tissue and resulted in strong positivity for Leishmania antibody. Myeloid metaplasia portion of mammary carcinoma in a mixed tumor may be, rarely, a site for diagnosis of Canine Visceral Leishmaniasis.

MARBOFLOXACIN INDUCES LEISHMANICIDAL ACTIVITY AND LESS INFLAMMATORY RESPONSE IN Leishmania chagasi INFECTED MACROPHAGES

Marbofloxacin has promising leishmanicidal activity due to the direct action on the Leishmania chagasi amastigotes. It was developed only for veterinary medicine uses, and it could be used as an drug for the treatment of Canine Visceral Leishmaniasis (CVL). In the present study, we evaluated the leishmanicidal effect and macrophage modulation of marbofloxacin in macrophages infected with amastigotes of L. chagasi. Macrophages were collected from peritoneum of BALB/c mice and infected with promastigotes of L. chagasi. After internalization and transformation into amastigotes forms, cells were treated with marbofloxacin in concentration of 100, 500 and 750 μg/mL for 18 hours. The leishmanicidal effect was evaluated by morphological spects of amastigotes inside of macrophages by phagocytosis assay and detection of death amastigotes and macrophages by TUNEL assay. Macrophage modulation was evaluated by release of cytotoxic metabolites and cytokine production. The results showed that L. chagasi-infected macrophages treated with the highest concentration of the drug showed lower amount of amastigotes into the macrophages (p&lt;0,0632) than untreated infected cells. There was pronounced presence of cellular vacuoles in the treated infected-cells, and more apoptotic amastigotes in alive macrophages. It was observed decreased levels of H2O2, IL-1β, IL-6, and TNF-α dose-dependent of marbofloxacin in infected macrophages. The results indicate the leishmanicidal effect of marbofloxacin in infected macrophages and immunomodulation actions in these cells.

Phase I and II Clinical Trial Comparing the LBSap, Leishmune®, and Leish-Tec® Vaccines against Canine Visceral Leishmaniasis

In this study, we performed a phase I and II clinical trial in dogs to evaluate the toxicity and immunogenicity of LBSap-vaccine prototype, in comparison to Leishmune® and Leish-Tec® vaccines. Twenty-eight dogs were classified in four groups: (i) control group received 1 mL of sterile 0.9% saline solution; (ii) LBSap group received 600 μg of Leishmania braziliensis promastigotes protein and 1 mg of saponin adjuvant; (iii) Leishmune®; and (iv) Leish-Tec®. The safety and toxicity of the vaccines were measured before and after three immunizations by clinical, biochemical, and hematological parameters. The clinical examinations revealed that some dogs of LBSap and Leishmune® groups presented changes at the site of vaccination inoculum, such as nodules, mild edema, and local pain, which were transient and disappeared seventy-two hours after vaccination, but these results indicate that adverse changes caused by the immunizations are tolerable. The immunogenicity results demonstrate an increase of B lymphocytes CD21+ regarding the Leishmune® group and monocytes CD14+ concerning LBSap and Leishmune® groups. In the in vitro analyses, an increase in lymphoproliferative activity in LBSap and Leishmune® groups was observed, with an increase of antigen-specific CD4+ and CD8+ T lymphocytes in the LBSap group. A second approach of in vitro assays aimed at evaluating the percentage of antigen-specific CD4+ and CD8+ T lymphocytes producers of IFN-γ and IL-4, where an increase in both IFN-γ producing subpopulations in the LBSap group was observed, also showed an increase in IFN-γ producers in CD8+ lymphocytes in the Leish-Tec® group. Our data regarding immunogenicity indicate that the vaccination process, especially with the LBSap vaccine, generated a protective immune response compatible with L. infantum parasite control. Based on the foregoing, the LBSap vaccine would be suitable for further studies of phase III clinical trial in endemic areas with high prevalence and incidence of canine visceral leishmaniasis (VL) cases.

Seroprevalence and risk factors associated with Leishmania infantum in dogs in Sardinia (Italy), an endemic island for leishmaniasis

Leishmaniasis is a widespread, vector-borne parasitosis causing clinical manifestations in animals and in humans. In dogs, Canine Leishmaniasis has been reported in as much as 50 countries and the Mediterranean basin is known to be one of the most affected zones. Within these areas, the Island of Sardinia (Italy) has long been considered endemic for leishmaniasis and the presence of two arthropod vectors has recently been reported there. Nevertheless, to date, no epidemiological surveys regarding CanL have been carried out on the island. Hence, for the first time, the seroprevalence and the risk factors were investigated. Blood samples, as well as clinical and general information from 1.147 dogs, were collected and analyzed. Dogs consisted of two distinct populations, namely “owned dogs” and “kennel dogs.” Anti-Leishmania IgG antibodies were detected using IFAT and samples were scored as positive at a cut-off dilution of 1:80. Data was analyzed using a Chi-squared test and bivariate and multivariate analyses were performed. Overall, 15.4% of dogs were found to be infected with CanL while only 44.1% of these animals exhibited clinical signs. Owned dogs (27.2%) were found to be infected more often than kennel dogs (10.6%); male dogs were found to be more frequently infected than female dogs and the number of infected animals increases with age. The present survey confirmed the endemic nature of leishmaniasis in Sardinia with a similar seroprevalence as mainland Italy. The results obtained serve as validation for the hypothesis that, in endemic areas, clinical CanL representations constitute only a fraction of the leishmaniasis cases.

Seasonal dynamics of phlebotomine sand flies and autochthonous transmission of Leishmania infantum in high-altitude ecosystems in southern Spain

Leishmaniasis is a vector-borne disease transmitted by sand flies. A dozen species have been involved in the transmission of Leishmania infantum in the Mediterranean region. Climate change may alter sand fly distribution at particular altitudes and latitudes. The objective of this study was to interrogate the existence of stable populations of sand flies in high-altitude ecosystems and evaluate if these populations are enough to support autochthonous transmission of leishmaniasis. These altitudinal conditions can be found in Sierra Nevada (southern Spain). Therefore, we have determined the sand fly population dynamics in different biotopes located at elevations above 1,300 m a.s.l. and searched for evidence of leishmaniasis transmission. Five collecting sites above 1,300 m a.s.l. containing large livestock concentrations were selected. Sand flies were caught using CDC light traps from May to November, annually from 2008 to 2013, and these were morphologically identified. Association between sand fly density or presence and temperature/humidity was estimated by linear and logistic regression, respectively. Leishmania infantum detection in female sand flies was performed by PCR. Diagnosis of canine leishmaniasis (CanL) was carried out by indirect immunofluorescence and PCR. A total of 2,973 specimens of 5 sand fly species were collected from June to October. Phlebotomus perniciosus was the most frequent (100%), abundant (80.1%) and densest species (9.8 sand flies/trap). The minimum temperature on the day of capture was the most important variable factor for sand fly presence and P. perniciosus density. An increase in altitude showed a negative effect over the sand fly diversity and activity period, driving changes in seasonal dynamics similar to those reported by latitudinal changes. CanL prevalence was 23%, a similar rate to previous surveys carried out on randomly selected dogs from towns in southern Spain. A successful host-vector-pathogen network was found at this altitude characterised by 9.9% L. infantum infection rate in non-blood fed P. perniciosus and Phlebotomus ariasi females and high CanL prevalence that entails an increase in the leishmaniasis risk area driven by sand fly colonization.