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Abstract Background Zoonotic leishmaniosis caused by Leishmania infantum is endemic in several countries of the Mediterranean Basin, Latin America, and Asia. Dogs are the main hosts and reservoirs of human infection. Thus, from a One Health perspective, early diagnosis of Leishmania infection in dogs is essential to control the dissemination of the parasite among other dogs and to humans. The aim of this study was to estimate the diagnosis accuracy of three serological tests to detect antibodies to Leishmania in dogs from two endemic settings using Bayesian latent class models (BLCM). Methods A total of 378 dogs from two Portuguese and Brazilian endemic areas of leishmaniosis (194 animals from Portugal and 184 from Brazil) were screened. Detection of anti-Leishmania antibodies was performed using two commercial ELISA (L. infantum IgG-ELISA® and EIE-LVC®) and a rapid immunochromatographic test (DPP-LVC®). Bayesian latent class models were used to estimate Leishmania infection prevalence, together with sensitivities and specificities of the three diagnostic tests, in the two dog populations simultaneously. Predictive values were also calculated. Credibility intervals (CI) were obtained, considering different types of prior information. Results A posterior median Leishmania seroprevalence of 13.4% (95% CI 9.0–18.7) and of 21.6% (15.0–28.3) was estimated to the Portuguese and Brazilian dog subpopulations, respectively. The Bayesian analysis indicated that all tests were highly specific (specificity above 90%), and that the DPP-LVC® was more sensitive (96.6%; 83.1–99.9) than both ELISAs in the Portuguese subpopulation, while in the Brazilian subpopulation, EIE-LVC® and L. infantum IgG-ELISA®, had the highest sensitivity (88.2%; 73.7–97.0) and specificity (98.7%; 95.1–99.9), respectively. Conclusions In general, the levels of diagnosis accuracy of the three serological tests to detect Leishmania antibodies assessed by BLCM indicate their utility in canine epidemiological studies. The same approach should be used to assess the performance of these techniques in the clinical management of infected and sick dogs using representative samples from the wide spectrum of clinical situations, namely from subclinical infection to manifest disease. The low positive predictive value of the serological tests used in the current protocol of the Brazilian Ministry of Health suggests that they should not be used individually and may not be sufficient to target reservoir-based control interventions. Graphical Abstract

Leishmanioses are of public health concern in Morocco, mainly the Zoonotic Cutaneous Leishmaniasis (ZCL) endemic in the Moroccan pre-Saharian area. Transmission of this disease depends on eco-epidemiological and socio-economic conditions. Therefore, a multivariable approach is required to delineate the risk and intensity of transmission. This will help outline main disease risk factors and understand interactions between all underlying factors acting on disease transmission at a local and regional scale. In this context, we propose a new conceptual model, the Biophysical-Drivers-Response-Zoonotic Cutaneous Leishmaniasis (BDRZCL), adapted to the Pre-Saharian area. The proposed model highlights how the physical and human drivers affect the environment and human health. The incidence of ZCL is linked to human activity and biophysical changes or by their interactions. The human response added to risk drivers are the main components that influence the biophysical part. This model improves our understanding of the cause-effect interactions and helps decision-makers and stakeholders react appropriately.

Abstract Background In endemic areas of zoonotic leishmaniosis caused by L. infantum, early detection of Leishmania infection in dogs is essential to control the dissemination of the parasite to humans. The aim of this study was to evaluate the serological and/or molecular diagnostic performance of minimally and non-invasive samples (conjunctiva cells (CS) and peripheral blood (PB)) for monitoring Leishmania infection/exposure to Phlebotomus perniciosus salivary antigens in dogs at the beginning and the end of sand fly seasonal activity (May and October, respectively) and to assess associated risks factors. Methods A total of 208 sheltered dogs from endemic areas of leishmaniosis were screened. Leishmania DNA detection in PB on filter paper and CS was performed by nested-PCR (nPCR), while the detection of anti-Leishmania antibodies was performed using IFAT and ELISA. The exposure to P. perniciosus salivary antigens (SGH, rSP01 and rSP03B + rSP01) was measured by ELISA. Results Ninety-seven (46.6%) and 116 (55.8%) of the 208 dogs were positive to Leishmania antibodies or DNA by at least one test at the beginning and end of the sand fly season, respectively. IFAT and ELISA presented a substantial agreement in the serodiagnosis of leishmaniosis. Discrepant PB nPCR results were obtained between sampling points. Leishmania DNA was detected in CS of 72 dogs at the end of the phlebotomine season. The presence of antibodies to the parasite measured by ELISA was significantly higher in dogs presenting clinical signs compatible with leishmaniosis at both sampling points. Phlebotomus perniciosus salivary antibodies were detected in 179 (86.1%) and 198 (95.2%) of the screened dogs at the beginning and end of the phlebotomine season, respectively. Conclusions The association between ELISA positivity and clinical signs suggests its usefulness to confirm a clinical suspicion. CS nPCR seems to be an effective and non-invasive method for assessing early exposure to the parasite. PB nPCR should not be used as the sole diagnostic tool to monitor Leishmania infection. The correlation between the levels of antibodies to P. perniciosus saliva and Leishmania antibodies suggests the use of a humoral response to sand fly salivary antigens as biomarkers of L. infantum infection.

Canine leishmaniosis (CanL) caused by Leishmania infantum is an important zoonosis in southwestern European countries where this disease is endemic, and dogs, as domestic animals in close contact with humans, are the reservoir hosts for the parasite. In Portugal, CanL is of relevant veterinary concern. The previous national study revealed an overall seroprevalence of 6.3%. Since then, new prophylactic measures, such as vaccines, have been introduced in Europe. The aim of this study was to update seroprevalence for Leishmania infection and reassess risk factors in Portugal. A cross-sectional study was conducted from January–March 2021 with 1860 client-owned dogs from continental Portugal. A questionnaire and whole blood samples on filter paper were collected and a direct agglutination test was used to calculate anti-Leishmania antibody titres. True seroprevalence was 12.5% (95% CI 10.3–13.2%). Potential risk factors associated with L. infantum infection in dogs were age ≥ 2 years (aOR = 1.68, 95% CI 1.1–2.6) and residing in the interior regions of the country (aOR = 1.92, 95% CI 1.3–2.9) and non-use of repellents (aOR = 1.75, 95% CI 1.2–2.5). The key to controlling CanL and its impact on Public Health in endemic areas lies in continuous implementation of prophylactic measures, through the correct use of repellents/insecticides and vaccines and early detection and monitoring of infected dogs.

Abstract Background Zoonotic leishmaniosis, caused by the protozoan Leishmania infantum, is a public and animal health problem in Asia, Central and South America, the Middle East and the Mediterranean Basin. Several phlebotomine sand fly species from the subgenus Larroussius are vectors of L. infantum. Data from dogs living in endemic areas of leishmaniosis advocate the use of antibody response to phlebotomine sand fly saliva as an epidemiological biomarker for monitoring vector exposure. The aim of this study was to analyse the exposure of cats to phlebotomine sand flies using detection of IgG antibodies to Phlebotomus perniciosus saliva. The association between phlebotomine sand fly exposure and the presence of Leishmania infection was also investigated. Results IgG antibodies to P. perniciosus saliva were detected in 167 (47.7%) out of 350 cats; higher antibody levels were present in sera collected during the period of phlebotomine sand fly seasonal activity (OR = 19.44, 95% CI: 9.84–38.41). Cats of 12–35 months had higher antibody levels than younger ones (OR = 3.56, 95% CI: 1.39–9.16); this difference was also significant with older cats (for 36–95 months-old, OR = 9.43, 95% CI: 3.62–24.48; for older than 95 months, OR = 9.68, 95% CI: 3.92–23.91). Leishmania spp. DNA was detected in the blood of 24 (6.9%) cats, while antibodies to L. infantum were detected in three (0.9%). Only one cat was positive to Leishmania by both techniques. Cats presenting IgG antibodies to P. perniciosus had a significantly higher risk of being positive for Leishmania infection. Conclusions To our knowledge, this is the first study demonstrating anti-sand fly saliva antibodies in cats. The evaluation of the contact of this animal species with the vector is important to the development of prophylactic measures directed to cats, with the aim of reducing the prevalence of infection in an endemic area. Therefore, studies evaluating whether the use of imidacloprid/flumethrin collars reduces the frequency of P. perniciosus bites in cats are needed. It is also important to evaluate if there is a correlation between the number of phlebotomine sand fly bites and IgG antibody levels.

Leishmaniasis remains one of the ten Neglected Tropical Diseases with significant morbidity and mortality in humans. Current treatment of visceral leishmaniasis is difficult due to a lack of effective, non-toxic, and non-extensive medications. This study aimed to evaluate the selectivity of 12 synthetic endoperoxides (1,2,4-trioxolanes; 1,2,4,5-tetraoxanes) and uncover their biochemical effects on Leishmania parasites responsible for visceral leishmaniasis. The compounds were screened for in vitro activity against L. infantum and L. donovani and for cytotoxicity in two monocytic cell lines (J774A.1 and THP-1) using the methyl thiazol tetrazolium assay. Reactive oxygen species formation, apoptosis, and mitochondrial impairment were measured by flow cytometry. The compounds exhibited fair to moderate anti-proliferative activity against promastigotes of the 2 Leishmania species, with IC50 values ranging from 13.0 ± 1.7 µM to 793.0 ± 37.2 µM. Tetraoxanes LC132 and LC138 demonstrated good leishmanicidal activity on L. infantum amastigotes (IC50 13.2 ± 5.2 and 23.9 ± 2.7 µM) with low cytotoxicity in mammalian cells (SIs 22.1 and 118.6), indicating selectivity towards the parasite. Furthermore, LC138 was able to induce late apoptosis and dose-dependent oxidative stress without affecting mithocondria. Compounds LC132 and LC138 can be further explored as potential antileishmanial chemotypes.

Cystoseira is a common brown algal genus widely distributed throughout the Atlantic and Mediterranean regions whose taxonomical assignment of specimens is often hampered by intra- and interspecific morphological variability. In this study, three mitochondrial regions, namely cytochrome oxidase subunit 1 (COI), 23S rDNA (23S), and 23S-tRNAVal intergenic spacer (mt-spacer) were used to analyse the phylogenetic relationships of 22 Cystoseira taxa (n = 93 samples). A total of 135 sequences (48 from COI, 43 from 23S and 44 from mt-spacer) were newly generated and analysed together with Cystoseira sequences (9 COI, 31 23S and 35 mt-spacer) from other authors. Phylogenetic analysis of these three markers identified 3 well-resolved clades and also corroborated the polyphyletic nature of the genus. The resolution of Cystoseira taxa within the three clades improves significantly when the inclusion of specimens of related genera was minimized. COI and mt-spacer markers resolved the phylogeny of some of the Cystoseira taxa, such as the C. baccata, C. foeniculacea and C. usneoides. Furthermore, trends between phylogeny, embryonic development and available chemotaxonomic classifications were identified, showing that phylogenetic, chemical and morphological data should be taken into account to study the evolutionary relationships among the algae currently classified as Cystoseira. The resolution of Cystoseira macroalgae into three well supported clades achieved here is relevant for a more accurate isolation and identification of natural compounds and the implementation of conservation measures for target species.

Canine leishmaniosis (CanL) is a vector-borne disease caused by the protozoan Leishmania (Leishmania) infantum species [syn. L. (L.) infantum chagasi species in the Americas] which is transmitted by the bite of a female phlebotomine sand fly. This parasitosis is endemic and affect millions of dogs in Asia, the Americas and the Mediterranean basin. Domestic dogs are the main hosts and the main reservoir hosts for human zoonotic leishmaniosis. The outcome of infection is a consequence of intricate interactions between the protozoan and the immunological and genetic background of the host. Clinical manifestations can range from subclinical infection to very severe disease. Early detection of infected dogs, their close surveillance and treatment are essential to control the dissemination of the parasite among other dogs, being also a pivotal element for the control of human zoonotic leishmaniosis. Hence, the identification of biomarkers for the confirmation of Leishmania infection, disease and determination of an appropriate treatment would represent an important tool to assist clinicians in diagnosis, monitoring and in giving a realistic prognosis to subclinical infected and sick dogs. Here, we review the recent advances in the identification of Leishmania infantum biomarkers, focusing on those related to parasite exposure, susceptibility to infection and disease development. Markers related to the pathogenesis of the disease and to monitoring the evolution of leishmaniosis and treatment outcome are also summarized. Data emphasizes the complexity of parasite-host interactions and that a single biomarker cannot be used alone for CanL diagnosis or prognosis. Nevertheless, results are encouraging and future research to explore the potential clinical application of biomarkers is warranted.

Leishmaniases are diseases caused by protozoa of the genus Leishmania that are phagocytic cells-obligated intracellular parasites. The aim of this study was to characterize the infection by Leishmania infantum on intradermally inoculated BALB/c mice, analyzing the evolution of parasitism and the immune responses developed. Parasite load was determined by real time PCR. Since day 7 post-infection, parasites were detected and visceral dissemination was observed 56 days post-infection. Lymphocytes from the animals of the infected group proliferated in response to antigenic stimulation, while peritoneal macrophages produced nitric oxide in the presence of Leishmania antigen. These results demonstrated that BALB/c mice intradermally inoculated are a good experimental model of visceral leishmaniasis.

Abstract Background Leishmaniosis, dirofilariosis and angiostrongylosis are parasitic diseases of established importance in dogs worldwide. The aim of the present report was to determine all together levels of infection with or exposure to Leishmania infantum, Dirofilaria immitis and Angiostrongylus vasorum in dogs from the Algarve region, in southern Portugal. Findings Serum samples were obtained from a total of 170 apparently healthy dogs. Antibodies to L. infantum were detected by the direct agglutination test; and antigens of D. immitis and of A. vasorum by an enzyme-linked immunosorbent assay and an immunochromatography test, respectively. Antibodies to L. infantum were found in 18.2% (31/170) of dogs, while D. immitis antigen was detected in 9.4% (16/170). None of 120 dogs tested out of the 170 under investigation had evidence of infection with A. vasorum. D. immitis infection was more prevalent in dogs aged 84–204 months than in those younger than 12 months. Three dogs were positive to both L. infantum and D. immitis. Conclusions Dogs living in southern Portugal are at risk of leishmaniosis and dirofilariosis. This scenario should make the veterinary community, local dog owners and also tourists from non-endemic countries coming on vacation with their pets aware of the need of effective prophylactic measures, in order to protect animals and public health.

Abstract Background The so-called canine vector-borne diseases (CVBD) are caused by a wide range of pathogens transmitted by arthropods. In addition to their veterinary importance, many of these canine vector-borne pathogens can also affect the human population due to their zoonotic potential, a situation that requires a One Health approach. As the prevalence of vector-borne pathogens in cats from southern Portugal has been recently evaluated, the aim of the present study was to assess if the same agents were present in dogs living in the same area, and to assess positivity-associated risk factors. Methods One thousand and ten dogs (521 domestic and 489 stray) from veterinary medical centres and animal shelters in southern Portugal were enrolled. Anaplasma spp./Ehrlichia spp., Bartonella spp., Borrelia burgdorferi sensu lato, Babesia spp., Hepatozoon spp. and Leishmania infantum infections were evaluated by polymerase chain reaction (PCR) assays in blood samples. Results Sixty-eight (6.7%) dogs were PCR-positive to at least one of the tested CVBD agent species, genera or complex, including one dog found positive to two different genera. Nineteen (1.9%) dogs were positive to Anaplasma spp./Ehrlichia spp., eight (0.8%) to B. burgdorferi s.l., 31 (3.1%) to Hepatozoon spp. and 11 (1.1%) to L. infantum. Anaplasma platys, Ehrlichia canis, B. burgdorferis.l. and Hepatozoon canis were identified by DNA sequencing, including one animal confirmed with both A. platys and H. canis. Furthermore, Wolbachia spp. was amplified in blood from four dogs. None of the tested dogs was positive by PCR for Bartonella spp. or Babesia spp. Conclusions The molecular identification of CVBD agents in southern Portugal, some of them with zoonotic concern, reinforces the importance to alert the veterinary community, owners and public health authorities to prevent the risk of transmission of vector-borne pathogens among dogs and to other vertebrate hosts including humans. The prevalence of the selected pathogens was lower than that previously found in cats from the same region, probably because veterinarians and owners are more aware of them in the canine population and control measures are used more often.

Abstract Background Zoonotic visceral leishmaniasis caused by Leishmania infantum which is transmitted by phlebotomine sand flies (Diptera, Psychodidae) is endemic in the Mediterranean basin. The main objectives of this study were to (i) detect Leishmania DNA and (ii) identify blood meal sources in wild caught female sand flies in the zoonotic leishmaniasis region of Algarve, Portugal/Southwestern Europe. Methods Phlebotomine sand flies were collected using CDC miniature light traps and sticky papers. Sand flies were identified morphologically and tested for Leishmania sp. by PCR using ITS-1 as the target sequence. The source of blood meal of the engorged females was determined using the cyt-b sequence. Results Out of the 4,971 (2,584 males and 2,387 females) collected sand flies, Leishmania DNA was detected by PCR in three females (0.13%), specifically in two specimens identified on the basis of morphological features as Sergentomyia minuta and one as Phlebotomus perniciosus. Haematic preferences, as defined by the analysis of cyt-b DNA amplified from the blood-meals detected in the engorged female specimens, showed that P. perniciosus fed on a wide range of domestic animals while human and lizard DNA was detected in engorged S. minuta. Conclusions The anthropophilic behavior of S. minuta together with the detection of Leishmania DNA highlights the need to determine the role played by this species in the transmission of Leishmania parasites to humans. In addition, on-going surveillance on Leishmania vectors is crucial as the increased migration and travelling flow elevate the risk of introduction and spread of infections by Leishmania species which are non-endemic.

The genus Onchocerca encompasses parasitic nematodes including Onchocerca volvulus, causative agent of river blindness in humans, and the zoonotic Onchocerca lupi infecting dogs and cats. In dogs, O. lupi adult worms cause ocular lesions of various degrees while humans may bear the brunt of zoonotic onchocercosis with patients requiring neurosurgical intervention because of central nervous system localization of nematodes. Though the zoonotic potential of O. lupi has been well recognized from human cases in Europe, the United States and the Middle East, a proper therapy for curing this parasitic infection in dogs is lacking. To evaluate the efficacy of oxfendazole, 11 out of the 21 client-owned dogs (21/123; 17.1%) positive for skin-dwelling O. lupi microfilariae (mfs), were enrolled in the efficacy study and were treated with oxfendazole (50 mg/kg) per OS once a day for 5 (G2) or 10 (G3) consecutive days or were left untreated (G1). The efficacy of oxfendazole in the reduction of O. lupi mfs was evaluated by microfilarial count and by assessing the percentage of mfs reduction and mean microfilaricidal efficacy, whereas the efficacy in the reduction of ocular lesions was evaluated by ultrasound imaging. All dogs where subjected to follow-ups at 30 (D30), 90 (D90) and 180 (D180) days post-treatment. The percentage of reduction of mfs was 78% for G2 and 12.5% for G3 at D180. The mean microfilaricidal efficacy of oxfendazole in the treatment of canine onchocercosis by O. lupi at D30, D90 and D180 was 41%, 81% and 90%, in G2 and 40%, 65% and 70%, in G3, respectively. Retrobulbar lesions did not reduce from D0 to D180 in control group (dogs in G1), whereas all treated dogs (in G2 and G3) had slightly decreased ocular lesions. Percentage of reduction of ocular lesions by ultrasound examination was 50% and 47.5% in G2 and G3 at D180, respectively. Despite the decrease in ocular lesions in all treated dogs (G2 and G3), oxfendazole was ineffective in reducing ocular lesions and skin-dwelling O. lupi mfs in treated dogs (G2 and G3) in a six-month follow-up period. Here we discuss the need for more reliable diagnostic techniques and efficient treatment protocols to better plan future intervention strategies.

Abstract Background Feline vector-borne diseases (FVBD) have emerged in recent years, showing a wider geographic distribution and increased global prevalence. In addition to their veterinary importance, domestic cats play a central role in the transmission cycles of some FVBD agents by acting as reservoirs and sentinels, a circumstance that requires a One Health approach. The aim of the present work was to molecularly detect feline vector-borne bacteria and protozoa with veterinary and zoonotic importance, and to assess associated risk factors in cats from southern Portugal. Methods Six hundred and forty-nine cats (320 domestic and 329 stray), from veterinary medical centres and animal shelters in southern Portugal, were studied. Anaplasma spp./Ehrlichia spp., Babesia spp., Bartonella spp., Borrelia burgdorferi sensu lato, Hepatozoon spp. and Leishmania spp. infections were evaluated by polymerase chain reaction (PCR) in blood samples. Results One hundred and ninety-four (29.9%) cats were PCR-positive to at least one of the tested genera or complex of FVBD agents. Sixty-four (9.9%) cats were positive to Leishmania spp., 56 (8.6%) to Hepatozoon spp., 43 (6.6%) to Babesia spp., 35 (5.4%) to Anaplasma spp./Ehrlichia spp., 19 (2.9%) to Bartonella spp. and 14 (2.2%) to B. burgdorferi s.l. Thirty-three (5.1%) cats were positive to two (n = 29) or three (n = 4) genera/complex. Babesia vogeli, Bartonella clarridgeiae, Bartonella henselae, Ehrlichia canis, Hepatozoon felis and Leishmania infantum were identified by DNA sequencing. Conclusions The occurrence of FVBD agents in southern Portugal, some of them with zoonotic character, emphasizes the need to alert the veterinary community, owners and public health authorities for the risk of infection. Control measures should be implemented to prevent the infection of cats, other vertebrate hosts and people.

Phlebotomine sand fly-borne pathogens such as Leishmania spp. and phleboviruses are emerging threats to humans and animals worldwide. The aim of this work was to evaluate the exposure of cats from Portugal to Toscana virus (TOSV) and Sandfly Fever Sicilian virus (SFSV) and assess the associated risk factors. The possible association between exposure to Phlebotomus perniciosus saliva with TOSV and SFSV was also investigated. Out of 369 cats tested, 18 (4.9%, n = 365) were seropositive for TOSV, and eight (2.2%, n = 367) were seropositive for SFSV. Multivariate logistic regression analysis showed that cats presenting clinical signs that were compatible with leishmaniosis and antibodies to TOSV had a significantly higher risk of being SFSV seropositive. The presence of antibodies to sand fly-borne viruses in cats indicate that these animals are frequently exposed to sand flies and transmitted pathogens. Data suggest that cats can be used to qualitatively monitor human exposure to TOSV and SFSV in endemic areas. The clinical impact of SFSV in cats’ health should be investigated. The identification of the sand fly species responsible for the circulation of TOSV and SFSV in nature and the evaluation of the vectorial competence of P. perniciosus to SFSV should also be addressed.

The Algarve Region (AR) in southern Portugal, which is an international tourist destination, has been considered an endemic region of zoonotic leishmaniasis caused by Leishmania infantum since the 1980s. In the present study, phlebotomine and canine surveys were conducted to identify sandfly blood meal sources and to update the occurrence of Leishmania infection in vectors and dogs. Four sandfly species were captured: Phlebotomus perniciosus, Phlebotomus ariasi, Phlebotomus sergenti and Sergentomyia minuta. In one P. perniciosus female, L. infantum DNA was detected. Blood meal tests showed that this species had no host preferences and was an opportunistic feeder. An overall canine leishmaniasis (CanL) seroprevalence of 16.06% was found; the seroprevalence was 3.88% in dogs housed in kennels and 40.63% in dogs that attended veterinary clinics. The simultaneous occurrence of dogs and P. perniciosus infected with L. infantum in the AR indicates that the region continues to be an endemic area for CanL. Our results reinforce the need for the systematic spatial distribution of phlebotomine populations and their Leishmania infection rates and the need to simultaneously perform pathogen monitoring in both invertebrate and vertebrate hosts to investigate the transmission, distribution and spreading of Leishmania infection.

BACKGROUND:The recent geographical expansion of phlebotomine vectors of Leishmania infantum in the Mediterranean subregion has been attributed to ongoing climate changes. At these latitudes, the activity of sand flies is typically seasonal; because seasonal phenomena are also sensitive to general variations in climate, current phenological data sets can provide a baseline for continuing investigations on sand fly population dynamics that may impact on future scenarios of leishmaniasis transmission. With this aim, in 2011-2013 a consortium of partners from eight Mediterranean countries carried out entomological investigations in sites where L. infantum transmission was recently reported. METHODS/PRINCIPAL FINDINGS:A common protocol for sand fly collection included monthly captures by CDC light traps, complemented by sticky traps in most of the sites. Collections were replicated for more than one season in order to reduce the effects of local weather events. In each site, the trapping effort was left unchanged throughout the survey to legitimate inter-seasonal comparisons. Data from 99,000 collected specimens were analyzed, resulting in the description of seasonal dynamics of 56,000 sand flies belonging to L. infantum vector species throughout a wide geographical area, namely P. perniciosus (Portugal, Spain and Italy), P. ariasi (France), P. neglectus (Greece), P. tobbi (Cyprus and Turkey), P. balcanicus and P. kandelakii (Georgia). Time of sand fly appearance/disappearance in collections differed between sites, and seasonal densities showed variations in each site. Significant correlations were found between latitude/mean annual temperature of sites and i) the first month of sand fly appearance, that ranged from early April to the first half of June; ii) the type of density trend, varying from a single peak in July/August to multiple peaks increasing in magnitude from May through September. A 3-modal trend, recorded for P. tobbi in Cyprus, represents a novel finding for a L. infantum vector. Adults ended the activity starting from mid September through November, without significant correlation with latitude/mean annual temperature of sites. The period of potential exposure to L.infantum in the Mediterranean subregion, as inferred by adult densities calculated from 3 years, 37 sites and 6 competent vector species, was associated to a regular bell-shaped density curve having a wide peak center encompassing the July-September period, and falling between early May to late October for more than 99% of values. Apparently no risk for leishmaniasis transmission took place from December through March in the years considered. We found a common pattern of nocturnal females activity, whose density peaked between 11 pm and 2 am. CONCLUSIONS:Despite annual variations, multiple collections performed over consecutive years provided homogeneous patterns of the potential behavior of leishmaniasis vectors in selected sites, which we propose may represent sentinel areas for future monitoring. In the investigated years, higher potential risk for L. infantum transmission in the Mediterranean was identified in the June-October period (97% relative vector density), however such risk was not equally distributed throughout the region, since density waves of adults occurred earlier and were more frequent in southern territories.

The risk for reintroduction of some exotic vector-borne diseases in Europe has become a hot topic, while the reality of others is neglected at the public health policy level. Leishmaniasis is endemic in all southern countries of Europe, with ≈700 autochthonous human cases reported each year (3,950 if Turkey is included). Asymptomatic cases have been estimated at 30–100/1 symptomatic case, and leishmaniasis has up to 25% seroprevalence in domestic dogs. Even though leishmaniasis is essentially associated with Leishmania infantum and visceral leishmaniasis, new species, such as L. donovani and L. tropica, might colonize European sand fly vectors. Drug-resistant L. infantum strains might be exported outside Europe through dogs. Despite this possibility, no coordinated surveillance of the disease exists at the European level. In this review of leishmaniasis importance in Europe, we would like to bridge the gap between research and surveillance and control.

Case summary This is the first clinical report of feline viscerocutaneous leishmaniosis caused by Leishmania infantum genotype E associated with an invasive squamous cell carcinoma (SCC) in a domestic cat from Portugal. Initially, the cat presented a single cutaneous lesion in the right nostril. A fine-needle aspiration was performed and Leishmania amastigotes were observed without the presence of cells compatible with neoplasia. Systemic treatment with allopurinol was started. One year later, the cat presented a crateriform non-encapsulated and badly delineated mass in the nasal planum, with naso-oral fistulation and nasal destruction. Histologically, the skin mass consisted on an ulcerative plaque-like lesion with a nasal SCC. Leishmania infantum MON-1 parasites were detected by histopathology, culture and PCR of the skin mass, submandibular and popliteal lymph nodes, liver and spleen. Restriction enzyme analysis revealed genotype E, previously identified in humans and dogs living in the same region. Relevance and novel information This is, to the best of our knowledge, the first clinical report of feline viscerocutaneous leishmaniosis caused by L infantum genotype E. The detection and isolation of parasites from a cat that are genetically identical to the ones obtained from humans and dogs with visceral leishmaniosis highlights the need to clarify whether cats play a role in the epidemiology of this parasitic zoonosis. From a clinical point of view, this case reinforces the importance of including leishmaniosis in the differential diagnoses of feline pathology, especially in cats with cutaneous lesions.

This work aimed to study the T helper type 1/2 (Th1/Th2) cytokine profile in a co-infection murine model of Plasmodium chabaudi chabaudi and Leishmania infantum. Expression of interferon-gamma (IFN-g) and interleukin-4 (IL-4) was analyzed, in spleen and liver of C57BL/6 mice, by reverse transcriptase-polymerase chain reaction. High levels of IFN-g expression did not prevent the progression of Leishmania in co-infected mice and Leishmania infection did not interfere with the Th1/Th2 switch necessary for Plasmodium control. The presence of IL-4 at day 28 in co-infected mice, essential for Plasmodium elimination, was probably a key factor on the exacerbation of the Leishmania infection.

Phlebotomine sandflies of the genus Sergentomyia are widely distributed throughout the Old World. It has been suggested that Sergentomyia spp are involved in the transmission of Leishmania in India and Africa, whereas Phlebotomus spp are thought to be the sole vectors of Leishmania in the Old World. In this study, Leishmania major DNA was detected in one Sergentomyia minuta specimen that was collected in the southern region of Portugal. This study challenges the dogma that Leishmania is exclusively transmitted by species of the genus Phlebotomus in the Old World.

Five mixed breed dogs were inoculated intradermally (ID) with cultured virulent stationary phase promastigotes of Leishmania infantum Nicole, 1908 stocks recently isolated. Parasite transformations in the skin of ID infected dogs were monitored from the moment of inoculation and for 48 h, by skin biopsies. Anti-Leishmania antibody levels were measured by indirect immunofluorescence assay, counterimmunoelectrophoresis and direct agglutination test, and clinical conditions were examined. Thirty minutes after ID inoculation the first amastigotes were visualised and 3 to 4 h after inoculation the promastigotes were phagocyted by neutrophils and by a few macrophages. These cells parasitised by amastigotes progressively disappeared from the skin and 24 h after inoculation parasites were no longer observed. Local granulomes were not observed, however, serological conversion for antibodies anti-Leishmania was achieved in all dogs. Direct agglutination test was the only technique positive in all inoculated dogs. Amastigotes were found in the popliteal lymph node in one dog three months after inoculation. This work demonstrates that, with this inoculum, the promastigotes were transformed into amastigotes and were up taken by neutrophils and macrophages. The surviving parasites may have been disseminated in the canine organism, eliciting a humoral response in all cases.

BackgroundVisceral Leishmaniasis (VL) caused by species from the Leishmania donovani complex is the most severe form of the disease, lethal if untreated. VL caused by Leishmania infantum is a zoonosis with an increasing number of human cases and millions of dogs infected in the Old and the New World. In this study, L. infantum (syn. L.chagasi) strains were isolated from human and canine VL cases. The strains were obtained from endemic areas from Brazil and Portugal and their genetic polymorphism was ascertained using the LSSP-PCR (Low-Stringency Single Specific Primer PCR) technique for analyzing the kinetoplastid DNA (kDNA) minicircles hypervariable region.Principal findingsKDNA genetic signatures obtained by minicircle LSSP-PCR analysis of forty L. infantum strains allowed the grouping of strains in several clades. Furthermore, LSSP-PCR profiles of L. infantum subpopulations were closely related to the host origin (human or canine). To our knowledge this is the first study which used this technique to compare genetic polymorphisms among strains of L. infantum originated from both the Old and the New World.ConclusionsLSSP-PCR profiles obtained by analysis of L. infantum kDNA hypervariable region of parasites isolated from human cases and infected dogs from Brazil and Portugal exhibited a genetic correlation among isolates originated from the same reservoir, human or canine. However, no association has been detected among the kDNA signatures and the geographical origin of L. infantum strains.

Leishmaniasis has reemerged in recent years showing a wider geographic distribution and increased global incidence of human and canine disease than previously known. Dogs are the main domestic/peridomestic reservoir hosts of zoonotic visceral leishmaniasis caused by Leishmania infantum. Since the evolution of leishmaniasis and clinical appearance is a consequence of complex interactions between the parasite and host immune response, a profound knowledge about the immune profile developed in dog's infection is crucial for vaccine and immunomodulatory therapy design. The main goal of this paper is to compile the recent advances made on cytokine and phenotypic cell profiles in different tissues and organs of dogs infected with L. infantum. This paper also stressed that the knowledge of the immune responses developed, namely, in liver, lymph node, and spleen is very limited. All data emphasizes that more research on canine leishmaniasis is necessary for the development of new and efficacious tools to control zoonotic leishmaniasis.

As leishmanioses são doenças causadas por protozoários do género Leishmania que são parasitas intracelulares obrigatórios das células fagocíticas. O objectivo deste estudo foi caracterizar a infecção por Leishmania infantum em murganhos BALB/c inoculados por via intradérmica, analisando a evolução do parasitismo e as respostas imunitárias desenvolvidas. A carga parasitária foi determinada por PCR em tempo real. Foram detectados parasitas desde o 7º dia pós‑infecção, verificando-se a disseminação visceral do parasita ao 56º dia pós-infecção. Os linfócitos dos animais do grupo infectado proliferaram em resposta à estimulação antigénica, enquanto que os macrófagos peritoneais produziram nitritos na presença do antigénio. Estes resultados demonstraram que os murganhos BALB/c inoculados por via intradérmica constituem um bom modelo experimental de leishmaniose visceral.

Leishmaniases, caused by the intracellular protozoan Leishmania infantum, are an endemic zoonosis in the Mediterranean basin. Dogs are considered the major host for these parasites, and the main reservoir for human visceral infection. Parasites are transmitted by the bite of phlebotomine sand flies, being Phlebotomus perniciosus and P. ariasi the proven vectors in Portugal. The global climate changes associated with a higher density and activity of sand flies during a larger period might enhance the number of days favorable for transmission of parasites to humans and animals with a concomitant increase of incidence. In our country, as in other countries in the south of Europe, visceral leishmaniasis was initially a pediatric disease but in the last years the number of cases in children has decreased with an increase of infection in adults, normally associated with HIV/AIDS. Between 2000 and 2009, 173 new cases of human visceral leishmaniasis (66 in immunocompetent adults and children and 107 in immunocompromised patients) were observed in the Leishmaniasis Unit - IHMT. After the introduction of antiretroviral therapy HAART for HIV, the number of co-infected cases reported from southern Europe has fallen dramatically in all countries except in Portugal. In this country the number of coinfection new cases did not decrease - neither the frequent relapses. Despite cutaneous leishmaniasis being barely known in Portugal, it should not be considered as a rare disease since about ten new cases per year are diagnosed. In addition, canine leishmaniasis has increased with a prevalence rate up to 20% found in endemic foci. In 2008 the Portuguese National Leishmaniasis Observatory was created. The development of national and international epidemiological networks will help the knowledge of leishmaniasis and will promote opportunities to advise health authorities about the most effective measures for prevention and control of this parasitosis. These networking initiatives and, importantly their integration, will, hopefully, contribute towards a significant reduction of the incidence/prevalence of human and canine visceral leishmaniasis.

Leishmania infantum is the causative agent of visceral and cutaneous leishmaniasis in the Mediterranean region, South America, and China. MON-1 L. infantum is the predominating zymodeme in all endemic regions, both in humans and dogs, the reservoir host. In order to answer important epidemiological questions it is essential to discriminate strains of MON-1.We have used a set of 14 microsatellite markers to analyse 141 strains of L. infantum mainly from Spain, Portugal, and Greece of which 107 strains were typed by MLEE as MON-1. The highly variable microsatellites have the potential to discriminate MON-1 strains from other L. infantum zymodemes and even within MON-1 strains. Model- and distance-based analysis detected a considerable amount of structure within European L. infantum. Two major monophyletic groups-MON-1 and non-MON-1-could be distinguished, with non-MON-1 being more polymorphic. Strains of MON-98, 77, and 108 were always part of the MON-1 group. Among MON-1, three geographically determined and genetically differentiated populations could be identified: (1) Greece; (2) Spain islands-Majorca/Ibiza; (3) mainland Portugal/Spain. All four populations showed a predominantly clonal structure; however, there are indications of occasional recombination events and gene flow even between MON-1 and non-MON-1. Sand fly vectors seem to play an important role in sustaining genetic diversity. No correlation was observed between Leishmania genotypes, host specificity, and clinical manifestation. In the case of relapse/re-infection, only re-infections by a strain with a different MLMT profile can be unequivocally identified, since not all strains have individual MLMT profiles.In the present study for the first time several key epidemiological questions could be addressed for the MON-1 zymodeme, because of the high discriminatory power of microsatellite markers, thus creating a basis for further epidemiological investigations.

Due to its morbility and disfiguring scars, cutaneous leishmaniasis is a serious Public Health problem. The incidence of the disease, world wide, is estimated to be of 1 million to 1.5 million cases per year. In Portugal, it has been considered a rare disease and sporadic cases have been described. In Southwest Europe, Leishmania infantum has been the only agent responsible for the autochthonous cases of cutaneous leishmaniasis. In 1994, in an portuguese clinical case, the parasite isolated from a cutaneous lesion was identified as L. infantum zymodeme MON-29. As described in this work cutaneous leishmaniasis should not be considered, any longer, a rare disease in Portugal.