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Background: In the Mediterranean basin, three Leishmania species have been identified: L. infantum, L. major and L. tropica, causing zoonotic visceral leishmaniasis (VL), zoonotic cutaneous leishmaniasis (CL) and anthroponotic CL, respectively. Despite animal models and genomic/transcriptomic studies provided important insights, the pathogenic determinants modulating the development of VL and CL are still poorly understood. This work aimed to identify host transcriptional signatures shared by cells infected with L. infantum, L. major, and L. tropica, as well as specific transcriptional signatures elicited by parasites causing VL (i.e., L. infantum) and parasites involved in CL (i.e., L. major, L. tropica). Methodology/Principal findings: U937 cells differentiated into macrophage-like cells were infected with L. infantum, L. major and L. tropica for 24h and 48h, and total RNA was extracted. RNA sequencing, performed on an Illumina NovaSeq 6000 platform, was used to evaluate the transcriptional signatures of infected cells with respect to non-infected cells at both time points. The EdgeR package was used to identify differentially expressed genes (fold change > 2 and FDR-adjusted p-values < 0.05). Then, functional enrichment analysis was employed to identify the enriched ontology terms in which these genes are involved. At 24h post-infection, a common signature of 463 dysregulated genes shared among all infection conditions was recognized, while at 48h post-infection the common signature was reduced to 120 genes. Aside from a common transcriptional response, we evidenced different upregulated functional pathways characterizing L. infantum-infected cells, such as VEGFA-VEGFR2 and NFE2L2-related pathways, indicating vascular remodeling and reduction of oxidative stress as potentially important factors for visceralization. Conclusions: The identification of pathways elicited by parasites causing VL or CL could lead to new therapeutic strategies for leishmaniasis, combining the canonical anti-leishmania compounds with host-directed therapy. Author summary: Leishmaniasis is a zoonosis caused by the intracellular parasite of the genus Leishmania. In the Mediterranean region, the human disease can be present mainly in two forms: cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL), which can be fatal if untreated. The clinical presentation of the disease depends on the parasite species and host characteristics. However, the molecular mechanisms underlying the development of the visceral form of leishmaniasis are not yet fully understood. In this study, we have determined the gene expression profile in cells infected by three Leishmania species (L. infantum, L. major, L. tropica) responsible for different forms of the disease. The results have allowed us to identify an expression profile (and the related pathways) shared by cells infected by all Leishmania species. Aside this common response, we also identified a transcriptional response distinguishing cells infected by L. infantum, L. major or L. tropica, as well as the relevant pathways in which these genes participate. Therefore, our results contribute to the knowledge of the host cell mechanisms that are activated along infection with different Leishmania species causing different forms of the disease and, therefore, may help develop new host-directed therapy for Leishmaniasis. [ABSTRACT FROM AUTHOR]

A letter to the editor is presented which discusses a case of feline leishmaniosis with a high parasitic burden.

Background: Leishmaniasis is a zoonotic disease endemic in the Mediterranean region where Leishmania infantum is the causative agent of human and canine infection. Characterization of this parasite at the subspecies level can be useful in epidemiological studies, to evaluate the clinical course of the disease (e.g. resistant strains, visceral and cutaneous forms of leishmaniasis) as well as to identify infection reservoirs. Multilocus enzyme electrophoresis (MLEE), a method currently recognized as the reference method for characterizing and identifying strains of Leishmania, is cumbersome and time-consuming and requires cultured parasites. These disadvantages have led to the development of other methods, such as multilocus microsatellite typing (MLMT) and multilocus sequence typing (MLST), for typing Leishmania parasites; however, these methods have not yet been applied for routine use. In this study, we first used MLST to identify informative polymorphisms on single-copy genes coding for metabolic enzymes, following which we developed two rapid genotyping assays based on high-resolution melting (HRM) analysis to explore these polymorphisms in L. infantum parasites. Methods: A customized sequencing panel targeting 14 housekeeping genes was designed and MLST analysis was performed on nine L. infantum canine and human strains/isolates. Two quantitative real-time PCR-HRM assays were designed to analyze two informative polymorphisms on malic enzyme (ME) and glucose-6-phosphate isomerase (GPI) genes (390T/G and 1831A/G, respectively). The two assays were applied to 73 clinical samples/isolates from central/southern Italy and Pantelleria island, and the results were confirmed by DNA sequencing in a subset of samples. Results: The MLST analysis, together with sequences available in the Genbank database, enabled the identification of two informative polymorphisms on the genes coding for ME and GPI. The fast screening of these polymorphisms using two HRM-based assays in 73 clinical samples/isolates resulted in the identification of seven genotypes. Overall, genotype 1 (sequence type 390T/1831G) was the most highly represented (45.2%) in the overall sample and correlated with the most common L. infantum zymodemes (MON-1, MON-72). Interestingly, in Pantelleria island, the most prevalent genotype (70.6%) was genotype 6 (sequence type 390T/1831A). Conclusions: Applying our HRM assays on clinical samples allowed us to identify seven different genotypes without the need for parasite isolation and cultivation. We have demonstrated that these assays could be used as fast, routine and inexpensive tools for epidemiological surveillance of L. infantum or for the identification of new infection reservoirs. [ABSTRACT FROM AUTHOR]

Most of the antileishmanial modern therapies are not satisfactory due to high toxicity or emergence of resistance and high cost of treatment. Previously, we observed that two compounds of a small library of trans-stilbene and terphenyl derivatives, ST18 and TR4, presented the best activity and safety profiles against Leishmania infantum promastigotes and amastigotes. In the present study we evaluated the effects of ST18 and the TR4 in 6 different species of Leishmania and the modifications induced by these two compounds in the production of 8 different cytokines from infected macrophages. We observed that TR4 was potently active in all Leishmania species tested in the study showing a leishmanicidal activity higher than that of ST18 and meglumine antimoniate in the most of the species. Moreover, TR4 was able to decrease the levels of IL-10, a cytokine able to render the host macrophage inactive allowing the persistence of parasites inside its phagolysosome, and increase the levels of IL-1β, a cytokine important for host resistance to Leishmania infection by inducible iNOS-mediated production of NO, and IL-18, a cytokine implicated in the development of Th1-type immune response. [ABSTRACT FROM AUTHOR]

Leishmaniasis are globally widespread parasitic diseases which often leads to death if left untreated. Currently available drugs present different drawbacks, so there is an urgent need to develop new, safe and cost-effective drugs against leishmaniasis. In this study we tested a small library of trans -stilbene and terphenyl derivatives against promastigote, amastigotes and intramacrophage amastigote forms of Leishmania infantum . Two compounds of the series, the trans -stilbene 3 and the terphenyl 11 , presented the best activity and safety profiles. Terphenyl 11 showed a leshmanicidal activity higher than pentostam and the ability to induce apoptosis selectively in Leishmania infantum while saving macrophages and primary epithelial cells. Our data indicate that terphenyl compounds, as well as stilbenes, are endowed with leishmanicidal activity, showing potential for further studies in the context of leishmanial therapy. [ABSTRACT FROM AUTHOR]

Background: Leishmaniases are a group of anthropo-zoonotic parasitic diseases caused by a protozoan of the Leishmania genus, affecting both humans and other vertebrates, including dogs. L. infantum is responsible for the visceral and occasionally cutaneous form of the disease in humans and canine leishmaniasis. Previously, we have shown that L. infantum induces a mild but significant increase in endoplasmic reticulum (ER) stress expression markers to promote parasites survival in human and murine infected macrophages. Moreover, we demonstrated that the miRNA hsa-miR-346, induced by the UPR-activated transcription factor sXBP1, was significantly upregulated in human macrophages infected with different L. infantum strains. However, the ER stress response in infected dogs, which represent an important reservoir for Leishmania parasite, was described once recently, whereas the miR-346 expression was not reported before. Therefore, this study aimed to investigate these pathways in the canine macrophage-like cell line DH82 infected by Leishmania spp. and to evaluate the presence of cfa-miR-346 in plasma of non-infected and infected dogs. The DH82 cells were infected with L. infantum and L. braziliensis parasites and the expression of cfa-mir-346 and several ER stress markers was evaluated by quantitative PCR (qPCR) at different time points. Furthermore, the cfa-miR-346 was monitored in plasma collected from non-infected dogs (n = 11) and dogs naturally infected by L. infantum (n = 18). Results: The results in DH82 cells showed that cfa-mir-346 was induced at both 24 h and 48 h post-infection with all Leishmania strains but not with tunicamycin, accounting for a mechanism of induction independent from sXBP1, unlike what was previously observed in human cell lines. Moreover, the cfa-miR-346 expression analysis on plasma revealed a significant increase in infected dogs compared to non-infected dogs. Conclusions: Here for the first time, we report the upregulation of cfa-miR-346 induced by Leishmania infection in canine macrophage-like cells and plasma samples of naturally infected dogs. According to our results, the cfa-miR-346 appears to be linked to infection, and understanding its role and identifying its target genes could contribute to elucidate the mechanisms underlying the host–pathogen interaction in leishmaniasis. [ABSTRACT FROM AUTHOR]

Background: Feline leishmaniosis caused by Leishmania infantum is often associated with feline immunodeficiency virus (FIV) infection; however, the role and clinical significance of this coinfection remain unknown. This study aimed to assess whether FIV is associated with L. infantum infection in cats from canine leishmaniosis endemic areas and to report the clinical signs and hematological alterations associated with coinfection. Methods: A retrospective matched case-control study (ratio 1:2) was conducted. Data of clinical examination and complete blood count (CBC) were selected from a cohort of 705 cats examined for epidemiological studies on feline leishmaniosis conducted between 2012 and 2019. Ninety-one FIV seropositive cases and 182 FIV seronegative control cats were selected. Matching was done according to age, sex, lifestyle and geographic provenience of case cats. Rapid ELISA devices were mainly used to detect anti-FIV antibodies. Anti-Leishmania IgG antibodies were detected by indirect-immunofluorescence test (IFAT). Leishmania DNA was searched in blood, oral and conjunctival swabs by quantitative real-time PCR. Results: Feline immunodeficiency virus seropositive cats had no hematological abnormalities suggestive of an advanced stage of FIV infection and were statistically more frequently IFAT positive, and their risk of being L. infantum antibody positive was 2.8 greater than in the FIV seronegatives. The association of FIV seropositivity with L. infantum antibody positivity was confirmed in the univariable model of logistic regression. A multivariate model found FIV infection and L. infantum PCR positivity as predictors of a positive L. infantum IFAT result. Male outdoor cats from rural or suburban areas were at risk for FIV and L. infantum antibody positivity. Clinical signs more frequently associated with the coinfection were oral lesions, pale mucous membranes and low body condition score (BCS). Conclusions: This study documents that FIV seropositive cats with no hematological abnormalities suggestive of an advanced stage of FIV infection are more prone to be L. infantum seroreactive by IFAT in endemic areas. Therefore, FIV seropositive cats should be tested for L. infantum antibodies and treated for preventing sand fly bites. Pale mucous membranes, low BCS and oral lesions but no CBC abnormalities were significantly associated with the coinfection. [ABSTRACT FROM AUTHOR]

The aim of this study was to evaluate, through qPCR, the prevalence of parasitemia in sick kennel dogs naturally infected by canine leishmaniasis. An evaluation of daily changes of the parasitic load in peripheral blood was also performed. A comprehensive clinical examination and the collection of several samples (blood, lymph node, skin, and conjunctiva) were performed in 140 dogs living in an endemic area. Among these, only the dogs with clinically evident leishmaniasis were enrolled (39/140; 27.9%). Twelve (30.8%) out of 39 showed parasitemia, with a low load (median: 4 Leishmania/ml) despite a high lymph node parasite load (median: 4000 Leishmania/ml) and high IFAT titers (≥ 1:640). Seven sick dogs were sampled every 4 h for 6 times during a 24-h period, in order to obtain light- and dark-span samples. Only one (14.3%) out of the seven serial sampled dogs showed Leishmania DNA in the peripheral blood in two samples (2/42; 4.8%). Surprisingly, Leishmania DNA was also detected in the peripheral blood of asymptomatic dogs, negative to both serology and PCR performed on samples other than blood (6/101; 5.9%). The present study confirms that in canine leishmaniasis parasitemia is uncommon and even transitory. Even if recommended, microscopic examination is confirmed as a low sensitivity method with a lower diagnostic utility in canine leishmaniasis than qPCR. Moreover, circulating Leishmania DNA can be found even in healthy dogs. This finding is important in clinical practice because in endemic areas it suggests a transfusion risk and a possible transmission to the vector. [ABSTRACT FROM AUTHOR]

Abstract: Visceral leishmaniasis (VL) is a life-threatening disease of medical, social and economic importance in endemic areas. Dogs are the main reservoir of Leishmania infantum. In this study, the authors investigated a group of 56 natural infected dogs to establish the relationship between parasite load and various clinical forms of leishmaniasis. The sick dogs were monitored at the beginning from clinical and physiological point of view. Leishmania load was measured by real-time PCR assay on whole blood samples and lymph node aspirates, collected at the time of diagnosis. Our results indicate that a higher quantity of Leishmania DNA was found in the lymph nodes of dogs characterized by maximum clinical score. This interesting finding indicates the presence of a positive relationship between Leishmania load and clinical manifestations in dogs showing a severe clinical form of leishmaniasis. [Copyright &y& Elsevier]

Background: During last decade Lampedusa island (Italy) has been interested by a deep social change caused by the massive arrival of migrants from north Africa. The goal of this study was to evaluate current CanL burden and risk factors for Visceral Leishmaniosis (VL) on Lampedusa, actually based on very few data obtained in a previous study performed fifteen years ago. Two hundred and forty-two dogs were enrolled for the detection of Leishmania infantum infection by serology. In addition, an entomological investigation was performed to confirm the presence of Leishmania-vectors. Results: Seroprevalence was of 54.13%. 223 sand flies specimens were collected. Among them, 4 species were identified: Phlebotomus perniciosus, P. papatasi, P. neglectus, Sergentomia minuta, with P. perniciosus the most abundant (67.7%; p < 0.01). Conclusion: The high proportion of seropositive dogs together with the presence of the most competent vector for L. infantum, P. perniciosus, demonstrate that L. infantum abundantly circulates in the island and may constitute a risk for people, particularly for hosted migrants. [ABSTRACT FROM AUTHOR]

Background: Visceral leishmaniasis (VL) caused by Leishmania infantum is an ongoing health problem in southern Europe, where dogs are considered the main reservoirs of the disease. Current data point to a northward spread of VL and canine leishmaniasis (CanL) in Italy, with new foci in northern regions previously regarded as non-endemic. Methodology/Principal findings: Multilocus microsatellite typing (MLMT) was performed to investigate genetic diversity and population structure of L. infantum on 55 samples from infected humans, dogs and sand flies of the E-R region between 2013 and 2017. E-R samples were compared with 10 L. infantum samples from VL cases in other Italian regions (extra E-R) and with 52 strains within the L. donovani complex. Data displayed significant microsatellite polymorphisms with low allelic heterozygosity. Forty-one unique and eight repeated MLMT profiles were recognized among the L. infantum samples from E-R, and ten unique MLMT profiles were assigned to the extra E-R samples. Bayesian analysis assigned E-R samples to two distinct populations, with further sub-structuring within each of them; all CanL samples belonged to one population, genetically related to Mediterranean MON-1 strains, while all but one VL cases as well as the isolate from the sand fly Phlebotomus perfiliewi fell under the second population. Conversely, VL samples from other Italian regions proved to be genetically similar to strains circulating in dogs. Conclusions/Significance: A peculiar epidemiological situation was observed in northeastern Italy, with the co-circulation of two distinct populations of L. infantum; one population mainly detected in dogs and the other population detected in humans and in a sand fly. While the classical cycle of CanL in Italy fits well into the data obtained for the first population, the population found in infected humans exhibits a different cycle, probably not involving a canine reservoir. This study can contribute to a better understanding of the population structure of L. infantum circulating in northeastern Italy, thus providing useful epidemiologic information for public health authorities. [ABSTRACT FROM AUTHOR]

Human leishmaniasis is an emerging problem in Italy and is on the increase in the Emilia-Romagna region, northeastern part of the country. Nevertheless, studies dealing with the molecular characterization of Leishmania spp. circulating in these areas are limited. In the present work, we explored the genetic polymorphism of Leishmania isolates from 28 cases of canine leishmaniasis and three cases of human visceral leishmaniasis (VL), which occurred in 2013-2014 in the Emilia-Romagna region. The characterization was carried out in comparison with nine human isolates of Leishmania from other VL endemic Italian regions and two reference strains. Nucleic acid from 31 Leishmania-positive phlebotomine sandfly pools, sampled in 2012-2013 in the Emilia-Romagna region, were also evaluated. DNA amplification and sequencing of the ribosomal internal transcribed spacer-1 and of a repetitive nuclear region on chromosome 31 were carried out for genotyping. Two size polymorphic targets were also analyzed by PCR, the cpb E/F-gene and the k26-gene. Altogether, the analysis showed the circulation of different Leishmania infantum genotypes in the Emilia-Romagna region: two genotypes found in dogs from public kennels were similar to VL isolates from other Italian regions, whereas a third genotype was detected in VL cases of the Emilia-Romagna region and in all but one of the sandfly pools. The combined molecular tools applied in this study can constitute a helpful support for parasite tracking ( e.g., in outbreak investigations) and for a better understanding of the epidemiological evolution of leishmaniasis in northeastern Italy. [ABSTRACT FROM AUTHOR]

The article provides information on a study which compared vascular pattern to polymerase chain reaction (PCR) value in cutaneous leishmaniasis and proposed the use of videodermatoscopy to improve the accuracy of clinical diagnosis of the disease. Topics discussed include multi-locus enzyme electrophoresis (MLEE), vascular structures identified, and factors affecting the clinical features of cutaneous leishmaniasis.

[Display omitted] • Feline Leishmania strains are phenotypically similar to those from humans and dogs. • Feline, canine, and human strains have an identical susceptibility to drugs. • Parasites isolated from cats can infect feline, canine, and human macrophages. • Feline macrophages are permissive to strains isolated from dogs and humans. Zoonotic visceral leishmaniosis is a worldwide severe disease caused by Leishmania infantum , a protozoan that has phlebotomine sand flies as vectors and dogs as primary reservoir hosts. Over the last few decades, cats have been regarded as an indisputable piece within the ecological system in which L. infantum is maintained indefinitely. However, little is known about feline strains, including their phenotypic plasticity and infectivity. In this study, the phenotypic behaviour of seven L. infantum feline strains was compared to those of well-characterised counterparts isolated from two dogs and two humans in terms of growth profile, adaptive capacity under several stress conditions, susceptibility to antileishmanial drugs, and infectivity to host cells. Feline strains displayed a similar growth profile, survival capacity, and ability to infect feline, canine, and human monocyte-derived primary macrophages. Furthermore, multivariate cluster analysis suggested that most strains studied did not display distinctive phenotypic features. To our knowledge, this is the first study to analyse the phenotypic behaviour of feline L. infantum strains. This study brings new insights into the hypothetical role of cats as reservoir hosts of L. infantum since the parasites found in them are phenotypically identical to those of dogs and humans. However, further studies on the transmission dynamics should be encouraged to fully establish the status of cats in the maintenance of L. infantum foci. [ABSTRACT FROM AUTHOR]