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In this study, different types of tissue sampling for PCR-based diagnosis and follow-up of canine visceral leishmaniosis were compared. Skin, whole blood and lymph node samples were collected from 95 naturally infected dogs living in South Italy, where the disease is endemic. Twenty-nine of these 95 dogs, treated with meglumine administered concurrently with allopurinol for 30 days, and then with allopurinol alone, were monitored during a period of 2 years. The DNA extracted from the clinical specimens was amplified by PCR using as target DNA a 116-bp fragment in the constant region of the kinetoplast DNA minicircle. PCR analysis was more sensitive than indirect immunofluorescence antibody test in detecting Leishmania infection in symptomatic dogs: 99% of lymph node samples resulted positive, whereas 94% of blood samples and 95% of skin samples gave a positive result. PCR analysis of samples from dogs followed up 2 years showed that: (1) all subjects resulted positive in at least one of the three types of samples; (2) all time the dogs had a relapse, PCR resulted positive in all three types of samples; (3) when dogs were apparently healthy, PCR analysis was positive on skin and lymph node samples, but not always on blood samples. Since lymph node sampling is invasive and sometimes difficult in healthy asymptomatic dogs, our results suggest that, independently from the presence or not of cutaneous lesions, skin biopsy represents a good substratum for PCR-based diagnosis and follow-up of canine visceral leishmaniosis. [Copyright &y& Elsevier]

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]

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]

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]

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]

Background: Leishmaniasis is one of the most important vector-borne diseases and it represents a serious world health problem affecting millions of people. High levels of Leishmania infections, affecting both humans and animals, are recognized among Italian regions. Among these, Sicily has one of the highest prevalence of Leishmania infection. Methodology/Principal Findings: Seventy-eight Leishmania strains isolated from human and animal samples across Sicily, were analyzed for the polymorphic k26-gene and genotypes were assigned according to the size of the PCR products. A multilocus microsatellite typing (MLMT) approach based on the analysis of 11 independent loci was used to investigate populations structure and genetic diversity of the isolated strains. Six L. infantum reference strains were included in the analysis for comparison. Bayesian clustering analysis of microsatellite data showed that all the isolated strains clustered in two genetically distinct populations, corresponding to human and canine isolates respectively. A further subdivision was observed between the two main groups, giving a good correlation between human strains and their geographic origin, conversely canine population showed a great genetic variability diffused in the territory. Conclusions/Significance: Among the 78 Leishmania isolates, K26 analysis detected 71 samples (91%) as MON-1 zymodeme, confirming it as the predominant strain in Mediterranean area and 7 human samples (9%) as non-MON-1. MLMT gives important insights into the epidemiology of leishmaniases and allows characterization of different strains to a higher resolution than possible with zymodeme typing. Two main populations presented a strong correlation respect to the different hosts, exhibiting a co-circulation of two distinct populations of L. infantum. The population found in infected humans exhibited a correlation with geographic origin. These clusters could represent a geographically restricted population of strains with the same or related genotypes. This study can contribute to an understanding of Leishmania epidemiology, including the spread of reservoirs and sand fly vectors in the different foci of infection, characterizing parasites within the different hosts. Author summary: High levels of Leishmania spp. infections affecting both humans and animals are recognized among Italian regions; in particular, Sicily is an endemic area for Leishmania infantum. In this study 78 Sicilian L. infantum strains isolated from humans and dogs were assessed to investigate their biodiversity by genetic tools. Results were compared with 6 L. infantum reference strains included in the analysis. The evaluation of K26 genetic markers identified 91% of samples as belonging to the MON-1 zymodeme, confirming it as the predominant strain in the Mediterranean area and 9% of the samples–all isolated from humans–as non-MON-1. Multilocus microsatellite typing has proven to be a powerful tool to discriminate strains showing all the isolated strains clustered in two genetically distinct populations, corresponding to human and canine isolates, respectively. A further subdivision was observed between the two main groups, giving in the human population a correlation between microsatellite profile and geographical origin. Our results demonstrate that genetic tools are able to discriminate Leishmania strains and to give useful insights into the epidemiology of leishmaniasis, raising questions on the role of dogs as main reservoirs for human leishmaniasis in the Sicily region. [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]

Simple Summary: Leishmaniosis caused by Leishmania infantum is a parasitic disease of people and animals transmitted by sand fly vectors. Although dogs in south and central Italy are most affected, in recent decades feline leishmaniosis (FeL) has become an emerging disease. This study aimed to update information on the presence of FeL in stray cats in Milan, in northern Italy; to compare these results with previous studies performed in the same area; and to report aspects of Leishmania infection in cats. A total of 117 stray cats were included and 10 (8.6%) had L. infantum infection. The parasite was identified in popliteal lymph nodes in five cats and five had antibodies against L. infantum. Most infected cats were from a specific area of Milan. Increased gammaglobulins were seen in infected cats, which also had antibodies against the pathogens Anaplasma phagocytophilum, Chlamydophila felis, and Toxoplasma gondii. There was a high prevalence of FeL in the surveyed area of northern Italy. Further studies are needed to understand if these infected cats are being imported from other areas, or if there is a real Leishmania focus in Milan. Investigation of the presence of sand fly vectors in Milan would be useful. Feline leishmaniosis (FeL) is an emerging vector-borne feline disease, with increasing numbers of cases reported and studies performed internationally. This study aimed to update the epidemiological status for FeL in stray cats in Milan, northern Italy; compare these results with previous studies in Northern Italy; and report clinicopathologic findings and coinfections in cats infected with Leishmania spp. A total of 117 cats were tested for L. infantum and retrovirus infection, hematological, and biochemical parameters. Demographic and clinical data were collected and FeL affected cats screened for selected coinfections. Overall, 10/117 (8.6%) cats tested positive for L. infantum: in five cats L. infantum DNA was found in popliteal lymph nodes and five were IFAT seropositive at titers from 1:80 to 1:160. Infected cats were concentrated in a specific area of Milan (p = 0.0154). No specific clinicopathologic abnormalities or retroviral infections were significantly linked to the infection, other than hypergammaglobulinemia (p = 0.0127). Seroreactivity to Anaplasma phagocytophilum, Chlamydophila felis, and Toxoplasma gondii was found in some infected cats. A high prevalence of FeL was found in a non-endemic area of northern Italy and future studies should continually monitor this data to understand whether these cases are imported or if Leishmania vectors are present in this area. [ABSTRACT FROM AUTHOR]

Abstract In recent years, several studies demonstrated the role of exosomes in intercellular communications, several Leishmania species belonging to subgenera Leishmania and Viannia have been demonstrated to release exosomes, and their role in parasite-macrophage interactions and in leishmaniasis development has been investigated. However, the release of exosomes by Leishmania infantum has not been studied so far. The aim of this study was to isolate and characterize L. infantum exosomes, and to investigate the biological activity of these exosomes in macrophage cultures. To this end, exosomes were collected from both amastigote and promastigote L. infantum conditioned medium by ultracentrifugation. Exosomes were then characterized by monitoring the presence of HSP70, HSP83/90 and acetylcholinesterase activity. Moreover, extracellular vesicles-tracking analysis revealed that promastigote and amastigote exosomes had mean diameter of 122 ± 56 nm and 115 ± 65 nm, respectively. Human monocytic cell line U937-derived macrophages treated with promastigote and amastigote exosomes showed an increase in motility and an overproduction of interleukin IL-10 and IL-18 reduction, involved in immune response. Since L. infantum exosomes demonstrated the capacity to modulate the chemotactic behaviour of the cells studied and cytokines production, they could contribute in the disease establishment and may be considered an appropriate candidate for a vaccine therapy in prophylaxis and treatment. Graphical abstract Image 1 Highlights • Exosomes are nanovesicles secreted from different cell types. • Characterization of Leishmania infantum exosomes are conducted in this study. • This study demonstrates the capacity of exosomes to regulate cytokinic production. • L. infantum exosomes create an environment permissive for early infection. [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]

Background: Leishmaniasis is a neglected disease caused by many Leishmania species, belonging to subgenera Leishmania (Leishmania) and Leishmania (Viannia). Several qPCR-based molecular diagnostic approaches have been reported for detection and quantification of Leishmania species. Many of these approaches use the kinetoplast DNA (kDNA) minicircles as the target sequence. These assays had potential cross-species amplification, due to sequence similarity between Leishmania species. Previous works demonstrated discrimination between L. (Leishmania) and L. (Viannia) by SYBR green-based qPCR assays designed on kDNA, followed by melting or high-resolution melt (HRM) analysis. Importantly, these approaches cannot fully distinguish L. (L.) infantum from L. (L.) amazonensis, which can coexist in the same geographical area. Methods: DNA from 18 strains/isolates of L. (L.) infantum, L. (L.) amazonensis, L. (V.) braziliensis, L. (V.) panamensis, L. (V.) guyanensis, and 62 clinical samples from L. (L.) infantum-infected dogs were amplified by a previously developed qPCR (qPCR-ML) and subjected to HRM analysis; selected PCR products were sequenced using an ABI PRISM 310 Genetic Analyzer. Based on the obtained sequences, a new SYBR-green qPCR assay (qPCR-ama) intended to amplify a minicircle subclass more abundant in L. (L.) amazonensis was designed. Results: The qPCR-ML followed by HRM analysis did not allow discrimination between L. (L.) amazonensis and L. (L.) infantum in 53.4% of cases. Hence, the novel SYBR green-based qPCR (qPCR-ama) has been tested. This assay achieved a detection limit of 0.1 pg of parasite DNA in samples spiked with host DNA and did not show cross amplification with Trypanosoma cruzi or host DNA. Although the qPCR-ama also amplified L. (L.) infantum strains, the Cq values were dramatically increased compared to qPCR-ML. Therefore, the combined analysis of Cq values from qPCR-ML and qPCR-ama allowed to distinguish L. (L.) infantum and L. (L.) amazonensis in 100% of tested samples. Conclusions: A new and affordable SYBR-green qPCR-based approach to distinguish between L. (L.) infantum and L. (L.) amazonensis was developed exploiting the major abundance of a minicircle sequence rather than targeting a hypothetical species-specific sequence. The fast and accurate discrimination between these species can be useful to provide adequate prognosis and treatment. [ABSTRACT FROM AUTHOR]

Background: Anti-Leishmania antibodies are increasingly investigated in cats for epidemiological studies or for the diagnosis of clinical feline leishmaniosis. The immunofluorescent antibody test (IFAT), the enzyme-linked immunosorbent assay (ELISA) and western blot (WB) are the serological tests more frequently used. The aim of the present study was to assess diagnostic performance of IFAT, ELISA and WB to detect anti-L. infantum antibodies in feline serum samples obtained from endemic (n = 76) and non-endemic (n = 64) areas and from cats affected by feline leishmaniosis (n = 21) by a Bayesian approach without a gold standard. Methods: Cut-offs were set at 80 titre for IFAT and 40 ELISA units for ELISA. WB was considered positive in presence of at least a 18 KDa band. Statistical analysis was performed through a written routine with MATLAB software in the Bayesian framework. The latent data and observations from the joint posterior were simulated in the Bayesian approach by an iterative Markov Chain Monte Carlo technique using the Gibbs sampler for estimating sensitivity and specificity of the three tests. Results: The median seroprevalence in the sample used for evaluating the performance of tests was estimated at 0.27 [credible interval (CI) = 0.20-0.34]. The median sensitivity of the three different methods was 0.97 (CI: 0.86-1.00), 0.75 (CI: 0.61-0.87) and 0.70 (CI: 0.56-0.83) for WB, IFAT and ELISA, respectively. Median specificity reached 0.99 (CI: 0.96-1.00) with WB, 0.97 (CI: 0.93-0.99) with IFAT and 0.98 (CI: 0.94-1.00) with ELISA. IFAT was more sensitive than ELISA (75 vs 70%) for the detection of subclinical infection while ELISA was better for diagnosing clinical leishmaniosis when compared with IFAT (98 vs 97%). Conclusions: The overall performance of all serological techniques was good and the most accurate test for anti-Leishmania antibody detection in feline serum samples was WB. [ABSTRACT FROM AUTHOR]

The Leishmaniases are a group of parasitic diseases caused by protozoa of the Leishmania genus affecting both humans and other vertebrates. Leishmania is an intracellular pathogen able to confer resistance to apoptosis in the early phase of macrophages infection by activation of host PI3K/Akt pathway and inhibition of caspase-3 activation. Intracellular pathogens hijack organelles such as ER to facilitate survival and replication, thus eliciting ER stress and activating/modulating the unfolded protein response (UPR) in the host cell. The UPR is aimed to mitigate ER stress, thereby promoting cell survival. However, prolonged ER stress will activate the apoptotic pathway. The aim of this study was to investigate the ER stress response in Leishmania-infected macrophages to gain insights about the mechanisms underlying the apoptosis resistance in parasitized cells. Macrophages differentiated from human monocytic cell lines (U937 and THP-1) and murine primary macrophages were infected with Leishmania infantum MHOM/TN/80/IPT1 (WHO international reference strain). Several ER stress/autophagy expression markers, as well as cell survival/apoptosis markers (phospho-Akt and cleaved caspase-3) were evaluated by qPCR and/or by western blotting. As ER stress positive control, cells were treated with tunicamycin or dithiothreitol (DTT). The gene expression analyses showed a mild but significant induction of the ER stress/autophagy markers. The western blot analyses revealed that the Leishmania infection induced Akt phosphorylation and significantly inhibited the induction of caspase-3 cleavage, eIF2α phosphorylation and DDIT3/CHOP expression in tunicamycin and DTT treated cells. The mild but significant increase in ER stress expression markers and the delay/attenuation of the effects of ER stress inducers in infected cells support the hypothesis that L. infantum could promote survival of host cells by inducing a mild ER stress response. The host ER stress response could be not only a common pathogenic mechanism among Leishmania species but also a target for development of new drugs. [ABSTRACT FROM AUTHOR]

Stray cats in the city of Milan, Italy, were tested for Leishmania infantum and other selected infections. Twenty-seven cats (30.0%) were seroreactive by indirect fluorescent antibody test (IFAT), with an antibody titer of 1:40 for 16 (17.7%) cats and 1:80 (cut-off for feline L. infantum infection) for 11 (12.2%) cats. One blood (1.1%) and one popliteal lymph node (1.1%) sample tested positive by real-time polymerase chain reaction; no oculoconjunctival swabs tested positive. Feline immunodeficiency virus, feline leukemia virus, and feline coronavirus (FCoV) seroprevalence determined by enzyme-linked immunosorbent assay was 6.1, 6.1, and 39.0%, respectively. Toxoplasma gondii , Bartonella henselae , and Chlamydophila felis prevalence determined by IFAT was 29.3, 17.1, and 17.1%, respectively. The frequency of seroreactivity to L. infantum was significantly higher in FCoV-seropositive cats (OR = 4.4, P = 0.04). L. infantum -infected stray cats in Milan have a high seropositivity rate, comparable to that of cats in areas endemic for leishmaniosis. [ABSTRACT FROM AUTHOR]

[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]

Abstract: In this study, we searched for a connection between Leishmania load and cytokine expression levels in the tissues of Leishmania infantum naturally infected dogs and the efficacy of treatment with miltefosine and allopurinol. To this purpose, we exploited a real-time PCR system to detect Leishmania load and the expression levels of IFN-γ and IL-4 mRNAs at the time of diagnosis and during the follow up post-treatment. In particular, we measured the amount of parasites in blood and lymph node samples, while the expression levels of IFN-γ and IL-4 cytokines were assessed in the blood of the animals. We employed different targeted real-time PCR assays on 20 naturally infected dogs with clinical signs. Three healthy dogs living in a non-endemic area were selected as negative controls. The overall results obtained demonstrate that the simultaneous evaluation of parasites and cytokine levels in different kinds of tissue might represent a reliable tool to evaluate the immune response, the efficacy of the therapy and to predict the relapses during the treatment. [Copyright &y& Elsevier]

Abstract: The factors responsible for the clinical progress of visceral leishmaniasis (VL) in dogs have not been yet established. The starting hypothesis was the possibility of associating the changing level of a specific type of cytokines with the evolution of the infection towards infection-manifested disease or resistant behaviour. For this purpose the authors have established a connection between Leishmania load, cytokine mRNA accumulation, and the progression of the disease in naturally infected asymptomatic dogs. We made use of real-time (RT) PCR system to detect the expression of cytokine mRNA levels during all the phases of the infection. In particular, we measured the amount of parasites in samples such as blood, lymph nodes and skin, and the expression levels of IFN-γ, IL-2, IL-4, IL-10, IL-12 and IL-18 cytokines in the blood. We employed different targeted real-time PCR assay on 40 naturally infected dogs, initially asymptomatic; 20 of these progressed to overt disease, and the 20 remaining dogs remained asymptomatic throughout the period of study (2 years). Two other groups included: 20 naturally infected dogs with clinical signs of VL, and 20 healthy dogs living in a non-endemic area. All these animals were employed as positive and negative controls, respectively. The overall results obtained demonstrate that the simultaneous evaluation of parasites and cytokine levels represents a reliable tool for predicting disease development, and thus for choosing the best treatment for the asymptomatic form of the disease. [Copyright &y& Elsevier]

Background: Leishmania infantum is a vector-borne pathogen endemic in countries in the Mediterranean basin, including Italy. Dogs act as the primary reservoir for this parasite, but other animal species may also be infected. Low-to-moderate seroprevalence levels of infection have been reported in apparent healthy equine populations in southern Europe, reinforcing the importance of exploring those species, including horses, that act as a food source for vectors and may thus participate in the epizoological scenario of canine leishmaniosis (CanL) and zoonotic visceral leishmaniosis (ZVL). Since little is known regarding the exposure to L. infantum in horses in Italy, we assessed the seroprevalence in healthy equine populations from different CanL endemic areas. Methods: The survey was conducted on 660 apparently healthy horses distributed throughout central and northern regions of Italy between 2016 and 2019. Blood samples were collected and the presence of anti-Leishmania antibodies (IgG) was investigated by the immunofluorescence antibody test. Information on the location and altitude of the stables, along with the horses' breed, age, sex, and reproductive status was obtained by filling in a questionnaire. This was then used for statistical analysis by generalized linear models to explore risk factors associated with seroreactivity to L. infantum. Results: An average seroprevalence of 13.9% was detected for L. infantum in the equine populations investigated, with statistically significant associations between seroprevalence, geographical variables (northern vs central Italy, origin and altitude) and individual factors (i.e. age and breed morphotype). Conclusions: Our results highlight that horses are frequently exposed to L. infantum. Further prevalence surveys in horses, also using direct methods (e.g. PCR), are warranted to clarify the role of these hosts in the epidemiology of Leishmania in Italy. [ABSTRACT FROM AUTHOR]

Background: Leishmania infantum is the aetiological agent of visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). Numerous strains and/or zymodemes have been identified and characterized by multilocus enzyme electrophoresis (MLEE). MLEE is considered the reference method for L. infantum parasite typing and it is based upon enzyme electrophoretic mobility analysis from promastigote cultures. However, the MLEE technique is cumbersome, time-consuming and does not detect silent genetic mutations or nucleotide changes that give rise to amino acid changes that do not alter electrophoretic mobility. As a result of these difficulties, many DNA-based typing methods have been developed over the past few years. However, relative to the enzymes utilized in MLEE analysis, we observed a shortage of DNA sequences available in the GenBank database or an absolute lack of sequences belonging to specific zymodemes. The aims of the present study were to (i) implement the number of sequences coding for metabolic enzymes used in MLEE; (ii) identify polymorphisms that characterize L. infantum zymodemes most prevalent in the Mediterranean basin; and (iii) exploit these polymorphisms to develop a rapid screening test that would give results comparable with existing MLEE typing. Results: Partial sequences of seven metabolic enzyme genes (malic enzyme, 6-phosphogluconate dehydrogenase, mitochondrial isocitrate dehydrogenase, glucose-6-phosphate isomerase, glucose-6-phosphate dehydrogenase, phosphoglucomutase and mannose phosphate isomerase) were obtained from 11 L. infantum strains. The comparison of these sequences with those obtained from GenBank allowed for the identification of a few polymorphisms that could distinguish several zymodemes. In particular, the polymorphism 390T>G in the malic enzyme gene has been exploited to develop a high-resolution melt (HRM)-based assay to rapidly differentiate the genotype 390T, associated with zymodemes MON-1, MON-72 and MON-201, evidencing a partial agreement between genotyping results and MLEE. The assay has been successfully applied to L. infantum clinical isolates and clinical samples. Conclusions: A HRM-based assay for rapid identification of genotypes associated with the most common L. infantum zymodemes in the Mediterranean basin has been developed and its potential application in epidemiological research for L. infantum population screening, without parasite isolation and culturing, has been demonstrated. [ABSTRACT FROM AUTHOR]