Infantum Infection Research Articles

Can letrozole be repurposed for the treatment of visceral leishmaniasis?

Visceral leishmaniasis, caused by Leishmania infantum in New World countries, is the most serious and potentially fatal form of leishmaniasis, if left untreated. There are currently no effective prophylactic measures, and therapeutic options are limited. Therefore, we investigated whether the aromatase inhibitor letrozole (LET), which is already used to treat breast cancer, has an antileishmanial activity and/or immunomodulatory potential and therefore may be used to treat L. infantum infection. LET was active against L. infantum promastigote and amastigote life cycle stages in an in vitro infection model using human THP-1 cell-derived macrophages. In human peripheral blood leukocytes ex vivo, LET reduced the internalized forms of L. infantum by classical monocytes and activated neutrophils. Concomitantly, LET stimulated the production of IL-12/TNF-α and decreased the production of IL-10/TGF-β by peripheral blood phagocytes, while in T and B cells, it promoted the production of TNF-α/IFN-γ and decreased that of IL-10. In a murine infection model, LET significantly reduced the parasite load in the liver after just 5 days and in the spleen after 15 days. During in vivo treatment with LET, the production of TNF-α/IFN-γ also increased. In addition, the proportion of developing granulomas decreased and that of mature granulomas increased in the liver, while there was no significant change in organ architecture in the spleen. Based on these data, repositioning of LET may be promising for the treatment of visceral leishmaniasis in humans.

Monitoring of Leishmania infantum in captive non-human primates in Spain

Cases of Leishmania infantum infection have recently been reported in non-human primates (NHPs) in Spain causing severe clinical disease in critically endangered orangutans (Pongo pygmaeus pygmaeus). The aim of this study was to determine exposure and risk factors associated with L. infantum infection in NHPs housed in zoos and wildlife rescue centers (WRC) in Spain. Between 2007 and 2023, sera from 252 NHPs belonging to 47 different species were collected at 15 centers. Indirect immunofluorescence was used to detect the presence of antibodies against L. infantum (cut-off ≥1:80). In addition, hair samples from 78 individuals were tested for Leishmania kDNA by real-time quantitative PCR (qPCR). Anti-Leishmania antibodies were detected in 4.0 % (10/252; 95 %CI: 1.6–6.4) of the NHPs tested at 26.7 % (4/15) of the centers sampled. Twenty-two NHPs were longitudinally sampled between 2010 and 2023: one ring-tailed lemur (Lemur catta) seroconverted and a seropositive orangutan increased antibody titers during the study period. Leishmania infantum kDNA was found in 62.8 % (49/78; 95 %CI: 52.1–73.6) of animals and at all centers sampled (100 %; 7/7). Phylogenetic analysis revealed high homology between the sequence obtained and strains previously isolated in humans, dogs and captive and free-living wildlife species in Spain. To the authors´ knowledge, this is the first report of Leishmania kDNA detection in NHP hair samples. The results indicate that hair samples could be a useful, non-invasive method of detection of L. infantum infection in these species. This is also the first large-scale survey of L. infantum conducted in NHP species in Europe. We report for the first time the presence of Leishmania kDNA in nine different NHP species belonging to the families Cercopithecidae, Lemuridae, and Hylobatidae, expanding the host range for this parasite. The main risk factors associated with L. infantum infection were: age (≥5 years old) and body size (large). Our results demonstrate widespread circulation of this parasite among NHPs housed in Spain, which could be of conservation and public health concern. Monitoring and control programs should be implemented in zoos and WRCs to minimize the risk of NHP exposure to L. infantum in endemic areas worldwide.

Kupffer Cells and Hepatocytes: A Key Relation in the Context of Canine Leishmaniasis.

Human zoonotic visceral leishmaniasis (ZVL) and canine leishmaniasis (CanL) constitute a major public and veterinary health concern and are both caused by the infection with the protozoan parasite Leishmania infantum. One of the main target organs in CanL is the liver. This complex organ, composed of various highly specialized cell types, has garnered significant attention from the scientific community as a crucial player in innate immune functions. In the context of CanL, liver infection by parasites and the host immune response generated strongly influence the disease outcome. Thus, taking advantage of a co-culture system involving canine hepatocytes and L. infantum-infected autologous Kupffer cells (KCs), allowing cell-to-cell interaction, the current report aims to shed light on the hepatocyte-KCs immune interaction. The co-culture of infected KCs with hepatocytes revealed a vital role of these cells in the activation of a local immune response against L. infantum parasites. Although KCs alone can be immunologically silenced by L. infantum infection, the cell-to-cell interaction with hepatocytes in co-culture can lead to local immune activation. In co-culture it was observed gene expression increased the number of innate immune receptors, specifically cell membrane TLR2 and cytoplasmatic NOD1 along with high TNF-α generation. Altogether, these results suggest that the immune response generated in co-culture could induce the recruitment of other circulating cells to contain and contribute to the resolution of the infection in the liver. This work also enhances our understanding of the liver as a vital organ in innate immunity within the context of CanL.

Adaptive and innate immune response of Leishmania infantum infection in Cirneco dell’Etna dog breed

Leishmania spp. are an intracellular protozoa present in many countries around the world. In Europe, both the parasite and the disease it causes, leishmaniasis, are endemic in the Mediterranean basin. Clinical signs and severity of disease are highly variable depending on the host in both humans and dogs, traditionally considered the main reservoir of the parasite. The reason for these differences is not known, but it has been speculated that some hosts present immune response, related to activation of Th1 and Th17, capable of controlling the spread of the parasite, and that these immune responses are related to the genetic background of the host. The Ibizan hound, an autochthonous canine breed of the Mediterranean basin, has been postulated as a breed resistant to infection, but other canine breeds evolutionarily close to it and native to this region have not been studied. One of them is the Cirneco dell’Etna, native to the island of Sicily in southern Italy. In this study, the immune response against L. infantum infection in this canine breed was analysed. The results showed that infected dogs of this breed present high levels of several cytokines related to Th1 and Th17 immune response, and significant correlation between serum levels of cytokines related to disease resistance. Further studies are necessary in this canine breed to determine the mechanisms of immune response and genetic background related to L. infantum infection control.

Severe COVID-19 in HIV/Leishmania infantum coinfected patient: a successfully managed case report

BackgroundCoronavirus disease 2019 originated in China and swiftly spread worldwide, posing a significant threat to public health. Caused by SARS-CoV-2, it manifests as a flu-like illness that can escalate to Acute Respiratory Distress Syndrome, potentially resulting in fatalities. In countries where HIV/Leishmania infantum is endemic, the occurrence of concurrent SARS-CoV-2/HIV/Leishmania infantum infections is a reality, prompting inquiries into appropriate clinical management.Case presentationWe present the case of a 48-year-old woman who was hospitalized for 36 days across three different hospitals in the state of Pernambuco, Brazil. She was diagnosed with SARS-CoV-2/HIV/L. infantum coinfection. The patient exhibited severe COVID-19 symptoms, including fever, productive cough, and dyspnea. Throughout her hospitalization, she experienced oxygen saturation levels of ≤ 93%, along with fluctuations in blood pressure, respiratory rate, and heart rate. Her blood tests revealed lymphopenia, leukopenia, and neutropenia, while laboratory results indicated abnormal levels of d-dimer, AST, ALT, lactate dehydrogenase, ferritin, and C-reactive protein. A computed tomography scan revealed 75% involvement of the lung parenchyma with patchy ground-glass opacities.ConclusionAgainst all odds, the patient was discharged. The leukopenia associated with HIV/L. infantum may have played a decisive role. Further studies are necessary to better understand diagnostic strategies and clinical management measures for HIV/L. infantum coinfected patients who are susceptible to SARS-CoV-2 infection.

Feline leishmaniosis in the Mediterranean Basin: a multicenter study

BackgroundCats are now recognized as competent hosts for Leishmania infantum and a blood source for sand fly vectors. Although canine leishmaniosis (CanL) is endemic in Mediterranean Basin countries, large-scale epidemiological studies are lacking for feline leishmaniosis (FeL). This study aimed to assess the prevalence of L. infantum infections, associated risk factors, clinical signs, and clinicopathological abnormalities in domestic cat populations from six Mediterranean Basin countries.MethodsFrom 2019 to 2022, blood and serum samples of cats (n = 2067) living in Italy (n = 300), Greece (n = 297), Portugal (n = 295), France (n = 231), Israel (n = 313), and Spain (n = 631) were collected along with animal data (i.e., age, sex, breed, housing conditions, and geographical origin), clinical signs, and laboratory blood test parameters. Cats were grouped according to their age as kittens (up to 1 year), young (older than 1 and younger than 7 years), mature (between 7 and 10 years), and senior (older than 10 years). Serum samples were tested for L. infantum by immunofluorescence antibody test (IFAT) and enzyme-linked immunosorbent assay (ELISA), and blood samples of seropositive cats were tested for L. infantum kinetoplast deoxyribonucleic acid (kDNA). Viral infection by feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) was molecularly addressed in all cats enrolled. Statistical analysis was performed to evaluate the association between the risk of L. infantum infection and independent variables, and among co-infection of L. infantum with FIV and/or FeLV, clinical signs, and clinicopathological abnormalities.ResultsOverall, 17.3% (358/2067) of cats scored positive for L. infantum by serological tests. Specifically, 24.7% were from Portugal, 23.2% from Greece, 16.6% from Israel, 15% from Spain, 13.3% from France, and 12.6% from Italy. Leishmania infantum DNA was detected in 15 seropositive animals. Housing condition and FIV infection proved to be risk factors for FeL. Leishmania seropositivity was significantly associated with weight loss, lymphadenomegaly, gingivostomatitis, and oral ulcers, as well as with reduced albumin and albumin/globulin ratio, increased total globulins and total proteins, leukocytosis, and thrombocytosis.ConclusionsThis study provides, for the first time, a large-scale epidemiological survey on FeL and its clinical presentation, revealing that L. infantum circulates among domestic cats, especially shelter/free-roaming and FIV-infected animals, living in CanL endemic countries of the Mediterranean Basin.Graphical

Analysis of HLA Alleles in Different Cohorts of Patients Infected by L. infantum from Southern Spain.

Leishmaniasis is an infectious disease caused by protozoa of the genus Leishmania, which is endemic in certain areas of Europe, such as southern Spain. The disease manifests in various clinical phenotypes, including visceral, cutaneous, mucosal, or asymptomatic leishmaniasis. This diversity in clinical outcomes may be influenced by the host immune response, with human leukocyte antigen (HLA) molecules playing a crucial role in determining susceptibility and progression of the infection. This study explores the association between specific HLA variants and Leishmania infantum infection. We recruited four cohorts: a control group, asymptomatic individuals, patients with symptomatic disease, and cohabitants of infected individuals. HLA typing was performed for all participants, followed by an association analysis with infection status and disease progression. Our findings indicate that the HLA-B*38 and HLA-C*03 alleles are associated with protection against L. infantum infection. These results contribute to a better understanding of the disease's progression, offer potential for new therapeutic approaches such as vaccines, and expand the existing knowledge in the literature.

Twenty-year evolution of Leishmania infantum infection in dogs in Valdeorras (Galicia, Northwestern Spain): implication of climatic factors and preventive measures

BackgroundAbiotic factors play a significant role in the evolution of Leishmania infantum infection due to its vectorial nature. This study aims to assess the evolution in the detection of new L. infantum infection cases in Valdeorras (Ourense, Northwestern Spain) over a 20-year period and how different climatic variables and preventive measures may have affected it.MethodsIndirect immunofluorescence antibody tests (IFAT) were performed on serum samples collected from dogs attending the ‘Servicios Veterinarios de Sil’ veterinary clinic (Valdeorras, Northwestern Spain) between May 2003 and April 2023 to detect L. infantum exposure. The percentage of new cases of L. infantum infection was calculated from May of one year to April of the following year. Climatic conditions in the region, global sales of ectoparasiticides and the number of vaccines against L. infantum delivered in the veterinary clinic from 2003 to 2022 were recorded. Statistical analyses were conducted to determine the associations between these factors and the percentage of new cases of L. infantum infection.ResultsA total of 2909 dogs were assessed, and 3785 IFAT tests were performed between May 2003 and April 2023. The mean percentage of new seropositive cases over the 20-year period studied was 21.65 ± 10.8%, with a decline from the beginning to the end of the period studied. The percentage was significantly higher between May 2003 and April 2008 compared with the other periods (May 2008 to April 2013, May 2013 to April 2018 and May 2018 to April 2023). There was a positive correlation between the percentage of new cases of L. infantum infection and the maximum relative humidity in winter. Conversely, there was a negative correlation between the percentage of new cases and sales of ectoparasiticides and vaccination against L. infantum.ConclusionsThis study is one of the longest evaluations of the evolution of L. infantum infection in a fixed location and its association with external factors including climatic conditions and preventive measures. The results confirm that Valdeorras is a high-risk area for L. infantum infection. The use of ectoparasiticides and vaccines against L. infantum has been shown to play a significant role in preventing L. infantum infection, highlighting the crucial role of veterinarians in the fight against this disease.Graphical

Identification of Lutzomyia longipalpis' using MALDI-TOF peptide/protein profiles

Sand flies are vectors of great public health importance, since they constitute a group of hematophagous insects responsible for etiological agents transmission of zoonotic diseases such a visceral leishmaniasis. In face of the expansion of these diseases, efficient control strategies are needed which depend on comprehending the sand fly eco-epidemiology. In this regard, MALDI-TOF mass spectrometry has been used for bacteria, fungi and yeast detection studies through peptide/protein profiles. However, little is known about interference of biological factors associated with vector ecology, such as blood meal preferences and even sand fly age on the peptide/protein profiles. Thus, the present study aimed to evaluate the differences in peptide/protein profiles of the sand fly Lutzomyia longipalpis, by means of MALDI-TOF, due to the sand fly's age, sex, blood meal source and Leishmania infantum infection. Sample preparation was made removing both head and last abdomen segments keeping the thorax, its appendices and the rest of the abdomen. Five specimens per pool were used to obtain peptide/protein extract of which 1 μL solution was deposited over 1 μL MALDI matrix dried. Characteristic spectra were analyzed using principal coordinate analysis as well as indicator species analysis to discriminate differences in sand flies's peptide/protein profile by sex, age, blood meal source and L. infantum infection. The results show that the evaluated variables produced distinct peptide/protein profiles, demonstrated by the identification of specific diagnostic ions. It was found that the interference of biological factors should be taken into account when using the MALDI-TOF analysis of sand fly species identification and eco-epidemiological applications in field studies. Based on our results, we believe that it is possible to identify infected specimens and the source of blood meal in a collection of wild sand flies, serving to measure infectivity and understand the dynamics of the vector's transmission chain. Our results may be useful for epidemiological studies that look at the ecology of sand flies and leishmaniasis, as well as for raising awareness of biological characteristics’ impact on peptide/protein profiles in sand fly species identification.

Genomic and epidemiological evidence for the emergence of a L. infantum/L. donovani hybrid with unusual epidemiology in northern Italy.

Leishmania (L.) infantum is one of the main causative agents of animal and human leishmaniasis across many endemic areas in South America, Europe, North Africa, and Asia. Despite its clinical significance, little is known about the genetic diversity of L. infantum circulating in a given endemic area. Here, we investigate this important open question by applying a comparative genomics approach to seven L. infantum isolates from different hosts and Italian regions, including the northern part of the country (Emilia-Romagna, RER), Sicily, and Sardinia, as an initial attempt to explore the breadth of parasite genetic heterogeneity in Italy. Additionally, microsatellite analysis was carried out to compare the isolates from RER with other 70 L. infantum strains from the same region as well as 65 strains belonging to the L. donovani complex from other countries. We revealed important karyotypic instability and identified strain-specific changes in gene dosage, which affected important virulence factors such as amastins and surface antigen-like proteins. Single nucleotide polymorphism-based clustering analysis of these genomes together with over 80 publicly available L. infantum and L. donovani genomes placed the Italian isolates into three geographically distinct clusters within the Mediterranean basin and uncovered three isolates clustering with putative L. infantum/L. donovani hybrids isolated in Cyprus. As judged by microsatellite profiling, these hybrid isolates are representative of a sub-population of parasites circulating in northern Italy that preferentially infect humans but not dogs. Our results place Italy at the crossroads of L. infantum infection in the Mediterranean and call attention to the public health risk represented by the introduction of non-European Leishmania species.IMPORTANCEThis study closes important knowledge gaps with respect to Leishmania (L.) infantum genetic heterogeneity in a given endemic country, as exemplified here for Italy, and reveals genetic hybridization as a main cause for re-emerging human leishmaniasis in northern Italy. The observed high diversity of Leishmania parasites on the Italian peninsula suggests different geographical origins, with genomic adaptation to various ecologies affecting both pathogenicity and transmission potential. This is documented by the discovery of a putative L. infantum/L. donovani hybrid strain, which has been shown to preferentially infect humans but not dogs. Our results provide important information to health authorities, which need to consider the public health risk represented by the introduction of new Leishmania species into EU countries due to population displacement or travel from countries where exotic/allochthonous parasite species are endemic.

Immunotherapy Combining Mimotopes Selected by Phage Display Plus Amphotericin B Is Effective for Treatment Against Visceral Leishmaniasis.

The treatment for visceral leishmaniasis (VL) causes toxicity in patients, entails high cost and/or leads to the emergence of resistant strains. No human vaccine exists, and diagnosis presents problems related to the sensitivity or specificity of the tests. Here, we tested two phage clones, B1 and D11, which were shown to be protective against Leishmania infantum infection in a murine model as immunotherapeutics to treat mice infected with this parasite species. The phages were used alone or with amphotericin B (AmpB), while other mice received saline, AmpB, a wild-type phage (WTP) or WTP/AmpB. Results showed that the B1/AmpB and D11/AmpB combinations induced polarised Th1-type cellular and humoral responses, which were primed by high levels of parasite-specific IFN-γ, IL-12, TNF-α, nitrite and IgG2a antibodies, which reflected in significant reductions in the parasite load in distinct organs of the animals when analyses were performed 1 and 30 days after the treatments. Reduced organic toxicity was also found in these animals, as compared with the controls. In conclusion, preliminary data suggest the potential of the B1/AmpB and D11/AmpB combinations as immunotherapeutics against L. infantum infection.

Comparative Study of Colorimetric In Situ Hybridization and Quantitative Real-Time Polymerase Chain Reaction for Diagnosis of Infection by Leishmania infantum in Dogs in Formalin-Fixed and Paraffin-Embedded Skin.

The zoonotic visceral leishmaniasis is caused by the protozoan Leishmania infantum and dogs are reservoirs for this parasite. For the diagnosis of Leishmania at the species level in dogs in formalin-fixed, paraffin-embedded skin (FFPES) samples, colorimetric in situ hybridization (CISH) and quantitative real-time polymerase chain reaction (qPCR) are options, but their sensitivities are not well established. Therefore, the aim of this study was to determine the sensitivity of these two techniques in FFPES for the diagnosis of the L. infantum infection in dogs using culture as the reference standard. The FFPES of 48 dogs with cutaneous infection by L. infantum confirmed by culture and by multilocus enzyme electrophoresis were examined by CISH and qPCR using specific probes for L. infantum. The sensitivities of qPCR, CISH and their combination were, respectively, 77.0%, 58.0% and 83.3%. The sensitivities of qPCR in dogs with and without clinical signs were, respectively, 74.2% and 82.4%. The sensitivities of CISH in dogs with and without clinical signs were, respectively, 61.3% and 52.9%. The CISH and qPCR showed satisfactory sensitivities for the diagnosis of L. infantum in the FFPES of dogs, even in dogs without clinical signs, and their combination increases the sensitivity for this diagnosis.

A case report of acute claudication due to vena cava thromboembolism in a dog naturally infected with Leishmania Infantum

Canine leishmaniosis is a vector-borne disease caused by Leishmania infantum, and clinical manifestations of infection range from absent or severe to fatal and result from immune-mediated mechanisms. In dogs, the most common clinical signs of leishmaniosis include skin lesions and lymphadenomegaly. However, the presence of other nontypical signs has been described, and diagnosing these cases can be challenging. The aim of the present short communication was to describe the impact of the formation of circulating immunocomplexes due to L. infantum in a dog with leishmaniosis affected by a massive venous thrombus of the caudal vena cava and external iliac veins. On admission, the dog presented bilateral cutaneous vasculopathy of the thigh and renal disease due to L. infantum infection. Two weeks after starting anti-Leishmania treatment based on meglumine antimoniate and allopurinol administration, the animal developed acute claudication of the hind limbs with the presence of a thrombus in the caudal vena cava and the external iliac veins and a high level of circulating immunocomplexes detected by enzyme-linked immunosorbent assay. Exacerbation of the humoral immune response, along with deposition of circulating immune complexes in the tissues and the concurrent presence of kidney and liver damage, might have contributed to an imbalance in haemostasis in this patient. Future studies should evaluate and analyse the pathological mechanisms contributing to thrombosis in dogs with leishmaniosis.

Exploring the impact of epidemiological and clinical factors on the progression of canine leishmaniosis by statistical and whole genome analyses: from breed predisposition to comorbidities

Canine leishmaniosis (CanL), caused by Leishmania infantum, is a complex disease of growing importance in Europe. Clinical manifestations result from the down-modulation of the host immune response through multiple host-parasite interactions. Although several factors might influence CanL progression, this is the first known study evaluating risk factors for its different clinical stages in a large referral hospital population (n = 35.669) from an endemic area, over a 20 year period. Genome-wide scans for selection signatures were also conducted to explore the genomic component of clinical susceptibility to L. infantum infection. The prevalence of CanL was 3.2% (16.7% stage I; 43.6% stage II; 32.1% stage III; 7.6% stage IV). Dog breed (crossbreed), bodyweight (<10 kg), living conditions (indoors), regular deworming treatment, and being vaccinated against Leishmania significantly decreased the transmission risk and the risk for developing severe clinical forms. Conversely, the detection of comorbidities was associated with advanced clinical forms, particularly chronic kidney disease, neoplasia, cryptorchidism, infectious tracheobronchitis and urate urolithiasis, although those did not impact the clinical outcome. Significant associations between an increased risk of severe clinical stages and findings in the anamnesis (renal or skin-related manifestations) and physical examination (ocular findings) were also detected, highlighting their diagnostic value in referred cases of CanL. Sixteen breeds were found to be significantly more susceptible to developing severe stages of leishmaniosis (e.g. Great Dane, Rottweiler, English Springer Spaniel, Boxer, American Staffordshire Terrier, Golden Retriever), while 20 breeds displayed a clinical resistantance phenotype and, thus, are more likely to mount an efficient immune response against L. infantum (e.g. Pointer, Samoyed, Spanish Mastiff, Spanish Greyhound, English Setter, Siberian Husky). Genomic analyses of these breeds retrieved 12 regions under selection, 63 candidate genes and pinpointed multiple biological pathways such as the IRE1 branch of the unfolded protein response, which could play a critical role in clinical susceptibility to L. infantum infection.

Intranasal delivery of LaAg vaccine improves immunity of aged mice against visceral Leishmaniasis

There are no approved vaccines yet for human visceral leishmaniasis (VL), the most severe form of the leishmaniasis clinical manifestations that is fatal in over 95 % of untreated cases. It is well-accepted that immunological changes during aging have deleterious impact on the efficacy of vaccines and response to infections. In this work, we compared the response of young and aged mice to intranasal vaccination with killed Leishmania amazonensis promastigote antigens (LaAg) that were then challenged with L. infantum infection, a species that causes visceral leishmaniasis. Intranasal vaccination with LaAg induced a similar reduction in parasitism and hepatosplenomegaly in both young and aged mice compared to their unvaccinated counterparts. Following infection, there was also a less prominent inflammatory profile particularly in the vaccinated aged group, with lower production of TNF-α and nitrite compared to the respective unvaccinated group. Interestingly, the LaAg intranasal vaccination promoted increased production of IFN-γ that was observed in both young- and aged vaccinated groups. Additionally, CD4+ and CD8+T cells from both vaccinated groups presented decreased expression of the inhibitory receptors PD-1 and KLRG1 compared to their unvaccinated controls. Interestingly, a strong positive correlation was observed between the expression of both inhibitory receptors PD-1 and KLRG1 and parasitism, which was more conspicuous in the unvaccinated-aged mice than in the others. Overall, this study helps define new strategies to improve vaccine effectiveness and provides a perspective for prophylactic alternatives against leishmaniasis.

Expressão de TLR-2, FOXP3 e COX-2 na membrana sinovial de cães com artrite induzida pela leishmaniose canina

ABSTRACT: Canine leishmaniasis (CanL) is a multifaceted disease triggered by the protozoan Leishmania infantum, characterized by diverse clinical presentations, including osteoarticular complications. Immune-mediated joint diseases invariably initiate at the synovial membrane, implicating its pivotal role in arthritis pathogenesis. This study aimed to investigate the influence of natural L. infantum infection on synovial fluid characteristics and the expression of immune markers, including TLR-2, FOXP3, and COX-2, in the synovial membrane. Twenty naturally infected dogs (NID) with L. infantum were sourced from the Zoonosis Surveillance Unit (ZSU). Clinical-orthopedic assessments were conducted, encompassing lameness, joint edema, crepitus, patellar luxation, and the drawer test. Synovial fluid (SF) parameters, including volume, appearance, viscosity, total nucleated cell count (TNC), neutrophil count, and total protein (TP) content, were determined. After anesthesia and euthanasia, synovial membrane specimens were obtained. SF protein concentrations categorized dogs into three groups: GI (2 to 2.5g/dL), GII (2.5 to 6.0g/dL), and GIII (&gt;6g/dL). Inflammatory infiltrates and synovial membrane changes were assessed, and immunohistochemistry evaluated TLR-2, FOXP3, and COX-2 marker expressions. Clinical evaluations revealed various osteoarticular abnormalities in NID dogs, including lameness (55%), joint edema (25%), crepitus (30%), patellar luxation (20%), and positive drawer test (25%). Post mortem examinations revealed bilateral subchondral bone, meniscus, and trochlea erosion in 30% of cases. Amastigotes of L. infantum were identified extracellularly and within macrophages (60%). An inflammatory infiltrate was predominant in 70% of dogs, with varying intensity among the groups. Mononuclear cells, chiefly macrophages and lymphocytes, and neutrophils comprised the infiltrate. TLR-2 and COX-2 expression levels were elevated in GIII compared to GII and GI. Conversely, FOXP3 showed moderate expression in GI and minimal expression in GII and GIII. This study underscores the contributory role of L. infantum infection in the development of joint lesions in CanL. Additionally, alterations in the expression of immune markers TLR2, FOXP3, and COX2 within the synovial membrane imply the perpetuation and exacerbation of the inflammatory processes, shedding light on the intricate pathogenesis of CanL-induced arthritis.

Nodular pyogranulomatous panniculitis due to Leishmania infantum infection in a domestic ferret (Mustela putorius furo)

Leishmaniosis is a vector-borne disease caused by different Leishmania species and transmitted by phlebotomine sand flies under natural conditions in Europe. Scientific information related to Leishmania infantum in dogs is extensive, where less information is available in cats and other companion animals. Recently, first clinical cases of L.infantum infection in domestic ferrrets (Mustela putorius furo) have been described. However, clinical information on leishmaniosis in this species is limited A 15-month-old male neutered domestic ferret was presented with chronic weight loss and the presence of coalescent, erythematous and firm subcutaneous nodules in the ventral abdominal subcutis. A fine-needle aspiration of these nodules was performed and the cytological examination revealed a granulomatous inflammation with the presence of macrophages contained a number of oval organisms with an eccentric nucleus and pale cytoplasm, compatible with Leishmania spp. amastigotes compatible with Leishmania spp. amastigotes. The nodules were surgically excised and histological examination showed a severe multifocal pyogranulomatous panniculitis. Specific immunohistochemistry and qPCR for L. infantum from excised nodules were positive. Additionally, L. infantum was cultured and isolated from the nodules by a fine-needle aspiration. An in-house Western Blot test for L. infantum was performed in serum sample and a positive result was obtained. This is the first reported case of nodular pyogranulomatous panniculitis due to L. infantum infection in a domestic ferret. Further studies are necessary to determine the relevance of domestic ferrets in the transmission of leishmaniosis. The description of new clinical forms of the disease is important as it can assist veterinarians in identifying these new clinical presentations.

Evaluation of Proinflammatory Chemokines in HIV Patients with Asymptomatic Leishmania Infantum Infection.

Asymptomatic Leishmania infantum, when associated with HIV, can become severe and potentially fatal. In this co-infection, the worst prognosis may be influenced by the host's immunological aspects, which are crucial in determining susceptibility. Chemokines play an important role in this process by influencing the cellular composition at affected sites and impacting the disease's outcome. Therefore, the aim of this study was to evaluate proinflammatory chemokines in HIV patients with the asymptomatic L. infantum infection. In this cross-sectional study, the levels of CCL2, CCL5, CXCL8, MIG, and IP-10 were measured in 160 serum samples from co-infected patients (n = 53), patients with HIV (n = 90), and negative controls (n = 17). Quantification was determined by flow cytometry. The obtained data were statistically analyzed using the Kruskal-Wallis test, followed by the Dunn's post-test and the Spearman's correlation coefficient. Significance was set at p < 0.05. The chemokines CCL2, CCL5, MIG, and IP-10 exhibited higher levels in the HIV group compared to co-infection. However, the elevated levels of all these chemokines and their increased connectivity in co-infected patients appear to be important in identifying proinflammatory immune responses associated with the asymptomatic condition. Furthermore, a weak negative correlation was observed between higher levels of CXCL8 and lower viral loads in co-infected patients.

Presence of anti-Leishmania infantum antibodies in sheep (Ovis aries) in Spain

Sandflies are the primary transmission vector for Leishmania spp parasite in endemic regions. The role of other animals, different from the dog, is under discussion in the leishmaniosis endemic countries. A limited number of reports have been published on the possible role of livestock in European countries for Leishmania maintenance and diffusion. The aim of the present study was to perform a serosurvey on sheep in areas of Spain that are endemic for zoonotic leishmaniosis and establish the possible role of sheep regarding Leishmania infantum infection in endemic areas. Three hundred and two serum samples were obtained from sheep and were evaluated for serological survey to detect L. infantum by using the in-house ELISA technique. Twenty-eight out of the 302 samples included in this study, were positive for L. infantum antibodies (9.27%). In the present study, a significant association was found between adult age and seropositivity (p = 0.006) and female gender and seropositivity (p = 0.02). This association has not been previously described in other European studies related to L. infantum infection in sheep. Our study reveals that domestic sheep in a European Mediterranean country are exposed to L. infantum. To our knowledge, this study demonstrates the presence of seropositive sheep in different regions of Spain for the first time. Further epidemiological studies focus on evaluating the rural cycle of this parasite to know if livestock could act as a potential reservoir are needed.

Exploring the Relationship between Neutrophil Activation and Different States of Canine L. infantum Infection: Nitroblue Tetrazolium Test and IFN-γ.

This study aimed to investigate the role of neutrophils in canine leishmaniosis by assessing neutrophil activation and its relationship with different states of L. infantum infection and antibody and IFN-γ production. Dogs were categorized into five groups: healthy-seronegative (n = 25), healthy-seropositive (n = 21), LeishVet-stage I (n = 25), Leishvet-stage II (n = 41), and LeishVet-stage III-IV (n = 16). Results of the nitroblue tetrazolium reduction test (NBT) showed significantly higher neutrophil activation in stage I (median:17.17, range: [7.33-31.50]%) compared to in healthy-seronegative (4.10 [1.20-18.00]%), healthy-seropositive (7.65 [3.98-21.74]%), stage II (6.50 [1.50-28.70]%), and stage III-IV (7.50 [3.00-16.75]%) groups (p < 0.0001). Healthy-seropositive dogs also displayed higher values than all groups except stage I. Stages II and III-IV did not show significant differences compared to healthy-seronegative. Regarding IFN-γ, stage I dogs had higher concentrations (median:127.90, range: [0-3998.00] pg/mL) than healthy-seronegative (0 [0-109.50] pg/mL) (p = 0.0002), stage II (9.00 [0-5086.00] pg/mL) (p = 0.045), and stage III-IV (3.50 [80.00-548.80] pg/mL) (p = 0.02) dogs. Stage II dogs showed increased IFN-γ compared to healthy-seronegative dogs (p = 0.015), while stage III-IV dogs had no significant differences compared to healthy-seronegative dogs (p = 0.12). Healthy-seropositive dogs had elevated IFN-γ concentrations compared to healthy-seronegative dogs (p = 0.001) and dogs in stage III-IV (p = 0.03). In conclusion, neutrophil activation was higher in dogs with mild disease and healthy-seropositive dogs, and a relationship between neutrophil activation and the production of IFN-γ was found.