Human Leishmaniosis Research Articles

Epidemiological surveillance of Leishmania infantum in wild lagomorphs in Spanish Mediterranean ecosystems

Wild lagomorphs play a key epidemiological role as reservoirs of Leishmania infantum, causative agent of the largest outbreak of human leishmaniosis in Europe to date. A large-scale survey study was conducted on wild rabbit (Oryctolagus cuniculus) and Iberian hare (Lepus granatensis) populations in Spanish Mediterranean ecosystems to evaluate the exposure of L. infantum and investigate potential risk factors associated with exposure to this zoonotic parasite. Between 2018 and 2021, a total of 631 wild lagomorphs (471 wild rabbits and 160 Iberian hares) were collected in Andalusia (southern Spain) and tested for antibodies against L. infantum using the indirect fluorescent antibody test (IFAT). Spleen samples from 563 of the wild lagomorphs sampled (441 wild rabbits and 122 Iberian hares) were also evaluated by real-time quantitative PCR (qPCR) for detection of Leishmania kDNA. Exposure to L. infantum (positive by IFAT and/or qPCR) was detected in 56.4 % (356/631; 95 %CI: 52.3–60.3) of the lagomorphs analyzed. Anti-Leishmania antibodies were found in 12.8 % (81/631; 95 %CI: 10.2–15.5) of the animals, and L. infantum kDNA was detected in 59.0 % (332/563; 95 %CI: 54.9–63.0) of the spleen samples tested. Phylogenetic analysis revealed high homology (99.9–100 %) between L. infantum sequences obtained and strains previously isolated from humans in Spain. While apparent seroprevalence was significantly higher in Iberian hares (19.4 %; 95 %CI: 13.3–25.5) compared to wild rabbits (10.6 %; 95 %CI: 7.9–13.4), no significant differences in prevalence were found between wild rabbits (61.0 %; 95 %CI: 56.5–65.6) and Iberian hares (51.6 %; 95 %CI: 42.8–60.5). At least one positive animal was found on 64.8 % (70/108) of the hunting grounds sampled, and a high-risk spatial cluster (P < 0.001) was also identified in central Andalusia. The multivariable analysis identified bioclimatic level (meso-Mediterranean climate) and the presence of goats on hunting grounds as risk factors potentially associated with L. infantum exposure in wild lagomorphs. This study shows high, widespread exposure, but heterogeneous distribution of L. infantum in wild lagomorph populations in Mediterranean ecosystems in southern Spain. The results point to the need to promote integrated surveillance programs for the detection of Leishmania spp. in wild lagomorphs in order to establish effective control measures against human leishmaniosis under a One Health approach.

Leishmania Seroprevalence in Dogs: Comparing Shelter and Domestic Communities.

Canine leishmaniosis (CanL) is a chronic, systemic, and often severe disease. The main causative agent of CanL is a protozoan parasite, Leishmania infantum, with phlebotomine sand flies acting as vectors. In Europe and other continents, L. infantum is also responsible for leishmaniosis in other animals, such as cats, horses, and humans. In Portugal, animal and human leishmaniosis is endemic, and high prevalence levels of infections and disease have been reported in dogs. There is a prejudice against stray animals and also those housed in shelters, assuming they have higher levels of infection with vector-borne pathogens, including L. infantum, when compared to domestic animals. In northeastern Portugal, serum samples were obtained from March to May 2022 in three shelters (n = 179) and thirteen veterinary clinics (n = 164), resulting in 343 dogs being analyzed for antibodies to Leishmania spp. by the direct agglutination test (DAT). The overall seroprevalence was 9.9%, with 15.2% seroprevalence in domestic dogs and 5.0% in the shelter ones (p = 0.003). The fact that shelter dogs had a lower seroprevalence could be explained by more regular veterinary care provided in shelters regarding preventive measures, including insecticides with an antifeeding effect, in comparison with domestic dogs.

Bayesian multivariate longitudinal model for immune responses to Leishmania: A tick-borne co-infection study.

While many Bayesian state-space models for infectious disease processes focus on population infection dynamics (eg, compartmental models), in this work we examine the evolution of infection processes and the complexities of the immune responses within the host using these techniques. We present a joint Bayesian state-space model to better understand how the immune system contributes to the control of Leishmania infantum infections over the disease course. We use longitudinal molecular diagnostic and clinical data of a cohort of dogs to describe population progression rates and present evidence for important drivers of clinical disease. Among these results, we find evidence for the importance of co-infection in disease progression. We also show that as dogs progress through the infection, parasite load is influenced by their age, ectoparasiticide treatment status, and serology. Furthermore, we present evidence that pathogen load information from an earlier point in time influences its future value and that the size of this effect varies depending on the clinical stage of the dog. In addition to characterizing the processes driving disease progression, we predict individual and aggregate patterns of Canine Leishmaniasis progression. Both our findings and the application to individual-level predictions are of direct clinical relevance, presenting possible opportunities for application in veterinary practice and motivating lines of additional investigation to better understand and predict disease progression. Finally, as an important zoonotic human pathogen, these results may support future efforts to prevent and treat human Leishmaniosis.

Climate Variables Related to the Incidence of Human Leishmaniosis in Montenegro in Southeastern Europe during Seven Decades (1945-2014).

Leishmaniosis (or leishmaniasis) is a neglected parasitosis most commonly transmitted by the sandfly bite. Changes in temperature, precipitation, and humidity can greatly affect the vectors and reservoir hosts. This study aimed to determine the association between temperature, air humidity, and weather conditions with the incidence of leishmaniasis in Montenegro during a seven-decade period (1945-2014) and to statistically compare and correlate the obtained data. In the studied period, there were 165 registered cases of leishmaniosis, 96.4%, in the coastal and central region of Montenegro, with an average incidence rate of 0.45/100.000. The visceral form of leishmaniosis predominated (99% of the cases), with only one case of cutaneous disease. Climate factors (average temperature, air humidity, and precipitation) had an impact on the occurrence of leishmaniosis in Montenegro. Air temperature elevated by 1 °C in all regions of Montenegro was significantly correlated with an increased incidence of leishmaniosis, by 0.150 (0.013 to 0.287; p < 0.05). In order to improve prevention and control of this disease, it is also necessary to investigate other factors with a possible impact on the number of cases of this neglected parasitosis.

Immunochromatographic tests for human and canine leishmaniosis diagnosis: a critical review and future prospects

Immunochromatographic tests for human and canine leishmaniosis diagnosis: a critical review and future prospects

Leishmania infantum infection serosurveillance in stray dogs inhabiting the Madrid community: 2007\u20132018

BackgroundLeishmaniosis is an endemic zoonotic disease in the Mediterranean basin caused by Leishmania infantum and transmitted by phlebotomine sandflies. While in dogs disease may be severe, leishmaniosis is also a public health concern as was shown in the largest outbreak of human leishmaniosis (HL) in Europe in 2009 occurring in the Madrid region. The aim of the present study was to assess the applicability of the Leishmaniosis Surveillance Program (LeishSP) established in Madrid in 1996 by examining trends in L. infantum seroprevalence and associated epidemiological risk factors based on data for the 2007–2018 period.MethodsThe study population consisted of 3225 stray dogs from 17 animal shelters collaborating with the LeishSP. Seroprevalences were recorded twice annually (April and November) from 2007 to 2018. In each yearly period, a minimum of 100 dogs were tested to detect dogs infected before and after the sandfly risk season in Madrid area. Each dog was subjected to the same protocol of blood sample collection and clinical examination to collect epidemiological data and clinical signs. Anti-Leishmania-specific IgG was determined by IFAT cut-off ≥ 1:100.ResultsOverall seroprevalence was 6.1% (198 positive dogs). Epidemiological data indicate a significantly higher seroprevalence in dogs > 4 years old, purebred dogs (Pit Bull and related breeds), and medium to large size dogs. There were no seroprevalence differences according to sex and/or season (April and November). In addition, no significant differences were observed according to whether dogs lived inside or outside the HL outbreak area. Remarkably, of 198 dogs testing positive for L. infantum, 64.6% had no clinical signs, indicating a high proportion of clinically healthy infected dogs that could be a potential source of infection.ConclusionsResults indicate a stable seroprevalence of L. infantum infection after 2006 in stray dogs in Madrid but with a recent slightly increasing trend. These observations support the need to continue with the LeishSP implemented by sanitary authorities of the Madrid Community as an early warning strategy for human and animal leishmaniosis and to enable continued assessment of the epidemiological role of dogs with subclinical infection in this important zoonotic disease.Graphical

Vaccinations for pregnant women and women attempting to become pregnant

Infektionen sind eine wichtige potenzielle Gefahrenquelle für Schwangere und ihre ungeborenen Kinder. Schwangere und Frauen mit Kinderwunsch sollten sich daher fachkundig darüber beraten lassen, welche Impfungen vor bzw. während einer Schwangerschaft sinnvoll sind. Zur Impfberatung gehören auch Fragestellungen zu Reiseimpfungen, insbesondere wenn das Reiseziel in einem Endemiegebiet für Erkrankungen wie Malaria, Gelbfieber, Tuberkulose, Hepatitis, Human-immunodeficiency-virus-assoziierte Erkrankungen, Leishmaniose, Toxoplasmose und Japanische Enzephalitis liegt. Grundsätzlich können alle Toxoidimpfstoffe, inaktivierten Impfstoffe und Immunglobuline in der Schwangerschaft verabreicht werden, Lebendimpfstoffe sind kontraindiziert. Empfehlenswerte Impfungen in der Schwangerschaft sind Tetanus, Diphtherie, Pertussis im Falle eines fehlenden Impfschutzes, die saisonale Influenzaimpfung und aktuell die Coronavirus-disease-19(COVID-19)-Impfung ab dem zweiten Trimenon. Zu den empfohlenen Reiseschutzimpfungen zählen alle Standardimpfungen, das heißt die Grundimmunisierung gegen Rotaviren, Tetanus, Diphtherie, Pertussis, Haemophilus influenzae Typ B, Poliomyelitis, Hepatitis B, Pneumokokken, Meningokokken, Masern, Mumps, Röteln, Varizellen, humanes Papillomavirus und Influenza. Darüber hinaus hat in der Reisemedizin der Immunschutz gegen Hepatitis A einen hohen Stellenwert. Nach den internationalen Gesundheitsempfehlungen der World Health Organization ist zur Einreise in bzw. Ausreise aus bestimmten Ländern eine zusätzliche spezifische Impfvorschrift für die Gelbfieber- und Poliomyelitisimpfung vorgesehen. Einzelne Länder haben darüber hinaus zusätzliche Impfvorschriften für die Ein- und Ausreise. „Indikationsimpfungen“ setzen ein entsprechendes lokales Risiko voraus.

Modulation of Host Immune Response during Leishmania infantum Natural Infection: A Whole-Transcriptome Analysis of the Popliteal Lymph Nodes in Dogs.

Leishmania infantum, the etiological agent of canine leishmaniosis (CanL) in Europe, was responsible of the largest outbreak of human leishmaniosis in Spain. The parasite infects and survives within myeloid lineage cells, causing a potentially fatal disease if left untreated. The only treatment option relies on chemotherapy, although immunotherapy strategies are being considered as novel approaches to prevent progression of the disease. To this aim, a deeper characterization of the molecular mechanisms behind the immunopathogenesis of leishmaniosis is necessary. Thus, we evaluated, for the first time, the host immune response during L. infantum infection through transcriptome sequencing of the popliteal lymph nodes aspirates of dogs with CanL. Differential expression and weighted gene co-expression network analyses were performed, resulting in the identification of 5,461 differentially expressed genes (DEGs) and four key modules in sick dogs, compared to controls. As expected, defense response was the highest enriched biological process in the DEGs, with six genes related to immune response against pathogens (CHI3L1, SLPI, ACOD1, CCL5, MPO, BPI) included among the ten most expressed genes; and two of the key co-expression modules were associated with regulation of immune response, which also positively correlated with clinical stage and blood monocyte concentration. In particular, sick dogs displayed significant changes in the expression of Th1, Th2, Th17 and Tr1 cytokines (e. g. TNF-α, IFN-γ, IL-21, IL-17, IL-15), markers of T cell and NK cell exhaustion (e. g. LAG3, CD244, Blimp-1, JUN), and B cell, monocyte and macrophage disrupted functionality (e. g. CD40LG, MAPK4, IL-1R, NLRP3, BCMA). In addition, we found an overexpression of XBP1 and some other genes involved in endoplasmic reticulum stress and the IRE1 branch of the unfolded protein response, as well as one co-expression module associated with these processes, which could be induced by L. infantum to prevent host cell apoptosis and modulate inflammation-induced lymphangiogenesis at lymph nodes. Moreover, 21 lncRNAs were differentially expressed in sick dogs, and one key co-expression module was associated with chromatin organization, suggesting that epigenetic mechanisms could also contribute to dampening host immune response during natural L. infantum infection in the lymph nodes of dogs suffering from clinical leishmaniosis.

Human Cutaneous Leishmaniosis in Iran, Up to Date-2019.

Cutaneous leishmaniasis is a neglected and parasitic vector borne diseases that is endemic in tropical and subtropical countries, including Iran. The aim of this study was to explain the present status of CL in Iran. This report is based on data that recorded by cutaneous leishmaniasis surveillance system in 2019, and evaluated in Center for Communicable Diseases Management in Ministry of Health in Iran. Iran has been considered an endemic area for cutaneous leishmaniasis in the world. Dependent to activities for cutaneous leishmaniasis control the number of cases decreased from 23202 in 2008 (Incidence rate 32 per 100000) to 13124 in 2019 (Incidence rate 15.8 per 100000), more cases reported from September to December, in 2019, 46% of cases had one lesion and 21% had 2 lesions, 85% of cases diagnosed when the diameter of lesions had 3 centimeters and bellows.Although the Leishmania control program began in 1977, the incidence of the disease has dropped dramatically since 2008 when the new cutaneous leishmaniasis control program have been implemented. Although in some areas the incidence of the disease increased, but the implementation of the new program has reduced the number of cases, in order to continue reducing the disease, permanent support for the control programs is needed.

Leishmaniosis in Rodents Caused by Leishmania infantum: A Review of Studies in the Mediterranean Area.

Leishmaniosis infection begins when a phlebotomine sand fly vector inoculates pathogenic protozoan parasites of the genus Leishmania into a mammalian host. In the case of Leishmania infantum, the domestic dog is considered to be the main parasite reservoir, and canine leishmaniosis (CanL) has a high mortality rate in untreated dogs. Hundreds of cases of human leishmaniosis (HL) are reported in the world each year, the incidence in Europe being relatively low. Leishmaniosis control is primarily focused on the dog, combining methods that prevent sand fly bites and boost host resistance to infection. However, these measures are only partially effective and new solutions need to be found. One of the main factors limiting CanL and HL control is the existence of a sylvatic Leishmania transmission cycle that interacts with the domestic cycle maintained by dogs. It is suspected that the main reservoir of infection in wildlife are rodents, whose expansion and rapid population growth worldwide is increasing the risk of human and zoonotic pathogen transfer. The aim of this review is therefore to analyze reports in the literature that may shed light on the potential role of rodents in the leishmaniosis transmission cycle in the Mediterranean area. Following the general methodology recommended for reviews, six databases (Google Scholar, Ovid, PubMed, Science Direct, Scopus and Web of Science) were explored for the period January 1995 to December 2020. The results extracted from 39 publications that met the established inclusion criteria were analyzed. It was found that 23 species of rodents have been studied in nine countries of the Mediterranean basin. Of the 3,643 specimens studied, 302 tested positive for L. infantum infection by serology, microscopy and/or molecular techniques.

Allopurinol y domperidona en el tratamiento de leishmaniosis visceral canina

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

A further investigation of the leishmaniosis outbreak in Madrid (Spain): low-infectivity phenotype of the Leishmania infantum BOS1FL1 isolate to establish infection in canine cells

Human leishmaniosis caused by Leishmania infantum is a zoonotic disease, with dogs as the main reservoir in Mediterranean Basin countries. The largest European outbreak of human leishmaniosis declared in the southwestern Madrid region (Spain) is characterized by unusual epidemiological and clinical features, such as the emergence of new wild reservoirs (hares and rabbits), whereas the seroprevalence, infection, and severity of canine leishmaniosis have not substantially changed since the first studies conducted in Madrid before the outbreak. Previous studies reported that L. infantum isolates from the Madrid leishmaniosis focus displayed elevated virulence in in vivo models of infection and increased infectivity in murine target cells. With the aim of studying whether changes in the host-parasite interaction and virulence profile have developed, we first assessed the behaviour of one circulating isolate of the outbreak, IPER/ES/2012/BOS1FL1 (BOS1FL1), compared to that of a well-characterized strain from canine leishmaniosis, MCAN/ES/1996/BCN150 (BCN150), in terms of infection capacity (percentage of infected cells, representing infectivity, and number of amastigotes per infected cell, representing the intensity of infection) in canine monocytes and macrophages. BCN150 displayed significantly higher infectivity (76.82 ± 4.40 vs 38.58 ± 2.19; P < 0.0001) and intensity of infection (3.64 ± 0.13 vs 1.83 ± 0.12; P < 0.0001) than BOS1FL1 when interacting with canine cells. Our ROS induction results did not differ significantly between the two isolates or with the responses previously described for other L. infantum isolates. Paradoxically, increased resilience to hydrogen peroxide exposure was observed for BOS1FL1 (% viability 40.62 ± 5.54 vs 26.37 ± 2.93; P = 0.039). Finally, we demonstrated that a decreased intracellular load of BOS1FL1 was associated with increased IFN-γ (261.21 ± 26.29 vs 69.80 ± 9.02; P = 0.0151) and decreased IL-10 production (165.06 ± 23.87 vs 264.41 ± 30.58; P = 0.0002). In this study, we provide the first detailed insight into the differences between the isolate BOS1FL1 from the outbreak in Madrid and the well-characterized strain BCN150 MON-1 obtained from a dog in their response to interacting with canine cells. However, further studies are necessary to shed light on the immune mechanisms resulting in BOS1FL1 exhibiting less virulent behaviour in canine cells than in cells derived from other host species.

Properties of virulence emergence of Leishmania infantum isolates from Phlebotomus perniciosus collected during the human leishmaniosis outbreak in Madrid, Spain. Hepatic histopathology and immunological parameters as virulence markers in the mouse model.

Recent anthropic activity related to the construction of the Bosquesur Green Park in a large urban setting in Madrid (Spain) has resulted in the largest reported community outbreak of human leishmaniosis in Europe. Previous phylogenetic and molecular-typing studies of parasite isolates have implicated the Leishmania infantum ITS-Lombardi genotype in this outbreak. In an unusual scenario, visceral leishmaniosis (VL) is affecting a significant number of individuals, suggesting that an increase in parasite virulence has occurred. In this work, using an in vivo BALB/c model of VL, we aimed to investigate the properties of emergent virulence of the L. infantum POL2FL7 and BOS1FL1 isolates obtained from Phlebotomus perniciosus collected in the outbreak area and compare them with those of the well-characterized strain BCN150 MON-1 isolated from a dog. The P. perniciosus specimens were collected during an entomological survey conducted in the transmission season of 2012. We observed a range of virulence phenotypes from moderately to highly aggressive after 5weeks of infection. IV challenge of mice with outbreak isolates from sand flies induced higher splenic and liver parasite burdens, higher serological titres of specific anti-Leishmania antibodies and impaired capacities to control infection, as revealed by the arginine metabolism and low ratios of Th1/Th2 cytokine profiles analysed, compared with the corresponding measures evaluated in mice infected with the BCN150 strain. The BOS1FL1 isolate showed the highest degree of virulence among the isolates, superior to that of POL2FL7, as evidenced by the analysed biomarkers and the histopathological severity of liver lesions. These results provide insight into how L. infantum isolates from sand flies collected in the outbreak area have been able to affect not only immunosuppressed patients but also middle-aged people with normal immunocompetence in the largest human VL outbreak in Europe.

Cross-sectional survey of canine leishmaniasis in Pantelleria island in Sicily.

Dogs are the major reservoir of Leishmania infantum, the causative agent of canine visceral and cutaneous human leishmaniasis in the Mediterranean basin. Canine and human leishmaniosis are endemic in Italy, particularly in central and southern regions, including islands. Here we show a preliminary, clinical, serological and molecular study carried out in Pantelleria island during 2017. In this study, we clinically examined 136 dogs for the presence of symptoms compatible with leishmaniasis, determined the titer of anti‑Leishmania antibodies, and investigated Leishmania DNA by real time PCR in blood and/or lymph node of each dog. The prevalence of disease was equal to 27% with 95% CI [21%; 32%], lower than prevalence obtained in the other Sicily islands (Lampedusa, Lipari). We observed that enlarged lymph nodes was more positively associated with canine leishmaniasis (CanL)than other clinical signs. The results obtained showed that in an endemic area, such as Sicily, diagnosis of CanL needs to be carried out by including an immunological, molecular clinical approach.

Serological and molecular survey of Leishmania infection in dogs from Venezuela.

Venezuela is a country where human and canine leishmaniosis due to Leishmania infantum, Leishmania braziliensis and other Leishmania spp. is endemic. However, only limited data is available on canine Leishmania infection in Venezuela. The aim of this cross-sectional study was to evaluate the prevalence of Leishmania infection in dogs (n=152) from the states of Lara (n=91) and Yaracuy (n=61) in Venezuela by means of serological and molecular methods. Physical examination was performed and blood samples were collected from all dogs. Serology for antibodies reactive with L. infantum and L. braziliensis antigens was assessed by the enzyme-linked immunosorbent assay (ELISA) and detection of Leishmania DNA from blood samples was evaluated by kinetoplast Leishmania real-time polymerase chain reaction (RT-PCR). In addition, Leishmania internal transcribed spacer (ITS-1) RT-PCR was performed on the samples positive by kinetoplast RT-PCR. The prevalence of Leishmania infection based on serological and/or molecular techniques was 11.8%. The seroprevalence for L. infantum and L. braziliensis antigens were 2.1% (3/144) and 8.3% (12/144), respectively. All dogs from the state of Yaracuy were serologically negative to L. infantum while 4.6% (4/86) of the dogs were reactive to L. braziliensis antigen. Fourteen percent (8/58) of the dogs from the state of Lara were positive to L. infantum and 5.2% (3/58) to L. braziliensis antigen. Three dogs were positive to both Leishmania spp. antigens. By RT-PCR, 6.5% (4/61) and 4.4% (4/91) of the dogs were positive for infection in the states of Lara and Yaracuy, respectively. The RT-PCR product of one dog from the state of Yaracuy was sequenced revealing a 100% identity with L. infantum. However, all RT-PCR positive dogs were seronegative to both Leishmania spp. antigens. In conclusion, the positivity for Leishmania spp. infections observed indicates that dogs are frequently infected by L. infantum, L. braziliensis or related Leishmania spp. in Venezuela.

The impact of canine leishmaniosis vaccination with Canileish® in Leishmania infantum infection seroprevalence studies

Effective vaccines against Leishmania parasites are a goal for the scientific community working with both canine and human leishmaniosis. However, possible side effects of vaccination should also be considered and evaluated, preferably before vaccine licensing and marketing. One of these possible effects is the cross-reaction of vaccine-induced antibodies with standard serological tests for detection of Leishmania infantum infection. Longitudinal studies were performed on the type of humoral profile induced by Brazilian marketed canine leishmaniosis vaccines, but little is known regarding the European situation. In this study, an annual follow-up of 85 CaniLeish® vaccinated dogs and 83 non-vaccinated control dogs was performed. Blood samples were taken for all animals at pre-determined time points: before vaccination; immediately before each one of the two following vaccine doses (at 21 days intervals); and then one, four, six, nine and 12 months after finishing the vaccination course. All samples were tested by an in-house ELISA, using a whole promastigote antigen, for the presence of anti-L. infantum antibodies. Humoral response detectable by the used serological diagnostic method was significantly higher in the vaccine group when compared with the control group (p < 0.01) until one-month post-vaccination. Results show that CaniLeish® vaccine-induced antibodies cross-react with a commonly used serological test for diagnosis of L. infantum natural infection. Implications of this interference are discussed, with special emphasis on a possible negative impact on canine leishmaniosis surveillance studies.

Genetic diversity and phylogenetic relationships between Leishmania infantum from dogs, humans and wildlife in south-east Spain.

Leishmania infantum causes human and canine leishmaniosis. The parasite, transmitted by phlebotomine sand flies, infects species other than dogs and people, including wildlife, although their role as reservoirs of infection remains unknown for most species. Molecular typing of parasites to investigate genetic variability and evolutionary proximity can help understand transmission cycles and designing control strategies. We investigated Leishmania DNA variability in kinetoplast (kDNA) and internal transcribed spacer 2 (ITS2) sequences in asymptomatically infected wildlife (n=58) and symptomatically and asymptomatically infected humans (n=38) and dogs (n=15) from south-east Spain, using single nucleotide polymorphisms (SNPs) and in silico restriction fragment length polymorphism (RFLP) analyses. All ITS2 sequences (n=76) displayed a 99%-100% nucleotide identity with a L.infantum reference sequence, except one with a 98% identity to a reference Leishmania panamensis sequence, from an Ecuadorian patient. No heterogeneity was recorded in the 73 L.infantum ITS2 sequences except for one SNP in a human parasite sequence. In contrast, kDNA analysis of 44 L.infantum sequences revealed 11 SNP genotypes (nucleotide variability up to 4.3%) and four RFLP genotypes including B, F and newly described S and T genotypes. Genotype frequency was significantly greater in symptomatic compared to asymptomatic individuals. Both methods similarly grouped parasites as predominantly or exclusively found in humans, in dogs, in wildlife or in all three of them. Accordingly, the phylogenetic analysis of kDNA sequences revealed three main clusters, two as a paraphyletic human parasites clade and a third including dogs, people and wildlife parasites. Results suggest that Leishmania infantum genetics is complex even in small geographical areas and that, probably, several independent transmission cycles take place simultaneously including some connecting animals and humans. Investigating these transmission networks may be useful in understanding the transmission dynamics, infection risk and therefore in planning L.infantum control strategies.

Leishmania infection in lagomorphs and minks in Greece.

Greece is an endemic country for human and canine leishmaniosis. Studies about the role of lagomorphs and minks in the epidemiology of the diseases are, so far, limited. The aim of the present study was to investigate the prevalence of Leishmania infection in these animals, in different areas of the country. Samples from 393 domestic and wild rabbits, 90 hares and 200 minks were collected and examined by cytology (spleen imprints) and serology (ELISA), while spleen samples of 116, 56 and 95 of the rabbits, hares and minks, respectively, were examined by a PCR assay targeting the ITS1 region. For every animal examined a form was created, recording information like date, area, animal species, sex, etc. All imprint smears examined were negative, while serology revealed infection in 7.6% (C.I. 5.0-10.3%) rabbits, 6.7% (C.I. 1.5-11.8%) hares and 20% (C.I. 14.5-25.5%) minks. Infection was confirmed by molecular methods in 2.6% (C.I. 0.0-5.5%), 3.6% (C.I. 0.0-8.4%) and 2.1% (C.I. 0.0-5.0%) of the animals, respectively. The statistical analysis showed that minks are most likely to be seropositive and that in rabbits, the breeding method (i.e. homestead reared animals) was associated with infection. Because of the proximity of lagomorphs and minks to humans and dogs it is necessary to further elucidate their role in the epidemiology of leishmaniosis.

Biomarkers Associated With Leishmania infantum Exposure, Infection, and Disease in Dogs.

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

Clinical and diagnostic aspects of feline cutaneous leishmaniosis in Venezuela

Venezuela is an endemic area for human and canine leishmaniosis due to Leishmania infantum and parasites of the Leishmania braziliensis and L. mexicana complexes. Limited data are available on feline leishmaniosis (FeL) in this region. The aim of this study was to describe clinical and diagnostic aspects of FeL in Venezuela. Thirty-one domestic cats from urban areas of Lara State in Venezuela were enrolled. Twenty-five were healthy. Six other cats had solitary or multiple nodular lesions, which were located on the nose, ears, tail and lower limbs. Cutaneous lesions were characterized by diffuse pyogranulomatous infiltrate in all sick cats with numerous intracellular and extracellular amastigotes, and immunohistochemistry was positive for Leishmania in five sick cats. All healthy cats were seronegative for L. infantum and L. braziliensis antigens by ELISA. Two out of five sick cats yielded a positive ELISA result to both Leishmania antigens with higher antibody levels to L. braziliensis compared to L. infantum. Significantly higher antibody levels by ELISA as well as a higher number of bands by Western blot (WB) were found for L. braziliensis when compared to L. infantum antigens in all sera from Venezuelan sick and healthy cats. All healthy cats were blood Leishmania spp. qPCR negative while three out of six sick cats were blood qPCR positive. All paraffin-embedded skin biopsies (n = 4) as well as cutaneous cytology (n = 3) were positive by Leishmania spp. qPCR in sick cats. Leishmania speciation was obtained only from the cutaneous lesion samples from cytological preparations of two out of three sick cats which were identified as infected with L. mexicana or a closely related specie. Feline leishmaniosis should be included in the differential diagnosis list of nodular-ulcerative lesions. The most reliable diagnostic technique in sick cats is cytological or histopathological examination along with immunohistochemistry, since blood PCR and serology by ELISA might be negative. WB appears to be more sensitive in detecting infection. Cats with leishmaniosis from Venezuela are most likely infected with species of L. mexicana or a closely related species or the L. braziliensis species complex and not with L. infantum.