Equine infection with Leishmania spp. in Costa Rica: Study of five cases

Background: Cutaneous forms of leishmaniosis due to Leishmania braziliensis have been reported in horses in the New World. Domestic animals play a role in the transmission of the disease. In Costa Rica, human cases of L. braziliensis, L. panamensis and L. infantum have been reported. Objectives: The present report describes five cases of equine cutaneous leishmaniosis in Costa Rica. The aetiological diagnosis was based on the presence of the parasite within the lesions. Methods: Skin biopsies were used to perform histopathological analyses of the lesions. Immunohistochemistry was used to detect the presence of the Leishmania spp. antigens in tissue sections. Laser-capture micro-dissection and quantitative real-time PCR techniques were carried out to detect the pathogen nucleic acid within the microscopic lesions. Results: Histopathological analyses showed a granulomatous inflammation within the dermis, with multi-nucleated giant cells, macrophages, lymphocytes and few neutrophils and eosinophils. We detected the parasite by immunohistochemistry, using a rabbit polyclonal antibody raised against Leishmania spp. However, we could not identify Leishmania spp. by quantitative real-time PCR in formalin-fixed paraffin-embedded tissues, using specific primers for the conserved region in the minicircle of the Leishmania DNA kinetoplast. Conclusions: Our results emphasise the importance of Leishmania spp. not only as a causative agent of equine cutaneous disease in the New World, but also as a possible emerging pathogen. Leishmaniosis is one of the most prevalent parasitic public health problems worldwide, and equines may have a role in the epidemiology of the disease.This study was supported by the Ayuntamiento de Madrid of the Comunidad de Madrid S2013/ABI-2747 (TAVS-CM) and by the Structural Funds of the European Union.S

Leishmania infantum UBC1 in Metacyclic Promastigotes from Phlebotomus perniciosus, a Vaccine Candidate for Zoonotic Visceral Leishmaniasis

Leishmania parasites cause outstanding levels of morbidity and mortality in many developing countries in tropical and subtropical regions. Numerous gene expression profiling studies have been performed comparing different Leishmania species' life-cycles and stage forms in regard to their distinct infective ability. Based on expression patterns, homology to human orthologues, in silico HLA-binding predictions, and annotated functions, we were able to select several vaccine candidates which are currently under study. One of these candidates is the Leishmania infantum ubiquitin-conjugating enzyme E2 (LiUBC1), whose relative levels, subcellular location, in vitro infectivity in the U937 myeloid human cell model, and protection levels in Syrian hamsters against L. infantum infection were studied herein. LiUBC1 displays a low level of similarity with the mammalian orthologs and relevant structure differences, such as the C-terminal domain, which is absent in the human ortholog. LiUBC1 is present in highly infective promastigotes. Knock-in parasites overexpressing the enzyme increased their infectivity, according to in vitro experiments. Syrian hamsters immunized with the recombinant LiUBC1 protein did not show any parasite burden in the spleen, unlike the infection control group. The IFN-γ transcript levels in splenocytes were significantly higher in the LiUBC1 immunized group. Therefore, LiUBC1 induced partial protection against L. infantum in the Syrian hamster model.This work was financed by a contract with CZ Vaccines, Porriño, Spain, and partially defrayed by a grant from the Fundación Ramón Areces. JL thanks CZ Vaccines for the fellowship.S

Factors affecting the performance of P22 ELISA for the diagnosis of caprine tuberculosis in milk samples

Caprine tuberculosis (TB) is a zoonosis caused by members of the Mycobacterium tuberculosis complex (MTBC). Caprine TB eradication programmes are based mainly on intradermal tuberculin tests and slaughterhouse surveillance. However, the use of serological test has been extended as a potential diagnostic tool in goats through the use of serum, plasma, or even milk samples. Milk production and the antibodies (Ab) present in milk can vary depending on several circumstances. In the present study, different factors that may affect the performance of humoral TB diagnosis were analysed using goat milk samples: 1) lactation stage, 2) a recent previous skin test (booster effect) and 3) the effect of freeze-thaw cycles on milk samples preserved with azidiol. TB-infected animals (n = 44) were selected to evaluate the evolution of the Ab levels during the 6-month lactation period, along with its potential effect on the P22 ELISA results. In general, no significant changes (p = 0.079) were observed throughout the study as regards Ab levels in milk samples between consecutive analysis although the reactivity to P22 ELISA decreased when samplings were performed at the last two months of the lactation. Regarding the booster effect, the quantitative results showed a significant variation (p < 0.001) for both milk and serum samples when serological tests were carried out 15 days after the skin test. Finally, there were no significant differences (p = 0.99) in the P22 ELISA results when using milk samples preserved with azidiol that had undergone freeze-thaw cycles.This study was funded by the Spanish Ministry of Science and Innovation through the project “Análisis del proceso de erradicación de la tuberculosis caprina a largo plazo y desarrollo de pruebas de diagnóstico y medidas de control para su mejora (GoaTBfree-UCM, reference PID2019-105155RB-C31) and the Spanish Ministry of Agriculture, Fisheries and Food. JO was supported by an FPU (Formación de Profesorado Universitario) contract-fellowship provided by the Spanish Ministerio de Ciencia, Innovación y Universidades (FPU18/05197).S

Detection of environmental SARS-CoV-2 RNA in a high prevalence setting in Spain.

Since March 2020, Spain (along with many other countries) has been severely affected by the ongoing coronavirus disease 19 (COVID-19) pandemic caused by the rapid spread of a new virus (severe acute respiratory syndrome coronavirus 2; SARS-CoV-2). As part of global efforts to improve disease surveillance, we investigated how readily SARS-CoV-2 RNA could be detected in environmental samples collected from an isolated rural community in Spain with a high COVID-19 prevalence (6% of the population of 883 inhabitants). The first diagnosis of COVID-19-compatible symptoms in the village was recorded on 3 March 2020, and the last known active case resolved on 5 June 2020. By 15 May, two months after strict movement constraints were imposed ('lockdown'), and the cumulative number of symptomatic cases had increased to 53. Of those cases, 22 (41%) had been tested and confirmed by RT-PCR. On 13 May and 5 June, samples were collected from high-use surfaces and clothes in the homes of 13 confirmed cases, from surfaces in nine public service sites (e.g. supermarket and petrol station) and from the wastewater of the village sewage system. SARS-CoV-2 RNA was detected in 7 of 57 (12%) samples, including three households and three public sites. While there is not yet sufficient evidence to recommend environmental surveillance as a standard approach for COVID-19 epidemiology, environmental surveillance research may contribute to advance knowledge about COVID-19 by further elucidating virus shedding dynamics and environmental contamination, including the potential identification of animal reservoirs.S

Evaluation of P22 ELISA for the Detection of Mycobacterium bovis-Specific Antibody in the Oral Fluid of Goats

The ante-mortem diagnosis of tuberculosis (TB) in ruminants is based mainly on the intradermal tuberculin test and the IFN-γ assay. Antibody (Ab)-based tests have emerged as potential tools for the detection of TB infected animals using serum, plasma, or even milk samples. Oral fluids have also been evaluated as alternative samples with which to detect specific Abs against Mycobacterium bovis in pigs or wild boars, but not in ruminants. The objective of this study was, therefore, to evaluate the performance of an in house-ELISA for TB diagnosis (P22 ELISA) in goats as an experimental model for the diagnosis of TB using oral fluid samples. Oral fluid samples from 64 goats from a TB-infected herd (n = 197) and all the animals from a TB-free herd (n = 113) were analyzed using the P22 ELISA. The estimated sensitivity (Se) and specificity (Sp) were 34.4% (95% CI: 22.4-45.6) and 100% (95% CI: 97.4-100), respectively. The optimal cut-off point was set at 100% according to the ROC analysis. Those animals with a higher level of Abs in their oral fluid attained a higher lesion score (p = 0.018). In fact, when taking into account only the setting of the animals with severe lesions (n = 16), the ELISA showed a Se of 75% (95% CI: 53.7-96.2). Results of the present study suggest that the P22 ELISA is highly specific but has a limited value detecting infected animals in oral fluid samples. Nevertheless, its performance is significantly higher in the presence of severe lesions.This study was funded by the Herramientas para alcanzar la erradicación de la tuberculosis caprina (GoaTBfree) project (PID2019-105155RB-C31) and the Spanish Government's Ministerio de Agricultura, Pesca y Alimentación. JO was supported by an FPU (Formación de Profesorado Universitario) contract-fellowship provided by the Spanish Ministerio de Ciencia, Innovación y Universidades (FPU18/05197).S

Seroepidemiology of tuberculosis in sheep in southern Spain

Tuberculosis (TB) is a multi-host infectious disease caused by members of the Mycobacterium tuberculosis complex (MTC). In Mediterranean ecosystems, where multiple animal hosts of TB are present, identifying the role of the different species involved in the epidemiology of TB is a key point to be able to implement proper control measures. Sheep are susceptible to MTC infection but have traditionally been considered a spillover host. However, the occurrence of outbreaks involving sheep in recent years evidences the need to better understand the role of this small ruminant species in the epidemiology of the disease. Here, we aimed to determine the seroprevalence and risk factors associated with MTC seropositivity in sheep in Andalusia (southern Spain), a region with one of the highest prevalence of MTC infection in both cattle and wild ungulates. A total of 2266 sheep from 83 flocks were tested for antibodies against MTC using an in-house indirect ELISA. Anti-MTC antibodies were detected in 16 (0.7%) of the 2266 sheep (adjusted true prevalence 0.29%, 95% posterior probability interval 0.01-1.05). Seropositivity was found in 14.5% (12/83; 95%CI: 6.9-22.0) of the sheep farms analyzed. A semi-extensive management system was identified as a risk factor associated with MTC seropositivity in sheep farms (OR = 3.7; p < 0.038; 95%CI: 1.1-12.4) in the study area. To the best of the authors' knowledge, this is the first active TB surveillance study carried out to assess MTC exposure in sheep. Our results indicate MTC circulation in sheep farms in southern Spain. However, the low individual seroprevalence obtained suggests that sheep may play a limited role in the epidemiology of TB in this region. Serosurveillance programs could be a valuable tool to detect MTC circulation in sheep in risk scenarios or target farms, in order to optimize control measures on TB animal in multi-host Mediterranean ecosystems.This study was partially funded by the Spanish Ministry of Economy and Competitiveness (MINECO) research grant (AGL2013-49159-C2-2-R). This research was also supported by CIBER -Consorcio Centro de Investigación Biomédica en Red- (CB 2021), Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación and Unión Europea – NextGenerationEUD. D. Jiménez-Martín holds a PhD contract granted by Own Research Plan of the University of Córdoba. Funding for open access charge: Universidad de Córdoba / CBUA.S

Método de obtención de anticuerpos anti-hHDC y aplicaciones de los mismos

La presente invención proporciona un método para la obtención de anticuerpos, mono y/o policlonales, que interaccionan frente a la histidina descarboxilasa humana (hHDC), así como los polinucleótidos y polipéptidos necesarios para llevarlo a cabo. Del mismo modo, los usos de los mencionados anticuerpos, así como los kits de diagnóstico de los que formen parte también son objeto de la presente invención.REIVINDICACIONES: 1. Polinucleótido, capaz de codificar un polinpéptido capaz de generar de anticuerpos o fragmentos de los mismos específicos frente a hHDC, cuya secuencia es elegida del grupo: a.Secuencia que comprende SEQ ID 1 b.Secuencia que consiste en SEQ ID 1 o comprende fragmentos de ésta. c.Secuencia que difiera de las secuencias a o b debido a la degeneración del código genético. d.Secuencias que compartan al menos un 80%, 90%, 95% ó 98% de homología con cualquiera de las secuencias anteriores. 2. Polipéptido, según la reivindicación 1, capaz de generar anticuerpos específicos frente a hHDC y cuya secuencia es elegida del grupo: a.Secuencia que comprende SEQ ID 2 b.Secuencia que consiste en SEQ ID 2 o comprende fragmentos de ésta. c.Secuencia que comparta al menos un 80%, 90%, 95% ó 98% de homología la secuencia a o b. 3. Anticuerpos o fragmentos de los mismos específicos frente a hHDC, en donde dichos anticuerpos interaccionan específicamente frente cualquiera de los polipéptidos de la reivindicación 2. 4. Uso de cualquiera de los polipéptidos según la reivindicación 2 para la generación de anticuerpos o fragmentos de los mismos específicos frente a hHDC. 5. Uso según la reivindicación 4 en donde los anticuerpos o fragmentos de los mismos son obtenidos por técnicas de generación de hibridomas. 6. Uso según la reivindicación 4 en donde los anticuerpos o fragmentos de los mismos son obtenidos por la tecnología de "phage display". 7. Uso según la reivindicación 4 en donde los anticuerpos o fragmentos de los mismos son obtenidos por inmunización de mamíferos no humanos. 8. Uso de los anticuerpos o fragmentos de los mismos según la reivindicación 3, para la elaboración de un medicamento. 9. Uso de los anticuerpos o fragmentos de los mismos según la reivindicación 3, para la elaboración de un medicamento para el tratamiento de la degeneración neurológica. 10. Uso de los anticuerpos o fragmentos de los mismos según la reivindicación 3, para la elaboración de un medicamento para el tratamiento de la anafilaxis y/o procesos alérgicos. 11. Uso de los anticuerpos o fragmentos de los mismos según la reivindicación 3, para la elaboración de un medicamento para el tratamiento la degeneración de epitelios digestivos. 12. Uso de los anticuerpos o fragmentos de los mismos según la reivindicación 3, para la elaboración de un medicamento para el tratamiento para el tratamiento de diferentes tipos de cáncer. 13. Uso de los anticuerpos o fragmentos de los mismos según la reivindicación 3, para la elaboración de un medicamento para el tratamiento de procesos infecciosos. 14. Uso de los anticuerpos o fragmentos de los mismos según la reivindicación 3 para el diagnóstico in vitro de enfermedades. 15. Kit de diagnóstico que comprende la utilización de anticuerpos o fragmentos de los mismos según la reivindicación 3.Cuando una patente se hace internacional, se puede encontrar en el idioma de cada país en que se ha solicitado. En Espacenet se tiene acceso a los documentos en cada idioma.Instituto de Salud Carlos III; Universidad de MálagaSolicitud de patent

Kinetic analysis of ex vivo human blood infection by Leishmania

The leishmanioses, vector-borne diseases caused by the trypanosomatid protozoan Leishmania, are transmitted to susceptible mammals by infected phlebotomine sand flies that inoculate promastigotes into hemorrhagic pools created in host skin. We assumed that promastigotes are delivered to a blood pool, and analyzed early promastigote interactions (0-5 min) with host components, which lead to parasite endocytosis by blood leukocytes, and to host infection. Promastigotes were incubated with NHS or with heparinized blood in near-physiological conditions, and we used cell radioimmunoassay and flow cytometry to measure the on-rate constants (k(+1)) of promastigote interactions with natural opsonins and erythrocytes. We obtained quantitative data for parasitized cells to determine the time-course of promastigote binding and internalization by blood leukocytes. In these reactions, promastigotes bind natural opsonins, immune adhere to erythrocytes and activate complement cytolysis, which kills approximately 95% of promastigotes by 2 min post-infection. C3-promastigote binding is a key step in opsonization; nascent C3-promastigotes are the substrate for two simultaneous reactions, C3-promastigote immune adherence (IA) to erythrocytes and complement-mediated promastigote killing. The k(+1) for IA was 75-fold greater than that for promastigote killing, showing that IA facilitates promastigote endocytosis and circumvents lysis. At 5 min post-infection, when reaction velocity is still linear and promastigote concentration is not limiting, 17.4% of granulocytes and 10.7% of monocytes had bound promastigotes, of which approximately 50% and approximately 25%, respectively, carried surface-bound (live) or internalized (live and dead) leishmanias. Of other leukocyte types, 8.5% of B cells bound but did not internalize promastigotes, and T cells, NK cells and CD209(+) dendritic cells did not bind parasites. These data show that, once in contact with blood, promastigote invasion of human leukocytes is an extremely rapid and efficient reaction, and suggest that the IA reaction constitutes a central strategy for this parasite in subverting host innate immune defenses.This work was supported by grants from the Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica (Ministerio de Educación y Ciencia) SAF2004-03094, the Instituto de Salud Carlos III (MPY 1198/02), the Comunidad de Madrid (P2009/AGR-1489) and the Fondo de Investigación Sanitaria (Ministerio de Sanidad y Consumo; PI040814). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.S

Serum Removal from Culture Induces Growth Arrest, Ploidy Alteration, Decrease in Infectivity and Differential Expression of Crucial Genes in Leishmania infantum Promastigotes

Leishmania infantum is one of the species responsible for visceral leishmaniasis. This species is distributed basically in the Mediterranean basin. A recent outbreak in humans has been reported in Spain. Axenic cultures are performed for most procedures with Leishmania spp. promastigotes. This model is stable and reproducible and mimics the conditions of the gut of the sand fly host, which is the natural environment of promastigote development. Culture media are undefined because they contain mammalian serum, which is a rich source of complex lipids and proteins. Serum deprivation slows down the growth kinetics and therefore, yield in biomass. In fact, we have confirmed that the growth rate decreases, as well as infectivity. Ploidy is also affected. Regarding the transcriptome, a high-throughput approach has revealed a low differential expression rate but important differentially regulated genes. The most remarkable profiles are: up-regulation of the GINS Psf3, the fatty acyl-CoA synthase (FAS1), the glyoxylase I (GLO1), the hydrophilic surface protein B (HASPB), the methylmalonyl-CoA epimerase (MMCE) and an amastin gene; and down-regulation of the gPEPCK and the arginase. Implications for metabolic adaptations, differentiation and infectivity are discussed herein.PA thanks CSIC for the I3P-BPD2003-1 grant and two contracts of employment at a position included in the A1 group (respectively developed from January 16th to July 23rd 2008 and from October 16th 2008 to April 15th 2009). MAD thanks the Spanish Ministry of Economy and Competitiveness for the FPI Pre-doctoral fellowship BES-2011-047361.S

Infection of dogs by Leishmania infantum elicits a general response of IgG subclasses.

Leishmania infantum is the etiological agent of zoonotic visceral leishmaniasis. In endemic areas, canine infections are considered the main source of infection for human populations. Therefore, any control of human leishmaniasis must include the control of canine infections. Chemotherapy of leishmaniasis is inadequate and canine immunoprophylaxis has important limitations. Reports on the response of infected dogs are abundant but no clear picture of immune events has emerged. To shed some light on these shortcomings the specific IgG subclass response was followed in 20 Beagle dogs experimentally infected with L. infantum using monoclonal antibodies (MAb) specific for canine IgG1, IgG2, IgG3 and IgG4, along with ELISA and flow cytometry. Results showed that parasitic infection elicits a general response of all IgG subclasses, with a predominant IgG1 response and without any evidence of IgG1/IgG2 dichotomy. These findings suggest that the inconsistent results reported previously could be related to the lack of specific reagents and not to the actual differences in the immune response of infected animals. Differential IgG subclass reactivity in ELISA and cytometry and the analysis of the reacting antigens could facilitate the diagnosis and prognosis of the disease and provide a useful tool for adequate therapeutics and vaccine development against leishmaniasis.This project has received partial funding from the European Union Seventh Framework Programme for research, technological development and demonstration under grant agreement n° 603240 (NMTrypI – New Medicines for Trypanosomatidic Infections).S