Wolbachia Promotes an Anti-Angiogenic Response Using an In Vitro Model of Vascular Endothelial Cells in Relation to Heartworm Disease.

Heartworm disease caused by Dirofilaria immitis is a vector-borne zoonotic disease responsible for the infection of mainly domestic dogs and cats, or these are those for which the most data are known. Humans are an accidental host where a benign, asymptomatic pulmonary nodule may originate. Dirofilaria immitis also harbours the endosymbiont bacteria of the genus Wolbachia, which play a role in moulting, embryogenesis, inflammatory pathology, and immune response. When Wolbachia sp. is released into the bloodstream, endothelial and pulmonary damage is exacerbated, further encouraging thrombus formation and pulmonary hypertension, facilitating congestive heart failure and death of the animal. Previous studies have shown that parasite excretory/secretory products are able to activate the pro-angiogenic pathway (formation of new vessels) to facilitate parasite survival. The aim of this study was to analyse the role of Wolbachia sp. and its relationship with the cellular processes and the angiogenic pathway in a model of human endothelial cells in vitro. The use of recombinant Wolbachia Surface Protein (rWSP) showed that its stimulation exerted an anti-angiogenic effect by detecting an increase in the production of VEGFR-1/sFlt1 and sEndoglin and did not affect the production of VEGFR-2 and mEndoglin (pro-angiogenic molecules). Furthermore, it did not stimulate cell proliferation or migration, although it did negatively stimulate the formation of pseudocapillaries, slowing down this process. These cellular processes are directly related to the angiogenic pathway so, with these results, we can conclude that Wolbachia sp. is related to the stimulation of the anti-angiogenic pathway, not facilitating the survival of D. immitis in vascular endothelium.

Involvement of the excretory/secretory and surface-associated antigens of Dirofilaria immitis adult worms in the angiogenic response in an in-vitro endothelial cell model.

Angiogenesis is a process by which new vessels are formed from pre-existing ones when the physiological conditions of the vascular endothelium are altered. Heartworm disease, caused by Dirofilaria immitis, causes changes in the vascular endothelium of the pulmonary arteries due to obstruction, friction, and hypoxia. The aim of this study was to analyze whether the excretory/secretory and surface-associated antigens of adult worms interact and modulates the angiogenic mechanism, viable cell number and cell migration, as well as the formation of pseudo-capillaries. Cultures of human vascular endothelial cells (HUVECs) stimulated with excretory/secretory antigens (DiES), surface-associated antigens (Cut) from D. immitis adult worms, VEFG-A (Vascular Endothelial Growth Factor A), as well as DiES+VEFG-A and Cut+VEFG-A were used. The production of VEFG-A and other proangiogenic [soluble VEFGR-2 (sVEFGR-2), membrane Endoglin (mEndoglin)] and antiangiogenic [VEFGR-1/soluble Flt (sFlt), soluble Endoglin (sEndoglin)] molecules was assessed using commercial ELISA kits. Cell viability was analyzed by live cell count and cytotoxicity assays by a commercial kit. In addition, viable cell number by MTT-based assay, cell migration by wound-healing assay carrying out scratched wounds, and the capacity of pseudo-capillary formation to analyze cell connections and cell groups in Matrigel cell cultures, were evaluated. In all cases, non­stimulated cultures were used as controls. DiES+VEFG-A and Cut+VEFG-A significantly increased the production of VEFG-A and sVEFGR-2, and only Cut+VEFG-A significantly increased the production of VEFGR-1/sFlt compared to other groups and non-stimulated cultures. Moreover, only DiES+VEFG-A produced a significant increase in viable cell number and significant decrease cell migration, as well as in the organization and number of cell connections. Excretory/secretory and surface-associated antigens of adult D. immitis activated the angiogenic mechanism by mainly stimulating the synthesis of proangiogenic factors, and only excretory/secretory antigens increased viable cell number, activated cell migration and the formation of pseudo-capillaries. These processes could lead to vascular endothelial remodeling of the infected host and favor the long-term survival of the parasite.

Effect of somatic antigens of Dirofilaria repens adult worms on angiogenesis, cell proliferation and migration and pseudo-capillary formation in human endothelial cells.

BACKGROUND: Angiogenesis is defined as the formation of new vessels by sprouting of endothelial cells from pre-existing vessels in response to stimuli, such as hypoxia or inflammation. Subcutaneous dirofilariasis, caused by Dirofilaria repens, is a zoonotic disease characterized by the formation of subcutaneous nodules with the presence of at least one encapsulated worm, showing perivascular vascularization around it. The aim of this study is to analyze whether the somatic antigen of adult D. repens worms interacts with and modulates the angiogenic mechanism, cell proliferation and migration, and formation of pseudo-capillaries. METHODS: The expression of VEGF-A, VEGFR-1/sFlt, VEGFR-2, mEnd and sEnd in cultures of human vascular endothelial cells stimulated with somatic antigen of adult worms of D. repens (DrSA), vascular endothelial growth factor (VEGF) and DrSA + VEGF were evaluated by using ELISA commercial kits. Cellular viability was analyzed by live cell count, cytotoxicity assays by using a commercial kit, cell proliferation by MTT-based assay, cell migration by wound-healing assay carried out by scratching wounds and capacity of formation of pseudo-capillaries analyzing cell connections and cell groups in Matrigel cell cultures. In all cases unstimulated cultures were used as controls. RESULTS: DrSA + VEGF significantly increased the expression of VEGF-A, VEGFR-2 and mEndoglin compared to other groups and unstimulated cultures. Moreover, DrSA + VEGF produced cell proliferation and migration and increased the formation of pseudo-capillaries. CONCLUSIONS: Somatic antigen of adult D. repens worms activated the proangiogenic mechanism, cell proliferation and cell migration as well as formation of pseudo-capillaries in this in vitro human endothelial cell model. These processes could be related to the survival of adult D. repens in subcutaneous nodules in infected hosts.

Oral fluoxetine treatment changes serotonergic sympatho-regulation in experimental type 1 diabetes.

AIMS: This study investigated whether fluoxetine treatment changes the 5-HT regulation on vascular sympathetic neurotransmission in type 1 diabetes. MAIN METHODS: Four-week diabetes was obtained by a single alloxan s.c. administration in male Wistar rats, administering fluoxetine for 14 days (10 mg/kg/day; p.o.). Systolic blood pressure, heart rate, glycaemia, body weight (BW) evolution, creatinine, and blood urea nitrogen (BUN) were monitored. Afterward, rats were pithed to perform the vascular sympathetic stimulation. 5-HT1A/1D/2A receptors expression was analysed by Western blot in thoracic aorta. Both i.v. norepinephrine and the electrical stimulation of the spinal sympathetic drive evoked vasoconstrictor responses. KEY FINDINGS: Fluoxetine treatment significantly reduced the BW gain, hyperglycaemia, creatinine, and BUN in diabetic rats. The electrical-produced vasopressor responses were greater in untreated than in fluoxetine-treated diabetic rats. 5-HT decreased the sympathetic-produced vasopressor responses. While 5-CT, 8-OH-DPAT and L-694,247 (5-HT1/7, 5-HT1A and 5-HT1D agonists, respectively) reproduced 5-HT-evoked inhibition, the 5-HT2 activation by α-methyl-5-HT augmented the vasoconstrictions. The 5-CT sympatho-inhibition was reversed by 5-HT1A plus 5-HT1D antagonists (WAY-100,635 and LY310762, respectively), whereas ritanserin (5-HT2A antagonist) blocked the α-methyl-5-HT potentiating effect. Norepinephrine-generated vasoconstrictions were increased or diminished by α-methyl-5-HT or 5-CT, respectively. 5-HT1A/1D/2A receptors were expressed at vascular level, being 5-HT1A expression increased by fluoxetine in diabetic rats. SIGNIFICANCE: Our findings suggest that fluoxetine improves metabolic and renal profiles, changes the vasopressor responses, and 5-HT receptors modulating sympathetic activity in diabetic rats: 5-HT1A/1D are involved in the sympatho-inhibition, while 5-HT2A is implicated in the sympatho-potentiation, being both effects pre and/or postjunctional in nature.

First Report of Zoonotic Genotype of Giardia duodenalis in Mussels (Mytilus edulis) from Patagonia Argentina.

Blue mussels (Mytilus edulis) are among the most consumed fishery products globally. Foodborne outbreaks of mussel-associated infections by viral, bacterial, and parasitic pathogens have been reported in the last years. In this study, we investigated the occurrence, genetic diversity, and zoonotic potential of the diarrhea-causing enteric protozoan Giardia duodenalis in blue mussels from Caleta Córdova in Chubut Province, southeast Patagonia, Argentina. A total of 344 free-living blue mussels were collected and distributed in 53 aliquots of pooled mussel tissue (each containing 5‒7 specimens) during the period 2015‒2018. Conventional optical microscopy was used as screening method for the detection of G. duodenalis cysts in pooled, homogenized tissues. Samples with a positive result were assessed by a multilocus sequence genotyping scheme based on the amplification of partial fragments of the glutamate dehydrogenase and ß-giardin genes of the parasite. G. duodenalis cysts were found in 30.2% (16/53) of the aliquots of pooled mussel tissue tested. PCR and sequencing analyses revealed the presence of G. duodenalis subassemblage BIV in selected aliquots. To the best of our knowledge, this is the first description of zoonotic subassemblage BIV from blue mussels in Argentina.

Parásitos zoonóticos caninos de los barrios costeros de Comodoro Rivadavia, Chubut, Argentina / Canine zoonotic parasites from two coastal neighborhoods in Comodoro Rivadavia, Chubut, Argentina

INTRODUCCIÓN: Las zoonosis son enfermedades compartidas entre animales vertebrados y humanos. La secuencia de transmisión comienza con la exposición del humano a elementos infectivos en el ambiente. Muchas patologías mantienen su reservorio en la población canina. Según estudios previos en la región, un 47% de las muestras de heces caninas ambientales (HCA) recolectadas en plazas y paseos públicos contenían al menos una especie parasitaria. El objetivo del trabajo fue actualizar la frecuencia de aparición de parásitos de importancia zoonótica en HCA recogidas en plazas de dos barrios costeros de la ciudad de Comodoro Rivadavia (Chubut) entre marzo y diciembre de 2018. MÉTODOS: Se realizó un estudio observacional, descriptivo y transversal en Caleta Córdova (BCC) y Stella Maris (BSM), donde se recolectaron por conglomerados en forma de guarda griega, aleatoria y sistemática, 156 muestras de HCA. RESULTADOS: Se concentraron y observaron muestras al microscopio, resultando positivas para parásitos 83%; 63% presentó más de un género parasitario. Se destacaron 15 géneros con capacidad para infectar al humano. Los parásitos más abundantes fueron Toxocara spp. y Blastocystis spp. en BCC y Toxocara spp. y Giardia spp. en BSM. En BCC se reportó Mesostephanus spp. por primera vez en Argentina. DISCUSIÓN: Es necesario dimensionar la contaminación ambiental por parásitos zoonóticos, a fin de implementar intervenciones de salud pública adecuadas a cada escenario y contexto epidemiológico.

Consumo de mejillones y presencia de Giardia spp. en humanos en dos barrios costeros de Comodoro Rivadavia, Chubut, Argentina / Consumption of mussels and presence of Giardia spp. in humans in two coastal neighborhoods of Comodoro Rivadavia, Chubut, Argentina

RESUMEN INTRODUCCIÓN Giardia spp. puede adquirirse por consumo de alimentos contaminados. Mytilus edulis es un organismo filtrador, y su recolección es frecuente en pobladores de costas patagónicas. El objetivo fue evaluar la asociación entre consumo de mejillones (Mytilus edulis) y presencia de Giardia spp. en humanos en dos barrios costeros de Comodoro Rivadavia, Chubut, entre marzo y diciembre de 2018. MÉTODOS Se realizó un estudio observacional, descriptivo y transversal en los barrios Caleta Córdova y Stella Maris. La selección de la población estudiada fue no probabilística de colección por casos consecutivos y cumplió los criterios de inclusión. Se colectaron 100 muestras fecales humanas (mfh) y 284 mejillones (46 muestras compuestas). Las muestras se procesaron por sedimentación, se observaron al microscopio óptico y se confirmaron por inmunofluorescencia directa (IFD). Se analizaron variables epidemiológicas y sociodemográficas. RESULTADOS Giardia spp. se detectó en 15% de las mfh y en 33% de los pools de mejillones, en ambos barrios. Las muestras se confirmaron por IFD. Se encontró asociación significativa Chi2=12,734 (2gl), p=0,008 y OR: 4,50 (IC: 1,24-3,20; p=0,027) entre presencia de Giardia spp. en mfh e ingesta de mejillones. DISCUSIÓN El presente constituye el primer reporte de presencia de Giardia spp. en mejillones en Argentina y demuestra que la ingesta de mejillones está relacionada con la presencia del parásito en la población estudiada.

ABSTRACT INTRODUCTION Giardia spp. may be acquired by consuming contaminated food. Mytilus edulis is a filtering organism, and the inhabitants of the Patagonian coasts frequently collect it. The aim of the research was to evaluate the association between mussel consumption and presence of Giardia spp. in humans in two coastal neighborhoods of Comodoro Rivadavia (Chubut province, Argentina) during March-December 2018. METHODS An observational, descriptive and cross-sectional study was carried out in Caleta Cordova and Stella Maris neighborhoods. The selection of the population under study was non-probabilistic for collection by consecutive cases and met the inclusion criteria. A total of 100 human fecal samples (hfs) and 284 mussels (46 pools) were collected. The samples, processed by sedimentation and analyzed by light microscope, were later confirmed by direct immunofluorescence (DIF). Epidemiological and sociodemographic variables were analyzed. RESULTS Giardia spp. was detected in 15% of the hfs and 33% of the mussel pools, in both neighborhoods. Samples were confirmed by DIF. There was a statistically significant association between intake of mussels and presence of Giardia spp. in hfs: Chi2=12.734 (2gl), p=0.008 and OR: 4.50 (CI: 1.24-3.20; p=0.027). DISCUSSION This is the first report of Giardia spp. in mussels in Argentina, and it shows that there is a relation between the mussel intake and the presence of the parasite in the population under study.

An eco-epidemiological study of contamination of soil with infective forms of intestinal parasites.

The objectives of the present work were to screen topsoil samples collected from public squares in two cities within the Argentine Patagonia for the presence of infective forms of intestinal parasites and to examine the possible relationship between positive findings and the environmental, socioeconomic, and cultural conditions of that region. For this purpose we studied 13 public squares, their 13 custodians, and 44 family groups within their respective surrounding areas. Of the 226 topsoil samples analyzed, 44.3% proved positive for infective forms of intestinal parasites, with 17.3% of these containing more than one species. The frequency of appearance of positive samples was dependent on the season of the year (p < 0.001), while presence of the parasites was related to the soil pH (p < 0.05) but independent of the soil relative humidity (p > 0.05). Some of the organisms detected are associated with zoonoses. We observed the presence of Capillaria spp. and Spirocerca spp. under cool desert climatic conditions. Within the group of custodians we detected hematologic alterations one positive serology for toxoplasmosis and documented behavior conducive to risk of infection with the parasites found in those squares. Within the family group an acquaintance with parasitic zoonoses and their prevention was an inconsistent finding, with toxocarosis and toxoplasmosis being the diseases associated with the greatest degree of ignorance. Furthermore, we consider the failure to de-parasitize pets and the practice of feeding them with raw meat, as typically found in our family survey, to be factors contributing to a greater likelihood of public square contamination. From the results obtained here, we propose a spatial organization approach for the purpose of detecting zones at risk of contracting zoonotic parasitoses within urban environments.

Contaminación biológica con heces caninas y parásitos intestinales en espacios públicos urbanos en dos ciudades de la Provincia del Chubut. Patagonia Argentina / Biological contamination with canine faeces and intestinal parasites in pub lic parks in two cities of chubut province, Patagonia Argentina

Se realizó un estudio longitudinal durante un año, para determinar la presencia de parásitos intestinales en heces caninas y su relación con las condiciones climáticas. 481 muestras de heces fueron recolectadas en 13 plazas de dos ciudades de la Provincia del Chubut, Patagonia Argentina. Las muestras fueron analizadas por medio de los métodos de Telemann y Willis. Los datos meteorológicos se registraron diariamente. La frecuencia de aparición de muestras positivas para parásitos intestinales estuvo comprendida entre el 34,9 por ciento y 51,2 por ciento período estudiado, resultando la misma independiente de la estación climática (p > 0,05). Se registró un predominio de helmintos sobre protozoos durante las cuatro estaciones. La frecuencia de aparición de Entamoeba spp. y Nematoda resultó dependiente de la estación estudiada (p < 0,05). El hallazgo de huevos de T. canis varió de un 12,7 por ciento en invierno a un 20,9 por ciento en primavera. Se detectaron patógenos para el humanos como T. canis, Taenia spp., Uncinaria spp. y Entamoeba spp. Se reportan por primera vez para ésta región Spirocerca spp. y Capillaria spp.

Epidemiología de Giardia duodenalis / Epidemiology of Giardia duodenalis

Giardia spp es el agente causal de giardiosis. Los humanos pueden adquirir la infección a través de varias rutas de transmisión; contacto directo con personas infectadas (transmisión persona a persona) o animales (transmisión zoonótica) e ingestión de agua o alimentos contaminados. El ambiente marino, es un medio expuesto a contaminantes, debido a que recibe descargas naturales y de la actividad humana. Estudiar la epidemiología de G. duodenalis en el ámbito geográfico del sureste de la Provincia de Chubut aportó datos de interés para abordar los fenómenos relacionados con salud, enfermedad y ambiente en el ámbito de esta parasitosis en la región. La exposición del ser humano a huevos, quistes y ooquistes infectivos de helmintos y protozoos, presentes en el ambiente es el primer eslabón en la cadena de infestación. En Argentina, la prevalencia de esta parasitosis está comprendida entre un 6% y un 36%, dependiendo de las condiciones sanitarias de cada región y de los hábitos de higiene personal. Giardia duodenalis se encuentra presente en humanos y animales en los barrios Caleta Córdova y Stella Maris de Comodoro Rivadavia, provincia de Chubut (Argentina). Este parásito se encuentra en los moluscos bivalvos mejillones y hay una asociación significativa entre estar infectado por Giardia y la ingesta de los mismos