Author Archives: Grupo MVeIT

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Proteomic analysis of the somatic and surface compartments from Dirofilaria immitis adult worms

Veterinary Parasitology. Volume 203, Issue 1-2, 2014, Pages 144-152

Morchón, R. , González-Miguel, J., Carretón, E., Kramer, L.H., Valero, L., Montoya-Alonso, J.A., Simón, F., Siles-Lucas, M.

Abstract

Dirofilaria immitis (hearthworm) is a filarial roundworm transmitted by mosquitoes to different vertebrate hosts (dogs, cats and humans, among others), causing dirofilariosis. The adult worms reside in the pulmonary arteries affecting vessels and tissues and resulting in different pathological manifestations. Worms migrate to the heart and surrounding major vessels in heavy infections. Dirofilariosis can result in serious damage to affected hosts. In the last few years, a re-emergence of the disease driven by the climate change has been pointed out. Very recently, the knowledge at molecular level of this parasite has been extended by the published studies on its genome and transcriptome. Nevertheless, studies on the expression of defined protein sets in different parasite compartments and the corresponding role of those proteins in the host-parasite relationship have been relatively scarce to date. These include the description of the adult worm secretome, and some of the proteins eliciting humoural immune responses and those related with plasminogen binding in secreted and surface extracts of the parasite. Here, we investigate by proteomics the somatic and surface compartments of the D. immitis adult worm, adding new information on protein expression and localization that would facilitate a deeper understanding of the host-parasite relationships in dirofilariosis.


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Proteomic analysis of the urine of Dirofilaria immitis infected dogs

Veterinary Parasitology. Volume 203, Issue 1-2, 2014, Pages 241-246
Hormaeche, M., Carretón, E., González-Miguel, J., Gussoni, S., Montoya-Alonso, J.A., Simón, F., Morchón, R.

Abstract

Canine cardiopulmonary dirofilariosis caused by Dirofilaria immitis habitually develops as a chronic disease affecting pulmonary arteries, lung parenchyma and heart. Other organs like kidneys can also be involved. Renal pathology is a consequence of glomerulonephritis whose main sign is proteinuria. The aim of the present work is to identify proteins excreted in the urine of D. immitis infected dogs showing proteinuria, and the possible contribution of their loss to heartworm disease. Proteinuria is higher in microfilaremic (mf+) than in amicrofilaremic (mf-) dogs. Using bidimensional electrophoresis and mass spectrometry 9 different proteins from Canis lupus familiaris in the urine of both mf- and mf+ dogs were identified (serotransferrin isoform 6, serum albumin precursor, albumin, immunoglobulin gamma heavy chain D, apolipoprotein A-I, immunoglobulin lambda-like polypeptide 5-like, arginine esterase precursor, inmunoglobulin gamma heavy chain B and hemoglobin subunit alpha). Furthermore, 3 additional proteins were identified only in the urine of mf+ dogs, corresponding to dog fibrinogen alpha chain and immunoglobulin gamma heavy chain A and actin 2 homologous to a protein of Brugia malayi. The loss of these proteins and other in the urine of D. immitis infected dogs could affect the general condition of parasitized dogs through the interference in the cholesterol metabolism and O2 transport, among other mechanisms.


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Enfermedades cardiorrespiratorias del perro Colección Canis y Felis (nº127, 2)

DIRECCIÓN DE LA MONOGRAFÍA: José Alberto Montoya-Alonso

AUTORES: José Alberto Montoya-Alonso, Elena Carretón Gómez, Susana García Pérez de Ayala, David Marbella Fernández, Alexis José Santana González, José Luis Villaluenga Besaya

EDITORIAL: Acalanthis

ÁREAS TERAPÉUTICAS: Aparato respiratorio, Cardiología

El corazón es una víscera mágica y ha sido así desde el inicio de los tiempos. Es elsímbolo del amor y de la fraternidad. El corazón nos fascina y se ha investigado y escrito mucho sobre él.

He elegido dos frases célebres, que a mi entender, son aplicables a la cardiología veterinaria La primera la del político francés Jacques Duclós, que decía: “El idioma del corazón es universal: sólo se necesita sensibilidad para entenderle y hablarle”. Pienso que a la clínica cardiorrespiratoria hay que perderle el miedo, y al corazón hay que escucharlo para poder entenderlo. Y la de Oliver Wendell Holmes, médico y poeta norteamericano, que escribió: “Aquello que sale del corazón, lleva el matiz y el calor de ………

Editorial (año): Acalanthis Comunicación y estrategia S.L.U. Madrid, 2014

ISSN: 1133-2751

Enlace a editorial Canis et Felis

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Structure of cyclin G-associated kinase (GAK) trapped in different conformations using nanobodies.

Biochemical Journal
Volume 459, Issue 1, 1 April 2014, Pages 59-69
Chaikuad, A., Keates, T., Vincke, C., Kaufholz, M., Zenn, M.d, Zimmermann, B., Gutiérrez, C., Zhang, R.-G., Hatzos-Skintges, C., Joachimiak, A.f, Muyldermans, S., Herberg, F.W., Knapp, S., Müller, S.

Abstract

GAK (cyclin G-associated kinase) is a key regulator of clathrin-coated vesicle trafficking and plays a central role during development. Additionally, due to the unusually high plasticity of its catalytic domain, it is a frequent ‘off-target’ of clinical kinase inhibitors associated with respiratory side effects of these drugs. In the present paper, we determined the crystal structure of the GAK catalytic domain alone and in complex with specific single-chain antibodies (nanobodies). GAK is constitutively active and weakly associates in solution. The GAK apo structure revealed a dimeric inactive state of the catalytic domain mediated by an unusual activation segment interaction. Co-crystallization with the nanobody NbGAK-4 trapped GAK in a dimeric arrangement similar to the one observed in the apo structure, whereas NbGAK-1 captured the activation segment of monomeric GAK in a well-ordered conformation, representing features of the active kinase. The presented structural and biochemical data provide insight into the domain plasticity of GAK and demonstrate the utility of nanobodies to gain insight into conformational changes of dynamic molecules. In addition, we present structural data on the binding mode of ATP mimetic inhibitors and enzyme kinetic data, which will support rational inhibitor design of inhibitors to reduce the off-target effect on GAK.


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Mycoplasma detection and isolation from one-humped camels (Camelus dromedarius).

Trop Anim Health Prod. 2014 Oct;46(7):1317-20. doi: 10.1007/s11250-014-0639-9. Epub 2014 Jul 26.

Mederos-Iriarte LE, Poveda JB, Poveda CG, Vega-Orellana OM, Gutiérrez C, Corbera JA, Ramírez AS.

Abstract

In scientific literature, a small amount of information is found concerning mycoplasmosis in camel species. Mycoplasma (M.) arginini, Acholeplasma (A.) laidlawii, and Acholeplasma oculi have been reported to be isolated from these host species. Serologically positive results have been reported for Mycoplasma mycoides subsp. mycoides SC type, Mycoplasma capricolum subsp. capripneumoniae, and M. mycoides subsp. capri. The aims of this study were to detect, isolate, and identify mycoplasmas from camels (Camelus dromedarius). Initially, saliva and ear smears plus conjunctival and vaginal secretions were taken from five female animals, but only conjunctival secretions in three male animals, all belonging to the same farm. An unknown mycoplasma was isolated from one of the vagina samples. Additionally, another unknown and uncultured mycoplasma was detected with molecular biology in the same sample. In the second stage, 23 vaginal secretions were taken from the same farm plus another secretion from a different one. Ten isolates of the same unknown and previously isolated mycoplasma were detected, nine of them recovered from the vagina of female camels. Some mycoplasmas have been related to reproductive disorders; however, there is no evidence that the isolated mycoplasmas are related to such disorders.


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Geo-environmental model for the prediction of potential transmission risk of Dirofilaria in an area with dry climate and extensive irrigated crops: The case of Spain

Veterinary Parasitology. Volume 200, Issue 3-4, 2014, Pages 257-264

Simón, L., Afonin, A., López-Díez, L.I., González-Miguel, J., Morchón, R., Carretón, E., Montoya-Alonso, J.A., Kartashev, V.d, Simón, F.

Abstract

Zoonotic filarioses caused by Dirofilaria immitis and Dirofilaria repens are transmitted by culicid mosquitoes. Therefore Dirofilaria transmission depends on climatic factors like temperature and humidity. In spite of the dry climate of most of the Spanish territory, there are extensive irrigated crops areas providing moist habitats favourable for mosquito breeding. A GIS model to predict the risk of Dirofilaria transmission in Spain, based on temperatures and rainfall data as well as in the distribution of irrigated crops areas, is constructed. The model predicts that potential risk of Dirofilaria transmission exists in all the Spanish territory. Highest transmission risk exists in several areas of Andalucía, Extremadura, Castilla-La Mancha, Murcia, Valencia, Aragón and Cataluña, where moderate/high temperatures coincide with extensive irrigated crops. High risk in Balearic Islands and in some points of Canary Islands, is also predicted. The lowest risk is predicted in Northern cold and scarcely or non-irrigated dry Southeastern areas. The existence of irrigations locally increases transmission risk in low rainfall areas of the Spanish territory. The model can contribute to implement rational preventive therapy guidelines in accordance with the transmission characteristics of each local area. Moreover, the use of humidity-related factors could be of interest in future predictions to be performed in countries with similar environmental characteristics.


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A transversal study on antibodies against selected pathogens in dromedary camels in the Canary Islands, Spain

Veterinary Microbiology
Volume 167, Issue 3-4, 27 December 2013, Pages 468-473
Mentaberre, G., Gutiérrez, C., Rodríguez, N.F., Joseph, S., González-Barrio, D., Cabezón, O., de la Fuente, J., Gortazar, C., Boadella, M.

Abstract

The Canary Islands contain the most important dromedary camel ( Camelus dromedarius) population in the European Union and are the main export point of dromedaries to continental Europe and Latin America. We investigated the presence of antibodies against relevant disease agents in 100 Canarian camel sera. Selected blood samples of the same animals were also tested by PCR. Sera were tested for antibodies against Bluetongue virus (BTV; 0%), Bovine Viral Diarrhoea virus (BVDV; 0%), Camelpox virus (CPV; 8% by serum neutralization, 16% by ELISA), Peste des Petits Ruminants virus (PPRV, 0%), Rift Valley Fever virus (RVFV; 0%) and West Nile Fever virus (WNV; 3%), the bacterial pathogens Anaplasma sp. (3%), Brucella sp. (1%), Coxiella burnetii (19%), Mycobacterium avium paratuberculosis (MAP; 22%), Mycobacterium tuberculosis complex (MTC; 10%) and Rickettsia sp. (83%), and the parasites Toxoplasma gondii (36%) and Neospora caninum (86%). The most remarkable findings were the detection of antibodies against CPV and the high antibody prevalence against C. burnetii, Rickettsia sp., T. gondii and N. caninum. By PCR, we found no C. burnetii, N. caninum and Anaplasma sp. DNA in the tested samples. However, Rickettsia sp. DNA was detected in six antibody positive tested samples. These results should be taken into consideration in order to implement adequate control measures and avoid a potential dissemination of infections to other territories.


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Utility of cardiac biomarkers during adulticide treatment of heartworm disease (Dirofilaria immitis) in dogs

Veterinary Parasitology. Volume 197, Issue 1-2, 18 October 2013, Pages 244-250
Carretón, E. , Morchón, R., González-Miguel, J., Juste, M.C., Simón, F., Montoya-Alonso, J.A.

Abstract

Heartworm disease ( Dirofilaria immitis) is a parasitic disease of dogs and other carnivores, characterized by the presence of adult worms in the pulmonary arteries and right ventricle, leading to pulmonary hypertension which may progress to congestive heart failure. Cardiac biomarkers are biological parameters that can be objectively measured as indicators of pathological processes, or to assess the response to therapeutic interventions. To evaluate the myocardial damage during the adulticide treatment in 15 heartworm-infected dogs with ivermectin, doxycycline and melarsomine, measurements of cardiac troponin I (cTnI), myoglobin, MB isoenzyme of creatine kinase (CK-MB) and aspartate aminotransferase (AST) were carried out on days 0, 60 and afterwards weekly on days 67, 75, 82, 91, 106, 113 and 120. Dogs were divided by low parasite burden (n= 9) and high parasite burden (n= 6). On day 0, dogs with high worm burden showed increased cTnI concentrations (3.62. ±. 4.78. ng/ml) while dogs with low worm burden had concentrations similar to those of healthy dogs (0.78. ±. 0.22. ng/ml), CK-MB concentrations were increased only in dogs with high parasite burden as well (54.4. ±. 54.2. U/l) and 26.6% (4/15) of the dogs showed pathological concentrations of myoglobin. On day 91, most dogs showed pathological concentrations of myoglobin, CK-MB and AST, probably due to the myositis associated to the intramuscular injection of melarsomine. The rest of the measurements made in the study, the biomarkers concentrations were within normal values, except for cTnI in dogs with high parasite burden, which remained above reference concentrations for healthy dogs during all the study. The evaluation of cardiac biomarkers seems to be a helpful test in the assessment of the myocardium in dogs with heartworm disease during the adulticide treatment.


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Middle East respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: A comparative serological study (Article)

The Lancet Infectious Diseases
Volume 13, Issue 10, October 2013, Pages 859-866
Reusken, C.B.E.M. , Haagmans, B.L., Müller, M.A., Gutierrez, C., Godeke, G.-J., Meyer, B., Muth, D., Raj, V.S., Vries, L.S.D., Corman, V.M., Drexler, J.-F., Smits, S.L., El Tahir, Y.E., De Sousa, R., van Beek, J., Nowotny, N., van Maanen, K., Hidalgo-Hermoso, E., Bosch, B.-J., Rottier, P., Osterhaus, A., Gortázar-Schmidt, C., Drosten, C., Koopmans, M.P.G.

Abstract

Background: A new betacoronavirus-Middle East respiratory syndrome coronavirus (MERS-CoV)-has been identified in patients with severe acute respiratory infection. Although related viruses infect bats, molecular clock analyses have been unable to identify direct ancestors of MERS-CoV. Anecdotal exposure histories suggest that patients had been in contact with dromedary camels or goats. We investigated possible animal reservoirs of MERS-CoV by assessing specific serum antibodies in livestock. Methods: We took sera from animals in the Middle East (Oman) and from elsewhere (Spain, Netherlands, Chile). Cattle (n=80), sheep (n=40), goats (n=40), dromedary camels (n=155), and various other camelid species (n=34) were tested for specific serum IgG by protein microarray using the receptor-binding S1 subunits of spike proteins of MERS-CoV, severe acute respiratory syndrome coronavirus, and human coronavirus OC43. Results were confirmed by virus neutralisation tests for MERS-CoV and bovine coronavirus. Findings: 50 of 50 (100%) sera from Omani camels and 15 of 105 (14%) from Spanish camels had protein-specific antibodies against MERS-CoV spike. Sera from European sheep, goats, cattle, and other camelids had no such antibodies. MERS-CoV neutralising antibody titres varied between 1/320 and 1/2560 for the Omani camel sera and between 1/20 and 1/320 for the Spanish camel sera. There was no evidence for cross-neutralisation by bovine coronavirus antibodies. Interpretation: MERS-CoV or a related virus has infected camel populations. Both titres and seroprevalences in sera from different locations in Oman suggest widespread infection. Funding: European Union, European Centre For Disease Prevention and Control, Deutsche Forschungsgemeinschaft.


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NanoPad: An integrated platform for bacterial production of camel nanobodies aimed at detecting environmental biomarkers.

Proteomics
Volume 13, Issue 18-19, October 2013, Pages 2766-2775
Fraile, S., Jiménez, J.I., Gutiérrez, C, De Lorenzo, V.

Abstract

The presence of given antigens in environmental samples (e.g. biodegradative enzymes) reports the quality and catalytic vigor of particular soils or aquatic ecosystems. In this context, we have developed the NanoPad system consisting of a complete platform for isolation, amplification, and extracellular production of specific antibodies against antigens that diagnose the occurrence of protein markers in crude environmental samples. The workflow starts with the inoculation of camels (Camelus dromedarius) with various proteins (e.g. catabolic enzymes) for generating a phage display library of variable heavy-chain antibody H fragment (VHH) domains that bind the different antigens. Instead of being subjected to a conventional panning, such a library is then probed with a Western-panning technique that allows direct isolation of specific binders of proteins blotted on membranes from polyacrylamide gels. Finally, VHHs are fused to the C-domain of hemolysin for secretion to the culture media as virtually pure dimeric proteins that can be used as a primary antibody without further processing. The value of NanoPad is shown with the selection of nanobodies for detection of biphenyl 2,3-dioxygenase, a key enzyme for biodegradation of polychlorinated biphenyls. The thereby generated anti-biphenyl 2,3-dioxygenase VHHs revealed the presence of this enzyme in the metaproteome of an oil refinery waste treatment plant.