Caracterización de acuaporinas de garrapata como posibles antígenos protectores frente a vectores artrópodos

Trabajo Fin de Máster presentado por: Marinela Contreras Rojo. Máster Universitario en Investigación Básica y Aplicada en Recursos Cinegéticos.Las garrapatas son ectoparásitos hematófagos obligados que actúan como vectores de enfermedades transmitidas al hombre y los animales. El control inmunológico mediante vacunas se está utilizando como alternativa al uso de acaricidas . Las únicas vacunas comerciales frente a garrapatas hasta la fecha son Gavac® y TickGARD® . Las garrapatas se someten a un elevado estrés osmoregulatorio ya que toman hasta 100 veces su peso corporal en sangre; por tanto, los canales de agua o acuaporinas, que participan en este transporte de agua podrían ser buenos objetivos para el desarrollo de vacunas utilizando como diana proteínas de función biológica importante y altamente conservados. Para ello, se alinearon las secuencias que codificaban para acuaporinas de diferentes especies de garrapata y definieron las regiones homólogas y expuestas en membrana mediante herramientas bioinformáticas. Se seleccionaron las secuencias de las acuaporinas de las garrapatas Ixodes scapularis e Ixodes ricinus (rAQPSIxs y rAQPSIxr) que se amplificaron y posteriormente se expresaron en E. coli. Se realizó un ensayo in vitro para evaluar el efecto de anticuerpos frente acuaporina sobre células de garrapata del género Ixodes. Los resultados preliminares obtenidos en el ensayo in vitro mostraron un efecto significativo de anticuerpos frente acuaporina de Ixodes sobre células de la especie I. scapularis. En este estudio se presenta una aproximación del efecto protector de AQPS sobre de artrópodos. Para determinar si las AQPS son o no buenos candidatos a antígenos protectores frente garrapatas se sugieren más estudios, como ensayos in vivo para evaluar el efecto de la acuaporina recombinante sobre garrapatas.Peer Reviewe

Genomic Resources Notes accepted 1 April 2014 - 31 May 2014

Genomic Resources Development Consortium.This article documents the public availability of a global transcriptome comparison between Lyme disease tick vectors, Ixodes scapularis and Ixodes ricinus.Peer Reviewe

The Correlation between Subolesin-Reactive Epitopes and Vaccine Efficacy

Vaccination is an environmentally-friendly alternative for tick control. The tick antigen Subolesin (SUB) has shown protection in vaccines for the control of multiple tick species in cattle. Additionally, recent approaches in quantum vaccinomics have predicted SUB-protective epitopes and the peptide sequences involved in protein–protein interactions in this tick antigen. Therefore, the identification of B-cell–reactive epitopes by epitope mapping using a SUB peptide array could be essential as a novel strategy for vaccine development. Subolesin can be used as a model to evaluate the effectiveness of these approaches for the identification of protective epitopes related to vaccine protection and efficacy. In this study, the mapping of B-cell linear epitopes of SUB from three different tick species common in Uganda (Rhipicephalus appendiculatus, R. decoloratus, and Amblyomma variegatum) was conducted using serum samples from two cattle breeds immunized with SUB-based vaccines. The results showed that in cattle immunized with SUB from R. appendiculatus (SUBra) all the reactive peptides (Z-score > 2) recognized by IgG were also significant (Z-ratio > 1.96) when compared to the control group. Additionally, some of the reactive peptides recognized by IgG from the control group were also recognized in SUB cocktail–immunized groups. As a significant result, cattle groups that showed the highest vaccine efficacy were Bos indicus immunized with a SUB cocktail (92%), and crossbred cattle were immunized with SUBra (90%) against R. appendiculatus ticks; the IgG from these groups recognized overlapping epitopes from the peptide SPTGLSPGLSPVRDQPLFTFRQVGLICERMMKERESQIRDEYDHVLSAKLAEQYDTFVKFTYDQKRFEGATPSYLS (Z-ratio > 1.96), which partially corresponded to a Q38 peptide and the SUB protein interaction domain. These identified epitopes could be related to the protection and efficacy of the SUB-based vaccines, and new chimeras containing these protective epitopes could be designed using this new approach.La vacunación es una alternativa ecológica para el control de las garrapatas. El antígeno de la garrapata Subolesin (SUB) ha mostrado protección en vacunas para el control de múltiples especies de garrapatas en el ganado. Además, los recientes enfoques de la vaccinómica cuántica han predicho los epítopos protectores de la SUB y las secuencias peptídicas implicadas en las interacciones proteína-proteína en este antígeno de garrapata. Por lo tanto, la Por lo tanto, la identificación de epítopos reactivos a las células B mediante el mapeo de epítopos utilizando una matriz de péptidos SUB podría ser esencial como estrategia novedosa para el desarrollo de vacunas. La subolesina puede utilizarse como modelo para evaluar la de estos enfoques para la identificación de epítopos protectores relacionados con la protección y la eficacia de la vacuna. y la eficacia de las vacunas. En este estudio, el mapeo de epítopos lineales de células B de SUB de tres especies diferentes de garrapatas especies de garrapatas comunes en Uganda (Rhipicephalus appendiculatus, R. decoloratus y Amblyomma variegatum) se llevó a cabo utilizando muestras de suero de dos razas de ganado inmunizadas con vacunas basadas en SUB. Los resultados mostraron que en el ganado inmunizado con SUB de R. appendiculatus (SUBra) todos los péptidos reactivos péptidos (Z-score > 2) reconocidos por la IgG fueron también significativos (Z-ratio > 1,96) en comparación con el grupo de control. Además, algunos de los péptidos reactivos reconocidos por la IgG del grupo de control del grupo de control también fueron reconocidos en los grupos inmunizados con el cóctel SUB. Como resultado significativo, los grupos de ganado que mostraron la mayor eficacia de la vacuna fueron los Bos indicus inmunizados con un cóctel SUB (92%), y de raza cruzada inmunizados con SUBra (90%) contra las garrapatas de R. appendiculatus; las IgG de estos grupos grupos reconocieron epítopos superpuestos del péptido SPTGLSPGLSPVRDQPLFTFRQVGLICERMMKERESQIRDEYDHVLSAKLAEQYDTFVKFTYDQKRFEGATPSYLS (Z-ratio > 1,96), que correspondía parcialmente correspondía a un péptido Q38 y al dominio de interacción proteica SUB. Estos epítopos epítopos identificados podrían estar relacionados con la protección y la eficacia de las vacunas basadas en SUB, y nuevas quimeras que contengan estos epítopos protectores podrían diseñarse utilizando este nuevo enfoque

Complete genome sequences of field isolates of Mycobacterium bovis and Mycobacterium caprae

Here we report the complete genome sequences of field isolates of Mycobacterium bovis and the related mycobacterial species, Mycobacterium caprae. The genomes of three M. bovis (MB1, MB3, MB4) and one M. caprae (MB2) field isolates with different virulence, prevalence, and host distribution phenotypes were sequenced.This research was supported by grant AGL2011-30041 from the Ministerio de Economía y Competitividad, Spain, by the Programa de Tecnologías Avanzadas en Vigilancia Sanitaria (TAVS) from the Comunidad de Madrid (ref. S2013/ABI-2747), the EU H2020 COllaborative Management Platform for detection and Analyses of (Re-) emerging and foodborne outbreaks in Europe (COMPARE) Grant 377/14, and by the EU FP7 grants ANTIGONE (project number 278976) and WildTBvac (project number 613779).Peer Reviewe

Oral Vaccination With a Formulation Combining Rhipicephalus microplus Subolesin With Heat Inactivated Mycobacterium bovis Reduces Tick Infestations in Cattle.

Vaccines are an environmentally friendly alternative to acaracides for the control of tick infestations, to reduce the risk for tick-borne diseases affecting human and animal health worldwide, and to improve animal welfare and production. Subolesin (SUB, also known as 4D8) is the functional homolog of Akirin2 involved in the regulation of development and innate immune response, and a proven protective antigen for the control of ectoparasite infestations and pathogen infection. Oral vaccination combining protein antigens with immunostimulants has proven efficacy with increased host welfare and safety, but has not been used for the control of tick infestations. Here we describe the efficacy of oral vaccination with a formulation combining Rhipicephalus microplus SUB and heat inactivated Mycobacterium bovis (IV) on cattle tick infestations and fertility. The levels of IgG antibody titers against SUB and M. bovis P22, and the expression of selected immune response genes were determined and analyzed as possible correlates of protection. We demonstrated that oral immunization with the SUB+IV formulation resulted in 51% reduction in the number of female ticks and 30% reduction in fertility with an overall efficacy of 65% in the control of R. microplus infestations by considering the cumulative effect on reducing tick survival and fertility in cattle. The akr2, IL-1ß, and C3 mRNA levels together with antibody levels against SUB correlated with vaccine efficacy. The effect of the oral immunization with SUB+IV in cattle on tick survival and fertility is essential to reduce tick infestations, and extended previous results on the effect of R. microplus SUB for the control of cattle tick infestations. These results support the development of oral vaccines formulations for the control of tick infestations and the incidence of tick-borne diseases

1,4-Bis(alkylamino)benzo[g]phthalazines able to form dinuclear complexes of Cu(II) which as free ligands behave as SOD inhibitors and show efficient in vitro activity against Trypanosoma cruzi

The synthesis of a new series of 1,4-bis(alkylamino)benzo[g]phthalazines 1–3 is reported, and their ability to form dinuclear complexes with Cu(II) assayed. The geometry of the complexes is dependent on the nature of the electron-donor sites at the sidechains. Compounds 1 and 2, that contain sp3 or sp2 nitrogens at the end of the alkylamino groups, originate monopodal dinuclear complexes which seem to include endogenous OH bridges, and the sidechains seem to actively participate in complexation. However, the substitution of nitrogen by oxygen in 3 leads to a tripodal dinuclear complex in which the sidechains are not involved. The in vitro antiparasitic activity on Trypanosoma cruzi epimastigotes and amastigotes and the SOD activity inhibition have been evaluated for compounds 1–3, and, as expected, 1 and 2 show in all cases relevant results, whereas 3 is always the less active among the three substrates tested.The authors thank the Spanish Comision Interministerial de Ciencia y Tecnologia for the economical support given to this work (SAF99-0066)

Towards a multidisciplinary approach to improve cattle health and production in Uganda.

A meeting and course supported by the Vice-Presidency for International Affairs of the Spanish National Research Council (CSIC) and the National Agricultural Research Organization of Uganda (NARO) were held at the National Livestock Resources Research Institute (NaLIRRI) in Nakyesasa, Wakiso, Uganda on September 2-9, 2019. The activities were conducted within the collaboration program between the Institute of Game and Wildlife Research (IREC, CSIC-UCLM-JCCM, Spain) and NARO for the development of vaccines and other interventions for the control of cattle ticks in Uganda

Comparative genomics of field isolates of Mycobacterium bovis and M. caprae provides evidence for possible correlates with bacterial viability and virulence

This is an open access article distributed under the terms of the Creative Commons Attribution License.-- et al.Mycobacteria of the Mycobacterium tuberculosis complex (MTBC) greatly affect humans and animals worldwide. The life cycle of mycobacteria is complex and the mechanisms resulting in pathogen infection and survival in host cells are not fully understood. Recently, comparative genomics analyses have provided new insights into the evolution and adaptation of the MTBC to survive inside the host. However, most of this information has been obtained using M. tuberculosis but not other members of the MTBC such as M. bovis and M. caprae. In this study, the genome of three M. bovis (MB1, MB3, MB4) and one M. caprae (MB2) field isolates with different lesion score, prevalence and host distribution phenotypes were sequenced. Genome sequence information was used for whole-genome and protein-targeted comparative genomics analysis with the aim of finding correlates with phenotypic variation with potential implications for tuberculosis (TB) disease risk assessment and control. At the whole-genome level the results of the first comparative genomics study of field isolates of M. bovis including M. caprae showed that as previously reported for M. tuberculosis, sequential chromosomal nucleotide substitutions were the main driver of the M. bovis genome evolution. The phylogenetic analysis provided a strong support for the M. bovis/M. caprae clade, but supported M. caprae as a separate species. The comparison of the MB1 and MB4 isolates revealed differences in genome sequence, including gene families that are important for bacterial infection and transmission, thus highlighting differences with functional implications between isolates otherwise classified with the same spoligotype. Strategic protein-targeted analysis using the ESX or type VII secretion system, proteins linking stress response with lipid metabolism, host T cell epitopes of mycobacteria, antigens and peptidoglycan assembly protein identified new genetic markers and candidate vaccine antigens that warrant further study to develop tools to evaluate risks for TB disease caused by M. bovis/M.caprae and for TB control in humans and animals.This research was supported by grants AGL2014-56305 and IPT-2011-0735-010000 from Ministerio de Economía y Competitividad, Spain, and the European Union FP7 ANTIGONE grant 278976 and Horizon 2020 COMPARE Grant 377/14.Peer Reviewe

Complete Genome Sequences of Field Isolates of Mycobacterium bovis and Mycobacterium caprae

Here we report the complete genome sequences of field isolates of Mycobacterium bovis and the related mycobacterial species, Mycobacterium caprae. The genomes of three M. bovis (MB1, MB3, MB4) and one M. caprae (MB2) field isolates with different virulence, prevalence, and host distribution phenotypes were sequenced

Monitoring the Subolesin Vaccine Field Trial for Safer Control of Cattle Ticks Amidst Increasing Acaricide Resistance in Uganda

A collaboration program was established between the group of Health and Biotechnology (SaBio) of the IREC Institute of Game and Wildlife Research (CSIC-UCLM-JCCM, Spain) and the National Agricultural Research Organization of Uganda (NARO) for the development of vaccines for the control of cattle ticks in Uganda. Controlled pen trials identified a tick protective antigen, Rhipicephalus appendiculatus Subolesin, and a cross-species-effective vaccine formulation. As the next step, a controlled vaccine field trial has been approved by Ugandan state regulatory authorities, the Uganda National Council for Science and Technology (UNCST) and the National Drug Authority (NDA), to evaluate the efficacy and effectiveness of the vaccine formulation for the control of cattle tick infestations under field conditions. The results of this trial may lead to the approval of the vaccine for application in Uganda to improve cattle health and production while reducing the use of acaricides.Se estableció un programa de colaboración entre el grupo de Sanidad y Biotecnología (SaBio) del Instituto de Investigación de la Caza y la Fauna Silvestre (CSIC-UCLM-JCCM, España) y la Organización Nacional de Investigación Agrícola de Uganda (NARO) para el desarrollo de vacunas para el control de las garrapatas del ganado en Uganda. Los ensayos controlados en corral identificaron un antígeno protector de las garrapatas, Rhipicephalus appendiculatus Subolesin, y una formulación de vacuna eficaz para todas las especies. Como siguiente paso, las autoridades reguladoras del estado ugandés, el Consejo Nacional de Ciencia y Tecnología de Uganda, han aprobado un ensayo controlado de campo de la vacuna. Consejo Nacional de Ciencia y Tecnología de Uganda (UNCST) y la Autoridad Nacional de Medicamentos (NDA), para evaluar la eficacia y efectividad de la formulación de la vacuna para el control de las infestaciones de garrapatas en el ganado. infestaciones por garrapatas en condiciones de campo. Los resultados de este ensayo pueden conducir a la aprobación de la vacuna de la vacuna para su aplicación en Uganda, con el fin de mejorar la salud y la producción del ganado y reducir el uso de acaricidas. de acaricidas