II Jornadas Científicas do Centro de Estudos de Ciência Animal-UP

The FCT R&D Unit 211: Centre for the Study of Animal Sciences-CECA-UP

About CECA

The Centre for the Study of Animal Sciences-CECA is a R&D unit of the University of Porto, recognized by FCT as Unit 211. This Centre is formed by 13 senior scientists from the University of Porto, the National Laboratory for Veterinary Research (LNIV/INRB, I.P.) and the National Institute of Health Dr. Ricardo Jorge (INSA, I.P.), and 3 senior collaborators, scientists working in the areas of zoonotic diseases and biomaterials. The senior scientists and their teams are allocated in 3 sub-units/departments: 1) Biotechnology in Animal Science; 2) Emergent and Zoonotic Diseases; 3) Experimental Surgery and Regenerative Medicine. Recognizing that human and animal health are inextricably linked, CECA scientific teams, seek to promote, improve and defend the health and well-being by enhancing cooperation and collaboration between physicians, veterinarians and other scientific health professionals. This CECA’s new era is a consequence of the evaluation process and the FCT nominated panel report, who visits us at January 7º 2008. CECA’s vision is to be globally recognized as a R&D unit of reference for risk assessment on food safety, animal health and welfare. Its ultimate objective is to protect public health, by developing activities aiming the integration of Veterinary Medicine and Human Medicine. It aims to be a responsive and trusted partner to increase the competitiveness of the livestock production systems, to improve standards of animal health and welfare, prevent and control animal pathologies with particular emphasis to emerging diseases and zoonosis.

II JORNADAS CIENTÍFICAS DO CECA – ICETA

PROGRAMA 9.00 Sessão de abertura: José Luís Costa Lima – Presidente do ICETA Carlos Cunha Pinto – Director do CSPGF do INSA, I.P. Nuno Canada – Director do LNIV / INRB, I.P. José Manuel Correia da Costa – INSA, e CC do CECA 9.30 “Investigação Veterinária - A perspectiva de um Laboratório de Estado” Nuno Canada – LNIV / INRB, I.P. 10.00 “Aportaciones al conocimiento de las protozoosis y helmintosis de importancia económica en ruminantes en Galicia” Mercedes Mezo – INGACAL, Centro de Investigacións Agrarias de Mabegondo (Xunta de Galicia) 10.30 “Achievements and future perspectives of research on Reproductive Biology” António Rocha, Graça Lopes, Tiago Guimarães – CECA e ICBAS 11.00 “Extra-fetal tissues used in Tissue Engineering of peripheral nerve” Ana Colette Maurício – CECA e ICBAS 11.20 “Development of a skeletal muscular tissue regeneration model”

Tiago Pereira – CECA e ICBAS 11.40 “Biomaterials for bone Regeneration Medicine: in vivo studies and clinical trials for future applications” Luís Atayde – CECA e ICBAS 12.00 “Bonelike® and surgical-grade calcium sulfate for bone regeneration in cortical defects in sheep” Paulo Cortez – CECA e ICBAS 12.20 “Application of artificial vascular polyvinyl alcohol hydrogel grafts in sheep – our experience to date” Nuno Alexandre & Ana Lúcia Luís – CECA, ICBAS e Universidade de Évora 14.00 “Doenças Emergentes e Zoonóticas” José Manuel Correia da Costa – INSA e CECA 14.20 “A água como factor estruturante e essencial na Saúde Humana e Animal” André Almeida & Carla Mendonça – CECA 15.00 “Development of new Biotechnological tools” António Castro – INSA e CECA 15.20 “A novel fusion system for soluble over-expression of recombinant proteins in Escherichia coli” Sofia Costa – INSA e UM 15.40 “Schistosomes and estrogen: an old tale with new characters” Mónica Botelho – INSA e CECA 16.00 “Salmonella in swine: prevalence, antimicrobial resistance and genetic characterization” Eduarda Neves – ICBAS e CECA 16.20 “Toxoplasmose , a waterborne disease?” Susana Sousa – IBMC e CECA 16.40 “Saúde ambiental” João Paulo Teixeira – INSA e CECA 17.00 “Poluentes emergentes” Catarina Mansilha – INSA e REQUIMTE 17.20 “El agua como vehículo de transmisión de la cryptosporidiosos y giardiosis” José António Castro Hermida – INGACAL, Centro de Investigacións Agrarias de Mabegondo (Xunta de Galicia) 17.40 ”Chlorine dioxide based products for water treatment"

Iwan Zatterquist (presentation by JM Correia da Costa) 18.00 Debate, Conclusões, Recomendações e Encerramento

Aportaciones al conocimiento de las protozoosis y helmintosis de importancia económica en rumiantes en Galicia Mercedes Mezo Menéndez

Laboratorio de Parasitología, Departamento de Producción Animal, Centro de Investigaciones Agrarias de Mabegondo, Instituto Galego da Calidade Alimentaria-Xunta de Galicia. Carretera AC-542 de Betanzos a Mesón do Vento, Km 7,5. CP: 15318. Abegondo (A Coruña), España. E-mail: mercedes.mezo.menendez@xunta.es

Las infecciones parasitarias son una de las principales causas de enfermedad y pérdida de productividad en las explotaciones ganaderas de todo el mundo y no existe ninguna duda de que su control es absolutamente necesario. En los países desarrollados, sin embargo, debido a la disponibilidad de antiparasitarios de alta eficacia y a la mejora de las condiciones higiénico-sanitarias y de manejo, las parasitosis clínicas son cada vez menos frecuentes, y el uso de antiparasitarios, muy generalizado, se dirige fundamentalmente a evitar las pérdidas económicas asociadas a infecciones subclínicas, que no causan enfermedad aparente. Es precisamente en estos casos en los que es difícil determinar si los tratamientos antiparasitarios están justificados, es decir, si el beneficio económico que reportan compensa los gastos que conllevan y los problemas de contaminación y resistencias que ocasionan.

En la ponencia se destacarán los resultados de nuestras investigaciones más actuales llevadas a cabo en enfermedades parasitarias que presentan elevada prevalencia en Galicia; concretamente, tres protozoosis – cryptosporidiosis, giardiosis y neosporosis bovina - y dos trematodosis – fasciolosis y paranfistomosis. CRYPTOSPORIDIOSIS Y GIARDIOSIS Se expondrán los resultados más destacados de diversos trabajos realizados en nuestro Laboratorio. Las referencias bibliográficas que se comentarán son las siguientes: Castro-Hermida, J.A.; Carro-Corral, C; González-Warleta, M.; Mezo, M. Prevalence and intensity of

infection of Cryptosporidium spp. and Giardia duodenalis in dairy cattle in Galicia (NW Spain). Journal of Veterinary Medicine Series B, 53: 244-246. 2006.

Castro-Hermida, J.A.; Almeida, A.; González-Warleta, M.; Correia da Costa, J. M. and Mezo, M. Prevalence and preliminary genetic characterization of Cryptosporidium spp. isolated from asymptomatic heifers in Galicia (NW, Spain). Journal of Eukaryotic Microbiology, 53: S22-23. 2006.

Castro-Hermida, J.A.; Almeida, A.; González-Warleta, M.; Correia da Costa, J. M. and Mezo, M. Prevalence and preliminary genetic analysis of Giardia isolated from adult sheep in Galicia (NW, Spain). Journal of Eukaryotic Microbiology, 53: S172-173. 2006.

Castro-Hermida, J.A.; González-Warleta, M.; Mezo, M. Natural infection by Cryptosporidium parvum and Giardia duodenalis in sheep and goats in Galicia (NW Spain). Small Ruminant Research, 72: 96-100. 2007.

Castro-Hermida, J.A.; Almeida, A.; González-Warleta, M.; Correia da Costa, J.M.; Rumbo-Lorenzo, C. and Mezo, M. Occurrence of Cryptosporidium parvum and Giardia duodenalis in healthy adult domestic ruminants. Parasitology Research, 101: 1443-1448. 2007.

NEOSPOROSIS BOVINA

Esta producida por el protozoo Apicomplexa Neospora caninum. Sus manifestaciones clínicas son el aborto y las muertes neonatales, aunque es frecuente que no aparezca ningún signo clínico. A pesar de que en las hembras infectadas, el riesgo de aborto aumenta entre 2 y 7 veces, la gestación puede llegar a término. No obstante, el 90% de la descendencia nace congénitamente infectada. Una misma hembra puede transmitir la infección en partos sucesivos y a través de sus hijas perpetuar la enfermedad en el rebaño durante varias generaciones. N. caninum tiene un ciclo biológico heteroxeno facultativo en el que el hospedador definitivo es el perro y otros cánidos. La transmisión puede realizarse a través de la vía horizontal o vertical transplacentaria. Se comentará un amplio estudio sobre la neosporosis en Galicia realizado en nuestro Laboratorio: González-Warleta, M.; Castro-Hermida, J. A.; Carro-Corral, C.; Cortizo-Mella, J.; Mezo, M.

Epidemiology of neosporosis in dairy cattle in Galicia (NW Spain). Parasitology Research, 102: 243-249. 2008.

FASCIOLOSIS Esta enfermedad parasitaria de distribución mundial, está producida por especies de trematodos

pertenecientes al Género Fasciola. La especie más importante en Europa es F. hepatica, que también infecta al hombre. El método de diagnóstico tradicional consiste en el recuento de huevos de F. hepatica en las heces. Sin embargo, esta técnica es tardía ya que no se detectan huevos hasta las 10-12 semanas posinfección, poco sensible, no hay correlación entre el número de huevos y la carga parasitaria, es muy laborioso y requiere personal especializado.

La detección de anticuerpos anti-F. hepatica en suero sanguíneo tiene mejor sensibilidad que la coprología. Sin embargo, son frecuentes las reacciones cruzadas con otros parásitos, sobre todo cuando se emplean antígenos parasitarios totales. Para mejorar el diagnóstico de la fasciolosis, es necesario desarrollar y validar nuevas técnicas que permitan: detectar la infección en sus fases más tempranas; predecir la carga parasitaria y evaluar la eficacia del tratamiento. Con este objetivo, en nuestro Laboratorio hemos identificado y purificado los antígenos parasitarios de utilidad para el diagnóstico, es decir, aquéllos que inducen una respuesta inmune temprana y específica. En la ponencia se comentarán los estudios que hemos llevado a cabo para desarrollar 2 test de inmunodiagnóstico: a) ELISA indirecto para detección de anticuerpos específicos en el suero (MM3-SERO ELISA) y b) ELISA captura para detección de antígenos parasitarios en las heces del hospedador (MM3-COPRO ELISA). Se comentarán los siguientes trabajos: Mezo, M.; González-Warleta, M.; Carro, M.C.; Ubeira, F.M. An ultrasensitive capture ELISA for

detection of Fasciola hepatica coproantigens in sheep and cattle using a new monoclonal antibody (MM3). Journal of Parasitology, 90: 845–852. 2004.

Mezo, M.; González-Warleta, M.; Ubeira, F.M. The use of MM3 monoclonal antibody for the early immunodiagnosis of ovine fascioliasis. Journal of Parasitology, 93: 65–72. 2007.

En relación con la epidemiología de la fasciolosis en Galicia, se comentará un amplio estudio que realizamos en nuestro Laboratorio de Parasitología: Mezo, M.; González-Warleta, M.; Castro-Hermida, J.A.; Ubeira, F.M. Evaluation of the flukicide

treatment policy for dairy cattle in Galicia (NW Spain). Veterinary Parasitology, 157: 235-243. 2008. PARANFISTOMOSIS

Está producida por especies de trematodos pertenecientes a la Familia Paramphistomidae. Actualmente, están identificados 4 géneros: Paramphistomum, Cotylophoron, Gigantocotyle y Calicophoron. Las formas adultas que parasitan el rumen y la redecilla no son muy patógenas; mientras que los estadios inmaduros, localizados en el duodeno e íleon son responsables de lesiones que ocasionan hemorragias, pérdidas de producción e incluso la muerte del hospedador. El ciclo biológico es similar al de F. hepatica. La paranfistomosis está asociada al pastoreo en zonas húmedas que constituyen el hábitat idóneo para los hospedadores intermediarios (caracoles acuáticos).

En los estudios preliminares realizados hasta el momento en nuestro Laboratorio, se han examinado los pre-estómagos de 871 vacas pertenecientes a 685 explotaciones seleccionadas al azar entre ganado bovino sacrificado en un matadero. Los paranfistómidos recogidos en distintas zonas de la redecilla y rumen de cada animal se cuantificaron e identificaron mediante técnicas morfométricas y moleculares. La prevalencia de Paramphistomidae spp. fue 19,7% y las cargas parasitarias oscilaron entre 1-21.152 (media = 1.118,4). Sólo se identificó Calicophoron daubneyi. Las altas intensidades de parasitación observadas en algunas vacas adultas podrían tener efectos negativos que deberían valorarse. Paralelamente, se seleccionaron dos granjas con alta prevalencia de paranfistomosis y se recogieron diferentes tipos de moluscos acuáticos en su entorno. Sólo se hallaron fases larvarias de Paramphistomidae spp. en G. truncatula.

Achievements and future perspectives of research on reproductive biology

António Rocha, Graça Lopes & Tiago Guimarães Biotechnology in Animal Reproduction, CECA-UP

In this summary a brief description of the research achievements obtained in the past by the small group of CECA’s investigators working on animal reproduction, is presented. Several research programs were carried out in collaboration with fellow researchers and veterinary practitioners of other institutions, allowing the execution of research projects in an extended area of subjects, much above the capabilities of the limited number of professionals officially included in the group.

Research objectives

The research aims of this group encompass the study of basic and applied reproductive biology with emphasis in domestic species, including in the area of diagnosis and treatment of reproductive pathologies, development and improvement of assisted reproductive technologies, including the ones related to conservation of genetic resources. Clinical activity and professional collaboration with veterinary practitioners, breeders and famers is considered essential to the definition of research priorities. Achievements

Wildlife: One of the first collaborative activities of the group was the study of the reproductive biology of the hare and wild rabbit. These species are important for the sport hunting industry but above all are vital to the survival of several ecosystems. During that work, the reproductive potential of the species was quantified, as well as its seasonality. Environmental factors affecting reproductive productivity were identified as well as specific physiological characteristics of the native populations. Data gathered provided tools for a more rational management of wild populations of these species.

Dog: Cryopreservation protocols of dog semen are less developed than in other domestic animals, due to lower utilization of artificial insemination (AI) with frozen semen in this species. However, new technical developments have contributed to an increased number of AIs with cryopreserved semen, but more important cryopreservation of dog semen is essential to the establishment of genetic banks, as so far embryo collection in the bitch is not feasible. The research project carried out allowed for the development of cryopreservation protocols that induce minimum damage to the frozen/thawed sperm cells. In vitro maturation and fertilization (IVM/F) constitute fundamental basic technologies for artificial breeding, cloning, and production of transgenic domestic animals. Nevertheless, the efficiency of IVM of canine oocytes is extremely low, and consequently all the reproductive technologies dependent on IVM have lagged behind. Therefore, a research project was undertaken to improve our knowledge on the factors that contribute to the limited success of oocyte IVM in the canine species. The ultra structural characteristics of dog cumulus-oocyte-complexes were defined in great detail and unusual oocyte organelles, whose function needs to be established, were identified. The most productive age of bitches as oocyte donors, as well as the influence of stage of estrous cycle on oocyte production was defined. The role of apoptosis on meiosis resumption and oocyte degeneration was clarified, metabolic pathways of energy utilization by IVM embryos were studied, and the effect of different protein supplements on oocyte maturation and DNA fragmentation was assessed. Taken together the data indicate that individual oocyte variation is a key factor to the development of oocyte meiotic competence, while deficient culture conditions (media, energy and protein supplementation or duration of the culture) appear to be more associated with the preservation of oocyte viability during the long period of in vitro culture. Results obtained will allow development of IVM protocols better adequated to the structural and biological characteristics of dog oocytes.

During clinical reproductive work, we received chilled dog semen with deficient quality due to failures of the containers specifically designed to its transport. Thus, a research project was developed, resulting in the identification of the capabilities and shortcomings of the most common commercial containers used to ship refrigerated semen, clarifying the potential and limitations of the different models used.

Livestock species: a close professional relationship with veterinary practitioners and organizations in the dairy industry prompted the first diagnosis of abortion outbreaks in the country due to Neospora caninum infection. Subsequent research projects on the epidemiology and immunology of the disease, on improved methods of diagnosis, and on the potential for venereal transmission were carried out by multidisciplinary teams. This disease that has no treatment and no effective vaccine proved to have a high

prevalence and to be the main diagnosed cause of abortion in dairy cattle. Detailed results of the different projects will certainly be covered by other groups’ reports.

Participation in clinical work in dairy cattle reproduction led the group, in collaboration with other research teams and farmers’ associations, to analyse in detail and by the first time in the country, the reproductive efficiency of the dairy herds in the main milk producing region of the country. From that study it was concluded that fertility at insemination was one of the highest reported in the world, but that overall productivity was medium/low mainly due to abysmal oestrus detection efficiency. This study was followed up by a second one on time trends of reproductive parameters, where it was concluded that most fertility parameters were deteriorating, with the important exception of oestrus detection rate. Overall, results obtained provided pertinent information to define a range of technical options to revert negative trends, and contributed to improve the data collection systems and management information provided to dairy farmers and their consultants. The results achieved also point to the urgent need of an efficient extension service to actively pass out and support farmers in the development of better systems of management.

Portugal has a high number of native small ruminant breeds. Some, like the Merino, were selected elsewhere and played a pivotal role in the worldwide industry of wool production. Given the decreased economic importance of small ruminants, most Portuguese breeds are now represented by small populations with several breeds considered endangered by F.A.O. As such, a four year project was developed in collaboration with the University of Évora and regional sectors of the Ministry of Agriculture, with the objective of developing knowledge that would allow the maximization of embryo production and cryopreservation in the Merino (black and standard) breed. At the end of the project, conclusions were obtained in reference to the effect of season on the quantity and quality of embryos recovered per donor ewe, the efficiency of superovulation protocols was augmented, and techniques of embryo cryopreservation were assessed allowing the best selection of protocols for each specific combination of man power (technical skills), laboratory characteristics and type of equipment available. Furthermore, several hundred embryos were produce for storage in a gene bank.

Horses: so far most of the work done in horses was in the clinical reproductive area or in services of assisted reproduction techniques, rather than in research. However, a study on the seasonality of the mare in northern Portugal was carried out, dispelling the idea that mares in Portugal start cycling much sooner than horses at the same latitude elsewhere. Our persistent presentations in workshops and discussions with equine practitioners and breeders contributed to the alertness that allowed the first diagnosis in Portugal of Contagious Equine Metritis. This disease, transmitted by Taylorela equigenitallis is considered the most serious venereal disease in the equine, and the only venereal bacterial condition included in the list of notifiable diseases to the O.I.E. (it is not notifiable in Portugal, despite the recent notification to the O.I.E. of its existence, by the competent Portuguese authorities).

Genetics of tropical breeds of cattle: a project was carried out in Mozambique, with the main objective of establishing the genetic relationship between several breeds of Sanga cattle in Austral Africa. One of the main outputs was the identification of Tete breed as a type of cattle that originated from Sanga x Zebu crossbreeding, rather than being an off-shot of the Mashona, a similar breed native to a closely located border region in neighboring Zimbabwe. These findings had major implications for the strategy of conservation of Tete cattle.

Future Areas of Research Future areas of research are dependent on the vagaries of research funding. However, the main

medium term (5 years) objectives of our research group are:

1. Development of methods to increase the fertilizing ability of chilled and frozen stallion semen 2. Assess efficiency of alternative methods of semen collection in the stallion 3. Diagnostic and control of bacterial venereal diseases in the horse 4. Improve the efficiency of the cryopreservation of extra-gonadal sperm reserves 5. Assess the effect of BVD virus in cattle embryos and develop methods to its detection in in vitro

embryo production systems 6. Immunization of cattle against neosporosis 7. Study the genetic components of fertility in dairy cattle

The future line of research in dog reproduction is currently under discussion. Presently, financing for

research of areas 1 to 3 and 6 have been granted. It is intended to keep research collaboration with several national and international (Swedish University of Agriculture Sciences, University of S. Paulo and University of Botucatu in Brasil, Louisiana State University in the U.S.A, University of Ghent in Belgium and University

of Stellenbosch in South Africa) institutions and depending on ability to grant further research funding, to extend this collaboration to other international institutions.

Funded research projects PTDC/CVT/108456/2008. FCT (Foundation for Science and Technology), Ministry of Science and

Technology – 2010-2013. Development of methods to increase the fertilizing ability of chilled and frozen stallion semen: a multidisciplinary approach. (138.596,00€) PDTC/CVT/115126/2009. FCT (Foundation for Science and Technology), Ministry of Science and Technology – 2010-2012. Study of the immune response and protective effect against neosporosis elicited by mucosal immunization with Neospora caninum antigens. (125.763,00€) (Collaboration).

Past research projects ICCTI (Institute for International Scientific and Technological Cooperation), Ministry of Science and

Technology. Genetic and reproductive characterization of the native breeds of cattle in Mozambique. 1999-2000.

University of Porto, Vice-Chancellor’s Office (2005). “Apoptosis in cumulus-oocyte complexes of bitches”.

FCT (Foundation for Science and Technology), Ministry of Science and Technology- 2000-2003. Neospora caninum infection: Aspects of diagnostic, immunobiology, epidemiology and therapeutics. (Collaboration).

FCT (Foundation for Science and Technology), Ministry of Science and Technology 2000-2003. Bovine Viral Diarrhea Virus (BVDV): diagnosis, genotype characterization, vaccine efficacy and transmission studies. (Collaboration).

FCT (Foundation for Science and Technology), Ministry of Science and Technology- 2000-2003. Ionic channels during in vitro maturation (IVM) and fertilization (IVF) of bovine oocytes. (Collaboration). FCT (Foundation for Science and Technology), Ministry of Science and Technology 2002-2005. Immunobiological studies of Neospora caninum infection in a mice model for the development of a new vaccination strategy. (Collaboration).

Regenerative Medicine and Experimental Surgery A Gärtner

a, S Amado

b; T. Pereira

a,d, PAS Armada da Silva

b, AL Luís

a,d, AP Veloso

b, JD Santos

e, ASP Varejão

f,

S Geunac, AC Maurício

a,d*.

a Centro de Estudos de Ciência Animal (CECA), Instituto de Ciências e Tecnologias Agrárias e Agro-

Alimentares (ICETA), Universidade do Porto (UP), Portugal. b Faculdade de Motricidade Humana (FMH), Universidade Técnica de Lisboa (UTL), Portugal.

c Department of Clinical and Biological Sciences, University of Turin, Italy.

d Departamento de Clínicas Veterinárias, Instituto de Ciências Biomédicas Abel Salazar (ICBAS),

Universidade do Porto (UP), Portugal. eFaculdade de Engenharia da Universidade do Porto (FEUP), Portugal

fDepartamento de Ciências Veterinárias, CETAV, Universidade de Trás-os-Montes e Alto Douro (UTAD), Portugal. *Prof. Ana Colette Maurício Campus Agrário de Vairão, Rua Padre Armando Quintas 4485-661 Vairão VCD, Portugal. Telephone: +351252660412 Mobile: +351919071286 Fax: +351252661780 Email: ana.colette@mail.icav.up.pt; ana.colette@hotmail.com

Recent advances in nerve Tissue Engineering have greatly promoted the generation of biomaterials, which may be implanted empty, or may be filled with growth factors, and / or stem cell auto or allografts for nerve regeneration. Mesenchymal stem cells (MSCs) comprise a rare population of multipotent progenitors capable of supporting hematopoiesis and differentiating into several lineages (osteogenic, neurogenic, myogenic, among others). Due to this ability confirmed by the results of both in vitro experiments and in vivo studies, MSCs appear to be an attractive tool in the context of Tissue Engineering and cell-based therapy. Currently, bone marrow represents the main source of MSCs for both experimental and clinical applications. However the number of bone marrow MSCs significantly decreases with age and the bone marrow HLA compatible donors are very difficult to find, which makes the search for adequate alternative sources of these cells necessary for autologous and allogenic use. The stem cells obtained from the blood and Wharton´s jelly of the umbilical cord are a promising source of stem cells: i) the number of stem cells per volume is higher than in bone marrow, ii) a complete or high HLA profile match for allogenic use is not necessary, which permits to greatly enlarge the number of available donors iii) the stem cells are easier to obtain, manipulate and criopreserve, and their collection in ethically approved by national and international laws. A multidisciplinary team, including Veterinaries, Engineers, Medical doctors like neurologists and surgeons through Experimental Surgery has a crucial role in the development of biomaterials associated to these extra-fetal cellular systems, and in testing the surgical techniques that involve their application, always considering animal welfare and the most appropriate animal model. Several biomaterials developed by our research group (including poly(L-lactide):poly(glycolide) polymers, hybrid chitosan, and collagen) have been tested associated to cellular systems to promote nerve regeneration after axonotmesis and neurotmesis injuries in the sciatic nerve experimental model. The cellular systems that have been studied in this context include an immortalized neural cell line N1E-115, stem cells obtained from the Wharton´s jelly of the umbilical cord, CD34+ stem cells from the umbilical cord blood, and MSCs from umbilical cord matrix. The cells isolated from the extra-fetal tissues were also multiplied in a bioreactor, in order to obtain higher cell doses to clinical application. The tube-guides associated to one of the cellular systems are tested in the rat sciatic nerve across a 10 mm-gap (neurotmesis) or in a 3 mm axonotmesis lesion. The cells introduced are able to produce growth factors in the local of the nerve injury, during the necessary healing period. For nerve reconstruction after neurotmesis or axonotmesis, the biodegradable tubes/membranes covered by the cellular system are used to bridge the nerve gap or to involve the axonotmesis lesion area, respectively. Motor functional recovery after the sciatic nerve reconstruction is assessed serially using video recording of the gait for biomechanical analysis, by measuring extensor postural thrust (EPT), sciatic functional index (SFI) and sciatic functional index under static conditions (SSI). The sensitive recovery is tested by the withdrawal reflex latency (WRL). The repaired nerves are processed for light and electronic microscope analysis, imunohistochemistry,

confocal microscopy and stereological studies. Functional and morphologic results obtained with these different biomaterials and cell lines are presented and discussed in terms of better improvement of nerve regeneration after axonotmesis and neurotmesis injuries in rat sciatic model.

Medicina Regenerativa e Cirurgia Experimental

Avanços recentes em Engenharia de Tecidos, nomeadamente do tecido nervoso periférico conduziram ao desenvolvimento de tubos-guia, que podem ser implantados vazios ou preenchidos com factores de crescimento ou auto ou aloenxertos. As células estaminais mesenquimais (MSCs) fazem parte de uma rara e valiosíssima população de células progenitoras multipotentes capazes de suportarem a hematopoiese e a diferenciação em várias linhagens celulares. Devido a esta capacidade confirmada por resultados obtidos de experimentação in vitro e em modelos animais, MSCs parecem ser uma arma atractiva no contexto da Medicina Regenerativa e das Terapias Celulares. Correntemente, a medula óssea representa a principal fonte de MSCs tanto para estudos experimentais quer aplicações clínicas. No entanto, o número de MSCs da medula óssea diminui significativamente com a idade e a sua utilização alogénica é extremamente difícil devido ao reduzido número de dadores HLA compatíveis. Deste modo, as MSCs obtidas do sangue e da geleia de Wharton do cordão umbilical são uma alternativa viável, para utilização autóloga e alogénica. Estas células têm ganho adeptos devido a vários factores: i) maior número de células estaminais por volume do que a medula óssea; ii) a taxa de rejeição é menor quando usadas em enxerto alogénicos; iii) não é necessária uma compatibilidade antigénica HLA tão exigente como para a medula óssea alargando o número de dadores disponíveis; e 4) são mais fáceis de obter, processar e armazenar e a sua colheita é eticamente aprovada e legislada pelas leis nacionais e internacionais. Neste aspecto, os Médicos Veterinários em coordenação com Engenheiros e Médicos, através da Cirurgia Experimental têm um papel primordial no desenvolvimento destes biomaterias associados a estes sistemas celulares e das técnicas cirúrgicas inerentes à sua aplicação, através da experimentação in vivo e respeitando os princípios de bem-estar animal. Tem sido realizado o estudo in vivo de vários biomateriais reabsorvíveis desenvolvidos por esta equipa, utilizados para o fabrico de tubos-guia, nomeadamente PLGA na proporção dos dois polímeros, poli(L-lactido): poli(glicólico) de 90:10 e um híbrido de quitosano, associados a células estaminais obtidas da geleia de Wharton diferenciadas in vitro em células neurais, para promover a regeneração do nervo periférico após lesões de axonotmese e neurotmese. Estas células estaminais têm sido alvo de estudo desta equipa, em que 3 dos elementos (incluindo o IR) fundaram e coordenam cientificamente o único banco privado de criopreservação do sangue do cordão umbilical nacional (www.criovida.pt) que segue estritamente as Directivas Comunitárias e as normas Netcord. A equipa deste projecto trabalha na área da Medicina Regenerativa há cerca de 6 anos, apresenta um vasto número de publicações e frutuosas colaborações com os principais grupos mundiais desta área de investigação. Estudos morfológicos (microscopia óptica, de confocal, histomorfometria e electrónica de varrimento) serão efectuados ao fim de 20 ou 12 semanas, nos grupos de neurotemese ou axonotemese, respectivamente. A avaliação funcional da recuperação do nervo ciático é feita durante este período, através da análise biomecânica da locomoção, do Índice de Funcionalidade do Ciático (SFI), do Índice de Funcionalidade do Ciático em Condições Estáticas (SSI) e do Reflexo de Reacção Postural de Extensão (RPE). A percepção sensorial será avaliada através do Reflexo Flexor de Latência (RFL) e recorrendo aos filamentos de von Frey.

Development of skeletal muscular tissue regeneration models

Pereira Ta,b

, Gärtner Aa,

, Luís ALa,b

, Maurício ACa,b

, Armada Pc

a Centro de Estudos de Ciência Animal (CECA), Instituto de Ciências e Tecnologias Agrárias e Agro-

Alimentares (ICETA), Universidade do Porto (UP), Portugal. b Departamento de Clínicas Veterinárias, Instituto de Ciências Biomédicas Abel Salazar (ICBAS),

Universidade do Porto (UP), Portugal. c Faculdade de Motricidade Humana (FMH), Universidade Técnica de Lisboa (UTL), Portugal.

This work is one of the initial steps in a project that will be carried out in three parts and will

involve the study of morphological and physiological skeletal muscle properties of distinct animal models. First of all, we are developing a skeletal muscular tissue regeneration model, which consists in inducing a muscular lesion in the rat. This will be the basis for testing regeneration enhancement methods in muscular

tissue lesions. Also, the disuse model of desinervation/reinervation in the rat will be used in this project and a detailed analysis of skeletal muscle morphology will be performed. These models will be the starting point for other studies associated with the use of stem cells for muscular tissue regeneration. In the second part, the healing enhancement after mesenchymal stem cells injection in muscular tissue will be tested in the colagenase induced lesion ovine model. The last part of the project will establish a connection between the previous studies and the clinical features of a specific pathology in the horse. For that purpose a clinical trial will be outlined for testing the therapeutical benefits of autologous mesenchymal stem cells injection in clinical cases of fibrotic myopathy affecting these animals.

Elaboração de modelos de regeneração de tecido muscular esquelético

Este trabalho é parte integrante da fase inicial de um projecto que será realizado em três partes e vai envolver o estudo das propriedades morfológicas e fisiológicas do músculo esquelético através de distintos modelos animais. Em primeiro lugar, estamos a desenvolver um modelo de regeneração do tecido muscular esquelético, que consiste na indução de uma lesão muscular em ratos. Esta será a base para testar métodos de melhoramento de regeneração em lesões do tecido muscular. Além disso, será utilizado neste projecto o modelo de desuso por desinervação/reinervação em ratos, nos quais irá ser realizada uma análise detalhada da morfologia do músculo esquelético. Estes modelos serão o ponto de partida para outros estudos relacionados com a utilização de células estaminais mesenquimatosas para a regeneração do tecido muscular. Na segunda parte, irão ser testados os benefícios da injecção de células mesenquimatosas no tecido muscular no modelo de lesão induzida por colagenase em ovinos. A última parte do projecto vai estabelecer uma ligação entre os estudos anteriores e as características clínicas de uma patologia específica em equinos. Para esse efeito, irá ser desenhado um ensaio clínico para testar os benefícios terapêuticos do transplante autólogo de células estaminais mesenquimatosas em casos clínicos de miopatia fibrótica.

Bonelike® and surgical-grade calcium sulfate for bone regeneration in cortical defects in sheep P. P. Cortez*

1,6, M. A. Lopes

2, M. Santos

3, M.A. Silva

3, A. Afonso

4, P. Armada-da-Silva

5, J.D. Santos

2, A. C.

Maurício1,6‡

1 Centro de Estudos de Ciência Animal (CECA), Instituto de Ciências e Tecnologias Agrárias e Agro-

Alimentares (ICETA), Universidade do Porto, Campus Agrário de Vairão, Rua P. Armando Quintas, 4485-661, Vairão, Portugal 2 CEMUC, Departamento de Engenharia Metalúrgica e Materiais, Universidade do Porto, Rua Dr. Roberto

Frias, 4200-465 Porto, Portugal 3 Faculdade de Engenharia, Universidade do Porto (FEUP), Secção de Materiais, DEMM, Rua Dr. Roberto

Frias, 4200-465 Porto, Portugal 4 Faculdade de Medicina Dentária, Universidade do Porto (FMD-UP), Rua Dr. Manuel Pereira da Silva, s/n

4200-393 Porto, Portugal 5 Faculdade de Motricidade Humana (FMH), Universidade Técnica de Lisboa (UTL), Estrada da Costa,

1499-002 Cruz Quebrada – Lisboa, Portugal 6 Instituto de Ciências Biomédicas Abel Salazar da Universidade do Porto (ICBAS-UP), Largo Prof. Abel

Salazar, 2, 4099-003, Porto, Portugal.

A glass-reinforced hydroxyapatite composite (Bonelike®) was developed by the authors for bone grafting. This biomaterial is composed of a modified hydroxyapatite matrix with α- and β-tricalcium phosphate secondary phases, resulting in higher solubility than hydroxyapatite. Several in vitro and in vivo studies demonstrated that Bonelike® has a highly bioactive behavior, which was also confirmed by employing granular forms of this biomaterial in orthopedics and dental applications. However, a fast consolidation vehicle was needed to promote the fixation of the Bonelike® granules if applied in larger defects or in unstable sites. Surgical-grade calcium sulfate, which is widely recognized as a well-tolerated and inexpensive material, was the chosen vehicle to improve the handling characteristics of Bonelike® as it can be used in the form of a powder that is mixed with a liquid to form a paste that sets in situ. After application in cortical defects in sheep, histological and scanning electron microscopy evaluations demonstrated that Bonelike® associated to surgical-grade calcium sulfate functioned as an excellent

scaffold for bone regeneration as it achieved synchronization of the in-growing bone with biomaterials resorption and subsequent preservation of the bone graft initial volume. Therefore, our results indicate that surgical-grade calcium sulfate is an effective vehicle for Bonelike® granules as it facilitates their application and does not interfere with their proven highly osteoconductive properties. In the opposite way, the incorporation of Bonelike® improves the bone regeneration capabilities of surgical-grade calcium sulfate.

Biomaterials for Bone Regenerative Medicine: In vivo studies and clinical trials for future applications Luís Miguel Atayde

a,b, Paulo Pegado Cortez

a,b, Tiago Pereira

a,b, José Domingos Santos

c, Ana Colette

Maurícioa,b

a

Centro de Estudos de Ciência Animal (CECA), Instituto de Ciências e Tecnologias Agrárias e Agro-Alimentares (ICETA), Universidade do Porto (UP), Portugal. b

Departamento de Clínicas Veterinárias, Instituto de Ciências Biomédicas Abel Salazar (ICBAS), Universidade do Porto (UP), Portugal. c Faculdade de Engenharia da Universidade do Porto, FEUP, Portugal

In this work we test a bonegraft, with a patent formulation called Bonelike®, prepared with a new

spherical formulation with 2 distinct sizes (250-500µm and 500-1000µm). To this bonegraft, we also associate bioresorbable polymers and hidrogels, like chitosan, chitosan-silicate hybrid gel and dextrin to promote bone regeneration and making local application easier. Adding these substances to Bonelike® allows controlled release of therapeutically agents and microelements, such as bone morphogenetic proteins (BMPs), with significant improvement in new bone formation and bone regeneration. This study will be carried out with in vitro and in vivo tests using a sheep model. For the in vivo study, the two sizes of spherical bonelike® are implanted in the sheep’s femur either associated with bioresorbable polymers and hidrogels or with blood. We will do a radiological exam on the day of the surgery and every 30 days. The different sheep are euthanized on different periods of time and a post mortem exam is performed, using macroscopic and microscopic analyzes. Bonelike® is also being tested as a bone growth promotor on arthrodesis surgeries. Before the clinical application of Bonelike® on arthrodesis surgeries, a test is done on a sheep model, by doing an arthrodesis on tarso-metatarsal joints of both hind limbs: in one of the joints we only use the convectional technique of arthrodesis, serving as a control, and on the other limb in addition to this technique we add Bonelike® in the joint space. We proceed with a radiological exam on the surgery day and then every 30 days. The different sheep are euthanized on different periods of time and a post mortem exam is performed, using macroscopic and microscopic analyzes. We also have clinical trials on various clinical cases of arthrodesis.

Application of artificial vascular polyvinyl alcohol hydrogel grafts in sheep - our experience to date Nuno Alexandre

d, Andrea Gärtner

a, Irina Amorim

c, José D Santos

e, MA Lopes

e, Ana C Maurício

a,b, Ana L

Luísa,b

. a Centro de Estudos de Ciência Animal (CECA), Instituto de Ciências e Tecnologias Agrárias e Agro-

Alimentares (ICETA), Universidade do Porto (UP), Portugal. bDepartamento de Clínicas Veterinárias, Instituto de Ciências Biomédicas Abel Salazar (ICBAS),

Universidade do Porto (UP), Portugal. cDepartamento de Patologia e Imunologia Molecular, Instituto de Ciências Biomédicas Abel Salazar

(ICBAS), Universidade do Porto (UP), Portugal. dDepartamento de Zootecnia, Universidade de Évora (UE), Portugal

e Faculdade de Engenharia da Universidade do Porto (FEUP), Porto, Portugal.

Researchers developed this project according with the tasks originally planned, so far focusing their research activities in the biocompatibility and functionality of vascular implants. The biocompatibility was studied by following the ISO standard 10993-6 for evaluation of medical devices through the evaluation and grading of inflammation caused by implants placed subcutaneously. The implants were placed in sheep

and collected randomly at different time points of the experimental period and subsequently processed histologically and classified according to Annex E to the standard mentioned above. The functionality of vascular implants has been evaluated to date by replacement of segments of the carotid by ePTFE implants with 5 cm long and 4 mm in diameter, coated or not with cultured stem cells. The deployment of these prostheses has been performed in carotid arteries of sheep and their functional performance measured by vascular ultrasound in Doppler and B mode by measuring parameters such as: peak systolic / diastolic blood flow velocity, vascular diameters at implantation and at the periphery of these. These measurements are performed at various time points of the experiment were folloed by euthanasia of sheep and immediately sample collection for the implementation of techniques like immunohistochemistry, morphometry and scanning electronic microscopy. Complications were observed throughout the experimental period include the formation of thrombi after at the transition implant-carotid region and vascular dilatation as the most frequent, respectively, in periods of short-and long-term experiment.

Emergent and Zoonotic Diseases CECA’ sub-unit presentation José Manuel Correia da Costa, INSA & CECA-UP

Emergent and Zoonotic Diseases CECA’ sub-unit has 4 senior scientists, 2 senior scientists’ collaborators, and 3 PhD students and other technical staff. All senior scientists come from State Laboratories. As a consequence of the evolution process and CECA restructuration in 2008, the precedent CECA’ sub-unit Microbiology and Molecular Biology originate in 2009 the Emergent and Zoonotic Diseases sub-unit. Since 2007 to the present, funds to EZD were obtained from external funds originated in FCT, FC Gulbenkian, Xunta da Galiza and qREN. The general objectives of EZD supported research are to advance our understanding of biological systems, improve the diagnosis and control of animal diseases, including zoonosis. EZD’ specific objectives aiming the molecular epidemiology, the development of methods to isolate the parasite, and to proceed to their genetic characterization, in a pre-harvest manner, and the creation of models of experimental infection with potential implications for vaccine development, and in the development and evaluation of new diagnostic tools, medicines and vaccines for the prevention and control of diseases across species. Main achievements in last year: a) consolidation of a collaborative national and international network; b) consolidation of one spin-off: Biognosis; c) Serotyping Toxoplasma gondii; determining the genetic characteristics of parasite isolates from humans, animals and food, by using synthetic peptides; d) Schistosoma haematobium as a possible promoter of tumorigenesis and bladder cancer; e) Risk assessment of cryptosporidiosis and giardiasis in several watersheds in the North of Portugal and Galiza; f) Development of genetic tools for the characterization of Prosorynchus species, a fish infecting parasite.

Cryptosporidium spp. and Giardia duodenalis: protozoa transmitted through water. Detection and genetic characterization – new approaches Almeida, A.

1,2,3, Soares, S.

3, Delgado, M. L.

1,3, Moreira, M. J.

3, Silva, E.

1,3, Castro, A.

1,3, Mendonça, C.

2,3,

Canada, N.2,3,4

, Costa, J. M.1,3

1 Instituto Nacional de Saúde Dr. Ricardo Jorge

2 Instituto de Ciências Biomédicas de Abel Salazar

3 Instituto de Ciências e Tecnologias Agrárias e Agroalimentares da UP

4 Laboratório Nacional de Investigação Veterinária

Cryptosporidium is a protozoan parasite with a worldwide distribution; it causes the disease known as cryptosporidiosis which is characterized by severe gastrointestinal disorders. The parasite belongs to the Apicomplexa phylum, although there is a controversy in this classification. It infects a large spectrum of hosts, from amphibians to birds and mammals, including man. In man, infection is usually self limited in healthy individuals, although in immunocompromised ones, it can even be fatal as there is no effective treatment available. Besides the ability of this parasite to infect and destroy the epithelial cells from the gastrointestinal tract, it was also reported cases of extra-gastrointestinal infection, in particular trachea, lung and gall bladder infections. There are several transmission routes for Cryptosporidium, including direct fecal-oral transmission (human-to human, and animal-to-human) and food-borne transmission; however, water represents the major transmission route. Even thought, cryptosporidiosis was considered an important disease only in immunocompromised individuals, due to its opportunistic behavior. However, this perception changed radically in 1993, when a major outbreak associated with the consumption of contaminated water occurred in Milwaukee, USA, affecting more than 400000 people and killing nearly 100. Since then, Cryptosporidium is considered as a serious public health problem, and many waterborne outbreaks of cryptosporidiosis have been reported worldwide. Giardia duodenalis is a flagellate protozoan parasite with a worldwide distribution; similar to Cryptosporidium, Giardia causes severe gastrointestinal disorders and malabsorption symptoms, which characterize the disease, known as giardiasis. Giardia duodenalis infects humans, although is also one of the most common parasites of pets (dogs, cats) and livestock (cows and sheep). It colonizes and reproduces in the small intestine where, by attaching to the surface of epithelium with its ventral disc and covering it, reduces the ability of nutrients absorption. Similar to Cryptosporidium, Giardia is transmitted by fecal-oral contacts or by ingestion of contaminated food, although water is the major transmission vehicle. The importance of water for transmission also depends on the fact that both parasites, due to the robust

cyst and oocyst structure, can persist in the environment for weeks to months, even at low temperatures. Furthermore, oocysts and cysts can resist to conventional treatments of water, such as chlorination.

Giardia infections are reported worldwide and have particular negative impact on children’s health by reducing the absorption of nutrients and compromising a regular development.

In routine diagnostic laboratories, the diagnosis of Giardia and Cryptosporidium relies upon the demonstration of oocysts and cysts in fecal material by microscopy, with or without staining; in research laboratories, immunofluorescence with monoclonal antibodies or molecular techniques are often used. The genus Cryptosporidium currently comprises 19 valid species based on morphological, biological and molecular data. Several species are able to be transmitted from animal to humans (zoonotic transmission) or from humans to humans (anthroponotic). C. parvum is the most important zoonotic species, and C. hominis is the human restricted species. Other species, such as C. bovis, are restricted to specific hosts and have no zoonotic potential. Giardia duodenalis is one of the six species currently recognized in the Giardia genus. In spite of the small morphological variation among members of Giardia duodenalis, genetic analysis supports the existence of at least 7 distinct genetic groups, commonly referred to as assemblages A to G. Assemblage A and B are associated with human infections, and the prevalence of each assemblage varies considerably from country to country, although assemblage B seems more common overall. The remaining assemblages are likely to be host specific: assemblage C and D have been identified in dogs, cats, coyotes, and wolves, assemblage E in calves, sheep, goats, pigs, water buffaloes and muflons, assemblage F in cats, and assemblage G in rats. The molecular approaches used to genotype Cryptosporidium and Giardia isolates are mainly based on conventional PCR and sequencing of several markers. For Giardia genotyping, β-giardin, glutamate dehydrogenase (gdh), actin, small subunit ribosomal DNA (ssu-rDNA) and triosephosphate isomerase (tpi) genes are commonly used, being useful for species and genotype assignment. For Cryptosporidium, heat shock protein (hsp), small subunit ribosomal DNA (ssu-rDNA), 60 kDa glycoprotein (gp60), Cryptosporidium oocyst wall protein (cowp) and actin are used for species assignment. In particular, the highly polymorphic gp60 gene is used for subtyping studies. These markers have been used worldwide and applied to many animal and human fecal samples to determine the species and genotypes circulating among different hosts, to evaluate the possible transmission routes, and transmission dynamics. This approach is also used for environmental samples, such as water samples, with the objective of determine the source of contamination and the risk for human or animal infection. The immunomagnetic and immunofluorescence based method 1623, established by the Environmental Protection Agency, USA, is used worldwide to detect Cryptosporidium and Giardia in different water samples. These methods are used worldwide and provide useful information to understand the epidemiology of these parasites. In Portugal, little is known about the prevalence of these parasites in humans and animals, and on the level of contamination in raw and treated water. Moreover, PCR and sequencing techniques should be applied to these samples, and sometimes optimized, in order to generate accurate information on the species and genotypes of these parasites.

Emerging Pollutants Catarina Mansilha

1,2

1- Instituto Nacional de Saúde Dr. Ricardo Jorge, Porto, Portugal

2- Requimte, Universidade do Porto, Porto, Portugal

catarinamansilha@gmail.com; catarina.mansilha@insa.min-saude.pt

Emerging Pollutants are a broad category of chemicals of environmental occurrence, mostly of anthropogenic origin, previously unknown or unrecognized as being of concern that are currently under increasing scientific scrutiny. They are generally not regulated, showing no maximum levels for specific environments. However, some of them are candidates for future regulation, depending on investigations of its potential effects on human and animal health and monitoring data and incidence.

Although current knowledge of this category of contaminants contains significant gaps, an increasing effort has been developed by the scientific community towards the knowledge of their toxicity, persistence, bioaccumulation processes, occurrence, transport and processing mechanisms. Examples of compounds that have recently emerged as particularly relevant are surfactants, pharmaceuticals, personal care products, gasoline additives, flame retardants, antiseptic, industrial additives, steroids and hormones, pesticides and byproducts of water disinfection.

The emerging pollutants are widespread in the environment and water is one of their main dissemination vehicles. They have been detected in surface water, groundwater and even in drinking water that is an important source of these contaminants to humans.

One of the most concerning group is that of the emerging contaminants, that are compounds that are capable of interfering with the mechanisms of endocrine regulation. The growing concern about the issue of adverse effects of endocrine disrupters has been considered a priority problem by international organizations including the World Health Organization, the European Commission, the Organization for Economic Cooperation and Development and the Environmental Protection Agency of the United States of America so, research has been made worldwide in order to verify the existence of significant evidence of harmful effects in humans and animals, assess their concentrations in environmental matrices and validate biomarkers.

Optimization and validation of a method based on a solid phase extraction methodology coupled with gas chromatography tandem mass spectrometry was developed by our research group and is used to analyze different environmental water samples to assess the contamination of surface and groundwater of vulnerable areas.

It is also being performed a study to assess the evidence for Bisphenol-A migration from different plastic containers of high density polyethylene (HDPE), polyethylene terephthalate (PETE) and polycarbonate in order to evaluate its concentration in the contents and, consequently, the exposition rate of population.

The increasing knowledge about the origin, processing and effects of this new generation of contaminants is essential to allow the definition / presentation of a more accurately official list of organic pollutants, to propose new mechanisms for water treatment and to ensure quality and allow new environmental remediation strategies and health promotion. U.S. Environmental Protection Agency Region/ORD Workshop on Emerging Pollutants, August 11-14, 2003. Report of the NORMAN Workshop on Emerging Environmental Pollutants: Key Issues and Challenges Stresa, Italy 19-20 June 2006. Martha J. M. W. et al., Emerging Pollutants. Water Environment Research, Volume 79, Number 10 - Copyright © 2007 Water Environment Federation. Monitoring Strategies for Chemicals of Emerging Concern (CECs) in Recycled Water, Final Report. Recommendations of a Science Advisory Panel. Convened by the State Water Resources Control Board June 25, 2010, Sacramento,California.

Poluentes emergentes

Os Poluentes Emergentes constituem uma ampla categoria de produtos químicos de ocorrência ambiental, na sua maioria de origem antropogénica, anteriormente desconhecidos ou não reconhecidos como sendo preocupantes e que estão actualmente sob crescente escrutínio científico. Em geral não são regulamentados, não apresentando níveis máximos admissíveis para ambientes específicos sendo, no entanto, alguns deles candidatos a regulamentação futura, dependendo para tal das investigações levadas a cabo relativamente aos seus efeitos potenciais para a saúde humana e animal e a dados de monitorização e incidência.

Embora o conhecimento actual desta categoria de contaminantes contenha lacunas significativas, um esforço crescente tem sido desenvolvido pela comunidade científica mundial no sentido do conhecimento da sua toxicidade, persistência, processos de bioacumulação, ocorrência, transporte e mecanismos de transformação.

Exemplos de compostos que emergiram recentemente como particularmente relevantes são os surfactantes, produtos farmacêuticos, produtos de cuidado pessoal, aditivos das gasolinas, retardantes de fogo, anti-sépticos, aditivos industriais, esteróides e hormonas, pesticidas e subprodutos da desinfecção da água.

Os poluentes emergentes encontram-se disseminados no ambiente, sendo a água um dos seus principais veículos de difusão. Têm sido detectados em fontes de abastecimento de água, águas subterrâneas e, inclusivamente, na água considerada potável, sendo esta uma importante fonte destes contaminantes para o Homem.

Um dos grupos de poluentes que tem suscitado maior preocupação corresponde ao dos compostos com capacidade de interferir com os mecanismos de regulação endócrina, considerados desreguladores endócrinos.

A preocupação crescente com a questão dos efeitos adversos dos desreguladores endócrinos fez com que várias organizações internacionais, nomeadamente a Organização Mundial de Saúde, a Comissão Europeia, a Organização para a Cooperação no Desenvolvimento e Economia e a Agência de Protecção Ambiental do Estados Unidos da América, a considerassem um problema prioritário. Desde então, a investigação tem vindo a desenvolver-se no sentido de verificar a existência de evidências significativas de efeitos deletérios em humanos e animais, avaliar as suas concentrações em matrizes ambientais e validar biomarcadores.

O trabalho até agora desenvolvido no INSA em colaboração com outras Unidades de Investigação centrou-se no desenvolvimento e validação de metodologias de análise e a implementação de programas de monitorização para a detecção, quantificação e conhecimento da distribuição de alguns desreguladores endócrinos em diversas matrizes ambientais de água.

As metodologias analíticas desenvolvidas têm vindo a ser aplicadas na avaliação da contaminação de águas superficiais e subterrâneas de zonas vulneráveis, bem como tem sido igualmente desenvolvido um trabalho de pesquisa de Bisfenol - A não só em matrizes ambientais como também resultante da migração de embalagens plásticas.

O incremento do conhecimento sobre a origem, transformação e os efeitos desta nova geração de contaminantes é fundamental de forma a permitir a definição/apresentação de uma lista oficial de poluentes orgânicos mais exacta e para que se possam propor novos mecanismos de tratamento de águas, de modo a garantir uma qualidade idónea e permitir estabelecer estratégias de remediação ambiental e prevenção e promoção da saúde.

El agua como vehículo de transmisión de la cryptosporidiosis y giardiosis José Antonio Castro-Hermida

Laboratorio de Parasitología, Departamento de Producción Animal, Centro de Investigaciones Agrarias de Mabegondo, Instituto Galego da Calidade Alimentaria-Xunta de Galicia. Carretera AC-542 de Betanzos a Mesón do Vento, Km 7,5. CP: 15318. Abegondo (A Coruña), España. E-mail: jose.antonio.castro.hermida@xunta.es

Entre los numerosos agentes infecciosos que pueden contaminar el agua, los protozoos parásitos transmitidos por vía oral adquieren especial relevancia tanto por su elevada prevalencia en los animales y en el hombre como por la capacidad de las formas infectantes (ooquistes/quistes) para superar los sistemas comunes de tratamiento del agua en las plantas depuradoras. En consecuencia, brotes epidémicos son frecuentes incluso en países desarrollados. La cryptosporidiosis y la giardiosis son enfermedades parasitarias de distribución mundial, producidas por protozoos incluidos en los Géneros Cryptosporidium y Giardia, que afectan a un amplio rango de aves y mamíferos. Algunas especies o genotipos presentan, además, características zoonóticas, por lo que tienen gran importancia sanitaria. Ambas parasitosis ocasionan un síndrome de mal absorción y diarrea que se manifiesta principalmente en hospedadores neonatos y en individuos inmunocomprometidos. En los rumiantes domésticos, ambas enfermedades parasitarias producen elevada morbilidad y mortalidad durante el primer mes de vida, por lo que ocasionan importantes pérdidas económicas en las explotaciones ganaderas. En el hombre, la prevalencia de ambas infecciones es especialmente elevada tanto en individuos con SIDA e inmunocomprometidos como en niños y ancianos. En cuanto a la presencia de formas infectantes en el medio ambiente debe señalarse que Cryptosporidium y Giardia tienen un ciclo biológico directo, en el que todas las especies infectadas actúan como hospedadores definitivos, contribuyendo a la contaminación ambiental con ooquistes/quistes, por lo que ambos enteropatógenos son muy ubicuos. En todos los casos, un alto porcentaje de los ooquistes/quistes excretados por los hospedadores definitivos contaminarán las fuentes de agua facilitando la difusión de estos protozoos. La transmisión hídrica de estas parasitosis está ampliamente documentada a nivel mundial. La magnitud de un brote de cryptosporidiosis o de giardiosis, asociado al consumo de agua, estará determinada no sólo por la eficacia del tratamiento de potabilización, sino también por los factores propios de ambos parásitos y del hospedador, representando lo que se conoce como dinámica de transmisión. Por otra parte, la importancia que ha adquirido la cryptosporidiosis en los últimos años como enfermedad de origen alimentario o hídrico y de origen medioambiental, ha

obligado a la Unión Europea a incluir esta parasitosis entre las enfermedades transmisibles que deben integrarse en la red comunitaria. Además, el Parlamento Europeo ha decidido desarrollar un sistema de alerta rápido con el fin de dar una respuesta temprana para la vigilancia y control de esta enfermedad. Teniendo en cuenta que la contaminación de fuentes de agua con residuos fecales de origen humano o animal es un tema concerniente a la salud pública, y que el mecanismo de transmisión más importante de la cryptosporidiosis y de la giardiosis es el agua de bebida, ya que puede afectar en muy poco tiempo a grandes núcleos de población, se comentará la Directiva Europea relativa a la calidad de las aguas destinadas al consumo humano y las posibles medidas para la detección y cuantificación de (oo)quistes en aguas. Para ello, se abordan las normas de la EPA (Environmental Protection Agency) aplicadas en EEUU, el método 1623 para Cryptosporidium y Giardia. Por tanto, la presencia de ooquistes de Cryptosporidium y quistes de Giardia en los suministros de agua suponen un problema de salud pública, ya que ambas formas parasitarias superan sin dificultad los sistemas de depuración. Debido a la enorme cantidad de (oo)quistes eliminados por las personas enfermas y animales durante el período de patencia, la escasa capacidad de sedimentación de las formas parasitarias en pantanos y depósitos, las prácticas de abonado de los pastos con purines, a veces no convenientemente madurados, la eliminación de aguas residuales depuradas a las cuencas hidrográficas y la resistencia a las condiciones ambientales de las formas infectantes, conlleva a que la presencia de Cryptosporidium y Giardia sean más ubicuas cada día, y en consecuencia la necesidad de investigar las fuentes cuantitativas primarias de ambos parásitos en el ambiente. En este sentido, en el Laboratorio de Parasitología del Centro de Investigaciones Agrarias de Mabegondo perteneciente al Instituto Galego da Calidade Alimentaria de la Xunta de Galicia, estamos llevando a cabo diversos estudios con el fin de conocer las especies de Cryptosporidium y los genotipos de G. duodenalis presentes en aguas –superficiales, potables, residuales y regeneradas– en esta Comunidad Autónoma. Estos trabajos se realizan con la colaboración del CECA-ICETA, Universidade do Porto (Portugal). En la ponencia se destacarán los resultados de nuestras investigaciones llevadas a cabo recientemente en este tema:

Castro-Hermida, J.A.; García-Presedo, I.; Almeida, A.; González-Warleta, M.; Correia da Costa, J.M. and Mezo, M. Contribution of treated wastewater to the contamination of recreational river areas with Cryptosporidium spp. and Giardia duodenalis. Water Research, 42: 3528-3538. 2008.

Castro-Hermida, J.A.; García-Presedo, I.; Almeida, A.; González-Warleta, M.; Correia da Costa, J.M. and Mezo, M. Presence of Cryptosporidium spp. and Giardia duodenalis through drinking water. Science of the Total Environment, 405: 45-53. 2008.

Castro-Hermida, J.A.; García-Presedo, I.; Almeida, A.; González-Warleta, M.; Correia da Costa, J.M. and Mezo, M. Detection of Cryptosporidium spp. and Giardia duodenalis in surface water: a health risk for humans and animals. Water Research, 43: 4133-4142. 2009.

Development of a novel fusion system for soluble protein overexpression and purification in Escherichia coli Sofia Costa

a,b, Pedro Silva

b,c, André Almeida

d,e, Antónia Conceição

c, Lucília Domingues

a and António

Castrob

a IBB - Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, Universidade do

Minho, Campus de Gualtar, 4710-057 Braga, Portugal b

INSARJ – Instituto Nacional de Saúde Dr. Ricardo Jorge, Porto

c CERNAS-ESAC – Escola Superior Agrária de Coimbra

d ICETA-CECA

eHitag Biotechnology Lda

Recombinant protein production is a useful technology for therapeutic and diagnostic

applications. The bacterium Escherichia coli is widely used for the bio-production of proteins, but still presents some drawbacks. Recombinant proteins with biomedical interest have proved difficult to express properly in this host system, resulting in insoluble protein aggregates. Fusion protein technology has been applied to overcome these drawbacks and to optimize protein expression in E. coli. A novel fusion system (Fh8 and H tags) was recently discovered and demonstrated to improve antigen production in E. coli [1], presenting also attractive features for the development of antibodies. This research aims at the evaluation of a novel fusion system (Fh8 and H tags) effects on both protein and antibody production. Target proteins

used in this work present biomedical interest and results obtained here showed that these novel fusion tags increased protein production yields, besides working as immunopotentiating agents for specific polyclonal antibody production. Taking into account these results, this novel fusion system appears to be a promising tool for the production of recombinant proteins with a diagnostic and therapeutic interest.

Toxoplasmosis, a waterborne disease? Susana Sousa

a,b; Daniel Ajzenberg

f; Nuno Canada

b,c,d; José Manuel Costa

b,e; Marie-Laure Dardé

f

aParasite Disease, IBMC-INEB Laboratório Associado, Porto, Portugal

bCentro de Estudos de Ciência Animal, Universidade do Porto. Campus Agrário de Vairão, Vairão.

Portugal.

cInstituto de Ciências Biomédicas de Abel Salazar, Universidade do Porto, Porto. Portugal.

dLaboratório Nacional de Investigação Veterinária, Vairão, Vila do Conde, Portugal.

eCentro de Imunologia e Biologia Parasitária, INSA, Porto, Portugal

fLaboratoire de Parasitologie-Mycologie, EA 3174-NETEC, Faculté de Médecine, Université de Limoges, Limoges, 87025, França e-mail : susana.sousa@ibmc.up.pt

Toxoplasmosis is caused by the Apicomplexa parasite Toxoplasma gondii. Most infections in humans are asymptomatic. However, severe complications may occur in consequence of a congenital infection or in immunocompromised patients due to an acquired infection. Outbreaks of human toxoplasmosis have been described, where immunocompetent individuals developed severe infections. Isolation of parasite associated with these clinical manifestations from different geographic regions revealed that T. gondii has a high genetic diversity. Most of the methods used for genetic characterization of this parasite require the previous isolation of the parasite or of the DNA. This limits the genetic studies to infections from which it is possible to collect biological samples, usually from symptomatic patients. Besides, isolation by mice bioassay may favour some strains and do not detect mixed infections. Serotyping is a simple typing method that consists of an immunoenzymatic assay (enzyme-linked immunosorbent assay [ELISA]) using synthetic polymorphic peptides derived from T. gondii antigens. The only biological material required is serum samples, which can be collected from both symptomatic and asymptomatic patients and eliminates the biases associated with genotyping based on parasite isolation. Serotyping of infections in humans with known strains showed that infections due to strains belonging to the archetypal lineages I, II and III can be serotyped with strain-specific peptides derived from GRA6 and GRA7 antigens. However, peptides specific of non-archetypal strains have a poor specificity or sensitivity. This method was also used to predict the serotype of patients with acute and chronic toxoplasmosis due to unknown genotypes from Europe, Africa, and Latin America. In Europe the prevalent serotype is type II, while in Africa and Latin America, serotype I/III prevails. Serotype II was found in Uruguay with a considerable frequency in areas with possible influence of genotypes until now described in Europe. No relation was established between serotype distribution and pathology. Serotyping of naturally infected domestic animals presented some limitations with the selected peptides. Besides the actual limitations, serotyping may be a promising method for typing strains. However, other peptides from different markers must be studied in order to discriminate archetypal from non-archetypal strains. In the future, serotyping may be valuable tool in epidemiologic studies, in the detection of a re-infection with a different strain and also in the detection of T. gondii infections by non-archetypal strains in animals for human consumption.

Toxoplasmose

A toxoplasmose é causada pelo parasita Apicomplexa Toxoplasma gondii. No homem, as infecções são habitualmente assintomáticas. No entanto, quadros clínicos graves podem ocorrer em consequência de uma infecção congénita ou em indivíduos immunodeprimidos. A toxoplasmose severa em doentes immunocompetentes, com quadros clínicos inabituais tem sido descrita em regiões da América do Sul. A caracterização genética das estirpes de Toxoplasma gondii associadas a estes casos revelou a existência de estirpes não arquétipos. A diversidade genética deste parasita é na realidade maior do que os arquétipos I, II e III previamente descritos. A caracterização genética do parasita requer o isolamento prévio do parasita ou do seu ADN, o que limita os estudos genéticos às infecções sintomáticas, onde é possível obter

amostras biológicas. O isolamento do parasita por bioensaio em ratinhos pode favorecer certas estirpes em detrimento de outras, no caso de infecções mistas.

A serotipagem é um método simples, que tem por base um ensaio imunoenzimático (enzyme-linked immunosorbent assay – ELISA) usando, como antigénio, péptidos sintéticos polimórficos desenhados a partir de genes de T. gondii. Neste método, os soros são as únicas amostras biológicas necessárias, que podem ser obtidas quer de doentes sintomáticos como assintomáticos. A serotipagem de humanos infectados com estirpes conhecidas demonstrou que as infecções provocadas por estirpes de genotipo I, II e III podem ser serotipadas com os péptidos derivados dos antigénios GRA6 e GRA7. Os péptidos específicos das estirpes não arquétipos evidenciaram uma baixa especificidade e sensibilidade. Este método foi também usado para avaliação predictiva de serotipos em doentes com toxoplasmose aguda e crónica, da Europa, Africa e América Latina, nos quais se desconhecia o genotipo infectante. Na Europa, o serotipo II predomina, enquanto em Africa e na América Latina, o serotipo I/III é mais frequente. O serotipo II foi encontrado com uma frequência considerável no Uruguai. Não se estabeleceu nenhuma relação entre o serotipo e a patologia. Os resultados da serotipagem de animais domésticos naturalmente infectados, apesar de algumas limitações, sugerem que este pode ser um método promissor. Outros péptidos de diferentes marcadores que permitam a diferenciação das estirpes arquétipos dos não arquétipos têm de ser estudados. No futuro, a serotipagem pode tornar-se um instrumento importante em estudos epidemiológicos, na detecção de re-infecções com estirpes diferentes, bem como na detecção de infecções por estirpes não arquétipos em animais destinados ao consumo humano.

Schistosomes and estrogen: an old tale with new characters. Mónica Catarina Botelho

a,b, *, Raquel Soares

c, Nuno Vale

d, Ricardo Ribeiro

e, Vânia Camilo

b, Raquel

Almeida b, Rui Medeiros

e, Paula Gomes

d, José Carlos Machado

b, f and José Manuel Correia da Costa

a

a – CIBP – Centre for Parasite Immunology and Biology, National Institute of Health, Porto, Portugal b – IPATIMUP – Institute of Pathology and Molecular Immunology of Porto University, Porto, Portugal c – FMUP – Department of Biochemistry (U38), Faculty of Medicine of Porto University, Porto, Portugal d – CIQUP – Centro de Investigação em Química da Universidade do Porto, Department of Chemistry of the Faculty of Science of the University of Porto, Portugal e – IPO – Molecular Carcinogenesis lab, Portuguese Institute of Oncology, Porto, Portugal f - FMUP – Department of Pathology, Faculty of Medicine of Porto University, Porto, Portugal Mónica C. Botelho National Institute of Health, CIBP Rua Alexandre Herculano, 321 4000-055 Porto, Portugal Phone: 00-351-223401100 Fax: 00-351-223401109 e-mail: monicabotelho@hotmail.com; monica.botelho@insa.min-saude.pt

We have previously identified the expression of an estradiol (E2)-related molecule by Schistosoma haematobium total antigen (Sh). We now show that this molecule has an antagonistic effect of estradiol in vitro. Our results are consistent with the existence of an estrogenic molecule that antagonizes the activity of estradiol. We found evidence for this molecule as we identified and characterized by mass spectrometry new estrogenic molecules previously unknown, present in schistosome worm extracts and sera of schistosome infected individuals. We also show that Sh is able to interact in vitro with Estrogen Receptor (ER), explaining how host endocrine system can favour the establishment of schistosomes. These findings highlight the exploitation of the host endocrine system by schistosomes and represent an additional regulatory component of schistosome development that defines a novel paradigm enabling host-parasite interactions. The identification of these molecules opens new ways for the development of alternative drugs to treat schistosomiasis.

Salmonella in swine: prevalence, antimicrobial resistance and genetic characterization Eduarda Gomes Neves

1,2

1 Centro de Estudos de Ciência Animal, Universidade do Porto. Campus Agrário de Vairão, Vairão.

Portugal.

2 Instituto de Ciências Biomédicas de Abel Salazar, Universidade do Porto, Porto. Portugal.

Salmonella is an important causing agent of food borne illness in humans in which farm animals

and foods of animal origin are the main transmitting vehicles. The role of pork as a source of human salmonellosis has been shown in a number of investigations. Slaughtering pigs can carry Salmonella in several tissues, particularly the digestive tract and associated lymphatic tissue. There is a strong association between the slaughter of Salmonella-positive pigs and carcass and meat contamination. Additionally, the European Food Safety Authority (EFSA) indicated in its ‘Community Summary Report on Trends and Sources of Zoonoses, Zoonotic agents, Antimicrobial Resistance and Food borne Outbreaks in the European Union in 2005’ that a relatively high proportion of Salmonella isolates from animals and food were resistant to antimicrobials commonly used in the treatment of human diseases. Food borne infections caused by these resistant bacteria pose a particular risk to humans due to possible treatment failure. The aim of our study was to investigate the prevalence of Salmonella-positive pigs in Portuguese abattoirs, and evaluate the occurrence of this food borne pathogen in meat handlers hands and animal samples (ileocolic lymph nodes, carcass swabs and meat) presenting higher potential of being contaminated during slaughter, cutting and dressing procedures. We also wanted to determine antimicrobial resistance profiles in the isolates and characterize the genetic determinants of antimicrobial resistance. The clonal relationships between strains of the same Salmonella serotype were also investigated for epidemiological purposes.