Avian influenza. Main challenges in poultry farming.

PRESENTATION BROCHURE

MAIN CHALLENGES IN P

ULTRY FARMING

Avian influenza Servet (División de Grupo Asís Biomedia S.L.) Centro Empresarial El Trovador, planta 8, oficina I Plaza Antonio Beltrán Martínez, 1 • 50002 Zaragoza (España) Tel.: +34 976 461 480 • Fax: +34 976 423 000 • www.grupoasis.com

Helena Lage Ferreira Taiana Pereira da Costa Cintia Hiromi Okino Shamus Patrick Keeler Muhammad Munir

The publishing strength of Grupo AsĂ­s Editorial Servet, a division of Grupo AsĂ­s, has become one of the reference publishing companies in the veterinary sector worldwide. More than 15 years of experience in the publishing of contents about veterinary medicine guarantees the quality of its work. With a wide national and international distribution, the books in its catalogue are present in many different countries and have been translated into nine languages to date: English, French, Portuguese, German, Italian, Turkish, Japanese, Russian and Chinese.

Its identifying characteristic is a large multidisciplinary team formed by doctors and graduates in Veterinary Medicine and Fine Arts, and specialised designers with a great knowledge of the sector in which they work. Every book is subject to thorough technical and linguistic reviews and analyses, which allow the creation of works with a unique design and excellent contents.

Servet works with the most renowned national and international authors to include the topics most demanded by veterinary surgeons in its catalogue. In addition to its own works, Servet also prepares books for companies and the main multinational companies in the sector are among its clients.

MAIN CHALLENGES IN POULTRY FARMING

Avian influenza

MAIN CHALLENGES IN P

ULTRY FARMING

Avian influenza Helena Lage Ferreira Taiana Pereira da Costa Cintia Hiromi Okino Shamus Patrick Keeler Muhammad Munir

AUTHORS: Helena Lage Ferreira, Taiana Pereira da

Costa, Cintia Hiromi Okino, Shamus Patrick Keeler, Muhammad Munir.

FORMAT: 17 x 11 cm.

NUMBER OF PAGES: 82.

NUMBER OF IMAGES: 47.

BINDING: paperback, wire-o.

RETAIL PRICE

35 €

Avian influenza (AI) is an infectious viral disease mainly affecting birds. It is often causing no apparent signs of illness. AI viruses can sometimes spread to domestic poultry and cause significant outbreaks of serious disease. The aim of this handbook is to provide the most updated information (aetiology, epidemiology, clinical signs, diagnosis, control…) of these viruses with a practical and visual approach. The handbook has been written by prestigious and renowned experts with a wide experience in this field. Numerous graphic resources have been included to complement the information provided and make the contents more understandable and accessible to readers.

Avian influenza

Presentation of the book Avian influenza (AI) is one of the most studied avian pathogens affecting the poultry industry worldwide. It was first described in 1987 by Edoardo Perroncito, during the Italian outbreak of “fowl plague�. AI infection is caused by influenza A viruses, an enveloped RNA virus found in mammals and birds. AI viruses are classified into low pathogenic avian influenza (LPAI) virus or highly pathogenic avian influenza (HPAI) virus, depending on the clinical outcome and diagnostic tests. Due to commercial trade concerns and possible restrictions, the notification of some strains of these viruses to the World Organisation for Animal Health (OIE) is mandatory.

The occurrence of several HPAI outbreaks in poultry and human infections over the past decades has changed our perception of AI and raised awareness among the medical and veterinary communities. This highlighted the importance of a structured diagnosis network, as well as biosecurity, control practices and an early response.

The aim of this book is to provide a general knowledge of AI in poultry in a practical and illustrative manner. The virus classification, immune response, pathogenesis, clinical signs, pathology, specimen sampling, and diagnostic methods, as well as prevention and control of the disease, are discussed. The knowledge gained from this book will certainly contribute to a thorough understanding of AI infection, which is essential to develop more efficient networks and enhance the control of outbreaks.

Helena Lage Ferreira

The authors Helena Lage Ferreira Dr. Ferreira has DVM degree at São Paulo State University (UNESP, Brazil), PhD in Genetics and Molecular Biology at University of Campinas (UNICAMP, Brazil), and Post-doctoral studies on Avian Influenza at the Veterinary and Agrochemical Research Centre (VAR, Belgium). She was research assistant at VAR and currently works as Assistant Professor at University of São Paulo, Brazil.

Her research interests are focused on epidemiology, diagnosis tests, protection and immunity afforded by vaccines of the most important diseases affecting the poultry industry, including avian influenza, Newcastle disease and avian metapneumovirus. She has been awarded with the Bunge Foundation Awards - Animal and Plant Health Protection - Youth category (2011), Best Poster Award - young Investigator category at the 7th International Symposium on Avian Influenza (2009). Dr. Ferreira is member of Committee for Poultry Health National Program at the Ministry of Agriculture, Brazil.

Taiana Pereira da Costa Dr. Costa is a veterinary surgeon (UNESP, Brazil) with a PhD in Veterinary Pathology (University of Georgia, USA) and Post-doctoral studies on Avian Virology and Pathology (Autonomous University of Barcelona, Spain).

Her research interests are focused on host factors that affect viral pathogenesis, particularly avian influenza viruses in poultry and wild birds. She has received awards for her work with avian virology, including the Best Poster Award at the Southern Conference on Avian Diseases/IPSF (2010), the Terry Amundson Student Presentation Award at the International Conference of the Wildlife Disease Association (2010), and the Second Place Poster Award - Graduate Student category at the 7th International Symposium on Avian Influenza (2009).

Cintia Hiromi Okino Graduated in Veterinary Medicine and concluded MS and PhD in Veterinary Pathology at São Paulo State University (UNESP- Brazil).

Dr. Okino works at the Avian Virology Laboratory, Embrapa Swine and Poultry, a Brazilian Agricultural Research Corporation, where she develops research on avian respiratory viruses (Avian Infectious Bronchitis virus, Avian Metapneumovirus, Newcastle disease virus and Avian Influenza virus). The research includes evaluation of Immune Responses and Molecular Diagnostics for these diseases. She has published articles about Avian Infectious Bronchitis virus in national and international journals and she won the “José Maria de Lammas Filho award” in 2013 during the Brazilian Poultry Sciences meeting (FACTA).

Avian influenza

Shamus Patrick Keeler He completed an MS and MEd in Biology from East Stroudsburg University (East Stroudsburg, PA, USA), and a PhD in infectious diseases at the University of Georgia (Athens, GA, USA). Dr. Keeler is a postdoctoral research scholar at Washington University School of Medicine in St. Louis, where he researches the innate immune response against influenza A virus. He has been involved in research on a wide range of wildlife pathogens including Avian Influenza virus, Avian Coccidiosis, and several bacterial pathogens of white-tailed deer.

He won the Wildlife Disease Association Graduate Student Scholarship Award in 2012 and has received postdoctoral fellowships from both the Morris Animal Foundation and the National Institutes of Health (NIH).

Muhammad Munir

Research of Dr. Munir’s Group aims to understand the innate immune responses in different avian hosts that range from sensing of viruses to terminal IFN effectors mediated by myriad interferon stimulated genes. They have adapted large-scale, genome-wide and high throughput lentivirus-based screening platforms to investigate, both generically and specifically, the interaction of viruses with host immune responses, deciphering differential host responses to diverse avian pathogen, and the bases for genetic resistance/susceptibility of different avian species. They are also investigating the strategies that viruses use to escape from host defenses. Using reverse-genetics systems for Newcastle disease virus and avian metapneumoviruses, they are applying genome-wide transcriptomics and proteomics approaches to understand the molecular determinants of viral pathogenesis and the ways that viruses have adapted to circumvent these responses. With his extensive experience in Veterinary Virology, Dr. Munir has published more than 55 articles, 4 books and 12 chapters in different books.

hkeita/shutterstock.com

After graduation in Veterinary Medicine, Dr. Munir has completed his PhD studies on the pathobiology of poultry viruses at Swedish University of Agricultural Sciences, Uppsala, Sweden. Dr. Munir is now Institute Fellow on Avian Innate Immunity and Host Genetic Diversity at The Pirbright Institute, UK.

Communication services Website Online visualisation of the sample chapter.

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Author´s CV. Sample chapter compatible with iPad.

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MAIN CHALLENGES IN P

ULTRY FARMING

Avian influenza Helena Lage Ferreira Taiana Pereira da Costa Cintia Hiromi Okino Shamus Patrick Keeler Muhammad Munir

Table of contents 1. Introduction

4. Pathogenesis

History and reports of influenza viruses

Shedding

Aetiology

Transmission

Hosts

Molecular pathogenesis

Notification to OIE

Receptor binding specificity

2. Classification of AIV Viral taxonomy AI subtypes AI nomenclature Species specific influenza A viruses AIV pathotypes

Host susceptibility

5. Clinical signs and pathology 6. Sampling and diagnosis Specimen sampling Diagnostic methods Virus identification

Antigenic variation of strains-drift and shift

Molecular methods

Additional AIV classifications

Immunohistochemistry (IHC) and immunofluorescence (IFIC)

3. Avian immune responses Innate immune responses Adaptive immune response Cell-mediated immunity (CMI) Antibody-mediated immunity

Cytokine storm Evasion of immune response by influenza viruses

Assessment of pathogenicity

Serological methods3

7. Prevention and control Prevention and control of AI outbreaks Education Biosecurity Diagnosis Surveillance Vaccination Transfer of passive immunity in birds

Eradication of AI

8. References

1

Introduction

Avian influenza

History and reports of influenza viruses » In 1878, in Italy, Edoardo Perroncito described avian influenza (AI) for the first time. The disease was then known as “fowl plague”.

» The first report of highly pathogenic avian influenza (HPAI) by a virus of H5 subtype was in 1959. Since then, the World Organisation for Animal Health (OIE) has registered about 35 HPAI epizootics, 11 of them reported after 2009 (5 caused by influenza A viruses of H7 subtype, and 6 by viruses of H5 subtype).

6

» Low pathogenic avian influenza viruses (LPAIV) are equally important owing to their potential to mutate and gain higher pathogenicity (i.e. HPAIV). 1

INTRODUCTION

» Most of the HPAI endemic infections or outbreaks have shown very limited spread, whereas others have caused substantial losses to the poultry industry worldwide. 2, 3, 4 In 2003, the HPAIV subtype H5N1 had spread from Asia to Europe and Africa, resulting infections in millions of poultry birds. This outbreak has lead to a change in our understanding of the epidemiology and evolution of avian influenza virus (AIV).

» AIV can occasionally infect humans. Since 2003, HPAI H5N1 virus has caused over 840 human cases and 447 deaths in more than a dozen countries in Asia, Africa, the Pacific and Europe. 5 Recently, a H7N9 LPAI virus in humans has also been reported in China. 6

» Synonyms: fowl plague, fowl pest, highly pathogenic avian influenza, bird flu, high pathogenicity avian influenza.

7

1

Introduction

Avian influenza

Aetiology » Influenza viruses have a complex structure and possess a lipid membrane derived from the host cell. The viral genome contains eight segments of single-stranded, negative-sense RNA that code for 12-14 proteins. 9

10

» Some of the RNA segments (e.g. haemagglutinin -HA-, neuraminidase -NA-) are encoded for a single protein, whereas other segments (e.g. M, NS) encode for more than one protein.

INTRODUCTION

PB2 PB1 PA NP ssRNA HA NA M1 M2 NEP

HAEMAGGLUTININ: » Responsible for viral attachment. » The primary target of neutralizing antibodies. » Its cleavage site determines the pathogenicity.

NEURAMINIDASE: » Release from cell. » Target of neutralizing antibodies. » Restricts virus spread.

Figure 1. AIV structure. The viral genome is composed of eight single stranded, negative-sense RNA segments that code for 12-14 proteins. 9, 10

11

1

Introduction

Avian influenza

Hosts » Influenza A viruses are found in mammals and birds.

» Wild waterfowl represent the

Canine H3

Marine mammals H1, H3, H4, H7, H13

natural reservoir of multiple antigenically diverse strains of influenza A virus. 4

» AI viruses do occasionally infect

Equine H3 (H7)

?

Feline H5N1

H2, H4, H5, H7, H9

Swine H1, H3

?

Wild birds H1-H16 (Gulls: H13 and H16)

H17-H18

people and other mammals.

Figure 2. Ecology of influenza A viruses.

AI viruses can be transmitted to other hosts such as poultry and mammals. Yellow arrows indicates sporadic AI infection. 4, 11, 12

?

12

H5, H7, H9

Poultry H1-H13

Human H1, (H2), H3

INTRODUCTION

Notification to OIE The pathogenicity of the virus determines whether or not an AI infection is notifiable.

Notification to OIE of influenza A viruses 13

Notifiable influenza A virus » HPAI in poultry and other birds, including wild birds. » H5 and H7 subtypes with low pathogenicity (LPAI) in poultry.

Non-notifiable influenza A virus » Low pathogenicity non-H5 and non-H7 influenza A viruses in birds (i.e. H1-H4, H6 and H8-H16).

Figure 3. The notification of AI infection in birds to the OIE by the competent authorities (Veterinary Services) in a country is based on the subtype and pathogenicity of the virus. 13

2

Classification of AIV

Avian influenza

Viral taxonomy » The classification and nomenclature of viruses is governed by the International Committee on Taxonomy of Viruses (ICTV). 14

Table 2. AIVs are a category within the species influenza A virus.

Family

» AIVs are influenza A viruses within the family Orthomyxoviridae. This family includes other influenza viruses and several tick-borne viruses, which have not yet been assigned to any taxonomic order.

Orthomyxoviridae

Genus

Species

Influenzavirus A

Influenza A virus

Influenzavirus B

Influenza B virus

Influenzavirus C

Influenza C virus

Isavirus

Infectious salmon anemia virus

Quaranjavirus

Thogotovirus

14

Quaranfil virus Johnston Atoll virus Thogoto virus Dhori virus

CLASSIFICATION OF AIV

AI subtypes » Influenza A viruses are further categorized into subtypes based on the viral surface glycoproteins, HA and NA, determined by serological or molecular methods. 15

» The HA and NA proteins are numbered in order of discovery, and subtypes are designated by alpha-numeric combinations identifying the two surface proteins of the virus (e.g. H1N1 or H3N2).

» There are currently 18 HA and 11 NA variants. Only H1-16 and N1-9 have been reported in avian species.

15

2

Classification of AIV

Avian influenza

NA

HA H2 H5 H1 H6 H17 (Bat) H18 (Bat) H8 H12 H9 H11 H13 H16 H4 H14 H3 H10 H7 H15 Influenza B virus HA

N6 N9 N7 N2 N3 N1 N4 N5 N8 N10 (Bat) N11 (Bat)

16

Figure 4. Phylogenetic trees of the influenza A virus HA and NA proteins demonstrating the relative relationship of various subtypes.

The trees were generated (Mega 6) using representative viral protein sequences from avian hosts unless otherwise designated.

CLASSIFICATION OF AIV

AI nomenclature Viral strains of influenza virus are formally designated using a nomenclature established by the World Health Organisation (1980). 16 The strain nomenclature includes the influenza species, species of origin, geographic origin, strain number, year of isolation, and subtype to create a unique moniker for each viral isolate.

HA

NA

A/Mallard/Minnesota/199036/99 (H3N2) Influenza species

Host species

Geographic origin

Figure 5. Example and explanation of the influenza virus strain designation.

Strain ID number

Isolation year

Subtype

17

2

Classification of AIV

Avian influenza

Species specific influenza A viruses » Influenza A viruses can be broadly categorized based on their host species. AIVs are viruses isolated from an avian host and/or adapted for replication in avian cells but other host specific categories of influenza A virus exist.

» The host-specific influenza virus categories are widely used. It is important to remember that these viruses are not restricted to the eponymous host type as evidenced by the frequent introduction of AIVs into swine populations and the emergence of viruses capable of infecting humans from both birds and pigs. 17

18

AIV pathotypes » AIVs can be categorized into two pathotypes either low pathogenic (LP) or highly pathogenic (HP) based on specific molecular and pathogenesis criteria. 4, 13

» The vast majority of AIVs are LP including the majority of the circulating H5 and H7 viruses but LP AIVs can evolve into HPAI viruses after cross species transmission between wild birds and poultry. 18

CLASSIFICATION OF AIV

Antigenic variation of strains-drift and shift The evolution of AIVs occurs due to two events: antigenic drift (minor changes) and antigenic shift (genetic reassortment). 19 Antigenic drift

Antigenic shift 19

Virus A

Cell

Virus B

Figure 6. Antigenic drift and antigenic shift events for evolution of AIVs.

Reassortant virus: mixed segments of virus A and B

2

Classification of AIV

Avian influenza

Additional AIV classifications » To facilitate international collaborative research and communication, it is often necessary to further classify AIVs into genetic lineages using phylogenetic methods.

Due to the segmented nature of the AIV genome, genetic lineages are specific to particular gene segments.

20

» These phylogenetic classifications use specific methodologies and defined criteria to place viral strains into phylogenetic trees and identify unique clades within those trees. 20

» An example of a widely used AI phylogenetic classification system is the HPAI H5N1 nomenclature system. 4, 20

CLASSIFICATION OF AIV

First order clade

2 1 9

Second order clade

8 2.3 6

Figure 7. A diagram representing the phylogenetic relationship of the Asian HPAI H5N1. 20 Each blue triangle represents a clade of viruses, which are numbered 0-9. Within each clade, the viruses can be further subdivided into additional clades as demonstrated by the inset. Based on the most recent research, the HPAI H5N1 genetic lineage is delineated down to fourth order clades. 20

5 7 4 3 0

2.2 2.5 2.1 2.4

21

3

Avian immune responses

Avian influenza

Influenza viruses induce potent effector mechanisms of both innate and adaptive immune responses, and also encode for proteins that circumvent these responses. An elegant balance between induction and evasion is essential to establish successful influenza infections in the susceptible hosts.

Innate immune responses » AIV accesses the susceptible avian hosts via oral or respiratory routes and has to breach the mucus layer of the respiratory tract to infect the underlying non-immune cells (e.g. epithelial cells) and immune cells (macrophages and dendritic cells).

22

» Innate immune signaling in these cells is initiated by at least three distinct types of pattern recognition receptors (PRRs) including retinoic acid inducible gene I (RIG-I)-like receptors (RIG-I in duck and MDA5/LGP2 in chicken), Toll-like receptors (TLR3, 7, 8), and NOD-like receptors. 21

» Type I interferons (IFNs) -mainly secreted by macrophages and dendritic cells- induce the transcription of interferon-stimulated genes (ISGs), which determine the antiviral status of the host.

» Influenza viruses have to breach these defenses for successful infections. An adaptive immune response would then be required for virus clearance.

AVIAN IMMUNE RESPONSES Influenza virus

Influenza virus Phagocytosis Macrophage

dsRNA

Endocytosis

Phagosome TLR3

Endosome

P IRF7 P IRF7

P IκB p50 p65

P IκB p50 p65

p50p65

p50 p65 NF-κB

NF-κB Type I IFN & ISGs Pro-inflammatory cytokine

Cytoplasm

Dendritic cells

TLR7 MyD88

TRIF IRF7

ssRNA

Nucleus

Figure 8. Induction of innate immune responses by influenza virus infections.

23 IRF7 P IRF7 P IRF7

3

Avian immune responses

Avian influenza

Adaptive immune response The adaptive immune response is composed of two arms: cell-mediated (virus- specific CD4+ and CD8+ T cells) and antibody-mediated (humoral or virus- specific antibodies) immunity.

Cell-mediated immunity (CMI)

24

» The influenza virus-induced CMI is mainly facilitated by CD4+ and CD8+ T cells. » Upon influenza infection, naïve CD4+ T cells are activated by the major histocompatibility complex (MHC) class II molecules and differentiate into CD4+ T helper 1 (Th1) or Th2 cells.

» Naïve CD8+ T cells are stimulated by the MHC class I molecules and terminate at the production of two apoptosis causing molecules, perforin and granzymes.

AVIAN IMMUNE RESPONSES

Memory Influenza virus reaction

Naïve CD8+ T cell

CTL

IL-2 IFN-γ

MHC Class I MHC Class II

Th1

Antigen-presenting cell

Memory Naïve CD4+ T cell

Phagocytosis

Figure 9. Celland antibodymediated immune responses against influenza viruses.

Th2

Antibodies

B cell Memory

IL-4 IL-5 IL-13

25

3

Avian immune responses

Avian influenza

Antibody-mediated immunity » Influenza induced humoral immune responses are primarily mediated against HA and NA proteins. 22 » Antibodies against HA are the most neutralizing but antibodies against NA can be effective at the terminal stages of the virus life cycle.

» Primary influenza infections cause elevated levels of IgM, IgA and IgY isotypes of antibodies in the serum.

Cytokine storm

26

» Influenza viruses replicate extensively in epithelial cells, which attract macrophages and other immune cells.

» This can result in excessive and uncontrolled pro-inflammatory cytokines, and chemoattractants from immune cells.

» These cytokines migrate to the lungs and cause an acute respiratory distress syndrome referred to as “cytokine storm”. 22

AVIAN IMMUNE RESPONSES

a

b

c

d

e

f

Figure 10. A high level of chicken ISGs (e.g. ZAP) expression (a and b) in influenza infected DF-1 cells, followed by high replication of influenza virus (A/turkey/England/50-92/91) within 12 hours of infections in (c) CEF and (d) DF-1 cells. Staining of the influenza particles with the nucleoprotein antibodies 6 hours post infection in (e) CEF and (f) DF-1.

27

Avian immune responses

Avian influenza

Evasion of immune response by influenza viruses » Influenza viruses have gained intrinsic mechanisms to counter the host immune responses. 21, 22, 23 » Among the immunomodulatory proteins of influenza viruses, NS1 protein is the most characterized and potent.

Figure 11. Briefly, NS1 protein of influenza virus sequesters dsRNA (1), interacts with RIG-I directly (2) or through TRIM25 (3), blocks activation and nuclear translocation of NF-kB, IRF3/7 and AP-1 (4), interferes with pre-mRNA capping by interacting with CPSF30 (5), or export of nuclear mRNA (6), interacts with SOCS1/SOCS3 (7), interferes with PKR (8) or 2´-5´OAS (9) or Akt signaling (10) and interacts with sStaufen and associated proteins to block translation of cellular mRNA (11).

28

IFN-β IFNR1

NS1 3NS1 3

1

Viral dsRNA

1

Viral dsRNA

PKR PACT

RIG-1

TRIM 25

2

RIG-1

TRIM 25

4 NF-kB IRF3/7 AP-1

NS1 2´5´OAS

9 NS1

NS1

Translation Translation

5´cap

NS1 elF2α

NS1 6 AAAAA

7

TYK2 JAK1

7

6

P

AAAAA mRNA nuclear export P mRNA nuclear export

RNaseL

SOCS1 SOCS3 SOCS1 SOCS3

STAT1 STAT2 IRF9

STAT1 STAT2 IRF9

5´cap

NF-kB IRF3/7 AP-1 CrkL p110

p65ß

11

5´cap

tau

PABP

hS

5´cap

elF4GI PABP elF4GI

P Akt P

fen

10 11

P Akt P

AAAAA

fen

NS1

p65ß CrkL p110

tau

NS1

10

elF2α

AAAAA

IFNR2

TYK2 JAK1 AVIAN IMMUNE RESPONSES

8 PKR PACT9 2´5´OAS 5´cap

2NS18

4

hS

3

AAAAA mature-mRNA RNaseL Posttranscriptional modifications 5´cap AAAAA mature-mRNA 5 Posttranscriptional PABPII modifications NS1 CPSF30 5 PABPII Pre-mRNA NS1 5´cap CPSF30 mRNA 5´cap Pre-mRNA Pol II G7 promoter

Cellular transcription

Pol II G7 promoter

Cellular transcription

mRNA

P P

29

The publishing strength of Grupo AsĂ­s Editorial Servet, a division of Grupo AsĂ­s, has become one of the reference publishing companies in the veterinary sector worldwide. More than 15 years of experience in the publishing of contents about veterinary medicine guarantees the quality of its work. With a wide national and international distribution, the books in its catalogue are present in many different countries and have been translated into nine languages to date: English, French, Portuguese, German, Italian, Turkish, Japanese, Russian and Chinese.

Its identifying characteristic is a large multidisciplinary team formed by doctors and graduates in Veterinary Medicine and Fine Arts, and specialised designers with a great knowledge of the sector in which they work. Every book is subject to thorough technical and linguistic reviews and analyses, which allow the creation of works with a unique design and excellent contents.

Servet works with the most renowned national and international authors to include the topics most demanded by veterinary surgeons in its catalogue. In addition to its own works, Servet also prepares books for companies and the main multinational companies in the sector are among its clients.

PRESENTATION BROCHURE

MAIN CHALLENGES IN P

ULTRY FARMING

Avian influenza Servet (División de Grupo Asís Biomedia S.L.) Centro Empresarial El Trovador, planta 8, oficina I Plaza Antonio Beltrán Martínez, 1 • 50002 Zaragoza (España) Tel.: +34 976 461 480 • Fax: +34 976 423 000 • www.grupoasis.com

Helena Lage Ferreira Taiana Pereira da Costa Cintia Hiromi Okino Shamus Patrick Keeler Muhammad Munir