LIVRO-Transmissible Diseases Handbook PDF

Transmissible Diseases Handbook

TABLE OF CONTENTS
I. INTRODUCTION J. Kaandorp
II. GENERAL CONSIDERATIONS J. Kaandorp
III. EUROPEAN UNION J. Kaandorp
1. EUROPEAN UNION
2. DATA ON EU MEMBER STATES
3. EU ANIMAL HEALTH STRATEGY AND ANIMAL

HEALTH ADVISORY COMMITTEE
4. EU ANIMAL HEALTH LAW
5. EAZA POSITION STATEMENT ON THE ANIMAL

HEALTH STRATEGY FOR THE EUROPEAN UNION
6. OIE
IV. LINKS J.Kaandorp
1. EU VETERINARY FACULTIES
2. USEFUL (VETERINARY) WEBLINKS
V. ANIMAL HEALTH LEGISLATION IN EUROPE P. Dollinger
VI. RECOMMENDATIONS FOR THE APPLICATION OF EAZWV – IDWG – DEFRA –

ANNEX C TO THE BALAI - DIRECTIVE BMVEL – RVV – DG SANCO
VII. POST-MORTEM PROCEDURES G. Dorrestein et al.
VIII. GUIDELINES FOR CLEANING AND DISINFECTION IN M. Kiupel et al.

ZOOLOGICAL GARDENS
IX. BLUETONGUE IN NON-DOMESTIC RUMINANTS: S. Sanderson

EXPERIENCES GAINED IN EAZA ZOOS DURING THE
2007 & 2008 BTV8 AND BTV1 EPIZOOTICS
X. TUBERCULOSIS IN ZOO SPECIES: DIAGNOSTIC EAZWV Tuberculosis Working

UPDATE AND MANAGEMENT ISSUES Group
1. ELEPHANTS (ELEPHAS MAXIMUS) W. Shaftenaar
XI. AVIAN INFLUENZA
1. VACCINATION OF NON-DOMESTIC AVIAN J. Philippa

SPECIES AGAINST HIGHLY PATHOGENIC AVIAN
INFLUENZA (HPAI) VIRUSES
2. WAZA/EAZA/EAZWV RECOMMENDATIONS ON WAZA – EAZA – EAZWV

AVIAN INFLUENZA
XII. VACCINATION OF NON-DOMESTIC CARNIVORES: A J. Philippa

REVIEW
XIII. LIST OF LABORATORIES
XIV. TEMPLATE FACT SHEET
XV. INSTRUCTIONS FOR AUTHORS
XVI. FACT SHEETS

I. INTRODUCTION
Jacques Kaandorp
Past - President EAZWV, IDWG chairman, DFZVO chairman, EAZA veterinary committee chairman, member
EAZA legislation committee
At the 2000 EAZWV congress in Paris there was a proposal to establish an Infectious
Diseases Working Group (IDWG) within the EAZWV. This idea emerged when the problems
of diagnosing and handling paratuberculosis were discussed in a presentation. It was obvious
that commonly agreed recommendations were urgently needed for improved control of the
infectious diseases that threaten our collections. Also, as Europe becomes more and more
united it is necessary to deal with European politics and legislation, and intensify international
collaboration.
The Foot and Mouth Disease (FMD) outbreak in 2001 clearly underlined the importance of
this initiative and made agreement on European standards a matter of even more urgency.
During this outbreak we saw how difficult it was to control a disease in domesticated animals -
because of public opinion, politics, money and difficulties in understanding the disease and
agreeing on legislation. With this in mind it is obvious that in lesser-studied animals, like our
exotics, even more misunderstandings may arise when dealing with such diseases. In 2006
Highly Pathogenic Avian Influenza spread across Europe and threatened zoo collections.
Outbreaks of Classical Swine Fever now and then occur. Blue Tongue has made serious
problems in the ability of transporting our hoof stock and thus jeopardizing our endangered
species programs. African Swine Fever, and maybe in the near future diseases like West Nile
Virus, African Horse Sickness, and Monkey pox may also threaten our collections. In recent
history even the SARS epidemic (related to an animal Corona virus) indeed confirmed the
above and made the concept and ideas of The Transmissible Diseases Handbook even more
important.
European laws (e.g. the Balai Directive) are still under discussion and in development.
Different attitudes in Europe (e.g. vaccination vs. non-vaccination against FMD and Avian
Influenza), different levels of veterinary faculties and laboratories, and thus differences in the
possibility of diagnosing diseases, pose further problems for European zoo veterinarians. As a
consequence, we have to deal with different approaches to infectious diseases. Standardising
our work is therefore in the interests of us all. To achieve this goal we need to work together
to define procedures for transmissible diseases and propose recommendations for people
dealing with exotic animals. Only in this way will it be possible to make comparisons, and to
statistically analyse our similarities and differences.
The IDWG brings together experienced zoo veterinarians and specialists in infectious
diseases from several European countries and even from countries on the other side of the
Atlantic. The idea of the Group is to combine our knowledge in order to help our animals by
dealing more efficiently with future disease outbreaks which may threaten our collections.
Putting efforts into the creation of a reference manual published under the umbrella of our
Association is an important step in the process of European standardisation, and should
provide a useful tool for zoo practitioners, zoo managers and European legislative authorities
dealing with wildlife and zoo animals.
II. GENERAL CONSIDERATIONS
Jacques Kaandorp
Past-President EAZWV, IDWG chairman, DFZVO chairman, EAZA veterinary committee chairman, member EAZA
legislation committee
This handbook on transmissible diseases, created by the IDWG, is not intended to provide
extensive descriptions of all transmissible diseases which may occur in a zoo collection, but
we hope that it will be a useful tool for every European zoo and/or wildlife veterinarian
encountering a problem related to such diseases. The handbook should help to find answers
to the question “what should I do” when an infectious disease is suspected. Furthermore, in
regard to inter-zoo exchanges, it is very important to standardise surveillance programs for
infectious diseases in European zoos. This handbook should help to overcome the
differences present within Europe and to attain the standardisation required by the Balai
Directive (92/65/EC). EAZWV recommendations for application of the directive have
therefore been included. These recommendations are accepted by SANCO in Brussels as a
guide for the authorities to audit the approvals of zoos under the Balai Directive.
(SANCO/10479/2005)
As mentioned above, the handbook is not supposed to replace good scientific books. On the
contrary, such books will still be necessary for finding detailed information. The handbook is
basically a review. It summarises information on various diseases: susceptible animals,
zoonotic potential, clinical symptoms, pathology, diagnostic methods, qualified laboratories,
treatment, prevention, experts who may be consulted, legislation (esp. European laws) and
relevant literature. The detailed table of contents and the standardised format of the fact
sheets will help the reader to find information quickly. Almost all the fact sheets were
reviewed by two experts. For a few fact sheets only one reviewer could be found. The
search engine on this CD-ROM will help the reader to find his way through the book.
The handbook should be a “living” book. Our European world is changing fast and the
information will soon be outdated if we don’t revise it regularly. A continuous updating
process is a prerequisite for a reliable and useful tool! Ideally, the handbook should include
all diseases mentioned in the Balai Directive (92/65/EC) as well as other diseases relevant
for exchanges of animals between zoos (OIE list A and B and certain zoonoses). Again it
looks like we have succeeded in this!
In addition to the fact sheets, there are general chapters about European legislation, the
integral texts of the relevant European regulations, the EAZWV recommendations on the
Balai Directive, post-mortem procedures, guidelines for cleaning and disinfection, vaccination
of non-domestic carnivores, Tuberculosis, Blue Tongue and lists of reference laboratories.
The authors of the various fact sheets have done their best to meet the requirements. Almost
all fact sheets from the second edition have been updated! Our living book needs to be
updated regularly. We therefore urge all readers to send us their comments for incorporation
in future editions.
II. General Considerations
The chapters on legislation, the list of reference laboratories as well as OIE list A and B have
been included to compensate for the empty sections in many fact sheets. Many European
legislation relevant for exotic animals are added in order to have a more complete dossier. A
list of IDWG members is not included in the handbook, because readers can address
questions directly to them if necessary, since addresses from the authors (read IDWG
members), can be found in the fact sheets themselves.
The legislation texts can be easily downloaded and printed as PDF-files in the various
European languages at http://eur-lex.europa.eu/
This time the IDWG has again chosen to bring this edition digitally on a CD-ROM or
downloadable at www.eazwv.org and www.eaza.net and not in print. This has reduced the
costs of producing the book and will also reduce the costs of spreading it enormously.
Another advantage was the easy expansion of the book, since the second edition already
comprised of 662 pages. This book contains more legislation and new chapters, thus a lot
more pages, and a printed version would become too massive to distribute and would also
be a lot more expensive. We also hope that the book in this format will find its way globally
more easily.
This fourth edition, like the previous editions, will be spread to all EAZWV members, all
EAZA zoos, Central Veterinary Officers of the 27 EU Member States, the O.I.E., and SANCO
in Brussels. Another advantage of this version is the search engine which is included on this
CD-ROM.
At this point I would like to thank the authors of all the fact sheets for their tremendous work.
Norin Chai did the editing of the fact sheets of this fourth edition. For the final editing Ayla
Bayens was of great help. I would also like to thank EAZA and EAZWV for their financial
support. All people involved understood the need to standardise some aspects of our work in
the context of a rapidly-changing Europe. Without their involvement it wouldn’t have been
possible to create this handbook. Let’s hope that all EAZWV members as well as the EAZA
zoo community will appreciate this work, make use of it, and help us to improve it!
For comments you are kindly requested to contact the chairman of the IDWG:
Jacques Kaandorp, e-mail: j.kaandorp@beeksebergen.nl
III. EUROPEAN UNION

Jacques Kaandorp
1. EUROPEAN UNION
2. DATA ON EU MEMBER STATES
3. EU ANIMAL HEALTH STRATEGY AND ANIMAL HEALTH
ADVISORY COMMITTEE
4. EU ANIMAL HEALTH LAW
5. EAZA POSITION STATEMENT ON THE ANIMAL HEALTH
STRATEGY FOR THE EUROPEAN UNION
6. OIE
1. European Union
The European Union is a unique economic and political partnership between 27 democratic
European countries aiming at peace, prosperity and freedom for its 498 million citizens. The
EU is run through bodies such as
 the European Parliament representing the people of Europe
 the Council of the European Union representing national governments
 the European Commission representing the common EU interest
 the Court of Justice making sure that EU law is interpreted and applied in the same
way in all EU countries
 the Court of Auditors checking that the EU’s funds are spent legally, economically
and for the intended purpose
III. European Union
2. Data on the 27 EU Member States
EU Member State Year of EU entry Political system Capital city Population Veterinarians Currency
Austria 1995 Federal republic Vienna 8.3 million 2704 Euro
Belgium Founding member Constitutional Monarchy Brussels 10.7 million 13709 Euro
Bulgaria 2007 Republic Sofia 7.6 million 3422 Lev
Cyprus 2004 Republic Nicosia 0.8 million 271 Euro
Czech Republic 2004 Republic Prague 10.4 million 6655 Czech koruna
Denmark 1973 Constitutional Monarchy Copenhagen 5.5 million 2330 Danish krone
Estonia 2004 Republic Talinn 1.3 million 1081 Estonian kroon
Finland 1995 Republic Helsinki 5.3 million 2124 Euro
France Founding member Republic Paris 63.8 million 27338 Euro
Germany Founding member Federal republic Berlin 82.2 million 35098 Euro
Greece 1981 Republic Athens 11.2 million 3547 Euro
Hungary 2004 Republic Budapest 10.1 million 3500 Forint
Ireland 1973 Republic Dublin 4.4 million 3887 Euro
Italy Founding member Republic Rome 59.6 million 22604 Euro
Latvia 2004 Republic Riga 2.3 million 2256 Lats
Lithuania 2004 Republic Vilnius 3.4 million 1923 Litas
Luxembourg Founding member Constitutional Monarchy Luxembourg 0.5 million 129 Euro
Malta 2004 Republic Valetta 0.4 million No data Euro
Netherlands Founding member Constitutional Monarchy Amsterdam 16.4 million 5978 Euro
Poland 2004 Republic Warsaw 38.1 million 9321 Zloty
Portugal 1986 Republic Lisbon 10.6 million 3665 Euro
Romania 2007 Republic Bucharest 21.5 million 9562 Romanian leu
Slovenia 2004 Republic Ljubljana 2.0 million 940 Euro
Slovakia 2004 Republic Bratislava 5.4 million No data Euro
Spain 1986 Constitutional Monarchy Madrid 45.3 million 9600 Euro
Sweden 1995 Constitutional Monarchy Stockholm 9.2 million 2553 Krona
United Kingdom 1973 Constitutional Monarchy London 61.2 million 29971 Pound sterling
III. European Union
3. The EU Animal Health Strategy (2007-2013) where “prevention is

better than cure” and the Animal Health Advisory Committee
The EU’s Animal Health Strategy provides the framework for EU animal health and welfare
measures until 2013. Give the devastating impact that serious disease outbreaks can have
on farmers, society and the economy, the strategy is based on the principle that “prevention
is better than cure”. The aim is to put greater focus on precautionary measures, disease
surveillance, controls and research, in order to reduce the incidence of animal disease and
minimise the impact of outbreaks when they do occur. However, the new strategy
encompasses much more than just the control of animal diseases. It also focuses on issues
which are inextricable linked to animal health, such as public health, food safety, animal
welfare, sustainable development and research.
The “Animal Health Advisory Committee” includes representatives from non governmental
organisations (including EAZA) across the animal health sector, consumers and
governments. It is aimed at gathering specialised inputs. It provides strategic guidance on
the appropriate/acceptable level of animal or public health protection, and on priorities for
action and communication. It also follows the Animal Health Strategy’s progress. The
Committee is consulted on all impact assessments and advises the European Commission
on the best means of delivering agreed outcomes.
www.one-health.eu
III. European Union
4. The EU’s Animal Health Law
Since its creation, the European Community has had as goal the creation of a single market
to allow the free circulation of different commodities, including live animals and products of
animal origin. To ensure the safe trade of live animals and animal products, harmonised
health requirements for all EU Member States had to be established.
The process of establishing the EU health requirements requires continual updates because
these requirements need to consider a number of evolving factors, such as new scientific
knowledge, and emerging animal diseases.
This process of continual updating of the legislation means that a number of different
regulatory measures are now in place. Currently, EU veterinary legislation includes more
than 400 different legislative acts. This large body of legislation has created difficulties for
different actors, such as the Member States’ veterinary authorities, commercial operators,
farmers, and breeders to understand the law. This is also true for the zoo community.
To tackle this problem, the European Commission decided to review the current EU animal
health legislation when it published its Animal Health Strategy in 2007. As part of its Strategy,
the European Commission decided that it should create a new Animal Health Law, which
would provide a single and clearer regulatory framework for all EU animal health regulation.
The aims of this legal framework would be to simplify, clarify and provide greater flexibility for
EU animal health legislation, and also to introduce important new elements, such as for
example a preventive approach to disease control. An extensive consultation process for the
EU Animal Health Law is launched end September 2009 and will end in December 2009.
The aim of this consultation is to gather the views of all concerned parties and to take these
comments into consideration when drafting the final legislative text. The target date for the
formal adoption of the proposal by the commission is the end of 2010.
III. European Union
5. EAZA Position Statement on the Animal Health Strategy for the

European Union
Introduction
This statement presents the position of the European

Association of Zoos and Aquaria (EAZA) on the Animal
Health Strategy for the European Union (2007-2013)
“Prevention is better than cure” and its resulting Action
Plan. It is also endorsed by the European Association of Zoo and Wildlife Veterinarians
(EAZWV) with approximately 700 individual members from zoos, zoo related research
institutions and universities.
In general EAZA is pleased with the strategy and would like to congratulate the European
Commission on its publication. The slogan “Prevention is better than cure” is much
appreciated and reflects the approach of EAZA and its member institutions. The flexible
approach to vaccination is a highly important aspect for the European zoo and aquarium
community as is the link between animal welfare and animal health. A clearer and simplified
regulatory framework is necessary, especially as EU animal health regulations are mostly
directed at agricultural animals and the impact of such legislation for zoo and aquarium
animals is often unclear or open to multiple interpretations. The development of one general
EU Animal Health Law is therefore strongly supported by EAZA.
The remainder of this statement will provide further detail on EAZA’s position.
EAZA’s current status and general position;

 As laid down in EAZA’s constitution the objects of the association are: a. to promote
co-operation for the furtherance of wildlife conservation, through internationally
coordinated breeding programmes of wild animals and in situ conservation; b. to
promote education, in particular environmental education; c. to promote scientific
study; d. to represent the interests of its members;
 EAZA represents 324 members from 35 countries, 300 of which maintain public
collections of animals. More than 280 institutions of the total EAZA membership are
located within the European Union. EAZA member institutions receive approximately
140 million visitors a year and house more than 250,000 animals, excluding fish and
invertebrates. EAZA member institutions employ 20,000 staff members, 5000 of
which are seasonal;
 The ‘EAZA Minimum Standards for the Accommodation and Care of Animals in Zoos
and Aquaria’ include extensive paragraphs on animal welfare, health, hygiene,
surveillance and veterinary aspects.
 EAZA members are often important economic drivers and cultural centres in their
local communities;
 EAZA has a significant social role in educating European citizens about animals, their
conservation, and overarching threat processes such as climate change. Zoos and
aquaria have been demonstrated to host a far more representative visitor social
spectrum than either museums or science centres;
III. European Union
 EAZA has adopted the World Zoo and Aquarium Conservation Strategy (2005) which
articulates the modern role of zoos and aquaria and their commitment to
conservation;
 EAZA institutions in the European Union comply with Council Directive 1999/22/EC
relating to the keeping of wild animals in zoos;
 EAZA encourages its member institutions to apply for approval under article 13 (2) of
Council Directive 92/65/EEC;
 EAZA has a Memorandum of Understanding with the European Association of Zoo
and Wildlife Veterinarians (EAZWV) and supported the publication of the
Transmissible Disease Handbook (2007). This is recognised as a key publication of
high relevance across all sectors concerned with public health. EAZA will also be
supporting the publication of the 2009 edition of this important document;
 All EAZA members must join the International Species Information System and use
the Animal Record Keeping System (ARKS) software to keep up to date records of
their animal collections;
 EAZA collections exchange approximately 25,000 animals annually;
 Emerging infectious disease outbreaks generally do not originate from zoo and
aquarium collections. EAZA zoos have highly trained specialised veterinarians and
therefore can quickly recognise newly arriving emerging infectious diseases, and thus
can serve as emergent disease sentinels. Zoos and aquariums can suffer from
outbreaks of diseases, through potential loss of stock, obstruction of transfers of
animals between collections as part of vital conservation breeding programmes and
loss of visitor revenue if the institution is within geographical areas of disease
outbreak and human movement restrictions occur;
 Populations of endangered species kept in EAZA collections are often irreplaceable
and some held in EAZA institutions are extinct in the wild.
We believe;
 Keeping and displaying healthy animals under good welfare conditions in EAZA
collections is of crucial importance to reach EAZA’s main objectives;
 Animal exchanges between EAZA member institutions (largely in the framework of
EAZA’s breeding programmes) are imperative to ensure healthy and sustainable
populations of wild animals in human care into the future;
 EAZA’s European Endangered species Programmes (EEPs) and European
studbooks (ESBs) should be managed independently, where appropriate, from ex situ
populations in other regions and from wild populations, unless specifically part of
global endangered species programmes. Nevertheless, occasional imports of
unrelated stock are important to ensure long-term genetic variability;
 Contamination risks in zoos and aquaria are significantly lower than in the agricultural
industry, e.g. because of surveillance, housing conditions, reduced numbers of
animals and individual care for most species;
 Compared to 4.3 million cattle, 21.7 million pigs and 794 million poultry traded
between EU member states in 2006 the number of animals exchanged between
EAZA members pose an extremely low health risk;
 Breeding programmes for endangered species are jeopardized by unclear legislation,
lack of uniform implementation of EU legislation by member states and slow decision
making processes making animal exchanges difficult or even impossible. This
obstruction can lead to compromised welfare conditions and obstruction of
conservation initiatives;
 EAZA’s breeding programmes and the Animal Record Keeping System (in the
process of being replaced by the Zoological Management Information System –
ZIMS) are suitable means for identification and traceability of zoo and aquarium
species.
III. European Union
What we would like to improve in the future;

 EAZA to be consulted at an early stage when the EU is formulating animal health
and welfare legislation to ensure that wild animal species as held in zoos and aquaria
are clearly and appropriately included in the regulatory definitions where relevant and
excluded where irrelevant;
 The position of EAZA zoos and aquaria to be fully considered when designing EU
animal health regulations;
 The health risks posed by animal collections of zoos and aquaria better evaluated in
light of the existing EU animal health regulations;
 Exchanges of animals in the framework of recognised EAZA breeding programmes
simplified and prioritised in relation to EU legislation pertaining to animal health;
 Implementation of the BALAI Directive 92/65 to be harmonized across EU Member
States;
 EAZA breeding programmes and ISIS ARKS registration formally recognised as
suitable means for identifying and tracing of zoo and aquarium animals;
 In relation to Council Regulation 1/2005 of 23 December 2004 animal transports
between zoos carried out only by the institutions themselves should not be
considered “commercial transports”. In a future review of Council Regulation 1/2005
the wide diversity of species to be transported between zoos and aquaria and
thereafter their differing and specific welfare needs should be recognised;,
 To continue to be able to vaccinate where appropriate in the case of emerging
disease outbreaks (refer to the ‘EAZA Minimum Standards for the Accommodation
and Care of Animals in Zoos and Aquaria’) ;
 To have, through approval under 13(2) of Council Directive 92/65, the possibility to
import wild animals from third countries where there is an agreed and demonstrable
programme need, particularly in relation to recognised and accountable EAZA
conservation breeding programmes, and where the import can be demonstrated to
not be of any detriment, either of welfare or persistence, to wild populations.
III. European Union
6. The O.I.E. (World Organisation for Animal Health) (Organisation

International des Epizooties)
The OIE is an intergovernmental organisation based in Paris, with a mandate from its 174
Member Countries and Territories to improve world animal health. In this capacity, the OIE is
responsible for ensuring transparency of the animal disease situation worldwide, including
diseases transmissible to humans, and the sanitary safety of world trade in animals and
animal products. The OIE publishes international standards in all fields covered by its
mandate, including animal welfare and consumer protection.
At the global level, the OIE has modernised its worldwide information system on animal
diseases (including zoonoses) with the creation of WAHIS, a mechanism whereby all
countries are linked on-line to a central server that collects all the compulsory notifications
sent to the OIE, covering 100 priority terrestrial and aquatic animal diseases, as well as any
emerging disease.
Together with the WHO (World Health Organisation) and the FAO (Food and Agricultural
Organisation of the United Nations), the OIE has created GLEWS, the Global Early Warning
System, a platform shared by the three organisations to improve early warning on animal
diseases and zoonoses worldwide.
The OIE, WHO and FAO (with the support of UNICEF, the United Nations System Influenza
Coordinator and the Worldbank) have prepared a consensus document on global measures
needed to coordinate medical and veterinary health policies more effectively, taking into
account new requirements to prevent and control zoonoses.
In this perspective veterinarians play a crucial role in protecting animals and society as a
whole from the negative effects of diseases such as Avian Influenza, Foot and Mouth
Disease and Bluetongue. Veterinarians play a crucial role at each stage of the food chain,
from “stable to table”. For example, by checking that only healthy animals are slaughtered for
human consumption, or by alerting the authorities at the first signs of disease on the farm or
in our zoos. Veterinarians across the European Union, through their work, help to ensure that
the goal of “One World, One Health” for all can be reached, encompassing both animals and
people in good health. The concept “One World, One Health” urges the international
community to consider the link between animal diseases and public health. It is well known
that 60% of known human infectious diseases have their source in animals, as do 75% of
emerging human diseases and 80% of the pathogens that could potentially be used in bio
terrorism. The unprecedented flow of commodities and people gives pathogens of all kinds
the opportunity to spread and multiply around the world to find genetic reassortment
opportunities, and climate change can enable them to expand in their range.
www.one-health.eu
IV. LINKS
Jacques Kaandorp
1. EU VETERINARY FACULTIES
2. USEFUL (VETERINARY) WEBLINKS
1. EU Veterinary Faculties
Austria
Veterinärmedizinische Universität Wien `
www.vu-wien.ac.at
Belgium
Université de Liège Faculté de Médecine Vétérinaire
www.ulg.ac.be/fmv
University of Ghent Faculty of Veterinary Medicine
www.ugent.be/di/en
Bulgaria
Trakia University. Faculty of Veterinary Medicine, Student’s Campus, Stara Zagora
www.uni-sz.bg
University of Forestry, Sofia. Faculty of Veterinary Medicine
www.edu.ltu.bg/typo3
Czech Republic
Veterinárni a Farmaceutická Universita
www.vfu.cz
Denmark
Københavns Universitet, Det Biovidenskablige Fakultet
www.life.ku.dk
Estonia
Estonian Agricultural University Faculty of Veterinary Medicine
Institute of Veterinary Medicine and Animal Sciences
www.eau.ee
Finland
University of Helsinki Faculty of Veterinary Medicine
www.vetmed.helsinki.fi
IV. Links
France
École Nationale Vétérinaire d’Alfort (ENVA)
www.vet-alfort.fr
École Nationale Vétérinaire de Lyon (ENVL)
www.vet-lyon.fr
École Nationale Vétérinaire de Nantes (ENVN)
www.vet-nantes.fr
École Nationale Vétérinaire de Toulouse (ENVT)
www.envt.fr
Germany
Veterinärmedizinische Fakultät der Freien Universität Berlin
www.fu-berlin.de
Fachbereich Veterinärmedizin der Universität Giessen
www.uni-giessen.de
Tierärztliche Hochschule Hannover
www.tiho-hannover.de
Veterinärmedizinische Fakultät der Universität Leipzig
www.uni-leipzig.de
Tierärztliche Fakultät der Ludwig-Maximillians Universität München
www.vetmed.uni-muenchen.de
Greece
Aristoteles University – Thessaloniki. School of Veterinary Medicine
University of Thessaly – Faculty of Veterinary Science
www.vet.uth.gr
Hungary
University of Veterinary Science in Budapest
www.univet.hu
Ireland
University College Dublin Faculty of Veterinary Medicine
www.ucd.ie
Italy
Facoltá di Medicina Veterinaria
www.uniba.it/
Universitá di Bologna Facoltá di Medicina Veterinaria
www.vet.unibo.it
Universitá di Camerino – Facoltá di Medicina Veterinaria
www.unicam.it
Universitá di Messina Facoltá di Medicina Veterinaria
www.unime.it
Universitá di Milano Facoltá di Medicina Veterinaria
www.veterinaria.unimi.it
Universitá di Napoli Federico Il Facoltá di Medicina Veterinaria
www.unina.it
Facoltá di Medicina Veterinaria Universitá degli studi di Padova
www.veterinaria.unipd.it
Universitá di Parma Facoltá di Medicina Veterinaria
www.unipr.it
Universitá di Perugia Facoltá di Medicina Veterinaria
www.unipg.it/-facvet
IV. Links
Universitá di Pisa Facoltá di Medicina Veterinaria

www.unipi.it
Universitá di Sassari Facoltá Medicina Veterinaria
www.uniss.it
Universitá degli Studi di Teramo
www.unite.it/
Universitá di Torino Facoltá Medicina Veterinaria
www.veter.unito.it/
Latvia
Latvia University of Agriculture Veterinârmedicìnas fakultâte
www.llu.lv/?mi=152
Lithuania
Lithuanian Veterinary Academy
www.lva.lt/en/
Netherlands
Universiteit Utrecht Faculty of Veterinary Medicine
www.vet.uu.nl
Poland
Uniwersytet Przyrodniczy w Lublinie – Wydzial Medycyny Weterynaryjnej
www.ar.lublin.pl
Uniwersytet Warmińsko – Mazurski w Olsztynie –Wydzial Medycyny Weterynaryjnej
www.uwm.edu.pl/wmw
Szkola Glówna Gospodarstwa Wiejskiego w Warszawie – Wydzial Medycyny Weterynaryjnej
www.sggw.pl
Uniwersytet Przyrodniczy we Wroclawiu - Wydzial Medycyny Weterynaryjnej
www.ar.wroc.pl
Portugal
Escola Universitária Vasco da Gama – Coimbra
www.euvg.net
Universidade de Évora
www.uevora.pt
Universidade Técnica de Lisboa – Faculdade de Medicina Veterinária
www.fmv.utl.pt
Universidade Lusófona de Humanidades e Tecnologias – Lisboa
Universidade do Porto – Instituto de Cièncias Biomédicas de Abel Salazar

www.up.pt
Universidade de Trás-os-Montes e Alto Douro – Vila Real
www.utad.pt
Romania
University of Agricultural Sciences and Veterinary Medicine Bucharest
www.fmvb.ro
Faculty of Veterinary Medicine, Cluj-Napoca
University of Agronomy and Veterinary Medicine – lasi
Faculty of Veterinary Medicine, Timisoara
Slovenia
University of Ljubljana
www.vf.uni-lj.si
IV. Links
Slovak Republic
University of Veterinary Medicine in Košice
www.uvm.sk/
Spain
Universitat Autònoma de Barcelona Facultat de Veterinària
www.uab.es/fac-veterinaria
Universidad de Córdoba Facultad de Veterinaria
www.uco.es/organiza/centros/veterinaria
Facultad de Veterinaria de Cáceres
www.veterinaria.unex.es
Universidad de León – Facultad de Veterinaria
www.unileon.es
Universidad de Murcia Facultad de Veterinaria
www.um.es/veterina/
Facultad de Veterinaria de las Palmas de Gran Canaria
www.vet.ulpgc.es
Universidad Complutense de Madrid, Facultad de Veterinaria
www.ucm.es/info/webvet
Universidade de Santiago de Compostella – Licenciado en Veterinaria (LUGO)
www.facveterinarialugo.org/
Universidad Politécnica de Valencia – Departamento de Ciencia Animal
www.www.dcam.upv.es/dcia/
Universidad de Zaragoza Facultad de Veterinaria
www.wzar.unizar.es/acad/fac/vete/unizar.html
Universidad Alfonso X (Madrid)
www.uax.es/oferta_docente/titulaciones/vet
Sweden
University of Agricultural Sciences Faculty of Veterinary Medicine
www.slu.se
United Kingdom
University of Bristol
www.bristol.ac.uk/fmus/
University of Cambridge Veterinary School
www.vet.cam.ac.uk
University of Edinburgh Royal (Dick) School of Veterinary Studies
www.vet.ed.ac.uk/
University of Glasgow Faculty of Veterinary Medicine
www.gla.ac.uk/faculties/vet/
University of Liverpool Faculty of Veterinary Science
www.liverpool.ac.uk/vets/
Royal Veterinary College
www.rvc.ac.uk
University of Nottingham
www.nottingham.ac.uk
IV. Links
2. Useful (Veterinary) Weblinks
Veterinary links
The World Organisation for Animal Health
www.oie.int
OIE Animal Disease Information Summaries
www.oie.int/eng/ressources/en_diseasecards.htm
OIE Terrestrial Animal Health Code
www.oie.int/eng/normes/mcode/en_index.htm
Food and Agricultural Organisation of the United Nations
www.fao.org
The World Health Organisation
www.who.int
WHO mediacentre
www.who.int/mediacentre/factsheets/en
Federation of Veterinarians of Europe
www.fve.org
A guide to the use of the Internet for Vets
www.vts.intute.ac.uk/he/tutorial/vet/
Veterinary Information Network
www.vin.com
International Veterinary Information Service
www.ivis.com
Veterinary Sciences Tomorrow
www.vetscite.org
World Veterinary Association
www.worldvet.org
Veterinary Events Worldwide
www.vetagenda.com
The World Small Animals Veterinary Association
www.wsava.org
The Federation of European Companion Animal Veterinary Associations
www.fecava.org
European Association for the Evaluation of Veterinary Establishments
www.eaeve.org
Centre for Disease Control and Prevention USA
www.cdc.gov/nczved/dfbmd/disease_listing.html
The Merck Veterinary Manual
www.merckvetmanual.com/mvm/index.jsp
EAZWV European Association of Zoo and Wildlife Veterinarians
www.eazwv.org
AAZV American Association of Zoo Veterinarians
www.aazv.org
EAZA European Association of Zoos and Aquaria
www.eaza.net
WAZA World Association of Zoos and Aquaria
www.waza.org
European communication platform for veterinary students
www.vetstart.org
International Veterinary Student Association
www.ivsa.org
IV. Links
European (Union) links

Gateway to the European Union
http://europa.eu
European Parliament
www.europarl.europa.eu
Council of the European Union
www.consilium.europa.eu
European Commission
http://ec.europa.eu
Directorate-General for Health and Consumers
http://ec.europa.eu/dgs/health_consumer/index_en.htm
Directorate-General for Agriculture and Rural Development
http://ec.europa.eu/dgs/agriculture/index_en.htm
Directorate-General for Maritime Affairs and Fisheries
http://ec.europa.eu/dgs/fisheries/index_en.htm
Directorate-General for Environment
http://ec.europa.eu/dgs/environment/index_en.htm
European Food Safety Authority
www.efsa.europa.eu
European Centre for Disease Prevention and Control
http://ecdc.europa.eu
European Medicines Agency
www.emea.europa.eu
Access to European law
http://eur-lex.europa.eu
VI. RECOMMENDATIONS FOR THE APPLICATION OF ANNEX C TO

COUNCIL DIRECTIVE 92/65 (“BALAI”) AS AMENDED BY
COUNCIL REGULATION (EC) NO 1282/2002 OF 15 JULY 2002
(OJ L 187/3) IN APPROVED ZOOS
1. Introduction
The amendment of 15 July 2002 to the Council Directive 92/65/EEC laying down animal
health requirements governing trade in and imports into the Community of animals, semen,
ova and embryos not subject to animal health requirements laid down in specific Community
rules referred to in Annex A (1) to Directive 90/425/EEC (“BALAI”) was made in the legal
form of a regulation. As a consequence, the Annexes A, C and E are directly applicable in
Member States. ANNEX C contains, however, a few points which leave considerable room
for interpretation. The present recommendations aim at contributing to a uniform
interpretation of these points, and thus at achieving the ultimate goal of this annex, namely to
facilitate the exchange of animals between approved zoos easily and without major health
risks.
These recommendations are the result of two meetings held on 15/16 September 2003 and 5
February 2004 at Cologne Zoo with the participation of representatives of the European
Commission (DG SANCO - Health and Consumer Protection), the Department for
Environment, Food & Rural Affairs (DEFRA), the Rijksdienst voor de Keuring van Vee en
Vlees (RVV), the Bundesministerium für Verbraucherschutz, Ernährung und Landwirtschaft
(BMVEL), the European Association of Zoo and Wildlife Veterinarians (EAZWV), the EAZWV
Infectious Diseases Working Group (IDWG) and zoo veterinarians from France, Germany,
Italy, The Netherlands and the United Kingdom representing also their respective
professional organisations at the national level.
The recommendations are meant to provide some practical guidance to both, Veterinary
Authorities and zoo veterinarians, throughout the European Union. EU veterinary legislation
applies also to the British Crown Dependencies (Channel Islands, Isle of Man), Andorra,
Monaco and San Marino. As Council Directive 92/65/EEC and Commission Regulation (EC)
No. 1282/2002 are texts with EEA relevance (Regulation 1282/2002 was incorporated into
the EEA Agreement by Decision of the EEA Joint Committee of 14 March 2003), and are
also part of the equivalent sector of the Bilateral Agreement on Agriculture with Switzerland
(Regulation 1282/2002 is included in the 2003 update), they apply in Liechtenstein, Norway,
and Switzerland too.
Note:
To make reading of this document easier, the term “Bodies, institutes and centres” is usually
replaced by the word “zoo”
2. Contents
A. The term ‘animals’
B. The approved veterinarian
C. The annual disease surveillance plan
VI. Recommendations
D. The added animals procedure

E. Quarantine / isolation requirements
F. The certificates
VI. Recommendations
A. The term ‘animals’
1. ‘Animals’ means specimens of animal species other than those referred to in

Directives 64/432/EEC, 90/426/EEC, 90/539/EEC, 91/67/EEC, 91/68/EEC, 91/492/EEC
and 91/493. (Directive 92/65/EEC Article 2 (b))
2. The following animal species are referred to in the Directives mentioned above:
Directive Article Species Comments

90/426/EE Art. 2 Equidae means wild or i.e. all Equidae species
C (b) domesticated animals of the equine
(including zebras) or asinine
species
64/432/EE Art. 2 Bovines including Bison bison Wild cattle, except
C (b) and (American bison) and Bubalus American bison, and wild
(c) bubalus (Domestic buffalo) and Suidae, including ‘feral
swine for slaughter, breeding or pigs’, do not fall under
production 64/432/EEC
91/68/EE Art. 2 (1 Ovine and caprine animals for Wild species do not fall
C and 2) slaughter, breeding and fattening under 91/68/EEC
90/539/EE Art. 2 Fowl, turkeys, guinea fowl, ducks ‘breeding’ in this context
C (1) geese, quails, pigeons, pheasants, means production of
partridges and ratites reared or kept hatching eggs for the
in captivity for breeding, the production of animals for
production of meat and eggs for breeding, the production of
consumption, or for re-stocking meat and eggs for
supplies of game consumption, or for re-
stocking supplies of game
91/67/EE Art. 2 ‘aquaculture animals’ i.e. live fishes, i.e. animals in a zoo or
C (1) crustaceans or molluscs coming aquarium do not fall under
from a farm, including those from 91/67/EEC if animals are
the wild intended for a farm transferred from a zoo to
another zoo, this transfer is
covered by 91/67.
3. Although the animals listed in the above tabulation do, in principle, not fall under
92/65/EEC, it would make no sense to exclude them from the health surveillance plan
(see Section C paragraph 3 of these recommendations).
VI. Recommendations
B. The approved veterinarian
1. In order to be granted official approval under Article 13 of the Directive, a zoo

must secure by contract or legal instrument the services of a veterinarian
approved by and under the control of the competent authority (ANNEX C paragraph
1.(g)).
2. The role of the approved veterinarian is to ensure that the requirements of the present
directive and other related legislation are complied with on a day to day basis.
3. Where this veterinarian is a member of a practice, other members of the same practice
may be included provided that they are also approved by the competent authority and
individually nominated in writing.
4. The approved veterinarians shall comply mutatis mutandis with the requirements
referred to in Article 14(3)(B) of Directive 64/432/EEC (ANNEX C paragraph 1.(g)(i)).
5. Taken directly from Directive 64/432- Article 14 (3)(b)
“According to this article, the approved veterinarians must:
i. meet the conditions for pursuing the veterinary profession;
ii. have no financial interest or family links with the owner of or person responsible for
the holding (but see point 6 below);
iii. possess particular knowledge in the field of animal health as it applies to animals of
the species concerned. This means that they must:
 regularly update their knowledge, especially as regards the relevant health
regulations,
 meet the requirements laid down by the competent authority to ensure the
proper functioning of the network,
 provide the owner of or person responsible for the holding with information and
assistance in order that all steps are taken to ensure that the holding's status is
maintained, particularly on the basis of programmes agreed with the competent
authority,
 ensure compliance with the requirements concerning:
(i) the identification and health certification of the animals of the collection, the
animals introduced and those traded;
(ii) compulsory reporting of infectious animal diseases and any other risk factor
for animal health or welfare, and for human health;
(iii) establishing as far as possible the cause of death of animals and where
they are to be consigned;
(iv) the hygiene conditions of the herd and of the livestock production units.”
6. For the purposes of the implementation of the present Directive point ii. above is of less
importance than it is in Directive 64/432/EEC as zoo animals have a conservation value
rather than an economic value and because, for the purposes of the present Directive,
the approved veterinarian is working under the supervision of the official veterinarian. It
is thus up to the official veterinarian to assess whether there could be a conflict of
interest, and whether the approved vet appointed by the zoo fulfils the requirements
above, and in particular has appropriate specialist knowledge in relation to zoo animals.
7. The competent authority shall draw up lists of approved veterinarians and of the
approved holdings participating in the network. If the competent authority finds that a
participant in the network no longer fulfils the conditions set out above, it shall suspend
or withdraw approval, without prejudice to any penalties that may be applied.
VI. Recommendations
C. The Annual Disease Surveillance Plan
1. The approved veterinarian has to draw up and implement an annual disease

surveillance plan (ANNEX C paragraph 1 (g) (ii) 1st indent). This plan is subject to
annual audits by an official veterinarian from the competent authority (ANNEX C
paragraph 2 (a) (ii).
2. For the purposes of approval under the present directive the surveillance plan must
cover those diseases listed in Annex A (and B if relevant).
3. The surveillance plan may also include other general measures as may be required
under Council Directive 1999/22/EC of 29 March 1999 relating to the keeping of wild
animals in zoos (“Zoo Directive”, O.J. L94/24 0f 09.04.1999), and specific measures for
individual taxonomic groups as may be agreed by the relevant Taxonomic Advisory
Group from the European Endangered Species Program (EEP) of the European
Association of Zoos and Aquaria (EAZA). As a general rule such specific measures
would be elaborated by the EAZWV Infectious Diseases Working Group and
subsequently be integrated into the Husbandry Guidelines for the taxon concerned.
4. Animals for the purpose of this surveillance programme mean those species that are
covered by article 13 of Directive 92/65/EEC; namely any species susceptible to the
diseases listed in Annex A or Annex B. In practice this means all mammals, all birds,
fishes of the salmonid group, and honey bees but not other invertebrates. Where
animals of the domestic species are kept within a zoo premises, for example in a
children's zoo, they will be regarded as part of the zoo collection and subject to all the
same conditions as the rest of the collection as far as approval is concerned, including
the surveillance programme. Note in this context that there may be specific requirements
for domestic animal species that are covered by other Directives. Reptiles and
amphibians and fishes other than salmonids are not included.
5. The Annual Disease Surveillance Plan and the measures based thereon must include
a. Immediate notification to the competent authority if there is any cause for suspicion
that animals may be affected by any disease, including zoonoses, that is notifiable
under Community legislation (including 92/65 and Directive 82/894 and other
relevant Community legislation) or national legislation.
b. Close observation of each animal at least once per day by suitably qualified staff,
under the direction of the approved veterinarian (in the case of large group species,
such as fish in an aquarium, the veterinarian may decide that observation of the
group is sufficient).
c. Immediate notification of the approved veterinarian by zoo staff if any animals
appear unwell or die (in the case of large group species, notification may be
triggered by mortality above an agreed, expected level).
d. Laboratory examination to establish the infective agent in any live animals that
appear to be affected by an infectious disease (in the case of large group species,
such as fish in an aquarium, the veterinarian may decide that a representative
sample is sufficient). In the case of suspicion of a disease that is listed on Annex A
and B and/or is notifiable under national legislation, the official veterinarian must be
informed immediately. The official veterinarian will be responsible for arranging
disease control precautions and further investigation for diseases notifiable under
national legislation, and may direct that samples should be taken and submitted to a
designated laboratory.
VI. Recommendations
e. Procedures for newly arrived and diseased animals, taking into account the relevant
risk factors and, therefore, including handling practices, clinical examination and
specific tests as appropriate.
f. Regular parasitological examination of faecal samples (individual or group samples,
depending on the housing system) in particular with regard to zoonotic parasites. It
is recommended by EAZWV that all relevant groups should be checked at least
once a year; the frequency of examination should be related to the prevalence of
parasites.
g. Opportunistic examination and taking of appropriate samples from immobilised or
otherwise restrained animals. EAZWV recommends all serum samples to be
retained and stored at –180 C or below.
h. Specific guidelines for the systematic testing of specific animal species may be
developed and recommended by the Infectious Diseases Working Group of
EAZWV.
i. Post mortem examination without unnecessary delay to check for significant
pathology, and as far as possible to establish the cause of death in every animal
that dies or foetus that is aborted (but the approved veterinarian may exercise
discretion where there is clearly no suspicion of infectious disease, such as obvious
trauma, or euthanasia of a healthy animal; and where it has been established that
an infectious disease is affecting a group, the veterinarian may decide, in
consultation with the competent authority if necessary, that a representative sample
is sufficient).
j. The vaccination programme should be based on the availability of safe vaccines. It
should take into account the species involved and the risk of diseases likely to occur
in the zoo, and may cover zoonotic diseases other than those mentioned in Annex A
or B, but these vaccinations must be in compliance with the applicable legislation.
k. Records must be kept in an easily accessible form, to be available as necessary for
audit purposes, and retained for at least 10 years, to show at least the following
information:
 All cases of disease, and treatment if applicable.
 Preventive actions such as vaccinations.
 Results of blood tests and other diagnostic procedures.
 Results of post mortem examinations including records of stillbirths.
 Observations during any periods of precautionary isolation.
 Reports to the veterinary authority of any suspicion of Annex A diseases or
diseases notifiable under national law.
l. Zoo vets should be aware that they will be asked for specific information on
diseases under the zoonosis directive and should therefore be able to extract this
information easily.
VI. Recommendations
D. The Added Animals Procedure
1. General
a. Only animals coming from another approved body, institute or centre may be
introduced into an approved zoo (ANNEX C paragraph 2 (b). There is, however
a derogation from this requirement provided that certain conditions are
respected (ANNEX C paragraph 3).
b. By way of derogation from Article 5(1) of the Directive, ANNEX C paragraph 3
allows also for the introduction of “apes” (i.e. non-human primates)1 from
non-approved sources.
c. Animals having an origin other than an approved body, institute or centre may
be introduced in an approved zoo provided they undergo a quarantine under
official control before being added to the collection (ANNEX C paragraph 3).
d. Animals from approved zoos and from non-approved sources should not be
transported together in the same container or vehicle.
e. The animals must be accompanied by transport permits or any other documentation
as may be required by national legislation. ANIMO requirements apply.
2. Animals from another approved establishment

a. Animals coming from another approved establishment in the same member state fall
outside the scope of Directive 92/65, and hence under Community legislation there
is no requirement for the animal to be accompanied by the model health certificate
in Annex E. However, national rules governing certification may apply. For the same
reason, there is no official requirement for post-arrival isolation, although the
establishment may choose to carry out isolation and/or testing for its own private
purposes.
b. If the animals are coming from an approved establishment in another Member State
they must be accompanied by the model health certificate in Annex E type 3.
Depending of the health situation there may be additional requirements imposed by
EU or national legislation.
3. Animals from a non-approved establishment in the same Member State

a. Animals coming from a non-approved establishment in the same member state fall
outside the scope of Directive 92/65, and hence under Community legislation there
is no requirement for the animal to be accompanied by the model health certificate
in Annex E. However, national rules governing certification may apply. However, in
accordance with Annex C of Directive 92/65, the animals must undergo post-arrival
isolation in the isolation area, designated in the terms of approval, for at least 30
days or such longer period as may be required by the approved veterinarian and/or
the competent authority to be satisfied that the health status of the animals is not
inferior to the health status of the other animals in the collection.
b. During isolation the animals may be required to undergo testing for any disease on
Annex A that the approved veterinarian and the competent authority consider
appropriate. They should have particular regard to diseases for which national
programmes are in place, such as tuberculosis, brucellosis, Aujeszky's disease. The
approved veterinarian may also wish to carry out specific testing for any diseases for
1
“apes” is zoologically not correct but it is the term used in the core text of the directive
VI. Recommendations
which the premises runs its own surveillance, control and eradication programme,
covering diseases other than those listed in Annex A.
c. The arrival of the new animals must be recorded by the receiving establishment, as
laid down in Annex C. There is no official requirement for any other health
certification.
4. Animals from a non-approved establishment in another Member State

a. Member states may, by way of derogation, allow the movement of animals from non
approved establishments in another member state. The Veterinary Service of the
receiving country has to be informed under paragraph 3 of ANNEX C and may lay
down specific conditions under which transfer must take place. In addition, in
accordance with Annex C of Directive 92/65, the animals must undergo post-arrival
isolation in the isolation area, designated in the terms of approval, for at least 30 days
or such longer period as may be required by the approved veterinarian and/or the
competent authority.
5. Animals from non-approved establishment to a non-approved establishment

a. Between establishments in the same Member State: National rules apply
b. Between establishments in different Member States: Not covered by Annex C of
Directive 92/65. This means that the rules laid down in the body of the Directive
apply in full. Therefore, where no provisions are laid down, the Member State of
destination can request specific requirements for introduction ( e.g. requests for
additional guarantees such as the status, negative tests or treatments see Article 6
of Directive 92/65 for details )
6. Animals from third countries

a. Animals being imported into the Community must fulfil the animal health conditions
as laid down in Directive 92/65. However where harmonised rules for a particular
species have not been laid down in the Directive, then national rules shall apply,
although these also should be based on the animal health principles laid down in the
Directive. EU rule, or if not applicable national rules, for licensing, health
certification, post-import quarantine and testing will apply, as appropriate for the
species. The importing zoo must apply for a specific import licence, which will
contain the conditions relevant to the species and place of origin.
(Please note that new legislation (amending Decision 79/542/EEC)is due to come into
force in the near future that will lay down a list of third countries and harmonised rules
(certification and animal health requirements) for the importation of all ungulates
(including all Artiodactyla). Because of these rules, imports of such animals will only be
allowed exclusively from a small number of third countries authorised for each species.
However a draft Decision is under discussion which foresees a particular regime for
importation of live animals originating in any third country but imported after a residency
period in St.Pierre and Miquelon (a little island in the Atlantic ocean close to Canada)
where they will spend a period in a quarantine station. During this period, specific testing
will be carried out on the animals. For the moment, these special conditions are limited
to the import of live Camelidae, but the intention is to extend this possibility to other
species.
Following introduction of this new legislation national rules will therefore no longer apply
to imports belonging to the order Artiodactyla.
VI. Recommendations
E. Quarantine / Isolation requirements
1. Definitions
a. ‘Isolation’ and 'quarantine' are not precisely defined in European Union legislation,
and one word is usually described by reference to the other. For example in the
poultry trade directive 90/539/EEC: ‘Quarantine station shall mean facilities where
the poultry is kept in complete isolation and away from direct or indirect contact with
other poultry, so as to permit long-term observation and testing’ (Council Directive
90/539/EC, Article 2).
b. The Office International des Epizooties (OIE) Terrestrial Animal Health Code defines
a quarantine station as 'a facility under the control of the veterinary authority where
a group of animals is maintained in isolation., with no direct or indirect contact with
other animals, in order to undergo observation for a specified length of time and, if
appropriate, testing and treatment.
c. In order to emphasize the difference between quarantine of the above types and
quarantine required for added animals under the BALAI Directive, the latter is
referred to as 'isolation' throughout this document.
d. The conditions below refer to isolation for added animals entering a BALAI approved
zoo from a non-approved source within the EU (or in Norway, Liechtenstein, and
Switzerland).
2. Principles
a. In order to be granted approval, zoos must have adequate means for isolating
animals, and have available adequate quarantine facilities and approved
procedures for animals from non-approved sources. (ANNEX C paragraph 1.
(b).
b. Incoming animals must be isolated as necessary (ANNEX C paragraph 1 (g) (iv).
c. Animals having an origin other than an approved body, institute or centre may
be introduced in an approved zoo provided they undergo quarantine under
official control before being added to the collection (ANNEX C paragraph 3).
d. For the purposes of the approval under the Directive only the diseases listed in
Annex A have to be taken into account. In addition, it should be considered that the
introduction of new animals susceptible to these diseases is only possible either
from within the EU or from listed third countries.
e. A risk analysis has to be made and the quarantine / isolation requirements must
cope with the risk. Quarantine requirements for comparable livestock could provide
some guidance. In this context it is noted that management procedures could be
adjusted easily to each individual case, but that the availability of suitable facilities is
a prerogative for approval and has to be seen without a specific case in mind.
f. For the purposes of the approval , i.e. for the introduction of animals from non-
approved sources within the European Union or from listed Third Countries where
such lists exist, the following information may be useful when considering, which
general requirements to apply:
There are three risk groups:
 Primates: can be imported from anywhere (no Third Countries List), they may be
carriers of zoonoses.
VI. Recommendations
 Birds: the introduction from areas where OIE list A diseases exist can not be
excluded (occurrence of NCD in wild birds), and the relevant diseases, NCD, AI
and Psittacosis are easily transmitted via the air or, in the case of West Nile
Virus, by mosquitoes.
 Mammals other than primates: introduction only from areas free from highly
contagious diseases, all relevant diseases not transmittable by air over a longer
distance, in most cases direct contact required.
g. Consequently, the following general requirements apply:
 Primates: The quarantine requirements laid down in the OIE Terrestrial
Animal Health Code (Chapter 2.11 and Appendix 3.5.1) shall be respected
(ANNEX C paragraph 3).
 Birds must be isolated in buildings and the possibility of disease transmission by
air or insects has to be taken into account. Windows should be kept closed.
EAZWV strongly recommends that the isolation rooms should be ventilated, and
the exhausted air should pass through a dust filter.
 Mammals should, as a general rule, be isolated indoors, but no special
precautions have to be taken regarding the exhausted air to cope with the
relevant diseases listed in Annex A of the Directive. If, for specific reasons,
mammals have to be isolated outdoors, the ground should be solid and easy to
disinfect. If this is not possible, the isolation enclosure should be relatively small
to allow for other treatment of the soil, e.g. removal of top soiling. No zoo will be
able to have specific isolation facilities for all mammalian taxa, which may include
a diverse range of species, including e.g. big cats, dolphins, elephants,
hippopotamuses. In such cases it should be possible to use the standard facility
for isolation purposes.
h. In order to be granted approval, zoos must have available adequate quarantine /
isolation facilities. This wording does not imply that the facilities are on the ground or
owned by the zoo concerned. In addition the option exists for several zoos to jointly
operate a facility, or have contracts among themselves. In this last case, the option
should be specified in the annual plan.
3. General conditions - Structural requirements

a. Location
The isolation quarters must be physically separated from other animal accommodation
by a reasonable distance, taking into account the species concerned and the ability of
the relevant viruses to spread on the air. This distance can be much reduced if the
exhausted air is filtered (for animals originating from within the EU or from listed Third
Countries the use of dust filters is sufficient, otherwise High Efficiency Particulate
Extraction (HEPA) may be required).
b. Demarcation
The limits of the isolation area must be clearly demarcated by walls or fences as
appropriate. This does not preclude the possibility that specific areas or pens within the
premises may be designated as isolation areas for a limited time and a particular
purpose, provided that they meet the general requirements.
c. Access
There must be a double door system to prevent escape at the entry/exit with sufficient
space between the doors to allow one to be closed before the other is opened. Entry/exit
VI. Recommendations
doors must be lockable and must display a notice stating: 'QUARANTINE: No Admission
to Unauthorised Persons'.
d. Hygiene barrier
Facilities must be available at the entry/exit point for attendants to change overalls, to
change and disinfect boots, to wash hands, and if appropriate to shower.
e. Loading/Unloading.
Suitable facilities must be available to load or unload animals between transport crates
and isolation pens without the risk of escape.
f. Restraint
Suitable crush or penning facilities should be available within reasonable access of the
isolation area, so that animals may be safely restrained for clinical and diagnostic
procedures such as blood sampling.
The route from isolation to restraint must not put other animals at risk of infection from
the introduced animals.
g. Inspection
The design of the pens or cages within the isolation area must be such that the animals
may be visually inspected at any time, with adequate light and ease of access.
h. Disinfection
The physical structure and all equipment must be made of such materials that they can
be effectively cleansed and disinfected, or destroyed after use.
i. Vermin
The design must be suitable to minimise access by rodents, wild birds and insects, as
appropriate for the species in question. Where drains are present, they must be fitted
with rodent proof covers.
j. Feed Store
The feed store must be suitably protected from vermin.
k. Waste Disposal
Adequate storage facilities must be available to contain the litter and animal waste
produced during the isolation period, and the storage facility must be bird and vermin
proof. There must be facilities to dispose of the waste either during or after the isolation
period in a way which will ensure that there is no risk of the spread of disease.
l. Post Mortem
Refrigeration facilities or equivalent must be available within the isolation area, or in a
suitably disease-protected location nearby, to hold carcases of animals that die until they
can be subject to post mortem examination. Procedures for conveying carcases safely to
the storage facility must be laid down in writing by the approved veterinarian.
4. General conditions - Management procedures

a. Surveillance
Every animal in isolation must be visually inspected at least once a day by suitably
competent staff. Any signs of illness must be recorded and reported immediately to the
responsible veterinarian, who should make a further examination of the affected animals
without any unreasonable delay.
b. Staff
VI. Recommendations
The premises must have designated staff who are present on a sufficiently regular
schedule to ensure surveillance of the animals on a daily basis, and more frequently if
appropriate.
c. Hygiene
Staff entering the isolation premises must always change into protective clothing and
footwear. On leaving, the overalls and footwear must be removed and left within the
isolation area, and the footwear must be disinfected. Hands must be washed, or
otherwise disinfected, on entering and leaving.
d. Equipment
None of the moveable items used in the isolation unit should be taken outside the unit,
or used with other stock outside the unit, for the entire duration of the isolation period.
e. Waste
Litter and waste material must be collected regularly, stored in the containers provided,
and disposed of either during or after the isolation period in such a way that disease
agents will not be spread.
f. Disinfection
Premises must have an effective programme, laid down in writing by the approved
veterinarian, for cleansing and disinfection after each isolation session; approved
disinfectants must be specified and used in the programme; and an appropriate resting
period (usually 7 days) must be specified after each cleansing and disinfection
operation.
g. Transport Crates
Crates or cages used for transport, if to be re-used, must be made of materials which
allow effective cleaning and disinfection, and this should be carried out within the
isolation unit. If not re-used, the crates and cages must be destroyed in such a way that
disease agents cannot be spread.
h. All-in, All-out
An ‘all-in, all-out’ policy should be followed in the isolation unit. If it is necessary to add
animals whilst others are already present in the unit, the isolation period of all of them
must be extended until the latest completion date of any of the animals.
i. Illness
If any animals become ill during isolation and the approved veterinarian considers that
they need to be moved to a specialised hospital facility for diagnosis or treatment,
he/she must ensure that this is done under his/her personal supervision in such a way
as to ensure no possible risk of disease spread. In particular the approved veterinarian
must personally supervise the arrangements for maintaining isolation throughout the
movement, and for disinfecting any vehicles, rooms and equipment with which the
animal has had contact.
j. Disease and Death
Any sign of any disease or death during isolation must be reported immediately to the
approved veterinarian. All suspicions of any infectious disease on Annex A and any
deaths in isolation must be reported immediately to the competent authority. Carcases of
animals, which die during isolation, and if necessary those that are dead on arrival, must
be submitted to a post mortem examination without unreasonable delay.
k. Designated Attendants
VI. Recommendations
The establishment must designate suitable staff to attend to the animals in isolation,
taking appropriate precautions to ensure that there is no risk of transferring infection
from the isolation unit to any other animals, and the arrangements must be agreed in
writing by the approved veterinarian.
l. Visitors
Visitors must not be allowed to enter the isolation unit. If personnel apart from the
designated attendants need to enter for essential maintenance etc., they must be
required to wash thoroughly on entering and leaving, and wear protective clothing which
shall be put on prior to entering and removed prior to leaving. There must be a visitors'
book to record the dates, names and addresses of all visitors.
m. Records
The person in charge of the isolation unit must keep the following records, which should
be retained for at least ten years
 the date, number and identification of animals entering and leaving the isolation
facility.
 copies of the export health certificates and border crossing certificates
accompanying imported animals.
 significant health observations, cases of illness and deaths on a daily basis.
 dates and results of testing
 dates and types of treatment
 dates and names and addresses of persons entering the isolation unit.
n. Duration
Isolation should normally last for at least 30 days, unless a longer period is required to
exclude specific risks such as rabies.
5. Additional requirements specifically for birds

a. Ventilation
Birds must always be isolated in buildings. There must be no possibility of access by
wild birds or by mosquitoes.
As a general rule, windows should be kept closed, the isolation rooms should be
ventilated, and the exhausted air should pass through a dust filter. If the isolation facility
is more than 250m away from other bird enclosures, this requirement can be waived and
ventilation through open windows can be permitted. In such case all ventilation openings
must be covered with a double layer of wire mesh.
b. Air Space
If there are separate units within the isolation facility, each unit must occupy a separate
airspace so as to be an isolated epidemiological unit. If this cannot be achieved, all the
birds in isolation must remain until the completion date of the last birds to enter.
6. Additional requirements specifically for ungulates

a. Fencing
If the isolation area includes open paddocks, situations where there may be stock in
adjacent paddocks must be avoided. The isolation paddocks must be surrounded by
VI. Recommendations
double fences allowing a suitable sanitary gap between the fences. A minimum gap of at
least 3 meters should normally be satisfactory, but taking account of the species
concerned, the competent authority may require a different standard. Both fences must
be escape-proof.
b. Herds in Isolation
If the isolation facility is intended to contain large groups of animals, there must be
additional provision so that any individual that appears to be unwell can be separated
and kept apart from the rest of the group, with facilities for testing and treatment as
appropriate.
7. Additional requirements specifically for primates

(based on the OIE Terrestrial Health Code, chapter 2.10.1. and Appendix 3.5.1. (2002
edition)
a. Zoonoses
Any biting or scratching incidents involving humans, or other events in which humans
are exposed to primate blood or saliva, are to be reported immediately to the responsible
zoo veterinarian, who should consult with medical authorities as appropriate.
b. Protection of Attendants
The overalls and boots provided for entry to the isolation facility should completely cover
the attendant's body, and suitable masks, visors, goggles and gloves should also be
provided (where this raises issues such as welfare and socialisation, the approved
veterinarian should consult with the competent authority who may agree to alternative
methods providing equivalent security).
c. Staff Training
The responsible veterinarian or physician should ensure that all attendants are fully
instructed in the procedures necessary to protect their own health, as well as the health
and welfare of the animals in isolation. Personnel must not eat, drink, smoke or store
food for human use within the isolation rooms.
d. Staff Health
Personnel working within the isolation area should be encouraged to provide baseline
serum samples, which would be stored for study and comparison if appropriate.
Additional serum samples may be collected periodically as an aid to epidemiological
investigations. Staff should be encouraged to report any signs of illness immediately to
their medical adviser.
e. Ventilation
If natural ventilation is used, the openings must be covered with a double layer of mesh,
each of which is individually strong and secure enough to prevent the escape of the
animals. Ventilation intakes and outlets must not be so close to any other animal holding
area as to present a disease risk. If forced ventilation with HEPA filtration is used, there
should be provision to maintain adequate ventilation in the event of a technical failure.
Separate units must be ventilated separately.
f. Washing facilities
Washing facilities with hot and cold running water should be available for personnel to
wash hands within each animal holding room. Personnel should wash or otherwise
disinfect hands at frequent intervals whilst working within the isolation premises.
VI. Recommendations
g. Footbaths
Footbaths should be available not only at the entrance/exit of the isolation premises, but
also between individual holding rooms within the premises. The footbaths should contain
an approved disinfectant agreed by the approved veterinarian. Personnel should use the
footbaths as they pass from one room to another.
h. Equipment
Each holding room should have its own complete range of dedicated equipment, and
equipment should not be transferred from one holding room to another. After use all
equipment including work surfaces should be effectively cleaned and disinfected.
Because of the aerosol risk power hoses should not be used, except with the agreement
of the approved veterinarian.
i. Group Separation
Separate groups entering the isolation premises from different sources or on different
occasions must remain physically and epidemiologically isolated from each other.
Separate groups must be accommodated in separate, isolated units. Animals may not
be transferred between groups. However where this raises issues such as welfare and
socialisation, the approved veterinarian in consultation with the competent authority may
agree to mixing animals, provided that isolation conditions then apply to all those in
contact with the introduced animals.
j. Cage Discipline
No animals may be removed from their cages, albeit within the self-contained isolation
premises, without the specific authority and supervision of the responsible zoo
veterinarian.
k. Duration
OIE recommends a quarantine duration of at least 30 days when the primate is sent
from another premises under veterinary supervision, and at least 12 weeks if it is coming
from circumstances without veterinary supervision or from the wild. In Great Britain
under rabies regulations the duration of quarantine for primates must be at least 6
months.
VI. Recommendations
F. The certificates
Where certificates are required

1. ‘Animals’ in the terms of article 2(b) of Directive 92/65/EEC must be accompanied by a
certificate corresponding to the specimen in Annex E (Directive 92/65, Article 5).
2. Bovines and swine falling under the scope of Directive 64/432/EEC must be
accompanied by a certificate conforming to the model set out in Annex F of that
Directive.
3. Ovines and caprines falling under the scope of directive 91/68/EEC must be
accompanied by a certificate conforming to the model set out in Annex E of that
Directive.
4. Equines (including zebras) must be accompanied by a certificate complying with Annex
C of Directive 90/426/EEC.
5. Poultry and ratites falling under the scope of Directive 90/539/EEC must be
accompanied by a certificate complying with Annex IV of that Directive.
V. ANIMAL HEALTH LEGISLATION IN EUROPE
Peter Dollinger
VDZ / zooschweiz / EAZWV Office

Berne, Switzerland
(Chapter updated in 2009)
1. Introduction
As a result of the Uruguay round, an Agreement on the Application of Sanitary and

Phytosanitary Measures (the "SPS Agreement") was developed and adopted. It entered
into force with the establishment of the World Trade Organization on 1 January 1995. It
concerns the application of food safety and animal and plant health regulations. The
Agreement allows countries to set their own standards. But it also says regulations must be
based on science. They should be applied only to the extent necessary to protect human,
animal or plant life or health. And they should not arbitrarily or unjustifiably discriminate
between countries where identical or similar conditions prevail. Member countries are
encouraged to use international standards, guidelines and recommendations where they
exist. However, members may use measures which result in higher standards if there is
scientific justification. They can also set higher standards based on appropriate assessment
of risks so long as the approach is consistent, not arbitrary.
All European countries have issued legislation to control the spread of transmissible
diseases within their territory and to protect livestock and the human population from the
introduction of exotic diseases. As a function of diseases occurring in a given country, of the
trade patterns prevailing, and of the desired level of protection, legal provisions may vary
from one country to another.
There are, however, two mechanisms leading in many respects to a standardisation of the
legal requirements throughout the whole of Europe:
a. all countries, except the Holy See (where hardly any animals are kept), Monaco and
San Marino (which have to apply EC legislation) are members of the Office
International des Epizooties, and
b. 27 countries (Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Ireland, Italy, Latvia, Lithuania,
Luxemburg, Malta, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden,
The Netherlands and the United Kingdom) are members of the European Union. EU
veterinary legislation applies also in the British Crown Dependencies (the Channel
Islands and the Isle of Man), the Faeroe Islands, Andorra (Protocol on veterinary
matters supplementary to the agreement in the form of an exchange of letters
between the European Economic Community and the Principality of Andorra - OJ L
148 06.06.1997 p.16) and San Marino (Decision No 1/94 of the EC-San Marino
Cooperation Committee of 28 June 1994 on Community veterinary regulations to be
adopted by the Republic of San Marino - OJ L 238 13.09.1994 p.25). Norway has
adopted Community legislation in the framework of the EEA Agreement (Iceland and
Liechtenstein are also members of the EEA Agreement, but have been exempted
V. Animal Health Legislation in Europe
from its veterinary provisions). Switzerland and the EU have concluded a bilateral
agreement by which the equivalence of the respective animal health legislation has
been mutually recognised (Agreement between the European Community and the
Swiss Confederation on Trade in Agricultural Products). This agreement entered into
force in spring 2002. In the meantime, Switzerland has been almost fully integrated
into the EU veterinary area, and Annex 11 of the agreement, dealing with veterinary
matters, has been made applicable also to Liechtenstein. As a consequence,
veterinary checks on shipments between Switzerland, Liechtenstein and the EU have
been waived, and Switzerland carries out EU Third Country checks at the two
intercontinental airports of Zurich and Geneva. Finally, there is a REGULATION (EC)
No 998/2003 on the animal health requirements applicable to the non-commercial
movement of pet animals and which addresses also the movement of pet animals
between the EU and the Holy See and Iceland
The present chapter is, therefore, limited to an introduction to OIE mechanisms and
standards and to the EU legislation on animal health.
2. The Office International des Epizooties (OIE)
The OIE is an independent world wide organisation whose membership is

made up from 172 sovereign states. The headquarters are located at Paris.
The organisation maintains an internet site at the URL http://www.oie.int
which provides up-to-date information on the animal health situation in
member countries and gives online access to some relevant OIE
publications.
OIE’s objectives are to
 Ensure transparency in the global animal disease situation

 Collect, analyse and disseminate veterinary scientific information
 Encourage international solidarity in the control of animal diseases
 Safeguard world trade by publishing health standards for international trade in animals
and animal products
 Improve the legal framework and resources of national Veterinary Services
 Provide a better guarantee of food of animal origin and to promote animal welfare
through a science-based approach
The OIE was originally founded to combat highly contagious cattle diseases, like rinderpest
and foot-and-mouth disease, and focuses today primarily on diseases affecting agricultural
livestock and which have either severe economic implications or a zoonotic potential. In the
OIE Terrestrial Animal Health Code, it makes recommendations to the veterinary
administrations of importing and exporting countries on how to regulate trade in animals in
order to prevent the introduction of transmissible diseases.
The complete text of the Terrestrial Animal Health Code 2008 can be found at the URL
http://www.oie.int/eng/normes/mcode/en_sommaire.htm. The Code contains a list of
diseases for which recommendations are made:
 Multiple species diseases: This section contains 26 diseases including
anthropozoonoses (e.g. brucellosis), parasitoses (e.g. echinococcosis) and viral
diseases affecting a wider range of taxa (e.g. foot-and-mouth disease).
 Cattle diseases contain 14 diseases including e.g. BVD and IBR/IPV, but also BSE and
bovine tuberculosis, which, interestingly, are not rated as multiple species diseases.
 Sheep and goat diseases include a list of eleven, like e.g. Maedi-Visna, scrapie, or
sheep and goat pox.
 There are also 11 equine diseases, including e.g. African horse sickness, equine
infectious anemia, or glanders.
 The seven swine diseases include traditionally well-known, highly contagious diseases,
such as African and classical swine fever, but also diseases, which were discovered, or
were considered of economic relevance, only recently such as Nipah virus or the porcine
reproductive and respiratory syndrome.
 Avian diseases include 14 traditionally well-known diseases. Avian flu is listed as
“Highly pathogenic avian influenza in birds and low pathogenicity notifiable avian
influenza in poultry”, meaning that low pathogenic avian influenza in wild birds does not
fall under the scope of the Code.
 The remaining three categories are made up by two Lagomorph diseases

(myxomatosis and rabbit haemorrhagic disease), seven diseases of bees, and two
other diseases, namely camel pox and leishmaniosis.
The Code recommends measures mainly with regard to diseases in domestic animals.
Wildlife is rarely explicitly mentioned, but the same criteria may be applied mutatis mutandis.
A separate standard which is also regularly updated is the International Aquatic Animal
Health Code. It currently refers to 10 fish, 7 crustaceans, and 7 mollusc diseases.
To improve the knowledge about the presence of infectious diseases in wildlife, however,
and to create awareness, an OIE Working Group on Wildlife Diseases has produced
annual reports since 1992. In 1996, an OIE-EAZWV Working Group began with the drafting
of a recommendation on zoonoses transmissible from non-human primates. In 1998, the
draft was adopted and included as Chapter 6.9 in the OIE Terrestrial Animal Health Code.
The chapter focuses on defining the health of non-human primates and on the practice of
protective measures against disease transmission. It emphasises the process of quarantining
after international transportation, and stresses that some degree of risk for zoonotic disease
transmission should always be recognised. In 1999, an annex on quarantine requirements
was adopted. It is now Chapter 5.9 of the Code.
For standards for testing non-human primates, the OIE Manual of Standards for
Diagnostic Tests and Vaccines (the full text of the Standards Manual is found at
http://www.oie.int/eng/normes/mmanual/a_summry.htm) recommends consulting the
following document: Health monitoring of non-human primate Colonies. Recommendations of
the Federation of European Laboratory Animal Science Associations (FELASA) Working
Group on Non-Human Primate Health accepted by the FELASA Board of Management, 21
November 1998 (http://www.lal.org.uk/pdffiles/LAfel5.pdf) .
3. The Veterinary Legislation of the European Union
In 1964, the European Union – at that time still as the European

Economic Community – started to issue legislation in the
veterinary field. The first of these legal acts has been amended
many times but is still in effect. Originally, the EU has produced
only Directives and Decisions in the veterinary field, as of
1990, also Regulations were issued, e.g. in the context of
BSE, animal by-products, pet animals, or circus animals.
As at March 1, 2009, there were 938 valid acts in the field of animal health and zootechnics.
Many of these have been repeatedly amended, and in a number of cases it had become
necessary to amalgamate the original version and all amendments to a consolidated version
serving as a documentation tool.
 Directives are addressed to the member states and must be implemented through
national legislation. This leaves the national authorities with some degree of freedom
regarding the ways by which they want to achieve the goals and policies set by the EU.
 Decisions must be implemented to the letter (examples: certificates, lists of approved
establishments).
 Regulations are addressed directly to EU citizens and companies, i.e. the authorities
have to follow them very closely.
If the EU has decided to control or eradicate a disease, the measures agreed upon are
binding for all member states. This means e.g. that an individual member state is not allowed
to vaccinate against a given disease if a non-vaccination policy is applied by the Community.
In areas not regulated by the EU, Member States are more or less free to set their own rules.
These must not lead to a distortion of intra-Community Trade. The countries may, however,
apply for additional guarantees if they have been able to eliminate a disease from their
territory or parts thereof.
The following are some EU acts of relevance either for the monitoring and control of
transmissible diseases or laying down conditions for the intra-community trade and import
from third countries. The text of these acts or other legislation can be found at the URL
http://eur-lex.europa.eu/. Most of the texts referred to are relevant also in the EEA context
(Norway, Liechtenstein) or under the bilateral agreement with Switzerland.
A. Trade and placing on the market
The legislation referred to in this section defines the conditions under which animals may be
moved between EU Member States. As a general rule, it is required that the animals come
from an area and from a holding which is free from certain diseases. The animals themselves
must be identified in agreement with prescribed marking systems (e.g. cattle, sheep and
goats must be double ear-tagged), or must be otherwise identifiable (e.g. horse passport),
and they must be healthy, in particular free from specified diseases, and fit for transport.
Most requirements apply to all Member States. As a result of a particularly favourable health
situation, certain member States are, however, allowed to request additional guarantees (e.g.
Denmark, Austria and others regarding IBR/IPV, or the majority of Member States regarding
Aujeszky’s Disease) and/or to take specific precautions (e.g. United Kingdom regarding
rabies).
 Council Directive 64/432/EEC of 26 June 1964 on animal health problems affecting intra-
Community trade in bovine animals and swine - OJ P 121 29.07.1964, p.1977.
 Council Directive 91/68/EEC of 28 January 1991 on animal health conditions governing intra-
Community trade in ovine and caprine animals - OJ L 46, 19. 2. 1991, p. 19.
 Council Directive 90/426/EEC of 26 June 1990 on animal health conditions governing the
movement and import from third countries of equidae - OJ L 224, 18. 8. 1990, p. 42.
 Commission Decision 93/623/EEC of 20 October 1993 establishing the identification document

(passport) accompanying registered equidae - OJ L 298, 3.12.1993, p. 45–55.
 Commission Regulation (EC) No 504/2008 of 6 June 2008 implementing Council Directives

90/426/EEC and 90/427/EEC as regards methods for the identification of equidae - OJ L 149,
7.6.2008, p. 3–32.
 Council Directive 90/539/EEC of 15 October 1990 on animal health conditions governing intra-
Community trade in, and imports from third countries of, poultry and hatching eggs - OJ L 303
31.10.1990, p.6.
 Commission Decision 2006/605/EC of 6 September 2006 on certain protection measures in
relation to intra-Community trade in poultry intended for restocking of wild game supplies - OJ
L 246, 8.9.2006, p. 12–14.
 Council Directive 91/67/EEC of 28 January 1991 concerning the animal health conditions
governing the placing on the market of aquaculture animals and products - OJ L 046
19.02.1991, p. 1.
 Council Directive 92/65/EEC of 13 July 1992 laying down animal health requirements governing
trade in and imports into the Community of animals, semen, ova and embryos not subject to
animal health requirements laid down in specific Community rules referred to in Annex A (I) to
Directive 90/425/EEC -OJ L 268, 14. 9. 1992, p. 54 – BALAI Directive.
 Regulation (EC) No 998/2003 of the European Parliament and of the Council of 26 May 2003 on
the animal health requirements applicable to the non-commercial movement of pet animals and
amending Council Directive 92/65/EEC – OJ L 146 13.06.2003, p.1.
 2003/803/EC: Commission Decision of 26 November 2003 establishing a model passport for the
intra-Community movements of dogs, cats and ferrets - OJ L 312, 27.11.2003, p. 1–13.
 Commission Regulation (EC) No 1739/2005 of 21 October 2005 laying down animal health
requirements for the movement of circus animals between Member States - OJ L 279
22.10.2005, p. 47.
 Regulation (EC) No 1760/2000 of the European Parliament and of the Council of 17 July 2000
establishing a system for the identification and registration of bovine animals and regarding the
labelling of beef and beef products and repealing Council Regulation (EC) No 820/97 - OJ L 204,
11.8.2000, p. 1–10.
 Commission Regulation (EC) No 644/2005 of 27 April 2005 authorising a special identification

system for bovine animals kept for cultural and historical purposes on approved premises as
provided for in Regulation (EC) No 1760/2000 of the European Parliament and of the Council -
OJ L 107 28.04.2005, p. 18.
 Commission Regulation (EC) No 509/1999 of 8 March 1999 concerning an extension of the

maximum period laid down for the application of ear-tags to bison (Bison bison spp.) - OJ L 60,
9.3.1999, p. 53.
 Council Regulation (EC) No 21/2004 of 17 December 2003 establishing a system for the
identification and registration of ovine and caprine animals and amending Regulation (EC) No
1782/2003 and Directives 92/102/EEC and 64/432/EEC - OJ L 005 09.01.2004, p. 8.
 Council Directive 2008/71/EC of 15 July 2008 on the identification and registration of pigs
(Codified version) - OJ L 213, 8.8.2008, p. 31–36.
After many years of lobbying by the zoo community and negotiations between EAZWV and
the EU Commission, the BALAI Directive was amended by Commission Regulation (EC)
No 1282/2002 of 15 July 2002 (OJ L 187/3). As a consequence, the Directive became more
acceptable to the zoo community and its Annexes A, C and E are now directly applicable in
Member States. As ANNEX C contains a few points which leave considerable room for
interpretation. EAZWV, in cooperation with the EU Commission and some national veterinary
services, developed recommendations aiming at contributing to a uniform application of the
Directive, and thus at achieving the ultimate goal of this annex, namely to facilitate the
exchange of animals between approved zoos easily and without major health risks. In spite
of this effort, differences between Members States in implementing the Directive remained,
and, in 2009, e.g. France still does not apply the Directive with regard to zoos.
The core body of Directive 92/65/EEC, the Regulation 1282/2002 and the recommendations
are annexed to this chapter.
B. Importation from third countries
A common import regime was established in 1972. On the basis of an assessment of the
health situation in Third Countries and of the reliability of their veterinary services, the EU
Commission has – for certain species – drawn up lists of countries eligible to export animals
to the Community starting. As far as harmonised import conditions exist, the animals must be
accompanied by an official veterinary health certificate which follows the models given in the
specific directives or decisions, they must undergo a border veterinary check at an approved
checkpoint on arrival, and they must undergo a quarantine period under supervision by the
official veterinarian. The original Council Directive 72/462/EEC applied to cattle, sheep, goats
and swine only. Successively other Directives regulating the import of other species were
adopted, and in 2004 the old 72/462/EEC was replaced by a new Directive, which adds all
wild even-toed ungulates, rhinos, tapirs and elephants to the list of regulated species.
 Council Directive 2004/68/EC of 26 April 2004 laying down animal health rules for the
importation into and transit through the Community of certain live ungulate animals, amending
Directives 90/426/EEC and 92/65/EEC and repealing Directive 72/462/EEC - OJ L 139
30.04.2004 p. 320.
Directive 2004/68/EC addresses the import and transit of even-toed ungulates of wild
species, and of rhinos, tapirs and elephants. Wild equids still fall under the scope of Directive
90/426/EEC. In Annex I, a list of the relevant taxa is given. Annex II defines the basic
conditions for a country being considered free from certain diseases. Annex III establishes
the basic requirements for veterinary certificates. Annex IV replaces Annex F of Directive
90/426/EEC, and Annex V contains a list of implementing rules for the import of live animals,
meat and meat products, which shall remain in force until replaced by measures adopted
under the new regulatory framework.
 Council Decision 79/542/EEC of 21 December 1979 drawing up a list of third countries from
which the Member States authorize imports of bovine animals, swine and fresh meat - OJ L 146
14.06.1979 p. 15.
Decision 79/542/EEC underwent frequent amendments – in 2008 alone it was amended six
times, and it is strongly recommended to consult the consolidated text, which now contains
also model certificates.
 Commission Decision 2004/212/EC of 6 January 2004 on Community health conditions on

imports of animals and fresh meat including minced meat from third countries and amending
Decisions 79/542/EEC, 2000/572/EC and 2000/585/ - OJ L 073 11.03.2004, p. 11.
 Commission Decision 2004/211/EC of 6 January 2004 establishing the list of third countries
and parts of territory thereof from which Member States authorise imports of live equidae and
semen, ova and embryos of the equine species, and amending Decisions 93/195/EEC and
94/63/EC - OJ L 073 11.03.2004, p. 1.
 2007/240/EC: Commission Decision of 16 April 2007 laying down new veterinary certificates
for importing live animals, semen, embryos, ova and products of animal origin into the Community
pursuant to Decisions 79/542/EEC, 92/260/EEC, 93/195/EEC, 93/196/EEC, 93/197/EEC,
95/328/EC, 96/333/EC, 96/539/EC, 96/540/EC, 2000/572/EC, 2000/585/EC, 2000/666/EC,
2002/613/EC, 2003/56/EC, 2003/779/EC, 2003/804/EC, 2003/858/EC, 2003/863/EC,
2003/881/EC, 2004/407/EC, 2004/438/EC, 2004/595/EC, 2004/639/EC and 2006/168/EC - OJ L
104, 21.4.2007, p. 37–50.
 Commission Regulation (EC) No 798/2008 of 8 August 2008 laying down a list of third
countries, territories, zones or compartments from which poultry and poultry products may be
imported into and transit through the Community and the veterinary certification requirements -
OJ L 226, 23.8.2008, p. 1–94.
 Council Directive 72/462/EEC of 12 December 1972 on health and veterinary inspection
problems upon importation of bovine animals and swine and fresh meat from third countries -
OJ L 302 31.12.1972, p.28.
 Commission Decision 93/198/EEC of 17 February 1993 laying down a model for the animal
health conditions and veterinary certification for the importation of domestic ovine and caprine
animals from third countries - OJ L 086 06.04.1993, p.34.
 Council Directive 90/426/EEC of 26 June 1990 on animal health conditions governing the
movement and import from third countries of equidae - OJ L 224 18.08.1990, p.42.
 Commission Decision 93/197/EEC of 5 February 1993 on animal health conditions and veterinary
certification for imports of registered equidae and equidae for breeding and production - OJ L 086
06.04.1993, p.1.
 Commission Decision 2003/459/EC of 20 June 2003 on certain protection measures with regard to
monkey pox virus - OJ L 154, 21.6.2003, p. 112–113).
Decision 2003/459/EC aims at preventing the introduction of monkey pox by the importation
of Prairie dogs (Cynomys spp.) from the United States.
 Council Directive 90/539/EEC of 15 October 1990 on animal health conditions governing intra-
Community trade in, and imports from third countries of, poultry and hatching eggs - OJ L 303
31.10.1990, p.6.
 Commission Decision 96/482/EC of 12 July 1996 laying down animal health conditions and
veterinary certificates for the importation of poultry and hatching eggs other than ratites and
eggs thereof from third countries including animal health measures to be applied after such
importation - OJ L 196 07.08.1996, p.13.
 Commission Regulation (EC) No 318/2007 of 23 March 2007 laying down animal health conditions
for imports of certain birds into the Community and the quarantine conditions thereof - OJ L 84,
24.3.2007, p. 7–29.
 Commission Decision 2006/696/EC of 28 August 2006 laying down a list of third countries from
which poultry, hatching eggs, day-old chicks, meat of poultry, ratites and wild game-birds,
eggs and egg products and specified pathogen-free eggs may be imported into and transit through
the Community and the applicable veterinary certification conditions, and amending Decisions
93/342/EEC, 2000/585/EC and 2003/812/EC - OJ L 295, 25.10.2006, p. 1–76.
 2003/881/EC: Commission Decision of 11 December 2003 concerning the animal health and
certification conditions for imports of bees (Apis mellifera and Bombus spp.) from certain third
countries and repealing Decision 2000/462/ - OJ L 328, 17.12.2003, p. 26–31.
 Commission Decision 2004/595/EC of 29 July 2004 establishing a model health certificate for the
importation into the Community for trade of dogs, cats and ferrets - OJ L 266 13.08.2004, p. 11.
 Commission Decision 2004/824/EC of 1 December 2004 establishing a model health certificate for
non-commercial movements of dogs, cats and ferrets from third countries into the Community -
OJ L 358 03.12.2004, p. 12.
 Commission Decision 2005/64/EC of 26 January 2005 implementing Council Directive 92/65/EEC
as regards import conditions for cats, dogs and ferrets for approved bodies, institutes or centres -
OJ L 027 29.01.2005, p. 48.
In the case of taxa not regulated by Community legislation, national rules apply. Please note
that new legislation is likely to come into force in the near future that will further harmonise
rules (certification and animal health requirements) for the importation of other wild animals.
Because of these rules, imports of such animals will only be allowed from a small number of
third countries authorised for each species. However a draft Decision has been discussed
which foresaw a particular regime for importation of live animals originating in any third
country but imported after a residency period in St. Pierre and Miquelon (a little island in the
Atlantic Ocean close to Canada) where they will spend a period in a quarantine station.
During this period, specific testing would be carried out on the animals. For the moment,
these special conditions are limited to the import of live Camelidae, but the intention has
been expressed to extend this possibility to other species.
In the event of a disease appearing in a country from which imports normally are permitted,
the Commission may decide on specific protective measures, including a temporary import
ban, e.g.:
 Commission Decision 2006/563/EC of 11 August 2006 concerning certain protection measures in
relation to highly pathogenic avian influenza of subtype H5N1 in wild birds in the Community and
repealing Decision 2006/115/EC - OJ L 222, 15.8.2006, p. 11–19.
 Commission Decision 2005/759/EC of 27 October 2005 concerning certain protection measures in
relation to highly pathogenic avian influenza in certain third countries and the movement from
third countries of birds accompanying their owners - OJ L 285 28.10.2005, p. 52.
C. Biosecurity measures
The following legislation contains specific rules for controlling and eradicating certain
diseases. In the case of the outbreak of a highly contagious or of an emerging disease, the
Commission will take specific Decisions ad hoc, e.g. defining the applicable infection and
surveillance zones and the specific trade restrictions to be observed.
 Council Directive 77/391/EEC of 17 May 1977 introducing Community measures for the
eradication of brucellosis, tuberculosis and leucosis in cattle - OJ L 145 13.06.1977, p.44.
 2004/226/EC: Commission Decision of 4 March 2004 approving tests for the detection of
antibodies against bovine brucellosis within the framework of Council Directive 64/432/EEC -
OJ L 68, 6.3.2004, p. 36–37.
 Council Directive 85/511/EEC of 18 November 1985 73/53/EEC: introducing Community
measures for the control of foot-and-mouth disease - OJ L 315, 26. 11. 1985, p. 11.
 Council Directive 90/423/EEC of 26 June 1990 amending Directive 85/511/EEC introducing
Community measures for the control of foot-and-mouth disease, Directive 64/432/EEC on
animal health problems affecting intra-Community trade in bovine animals and swine and
Directive 72/462/EEC on health and veterinary inspection problems upon importation of bovine,
ovine and caprine animals and swine, fresh meat or meat products from third countries - OJ L
224, 18. 8. 1990, p. 13.
 Commission Decision 2001/303/EC of 11 April 2001 laying down the conditions for the control
and eradication of foot-and-mouth disease in endangered species in application of Article 13
of Directive 85/511/EEC - OJ L 104 , 13/04/2001, p.3.
 Council Directive 2000/75/EC of 20 November 2000 laying down specific provisions for the
control and eradication of bluetongue – OJ L327 22.12.2000, p.74.
 Commission Decision 2004/558/EC of 15 July 2004 implementing Council Directive 64/432/EEC

as regards additional guarantees for intra-Community trade in bovine animals relating to
infectious bovine rhinotracheitis and the approval of the eradication programmes presented by
certain Member States - OJ L 249, 23.7.2004, p. 20–25.
 Regulation (EC) No 999/2001 of the European Parliament and of the Council of 22 May 2001
laying down rules for the prevention, control and eradication of certain transmissible
spongiform encephalopathies – OJ L147 31.05.2001, p.1. This regulation has been amended
very frequently, for the last time by Commission Regulation (EC) No 163/2009 of 26 February
2009.
 Commission Decision 2007/453/EC of 29 June 2007 establishing the BSE status of Member
States or third countries or regions thereof according to their BSE risk - OJ L 172, 30.6.2007, p.
84–86.
 Commission Regulation (EC) No 546/2006 of 31 March 2006 implementing Regulation (EC)
No 999/2001 of the European Parliament and of the Council as regards national scrapie control
programmes and additional guarantees and derogating from certain requirements of Decision
2003/100/EC and repealing Regulation (EC) No 1874/2003 - OJ L 94, 1.4.2006, p. 28–31.
 Council Directive 92/119/EEC of 17 December 1992 introducing general Community measures
for the control of certain animal diseases and specific measures relating to swine vesicular
disease - OJ L 62, 15. 3. 1993, p. 69.
 Council Directive 2001/89/EC of 23 October 2001 on Community measures for the control of
classical swine fever – OJ L316 01.12.2001, p.5 (note: this directive repeals 80/217/EEC).
 Council Directive 2002/60/EC of 27 June 2002 laying down specific provisions for the control of
African swine fever and amending Directive 92/119/EEC as regards Teschen disease and
African swine fever - OJ L 192, 20.7.2002, p. 27–46.
 Commission Decision 2008/185/EC of 21 February 2008 on additional guarantees in intra-
Community trade of pigs relating to Aujeszky’s disease and criteria to provide information on
this disease (notified under document number C(2008) 669) (Codified version) (Text with EEA
relevance)
- OJ L 59, 4.3.2008, p. 19–30.
 Council Directive 92/35/EEC of 29 April 1992 laying down control rules and measures to combat
African horse sickness - OJ L 157, 10. 6. 1992, p. 19.
 Council Directive 2005/94/EC of 20 December 2005 on Community measures for the control of
avian influenza and repealing Directive 92/40/EEC - OJ L 10, 14. 1. 2006, p. 16.
 Commission Decision 2005/464/EC of 21 June 2005 on the implementation of survey
programmes for avian influenza in poultry and wild birds to be carried out in the Member States -
OJ L 164 24.06.2005, p. 52.
 Commission Decision 2005/731/EC of 17 October 2005 laying down additional requirements for
the surveillance of avian influenza in wild birds - OJ L 274 20.10.2005, p. 93. Validity extended
until 31 December 2009 by Commission Decision 2009/6/EC of 17 December 2008.
 Commission Decision 2005/734/EC of 19 October 2005 laying down biosecurity measures to
reduce the risk of transmission of highly pathogenic avian influenza caused by Influenza virus A
subtype H5N1 from birds living in the wild to poultry and other captive birds and providing for an
early detection system in areas at particular risk - OJ L 274 20.10.2005, p. 105.
 2006/415/EC: Commission Decision of 14 June 2006 concerning certain protection measures in
relation to highly pathogenic avian influenza of the subtype H5N1 in poultry in the Community
and repealing Decision 2006/135/EC - OJ L 164, 16.6.2006, p. 51–60.
 2006/563/EC: Commission Decision of 11 August 2006 concerning certain protection measures
in relation to highly pathogenic avian influenza of subtype H5N1 in wild birds in the
Community and repealing Decision 2006/115/EC - OJ L 222, 15.8.2006, p. 11–19.
 2007/598/EC: Commission Decision of 28 August 2007 concerning measures to prevent the
spread of highly pathogenic avian influenza to other captive birds kept in zoos and approved
bodies, institutes or centres in the Member States - OJ L 230, 1.9.2007, p. 20–26.
 Council Directive 92/66/EEC of 14 July 1992 introducing Community measures for the control of
Newcastle disease - OJ L 260, 5. 9. 1992, p. 1.
 Council Directive 2006/88/EC of 24 October 2006 on animal health requirements for aquaculture
animals and products thereof, and on the prevention and control of certain diseases in aquatic
animals - OJ L 328, 24.11.2006, p. 14–56.
D. Notification of diseases
Disease notification is the basis for disease control. Any veterinarian suspecting the
presence of a disease which is notifiable under EU or national legislation is under an
obligation to immediately contact the competent official veterinarian or the competent
veterinary office/service and to communicate their suspicion.
 Council Directive 82/894/EEC of 21 December 1982 on the notification of animal diseases within
the Community - OJ L 378, 31. 12. 1982, p. 58.
 Commission Decision 2005/176/EC of 1 March 2005 laying down the codified form and the
codes for the notification of animal diseases pursuant to Council Directive 82/894/EEC - OJ L
059 05.03.2005, p. 40. Amended by Decisions 924/2006/EC and 2008/755/EC (no consolidated
text available).
Zoo veterinarians must be particularly aware of the list of notifiable diseases contained in
Annex a of Directive 92/65/EEC (BALAI).
E. Mixed texts
 Directive 2003/99/EC of the European Parliament and of the Council of 17 November 2003 on the
monitoring of zoonoses and zoonotic agents, amending Council Decision 90/424/EEC and
repealing Council Directive 92/117/ - OJ L 325 12.12.2003, p.31.
 Council Directive 89/608/EEC of 21 November 1989 on mutual assistance between the

administrative authorities of the Member States and cooperation between the latter and the
Commission to ensure the correct application of legislation on veterinary and zootechnical matters
- OJ L 351, 2. 12. 1989, p. 34.
 Regulation (EC) No 1774/2002 of the European Parliament and of the Council of 3 October 2002
laying down health rules concerning animal by-products not intended for human consumption
- OJ L 273 10.10.2002, p.1.
 Commission Decision 2003/322/EC of 12 May 2003 implementing Regulation (EC) No 1774/2002 of

the European Parliament and of the Council as regards the feeding of certain necrophagous birds
with certain category 1 materials - OJ L 117, 13.5.2003, p. 32–34.
Regulation 1774/2002 is extremely complex, sometimes contradicting itself, and the public
interest in some of the provisions is not evident. Within six years, there have been 49
amendments, 33 derogations, two corrections and nine consolidated versions. In addition,
the Regulation was affected by four court cases. From a zoo perspective, in particular the
definition of Category 1 material contained in Article 4 (1) a) is not acceptable and cannot be
implemented. Dead animals other than farmed animals and wild animals, including in
particular pet animals, zoo animals and circus animals, and experimental animals are
considered to fall under this category and are assumed to be directly disposed of as waste
by incineration in an incineration plant. Therefore, it is theoretically not permitted to feed
guinea pigs, laboratory rats or laboratory mice to reptiles, owls, raptors or small carnivores,
which may create a conflict with national animal welfare requirements. While the regulation
permits a zoo to feed to its carnivores or raptors a sick or wounded animal that perished in
the wild, it prevents the feeding of healthy surplus animals shot or euthanised for
management reasons at a zoo. If a zoo, however, were to declare itself being a game farm,
the same deer could be killed for human consumption.
Under Commission Decision 2003/322/EC the feeding of free-living vultures in France,

Greece, Italy, Portugal and Spain with certain category 1-materials is allowed. It could be
argued that the same should be allowable in the case of vultures kept by zoos.
F. Veterinary checks
The following Directives and Decisions describe the veterinary checks applicable in intra-
community trade and on importation, and they define the list of approved border checkpoints.
The most important recent development in this field is the introduction of the TRACES
System on April 1, 2004. This TRAde Control and Expert System combines the functions of
the previous ANIMO and SHIFT systems by creating a single central database to track the
movement of animals and certain types of products both within the EU and from outside the
EU. Consequently the duplication of data is avoided. TRACES is designed to be used
directly by economic operators under the control of the competent veterinary authorities, so
relevant information can easily be shared with customs authorities.
 Council Directive 89/662/EEC of 11 December 1989 concerning veterinary checks in intra-

Community trade with a view to the completion of the internal market OJ L 395, 30.12.1989, p.
13–22.
 Council Directive 90/425/EEC of 26 June 1990 concerning veterinary and zootechnical checks
applicable in intra-Community trade in certain live animals and products with a view to the
completion of the internal market - OJ L 224, 18. 8. 1990, p. 29.
 Commission Decision 94/339/EC of 25 May 1994 laying down detailed rules for the application of
Article 9.1 of Council Directive 90/425/EEC concerning veterinary and zootechnical checks
applicable in intra-Community trade in certain live animals and products with a view to the
completion of the internal market - OJ L 151 17.06.1994, p.38.
 Commission Regulation (EC) No 599/2004 of 30 March 2004 concerning the adoption of a

harmonised model certificate and inspection report linked to intra-Community trade in animals
and products of animal origin - OJ L 094 31.03.2004, p. 44.
 Council Directive 91/496/EEC of 15 July 1991 laying down the principles governing the
organization of veterinary checks on animals entering the Community from third countries and
amending Directives 89/662/EEC, 90/425/EEC and 90/675/EEC - OJ L 268, 24. 9. 1991, p. 56.
 Commission Decision 2001/812/EC of 21 November 2001 laying down the requirements for the
approval of border inspection posts responsible for veterinary checks on products introduced
into the Community from third countries – OJ L306 23.11.2001, p.28.
 Commission Decision 2001/881/EC of 7 December 2001 drawing up a list of border inspection

posts agreed for veterinary checks on animals and animal products from third countries and
updating the detailed rules concerning the checks to be carried out by the experts of the
Commission – OJ L326 11.12.2001, p.44. Amended by 2003/506/EC - OJ L 172 10.07.2003, p.16.
 Commission Regulation (EC) No 136/2004 of 22 January 2004 laying down procedures for
veterinary checks at Community border inspection posts on products imported from third
countries - OJ L 021 28.01.2004, p. 11.
 2007/275/EC: Commission Decision of 17 April 2007 concerning lists of animals and products to
be subject to controls at border inspection posts under Council Directives 91/496/EEC and
97/78/EC - OJ L 116, 4.5.2007, p. 9–33.
 Commission Decision 97/794/EC of 12 November 1997 laying down certain detailed rules for the
application of Council Directive 91/496/EEC as regards veterinary checks on live animals to be
imported from third countries - OJ L 323, 26.11.1997, p. 31–36.
 Commission Regulation (EC) No 282/2004 of 18 February 2004 introducing a document for the
declaration of, and veterinary checks on, animals from third countries entering the Community -
OJ L 049 19.02.2004, p. 11.
 Regulation (EC) No 882/2004 of the European Parliament and of the Council of 29 April 2004 on
official controls performed to ensure the verification of compliance with feed and food law, animal
health and animal welfare rules - OJ L 165, 30.4.2004, p. 1–141.
 Commission Decision 2003/623/EC of 19 August 2003 concerning the development of an

integrated computerised veterinary system known as Traces - OJ L 216 28.08.2003, p. 58.
 Commission Decision 2004/292/EC of 30 March 2004 on the introduction of the Traces system
and amending Decision 92/486/EEC (OJ L 094 31.03.2004, p. 63), amended by Commission
Decision 2005/515/EC of 14 July 2005 - OJ L 187 19.07.2005, p. 29.
4. Zoonoses – a special problem

Only a small proportion of the more than 200 communicable diseases known to be common
to man and animals are contained in the lists of the OIE Terrestrial Animal Health Code. With
a focus mainly on food-borne diseases, the European Union obliges Members States to
ensure that data on the occurrence of zoonoses and zoonotic agents and antimicrobial
resistance related thereto are collected, analysed and published without delay. Annex I of
Directive 2003/99/EC contains a list of 8 diseases or agents, which should be included in
monitoring, and another 16 diseases which should be monitored according to the
epidemiological situation. The
zoonoses covered by the second list may not always be regulated by national legislations.
The consequence of this situation is that zoonoses often are only detected after an animal
has been introduced into the collection and has either fallen sick and died, or other animals
or humans have been infected. This situation is largely due to veterinary administrations
primarily addressing diseases of agricultural livestock. Apart from a few zoonoses addressed
by veterinary services in all countries, like rabies, brucellosis, bovine tuberculosis etc., there
is no official network of officially approved diagnostic and reference laboratories for the bulk
of zoonotic diseases. When confronted with an import application for zoo animals, import
conditions are often established on an ad hoc basis which may not necessarily be
scientifically sound. To reduce the risk of introducing zoonoses by international trade and of
their spreading in zoo collections and to zoo staff, measures have to be taken at several
levels.
Measures by veterinary administrations
Import requirements for zoo animals should be defined in compliance with the OIE Code.
Where no such standards exist, a sound risk assessment has to be made, or quarantine
procedures of national zoo organisations may be followed if these are available. Certification
requirements should not be overemphasised, but proper quarantine should be ensured at the
importing zoos. Veterinary supervision of zoos should be mandatory, and this could be
best achieved by subjecting the operation of a zoo to licensing and to approval under the
BALAI Directive (92/65/EEC).
Measures by the zoological gardens
Zoos should keep high hygienic standards for animals, keepers and food, implement
veterinary controlled quarantine for all incoming animals (if there are no other requirements
usually 30 days, unless the judgement of the veterinarian allows for shortening this period of
time), avoid contact to neighbouring farms, implement a control programme for rats and
mice, and attempt to exclude other local free-ranging wild mammals from the zoo, as these
may be potential carriers of zoonoses. There should be close clinical, parasitological and
post-mortem surveillance of the collections and of free roaming wild animals, treatment
or vaccination of susceptible animals for relevant zoonoses, collection of blood samples for
direct diagnosis and for establishing serum banks. Exposed staff should be included in the
surveillance and prophylactic measures.
In Children's Zoos, there should be educational signage about how to behave for minimising
the risk of disease transmission, hand-washing stations should be available and eating and
drinking should not be allowed in the contact area.
Measures by the zoo and wildlife veterinarians’ organisations
Zoonoses should be a prominent feature in scientific venues. Regional organisations

should co-operate with the OIE Working Group on Wildlife Diseases, and should follow the
example of the European Wildlife Disease Association (EWDA) in producing regional
reports. As with the AAZV (American Association of Zoo Veterinarians) in North America,
they should co-operate with the zoo organisations of their region in establishing procedures
for minimising the zoonosis risk. EAZWV should continue to co-operate with OIE in improving
international standards, and should involve other groups from within the WAWV (World
Association of Wildlife Veterinarians) family.
5. Certification procedures
International movements of many animal species are only possible if the animal is
accompanied by a veterinary certificate. Unless otherwise defined, such certificates have to
be issued by an official veterinarian who, however, would often have to base his statement
on the findings of another veterinarian. Hence it follows that zoo or institute veterinarians
may be required to certify certain facts to the official veterinarian, who in turn will use the
information received for issuing an official certificate.
Under the revised BALAI Directive, the veterinarian of an approved zoo or institute is
authorised to issue certificates for certain species when moved between EU Members States
or between the EU and a Third Country under a bilateral agreement.
In order to maintain confidence in the certification process, it is necessary that certification is

based on the highest possible ethical standards, the most important of which is that the
professional integrity of the certifying veterinarian must be respected and safeguarded.
It is essential not to include in the requirements additional specific matters which cannot be
accurately and honestly signed by a veterinarian. For example these requirements should
not include certification of an area as being free from non-notifiable diseases, the occurrence
of which the signing veterinarian is not necessarily informed about. Equally, to ask
certification for events which will take place after the document is signed is unacceptable
when these events are not under the direct control and supervision of the signing
veterinarian.
Guidelines for certifying veterinarians have been drawn up by professional organisations, the
certification process is described in the OIE Terrestrial Animal Health Code, and the EU also
has defined requirements. All these texts are very similar to each other.
The following is the text of the articles 5.2.2 and 5.2.3 of the OIE Terrestrial Animal Health
Code.
Preparation of international veterinary certificates

Certificates should be drawn up in accordance with the following principles:
1. Certificates should be designed so as to minimize the potential for fraud including use of
a unique identification number, or other appropriate means to ensure security. Paper
certificates should bear the official identifier of the issuing Veterinary Authority. Each
page of a multiple page certificate should bear the unique certificate number and a
number indicating the number of the page out of the total number of pages. Electronic
certification procedures should include equivalent safeguards.
2. They should be written in terms that are as simple, unambiguous and easy to
understand as possible, without losing their legal meaning.
3. If so required, they should be written in the language of the importing country. In such
circumstances, they should also be written in a language understood by the certifying
veterinarian.
4. They should require appropriate identification of animals and animal products except
where this is impractical (e.g. day-old birds).
5. They should not require a veterinarian to certify matters that are outside his/her
knowledge or which he/she cannot ascertain and verify.
6. Where appropriate, they should be accompanied, when presented to the certifying
veterinarian, by notes of guidance indicating the extent of enquiries, tests or
examinations expected to be carried out before the certificate is signed.
7. Their text should not be amended except by deletions which must be signed and
stamped by the certifying veterinarian. The signature and stamp must be in a colour
different to that of the printing of the certificate.
8. Replacement certificates may be issued by a Veterinary Authority to replace certificates
that have been, for example, lost, damaged, contain errors, or where the original
information is no longer correct. These must be clearly marked to indicate that they are
replacing the original certificate. A replacement certificate should reference the number
and the issue date of the certificate that it supersedes. The superseded certificate should
be cancelled and where possible, returned to the issuing authority.
9. Only original certificates are acceptable.
Certifying veterinarians
Certifying veterinarians should:
1. be authorised by the Veterinary Administration of the exporting country to sign
international veterinary certificates;
2. only certify matters that are within their own knowledge at the time of signing the
certificate, or that have been separately attested by another competent party;
3. sign only at the appropriate time certificates that have been completed fully and
correctly; where a certificate is signed on the basis of supporting documentation, the
certifying veterinarian should be in possession of that documentation before signing;
4. have no conflict of interest in the commercial aspects of the animals or animal products
being certified and be independent from the commercial parties.
Annex: Diseases listed in the OIE Terrestrial Animal Health Code

and applicable EU legislation
Disease OIE List Notifiable under 82/894/EEC (yes/no)
- Zoonosis under 2003/99/EC (A/B)*
- other relevant EU law**
Anthrax Multiple species no - 64/432/EEC (E-I) - 91/68/EEC (B-
I) - 92/65/EEC (A)
Aujeszky's disease Multiple species no - 64/432/EEC (E-II)- 2008/185/EC
Bluetongue Multiple species yes - 92/65/EEC (A) - 2000/75/EC -
2004/68/EC
(Bovine) brucellosis (Brucella abortus) Multiple species no – A - 64/432/EEC (E-I) -
77/391/EEC - 92/65/EEC (A) -
2004/226/EC
(Caprine/ovine) brucellosis (Brucella melitensis) Multiple species no - A -91/68/EEC (B-I) - 92/65/EEC
(A)
(Porcine) brucellosis (Brucella suis) Multiple species no - A - 64/432/EEC (E-I) - 92/65/EEC
(A)
Crimean Congo haemorrhagic fever Multiple species no - 2006/696/EC -
Echinococcosis/hydatidosis Multiple species no - A
Epizootic haemorrhagic disease Multiple species no - 92/119/EEC
Equine encephalomyelitis (Eastern) Multiple species yes - 90/426/EEC
Foot and mouth disease Multiple species yes - 64/432/EEC (E-I) - 2003/85/EC
(repealing 85/511/EEC) - 90/423/EEC -
91/68/EEC (B-I) - 92/65/EEC (A) -
2001/303/EC - 2004/68/EC
Heartwater Multiple species no
Japanese encephalitis Multiple species yes - 90/426/EEC
Leptospirosis Multiple species no
New world screwworm (Cochliomyia hominivorax) Multiple species no
Old world screwworm (Chrysomya bezziana) Multiple species no
Paratuberculosis Multiple species no - 91/68/EEC (B-III)
Q fever Multiple species No
Rabies Multiple species no - B - 64/432/EEC (E-I) - 91/68/EEC
(B-I) - 92/65/EC
Rift Valley fever Multiple species yes - 92/65/EEC (A) - 2004/68/EC
Rinderpest (cattle plague) Multiple species yes - 92/65/EEC (A) - 2004/68/EC
Surra (Trypanosoma evansi) Multiple species no -
Trichinellosis Multiple species no - A - 377/96/EEC
Tularemia Multiple species no - 92/65/EEC
Vesicular stomatitis Multiple species yes - 90/426/EEC - 92/65/EEC (A) -
2004/68/EC
West Nile fever Multiple species no
Bovine anaplasmosis Cattle no
Bovine babesiosis Cattle no
Bovine genital campylobacteriosis Cattle no - A
Bovine spongiform encephalopathy Cattle yes -92/65/EEC (A) (all TSE)

2001/9/EC - R(EC)999/2001
Bovine tuberculosis (Mycobacterium bovis) Cattle no - A - 64/432/EEC (E-I) - 77/391/EEC
- 92/65/EEC (A) - 2000/504/EC
Bovine viral diarrhea Cattle no
Contagious bovine pleuropneumonia Cattle yes - 64/432/EEC (E-I) - 92/65/EEC (A)
- 2004/68/EC
Enzootic bovine leukosis Cattle no - 64/432/EEC (E-I) - 77/391/EEC
Haemorrhagic septicaemia Cattle no
Infectious bovine rhinotracheitis/infectious pustular Cattle no - 64/432/EEC (E-II) - 2000/502/EC
vulvovaginitis
Lumpy skin disease Cattle yes 64/432/EEC - 92/65/EEC (A) -
2004/68/EC
Theileriosis Cattle no
Trichomonosis Cattle no
Trypanosomosis (tsetse-transmitted) Cattle no
Caprine viral arthritis/encephalitis Sheep and goat no - 91/68/EEC (B-III)
Contagious agalactia Sheep and goat no - 91/68/EEC (B-III)
Contagious caprine pleuropneumonia Sheep and goat no
Enzootic abortion of ewes (ovine chlamydiosis) Sheep and goat no
Maedi-visna Sheep and goat no - 91/68/EEC (B-III)
Nairobi sheep disease Sheep and goat no
Ovine epididymitis (Brucella ovis) Sheep and goat no - A - 91/68/EEC (B-I) - 92/65/EEC
(A)
Peste des petits ruminants Sheep and goat yes - 92/65/EEC (A) (concerns also
Suidae) - 2004/68/EC
Salmonellosis (S. abortusovis) Sheep and goat no - A
Scrapie Sheep and goat no - 91/68/EEC - 92/65/EEC (A) -
2001/9/EC - R(EC)999/2001
Sheep pox and goat pox Sheep and goat yes - 92/65/EEC (A) - 2004/68/EC
African horse sickness Equine yes - 92/35/EEC, 92/36/EEC,
92/65/EEC (A)
Contagious equine metritis Equine no -
Dourine Equine yes - 90/426/EEC
Equine encephalomyelitis (Western) Equine yes - 90/426/EEC
Equine infectious anaemia Equine yes - 90/426/EEC
Equine influenza Equine no -
Equine piroplasmosis Equine no -
Equine rhinopneumonitis Equine no -
Equine viral arteritis Equine no -
Glanders Equine yes - 90/426/EEC
Venezuelan equine encephalomyelitis Equine yes - 90/426/EEC
African swine fever Swine yes - 64/432/EEC (E-I) - 92/65/EEC (A)
- 2002/60/EC - 2004/68/EC
Classical swine fever Swine yes - 64/432/EEC (E-I) - 92/65/EEC (A)
- 2001/89/EC - 2004/68/EC
Nipah virus encephalitis Swine no

Porcine cysticercosis Swine no
Porcine reproductive and respiratory syndrome Swine no
Swine vesicular disease Swine yes - 64/432/EEC (E-I) - 92/65/EEC (A)
- 92/119/EEC - 2004/68/EC
Transmissible gastroenteritis Swine no - 64/432/EEC (E-II)
Avian chlamydiosis Avian no - B - 92/65/EEC (A) (in
Psittaciformes)
Avian infectious bronchitis Avian no -
Avian infectious laryngotracheitis Avian no -
Avian mycoplasmosis (M. gallisepticum) Avian no - 90/539/EEC
Avian mycoplasmosis (M. synoviae) Avian no
Duck virus hepatitis Avian no
Fowl cholera (Avian pasteurellosis) Avian no
Fowl typhoid (Salmonella gallinarum) Avian no - A - 90/539/EEC
Highly pathogenic avian influenza in birds and low Avian yes - 92/65/EEC (A) - 2005/94/EC -
pathogenic notifiable influenza in poultry as defined 2005/464/EC - 2005/731/EC -
in Chapter 10.4 2005/734/EC - 2005/759/EC -
2006/415/EC - 2006/563/EC -
2006/598/EC
Infectious bursal disease (Gumboro disease) Avian no
Marek's disease Avian no
Newcastle disease Avian yes - 92/65/EEC (A) - 92/66/EEC
Pullorum disease Avian no - A - 90/539/EEC
Turkey rhinotracheitis Avian no
Myxomatosis Lagomorph no - 92/65/EEC
Rabbit haemorrhagic disease Lagomorph no - 92/65/EEC
Acarapisosis of bees Bee no - 92/65/EEC
American foulbrood of honey bees Bee no - 92/65/EEC (A)
European foulbrood of honey bees Bee no
Small hive beetle infestation (Aethina tumida) Bee yes - 92/65/EEC (A)
Tropilaelaps infestation of honey bees Bee yes - 92/65/EEC (A)
Varroosis of honey bees Bee no - 92/65/EEC
Camel pox Other no
Leishmaniosis Other no
Ovine pulmonary adenomatosis -- no - 91/68/EEC (B-III)
Caseous lymphadenitis -- no - 91/68/EEC (B-III)
Ebola in non-human primates -- 92/65/EEC (A)
Monkey pox in rodents and non-human primates -- 92/65/EEC (A) - 2003/459/EC
Porcine enterovirus encephalomyelitis (Teschen) -- 92/65/EEC (A)
Avian tuberculosis -- no - A
Listeriosis -- no - A
Verotoxigenic Escherichia coli -- no – A
Calicivirus -- no – B
Hepatitis A virus -- no – B
Influenza virus -- no – B
Viruses transmitted by arthropodes -- no – B
Borreliosis and agents thereof -- no – B
Botulism and agents thereof -- no – B
Leptospirosis and agents thereof -- no – B
Tuberculosis other than mentioned above -- no – B
Vibriosis and agents thereof -- no – B
Yersiniosis -- no – B
Anisakiasis and agents thereof -- no – B
Cryptosporidiosis and agents thereof -- no – B
Cysticercosis and agents thereof -- no – B
Toxoplasmosis and agents thereof -- no – B
Other zoonoses an zoonotic agents -- no – B
* A = Monitoring is mandatory, B = To be monitored according to the epidemiological situation:

** 64/432/EEC: (E-I) = Compulsorily notifiable, (E-II) = Member States may have national control or monitoring
programmes
91/68/EEC: (B-I) = Compulsorily notifiable, (B-III) = Member States may have national control or monitoring
programmes
92/65/EEC (A) = Notifiable in the context of the BALAI Directive
VII. POST-MORTEM PROCEDURES
Gerry M. Dorrestein and Marein M. van der Hage
Diagnostic Pathology Laboratory

Dutch Research Institute for Avian and Exotic Animals (NOIVBD)
Veldhoven,The Netherlands
1. Introduction
Pathology is not a science for pathologists alone. The findings of the pathologist should be
one of the bases of the clinician’s understanding of disease and the pathophysiology of
disease. Changes in anatomic pathology are the foundation of disease, and understanding
these changes will give the clinician an advantage in selecting the right diagnostic tools and
therapeutic approach.
Determination of the cause of death in zoo animals is often difficult and may require the close
co-operation of a number of disciplines. Some zoo veterinarians perform a post-mortem
examination in the zoo. But in doing the post-mortem, careless observation and sampling
can result in useless, and sometimes even harmful, information. Those who perform post-
mortem examinations in this manner are running a risk, as overlooking fundamental changes
in tissue can be costly to both pocketbook and intellectual development (Cheville, N.F., 1999,
In: Introduction to Veterinary Pathology, Iowa State University Press/Ames).
When a post-mortem is performed in the zoo itself, or samples are collected for diagnostic
purposes, the results and information deriving from this activity are highly protocol sensitive.
For a diagnostic laboratory to contribute fully to the final diagnosis, the specimen(s) collected
must be selected carefully and preserved in suitable conditions. A thorough post-mortem
examination of animals that die or are euthanised is a necessary adjunct to any good clinical
practice.
The purpose of this chapter is to assist the zoo veterinarian in the performance of a thorough
post-mortem examination and in the correct selection, preservation and transportation of
pathological and biological specimens. This chapter will not be a complete protocol, but more
of a guide. As with any activity, it is essential to prepare yourself before you begin. This
includes reading and preparing your protocols and setting up an area or room, with the
proper equipment, where you can perform the post-mortem. Keep in mind that the facilities
need to be “infectious disease proof”; every post-mortem should be treated as an infectious
problem until proven otherwise.
As the basis for this article we have used the excellent booklet, “Post-mortem procedures for
wildlife veterinarians and field biologists” written by M.H. Woodford, D.F. Keet and R.G.
Bengis (2000) and published jointly by the Office International des Epizooties (O.I.E.), Care
for the Wild International and the Veterinary Specialist Group/Species Survival Commission
of the World Conservation Union (IUCN). This little 55 page booklet is very comprehensive
and should be present in every zoo where post-mortems are performed. It is available
through the OIE, 12 Rue de Prony, 75017, Paris France (ISBN 92-9044-419-6).
VII. Post-Mortem Procedures
We will not try to rewrite this booklet here, but several parts will be completely reproduced in
this chapter, as is allowed by the OIE.
There are several reasons for performing a post-mortem or having one done. These can
include: finding the cause of death, confirming a diagnosis, investigating unsuccessful ther-
apy, increasing knowledge, or simply satisfying curiosity. In the zoo. Dead animals can also
function as sentinels, indicating the presence of subclinical infectious diseases which may
not be immediately related to the cause of death. Diagnostic pathology is not limited to a
post-mortem. The pathologist uses the clinical history (including haematology, blood
chemistry and therapeutic measurements), the gross description, culture results and other
data, as well as the cytological and histological appearance of the lesions, to make a
diagnosis. Absence of any of these, or incorrect submission of tissues, will hamper this
process. And remember: it is better to store and preserve too much material than to realise
after some time that the proper material required for a diagnosis has already been discarded.
We also consider it one of the functions of a “scientifically-minded” zoo to make optimal use
of their animals and associated data. This includes gathering scientific information about the
live animals regarding housing, feeding, behaviour, breeding, etc. Ideally, this would include
maintaining a data bank of sera and organs or tissues, for future retrospective studies of
diseases and their agents.
2. Submission of carcasses or specimens

When a zoo veterinarian has access to the services of a pathological institute that will
perform the post-mortem, procedures should be established for the submission of the intact
carcass. These specialists have the necessary experience and training, and their work will
yield the best results. In some cases the zoo veterinarian or technician will perform the post-
mortem examination and submit the appropriate tissue to a diagnostic laboratory. Based on
the gross post-mortem findings, material will be collected and sampled for follow-up
investigation. The quality of information received from such an examination is directly propor-
tionate to the quality and choice of the specimens submitted and the information that
accompanies them. When in doubt about “what” and “how” samples should be collected and
packed, the laboratory should always be contacted before sending in any materials.
In many situations it is not possible to start a post-mortem immediately after death. To

promote the rapid cooling of a small carcass, the fur or plumage should be thoroughly
soaked with cold water to which a small amount of soap or detergent has been added to aid
complete wetting of the coat or plumage and skin. The carcass should be placed in a plastic
bag, all excess air squeezed out, the bag sealed or tied, and then refrigerated. Larger
animals should be stored in a cool environment as soon as possible. Big animals will not cool
down quickly enough to prevent extensive autolytic post-mortem changes and should be
necropsied as soon as possible. When this is not possible, then opening the abdomen can
be helpful in lowering the core body temperature.
The animal should be kept refrigerated until the post-mortem is performed or the carcass has
been shipped to the laboratory. In general, providing the carcass has been cooled
immediately upon death and can be delivered to the laboratory within 72 to 96 hours of the
time of death, it should be refrigerated (not frozen). Small animals can be packed with
sufficient ice or cool packs to keep the carcass cold until arrival at the laboratory. If delivery
to a laboratory is expected to be delayed beyond 96 hours post-mortem the carcass should
be frozen immediately rather than simply refrigerated. Frozen tissue specimens or carcasses
must be packed with sufficient ice to keep them frozen until arrival at the laboratory.
Refrigerated or frozen specimens should be packed in a sturdy, insulated (Styrofoam®) box,

preferably in a leak-proof sealbag, and shipped to the laboratory by a private courier service,
which guarantees same- or next-day delivery to the laboratory.
Most laboratories cannot receive specimens over the weekend; it is thus advisable not to
ship refrigerated or frozen specimens on Fridays or weekends. Remember, it is crucial that
sufficient refrigerant be packed with the specimen and that it be adequately insulated to
insure that it will remain cold (or frozen) until it is received by the laboratory personnel.
In instances where the carcass is extremely small, such as embryos, nestlings or very small
adult animals, the entire carcass may be submitted for histological examination. This is best
accomplished by opening the body cavity, gently separating the viscera and fixing the entire
carcass in formalin solution.
When you perform a post-mortem yourself or collect diagnostic material it must be done
systematically. The correct selection of material for further examination, and the correct
sampling, storage and shipping of material, will increase the quality of results tremendously.
A written report of the post-mortem findings will help the zoo veterinarian to keep track of the
disease status of the zoo collection.
EVEN A NEGATIVE FINDING IS A FINDING, SINCE IT MEANS THAT THE

LESIONS/CHANGES YOU WERE LOOKING FOR ARE NOT PRESENT.
3. Post-mortem site, protective clothing and equipment

For details see Woodford et al. (2000), Section I: Preparing for a post-mortem examination.
It is helpful to have a set of instruments designated for post-mortem examinations. These

should be thoroughly cleaned and sterilised after use. A separate room to perform the post-
mortem is also advisable. Instruments that are used for post mortem examinations should
not be used for living animals. It is important to wear adequate protective clothing.
The instrument pack should include post-mortem knives, forceps, two scalpel handles (one
for cutting, one for burning organ surfaces before taking a microbiology sample), stout
scissors and/or poultry shears (for cutting bones), and fine scissors for dissection. Tiny
animals such as finches, lizards or rodents require fine instruments such as iris scissors. For
large animal post-mortem examinations special instruments such as a vibrating (cast-cutting)
saw may be used.
Other useful equipment includes a (gram) scale, a hand-lens or dissecting microscope, and
paper tissues.
In addition to instruments, one should have at hand:

- 10% neutral buffered formalin (= 4% formaldehyde),
- 70% alcohol for wetting and disinfecting the skin,
- 96-100% ethyl alcohol (for fixing specimens suspected of having gout, and 100mg/1ml for
PCR testing),
- a bottle with normal saline (0.9% NaCl) with a pipette (for parasitological examination),
and
- appropriate containers.
Other material for ancillary diagnostic procedures include:

- syringes and needles to obtain samples for serology, haematology, or cytology,
- clean glass slides for impression smears,
- a stain set for cytology (e.g. "Diff-QuickR, Hemacolor, Stamp or Machiavelli),

- clean glass slides and coverslips for wet mounts (parasitology),
- burner for heating and sterilising a scalpel blade before taking a sample for microbiology,
- sterile swabs or culture tubes with appropriate transport media for bacterial, or fungal
culture,
- transport media (96% ethanol or buffered 4 M guanidine isothiocyanate) for PCR testing
(viruses, mycobacteria and chlamydia)
- petri dishes or freezer-proof tubes for submission of tissues for viral isolation.
It may also be helpful to have a camera available for documentation of gross lesions.
A standard checklist and post-mortem report form will assist in recording observations.
4. Euthanasia
The method of euthanasia may affect specimens submitted to the pathologist. High doses of
barbiturates are caustic to tissues and cause crystallisation in and on organs. Such changes
may be mistaken for early gout, but will also change and mask macroscopic and microscopic
lesions. When euthanasia solutions are used at an appropriate dosage, e.g. pentobarbital
200 mg/kg bw intraperitoneally or T61 0.5 ml/kg intramuscular, few alterations are seen
The euthanasia agent can also be administered intravenously, or into the spinal cord at the
base of the skull with the head flexed (especially in larger birds). Administering such agents
slowly to effect is helpful to prevent undesired artificial changes.
Serum or heparinised haematological samples should be collected prior to euthanasia. The

blood may be centrifuged and serum or plasma submitted or saved and frozen pending post-
mortem results. This may be helpful in diagnosis of endocrine disorders or viral infections.
Routine haematological tests may also be performed on these samples.
5. Impression Smears
Impression smears of fresh cut organs or altered surfaces are not common practice at post-
mortems. They are a useful and often underestimated adjunct to a complete post-mortem
examination. In our protocol, two sets of impression smears are made at every post-mortem
from liver, spleen, lung and rectum. Organs with pathological changes are automatically
added to this list. For a first impression of the presence of bacteria, yeasts or protozoa, this
technique is very useful. Tissue phases of parasites such as Atoxoplasma spp, Toxoplasma
spp., Plasmodium spp, Hemoproteus spp., Leucocytozoon spp, and Trypanosoma spp. are
mostly readily identified in impression smears of liver, spleen and lungs. Immunofluorescent
staining for Chlamydia spp. can be carried out in specific laboratories on the impression
smears of these organs in all post mortem examinations of suspected cases in reptiles and
birds especially within the families Columbiformes and Psittaciformes. Immuno-
histochemistry staining on fixed paraffin-embedded histological sections is a good alternative
when available. The now-days confirmation of Chlamydiosis is by PCR. Also, the cell-type of
lymphoreticular and haematopoietic neoplasms is easier to diagnose from impressions of
liver, spleen and bone marrow than from histology.
To make a good impression smear (actually a touch preparation), it may be easier to hold the
slide when touching with the tissue. Grasp a small piece of the tissue with forceps so that a
fresh cut, well-blotted surface faces downward. Lower the tissue to the clean slide touching it
lightly. Retract quickly without dragging the tissue across the slide. Make several "touch
preps" on each slide. Impressions are generally more useful when air-dried. If other fixation
is necessary (e.g. heat fixation for acid-fast stains), it can be done after air-drying .
Exudate or any other fluids may be prepared for cytological evaluation by having a thick drop
air-dried.
6. Fixation for Histopathology

Several philosophies may determine the choice of tissues for histopathological examination:
1. Economic reasons; these are poor grounds for decision-making, but in this case it is better
to collect the tissues and, after consulting the pathologist, the selected tissues should be
sent in but additional tissue samples should be retained "just in case."
2. Completeness; this is especially valid for a scientific, research situation. Collect all tissues
listed in table 1.
3. A standard selection completed with a choice based on the post-mortem findings. This list
is practical and will in most cases lead to sufficient diagnostic support. In table 1 the
standard selection is marked with an asterisk (*).
Normally, selected tissues are fixed in neutral-buffered formalin for histopathological

examination. If the formalin solution is not freshly prepared on a frequent basis, formic acid
will be formed. A layer of pieces of marble at the bottom of the container or bottle will bind
the formic acid to a precipitate, keeping the formalin neutral. This prevents the formation of
“formalin pigment” in histological specimens; a confusing, annoying and unnecessary artefact
that can be present in improperly fixed tissue samples.
Ten percent buffered formalin penetrates only about 2-5 mm in 24 hours, so specimens must
be less than 10 mm thick. Penetration is slower in very bloody, dense tissues (e.g. congested
spleen or liver) and more rapid in relatively porous tissue (e.g. lung). Formalin will not
penetrate well into the brain through the unopened calvarium or into bone marrow unless the
bone has been cracked. The biggest problem seen with submission of fixed tissues is
inadequate fixation due to prior severe autolysis or an inadequate volume of fixative allowing
continuing decomposition. Proper initial fixation is achieved if at least ten times the volume of
formalin as volume of tissue is used. When preparing specimens for mailing the amount of
formalin may be reduced after tissues have been fixed for 12-24 hours. Wet formalin-fixed
tissue may be conveniently stored and shipped in heat-sealed plastic bags.
Other fixatives, such as those required for electron microscopy (EM), are not usually
necessary, since formalin fixed tissue is easily refixed with glutaraldehyde and the main
structures (including viruses) are preserved. For EM fixation very fresh tissue in tiny parts (1-
2 mm3) is essential.
The number of tissue specimens submitted to the histopathology laboratory may depend on
the cost per sample. If you do not send the complete set of specimens, it is prudent to save
the rest in formalin while awaiting a diagnosis. If only grossly visible lesions or limited tissue
specimens are submitted, a diagnosis may not be possible. When specific lesions are
observed at post-mortem, the tissue specimens collected should include a small margin of
normal tissue adjacent to the lesion. Too often, the limited tissue specimens submitted
suggest a diagnosis, which cannot be confirmed because other tissues have already been
discarded.
Tissue specimens for histopathology should not be frozen. Freezing creates crystals and
ruptures cells, making histopathology virtually useless.
Tissues for toxicological analysis should be frozen. They may be frozen at -20oC after being
wrapped in aluminium foil. The optimum temperature for freezing tissues for virus isolation is
-70oC. If this cannot be accomplished, the tissues for viral isolation should be sent (by rapid
mail) in sterile containers on wet ice to the laboratory.
For detailed information on sampling etc. see Woodford et al. (2000), Section III: “The
collection and field preservation of biological and pathological specimens” and the
Appendices.
Table 1: Tissues routinely collected for histopathology.

skin thyroid glands rectum ovary & oviduct
(feather follicles) parathyroid glands caeca testes (male)
trachea oesophagus (cloaca) pectoral muscle
lung* (crop) spleen* bone marrow
* *
(air sac) (proventriculus ) liver thymus
* *
heart stomach/ventriculus gall bladder brain
kidneys* duodenum* pancreas* spinal cord
*
adrenal glands small intestine (cloacal bursa ) (ischiatic nerve)
*
Standard selection of tissues for routine histopathological examination.
(..) Tissue specimens from birds. Selection of additional tissue specimens will depend upon
gross lesions observed at post-mortem.
7. Autopsy protocol
There are probably as many ways to dissect an animal as there are pathologists. One should
choose a procedure with which one is familiar and feels comfortable, and then use it consis-
tently. No matter what procedure is used, each post-mortem should be performed in as
regular and thorough a manner as can be accomplished by the prosector and a "complete"
set of tissues and specimens be collected for subsequent histopathological, parasitological,
toxicological, serological, and biochemical examination. The veterinarian should review the
appended detailed checklist of organs to be examined, observations to be made, ancillary
tests to be performed and specimens to be collected, prior to disposal of the remains.
The following procedure and checklist is used for avian species at the Diagnostic Pathology
Laboratory of the Dutch Research Institute of Avian and Exotic Animals (NOIVBD) in
Veldhoven, The Netherlands (www.noivbd.nl).
For a protocol for mammals see Woodford et al. (2000), Section II: “Post-mortem
procedures”.
Avian Necropsy Protocol
Gerry M. Dorrestein1, Marein M. van der Hage, and Marja J. L. Kik2

1
Diagnostic Laboratory of the NOIVBD,
Veldhoven, The Netherlands
and
2
Department of Veterinary Pathobiology,
Utrecht University, The Netherlands
Before starting the necropsy procedure (see also reference list), the packing material is to be
inspected for the presence of mites or lice!
I. History
Read the history - including identification, physical findings, medical history and pertinent
laboratory data - and summarise the most relevant data on your work sheet. Make a note
of leg band numbers, transponders or other identifying marks.
II. External examination of the carcass
First make a carcass identification based upon identification: species, age, and colour
pattern as well as leg band, tattoo or microchip implant data.
Record information about general bodily condition, weight, muscle mass, joints, integu-
ment (incl. beak and nails), plumage, (for defects, ectoparasites, faeces), body orifices
(eyes, ears, nostrils and vent), uropygial gland, traumata, and abnormalities. Palpate the
skeleton.
The feeding status can be judged based upon the muscles on the keel and the filling of
the crop and intestines.
When heavy metals are suspected (e.g. rifle bullets or ingested lead) survey radiographs
may be taken.
Examples (of alterations found at external examination):

- Broken feathers due to feather picking; diagnosis: normal feathers on the head.
- Altered feathers with constrictions at the base caused by PBFD; diagnosis: histology
of skin with feather-follicles; PCR test.
- thickened dry skin caused by Malassezia sp; diagnosis: cytology skin scraping.
- Look for feather and skin parasites.
- Swelling above the eye or dilated nostrils with a plug in parrots due to vitamin A
deficiency; diagnose: histological examination with metaplastic changes in salivary
glands.
- Conjunctivitis and sinusitis related to ornithosis, chlamydiosis or psittacosis
Conjunctivitis with pox-lesions; diagnosis: cytology, histology and culture.
- Abdominal or other swellings, tumours, egg-related peritonitis: diagnosis: histology
- Cloacal mucosal prolapse, papilloma; diagnosis: histology.
III. Preparation of the bird
Small birds are wetted and plucked, all other birds should be wetted with alcohol 70%
before the necropsy. This is done to allow better visualisation of the skin, to part the
feathers to permit incision of the skin and to prevent loose feathers from irritating or
harming the prosector (zoonosis) or contaminating the viscera.
The bird is positioned on its back, in small birds the wings and legs are pinned to a
dissecting board with nails or needles, large birds are fixed on a metal tray with pieces of
rope.
IV. Post mortem examination
General remarks:
- use a gram-scale for body weight and measuring the size of organs,
- open all tube-like structures,
- cut all parenchymatous organs in slices to find small focal lesions,
- tissue for optimal formalin fixation should preferably not exceed 3 to 4 mm in
thickness (5 mm maximum),
- ratio of tissue to formalin required for adequate fixation is 1:10,
- collect tissue samples during the necropsy to prevent desiccation. Do not wait till
the gross examination is finished,
- remember to collect and submit specimens from a broad spectrum of organs and
systems,
- collect at least heart, lung, liver, spleen, kidney, gonad, and adrenal, and a piece of
intestine (duodenum and pancreas) for histopathology,
- when suspecting a viral problem, collect 100mg tissue in 1ml ethanol 96% for a
PCR, freeze tissue as soon as possible at -20oC, or collect tissue on wet ice till
shipment.
- when you suspect a bacteriological problem, make a impression smear and look
before you select you culture media or send an organ or swab to a laboratory for
culturing.
1. An incision is made in the skin along the ventral midline from the mandible over
the sternum to the cloaca. The skin is reflected to expose the subcutis, crop,
pectoral muscles, keel, abdominal wall, leg muscles and fat.
Watch for colour of the muscles, parasites, haemorrhages, and oedema. Judge the
amount of food in the crop. In pigeons a vascular plexus in the deep layers of the
cutis of the cervical region can be seen, the plexus venosus intracutaneous collaris.
This plexus can be mistaken for an extensive haemorrhage.
Examples
- Stripes in leg- or breast-muscle; sarcosporidiosis; diagnosis: cytology of such a
stripe reveals the bradyzoites.
- A large dark spot distal to the keel; swollen liver: diagnosis: see under 3.
- Changes of the skin; cnemidocoptes, yeast-infection; diagnosis: wet mount
and cytology smear.
2. Make an incision through the pectoral muscle along the sides and around the
posterior border of the sternum through the abdominal muscles; cut with heavy
rongeurs, scissors or poultry shears through the ribs, coracoid bones, and
clavicle to remove the sternum.
Examine the inside of the sernum, the air sacs and pericardial sac, and make impres-
sion smears (when abnormalities or inflammations are seen).
During dissection of the keel the air sacs are easily seen. Normal air sacs appear as
glistening transparent membranes.
Examples
- Opaque air sacs or (fibrinous) inflammation: chlamydiosis; diagnosis: cytology
with special staining, PCR
- Opaque air sacs or obvious inflammation: bacterial infection; diagnosis: rods or
cocci in cytology smear; culture and sensitivity test.
- Air sacs covered with white/yellow plaques: fungal infection; diagnosis: wet
mount (heated with chlorallactophenol), showing hyphae, culture.
- Air sacs solid with white/yellow material; chronic fungal infection, mostly
aspergillosis; diagnosis: wet mount showing hypha, culture.
- Air sacs, esp. cervical and prescapular, with small black dots in passerines and
small psittacines; Sternostoma tracheocolum infestation; diagnosis:
magnifying-glass and wet mount.
- Air sacs filled with food: forced feeding; diagnosis: wet mount and histology.
- Pericardial sac filled with fluid: inanition, cachexia; diagnosis: muscle wasting,
oedema and gelatinous fat-tissue.
- Pericardium covered with white chalky deposits: visceral gout; diagnosis: wet
mount with crystals; often in combination with nephritis.
3. Identify the (para)thyroids cranial and lateral to the syrinx along the carotid
arteries. Remove the thyroids when required. Look for the thymus along the neck in
juvenile birds. The liver is examined in situ (examples see 5).
Examples
- In budgerigars enlarged thyroid glands; diagnosis: histology.
- Parrots (especially African greys) hyperparathyroidism; diagnosis: histology.
- "Abscesses"; Salmonella or E.coli infections; diagnosis: rod shaped bacteria in
cytology, culture.
4. Remove the heart with the carotids and thyroids attached and cut across the
apex to check for an "open" lumen and to assess the thickness of the ventricle
walls. Open the heart and large vessels and examine the valves and endocardial
surface. Keep in mind that the right atrioventricular valve in birds is a muscular
structure.
Examples
- Yellow plaques on the wall inside the large vessels; the vessels are stiff:
atherosclerosis: diagnosis: macroscopic (gross) examination, histology.
- Epi- or endocardial haemorrhages: septicaemia or agonal event; diagnosis:
continue post mortem.
- Gelatinous, serous pericardial fat; starvation, chronical illness: diagnosis;
continue post mortem.
- Changes (inflammation, necrosis) in the myocardium: myocarditis; diagnosis:
cytology, histology, microbiological isolation, continue post mortem.
- Cardiomyopathy with muscle cysts: sarcocystis; diagnosis: cytology, histology.
- An enlarged lumen of the left ventricle and only slight difference in thickness of
the ventricle walls: heart failure; diagnosis: congestion of the lungs and/or liver.
5. Examine and measure the liver.

Take a sample for cytology and histology. Decide if you want to do a bacteriological
culturing or freeze piece of liver-tissue for virological or toxicological testing.
Separate the rest of the liver from the viscera by holding the ligaments in the forceps
and cutting them with scissors, examine the gallbladder (if present). For a thorough
examination, slice the liver at regular intervals.
Examples
- Enlarged red variegated liver with pale areas: hepatitis; diagnosis: cytology with
many inflammatory cells; histology.
- Enlarged liver with necrotic areas: hepatitis by chlamydiosis, herpesvirusin-
fection; diagnosis: imprints, culture, histology, PCR.
- Very extensive acute liver necrosis: suspected for peracute or acute hepatitis
by bacterial septicaemia, polyoma-, herpes- adeno- or reovirus; in juvenile
African grey parrots acute circovirus infection diagnosis: macroscopic (gross)
examination, cytology, histology, virology (PCR), culture.
- Focal yellow proliferation with often central necrosis; tuberculosis; diagnosis:
see above.
- Small round yellow necrotic foci: salmonellosis or yersiniosis; diagnosis:
imprints with rod-shaped bacteria; culture.
- Evenly enlarged, often variegated, pale liver: leucosis; diagnosis: macroscopic
(gross) examination; included often other organs; cytology and histology.
- Evenly enlarged, often variegated, pale soft liver: degeneration; diagnosis:
cytology hepatocytes with vacuoles; histology.
- Enlarged orange, yellow liver: fatty liver; diagnosis: macroscopic (gross)
examination, cytology, histology with Sudan III stain.
- Liver with necrotic ulcer: histomoniasis (black head); diagnosis: histology.
6. The spleen can be found by cutting the oesophagus in the bifurcation of the
trachea and with combined blunt and sharp dissection remove the viscera leaving the
lungs and kidneys.
Do not cut the cloaca, but bend the viscera caudally. This exposes the spleen in the
angle between the proventriculus, gizzard (and liver). Examine, remove and measure
the spleen; make impression smears from a fresh cut surface after blotting to remove
excess blood.
Examples
- Spleen-swelling together with air sac opacity: chlamydiosis; diagnosis: see
above.
- Very large swollen and cherry red spleen in parrots watch for herpesvirus
infection (= Pacheco's) or sarcocystis; diagnosis: liver necrosis with
intranuclear inclusion bodies or protozoa, cytology, histology, IFT, PCR, virus
isolation.
- Very large swollen and cherry red spleen in penguins and some other species;
Plasmodium infection; diagnosis: cytology for parasites in macrophages and
severe pneumonia, histology
- Swollen and pale: (bacterial) septicaemia; diagnosis: cytology with bacteria,
culture.
- Multiple irregular yellow foci in the spleen; tuberculosis; diagnosis:
the same foci in other organs, in imprint non-staining rods, acid-fast.
Differentiation avian/bovine strains by culture or PCR.
- Large firm spleen: tumour; diagnosis: histology.
- Enlarged friable spleen with multiple, milliary necrotic foci: salmonellosis,
yersiniosis; diagnosis: the same foci in liver and caeca; imprint with rod shaped
bacteria; culture.
- Homogeneous red enlarged spleen in canaries and finches: atoxoplasmosis;
diagnosis: cytology.
- Small, grey spleen: lymphoid depletion; stress, viral infection; diagnosis:

cytology, histology, virus isolation.
7. Examine the adrenals, gonads (determine sex) and genital tract, and the kidney
with ureters in situ. Remove the kidneys by applying gentle traction to the cranial
vessels. Notice the adrenals and look for the sciatic nerve in the middle division of the
kidneys. In our laboratory the kidneys are not routinely screened in cytology, but only
when pathological changes are seen.
Examples
- A swelling inside the oviduct: egg-binding, egg concrements; diagnosis: open
the oviduct.
- Irregular swellings related to kidney or gonads: tumour; diagnosis: macroscopic
(gross) examination and histology.
- Pale swollen kidneys with white striation: urate congestion; diagnosis:
dehydration; histology (fixation 100% alcohol!!).
- Irregular pale swollen kidney with white foci: nephitis with "renal gout"; diag-
nosis: histology (fixation 100% alcohol).
- Irregular swollen kidney with multifocal abscessation: bacterial infection;
diagnosis: histology, cytology, culture.
- Enlarged red kidneys: acute nephritis; diagnosis: histology.
- Pale swollen friable kidneys: kidney degeneration; diagnosis: histology.
- White, firm small kidneys: chronic kidney fibrosis; diagnosis: macroscopic
(gross) examination
- NB the adrenals are important for the histological diagnosis of proventricular
dilatation disease (PDD, avian bornavirus infection) in psittacines.
8. Free the lungs by applying gentle traction to the trachea and oesophagus and
cut the attachment to the ventral ribs and backbone at the thoracic inlet. This may be
difficult as there is no pleural space in birds. Using blunt and sharp dissection will free
the lungs. Inspect the lungs. Open the oesophagus. To open the syrinx, trachea and
main bronchi a strip has to be cut out; cut through the lungs at intervals; make an
impression smear from the lungs.
Examples
- Dark coloured grey lungs: lung oedema; diagnosis: on cut surface clear serosal
fluid.
- Dark coloured wet red lungs: lung congestion; diagnosis: from a cut surface
only blood; the lungs are supple and evenly bright red; watch for congestion in
other organs and alterations of the heart. Think also of polytetrafluoroethylene
(TeflonR) toxicosis, acute mycotic infection, Plasmodium and sarcocystis.
- Dark firm lungs often variegated and focal changes: pneumonic foci; diagnosis:
cut surface; cytology (inflammation cells); histology.
- Dark, supple, dry lungs: atelectasis; diagnosis: on cut surface only a dark colour
of the surface of the lung and dried up.
- Scattered through the lungs white/yellow foci: aspergillosis, tuberculosis;
diagnosis: wet mount with hyphae (aspergillosis), acid fast rods (in routine quick
staining, non-stained rods) (tuberculosis); culture and histology.
- Irregular scattered pneumonic foci: bacterial pneumonia; eg. Salmonella spp. or
Yersinia spp.; diagnosis: cytology and culture.
- In the syrinx of parrots white material: syringeal mycosis; based on a
metaplasia due to vitamin A deficiency; diagnosis: see aspergillosis.
- In the trachea red worms: Syngamus spp, black dots: Sternostoma mites;
mucous and fibrin: avipoxvirus, cytomegalovirus
9. Examine the "abdominal" viscera.

Open and inspect the proventriculus and gizzard (with koilin layer). Examine the
contents for foreign bodies and heavy metals.
The bowel should be opened by making cuts at intervals to examine the contents and
the wall for changes and parasites. Open and inspect the caeca when present. Look
for the pancreas, bursa and umbilical sac.
Take the following samples:
---> contents from the duodenum and rectum for parasitology
(including direct examination on very fresh specimens for flagellates as well as
other techniques)
---> contents from the rectum for a smear for staining (Diff Quick®)
---> contents from the rectum for microbiology
Examples
Crop
- Thickened wall with white material: yeast infection; diagnosis: smear of the
material; culture.
- Thickened wall with mucous material: capillaria infection; diagnosis: smear of
scraping of the epithelium; histology.
- Thickened wall with grey/yellow material, sometimes with trapped air bubbles;
trichomoniasis; diagnosis: wet mount; cytology; histology.
- Local yellow necrotic ulceration: pox-lesions; diagnosis: macroscopic (gross)
examination; histology; virusculture.
- Local red mucosal thickening: papillomas: diagnosis: histology.
Stomach (proventriculus and ventriculus)

- Dilated proventriculus and gizzard, often stuffed with seeds (sunflower): gastric
dilatation syndrome; diagnosis: histology; (ganglio)neuritis, lymfoid infiltrates in
the adrenals..
- An empty proventriculus with excess of mucous: Macrorhabdes ornithogaster
(formally "megabacteria"); diagnosis: wet mount and cytology.
- Swollen red glands in proventriculus: Tetrameres spp; diagnosis: parasitologic
examination.
Intestines
- Haemorrhagic contents duodenum: coccidiosis; diagnosis: wet mount, cytology.
- Haemorrhagic, black contents in the entire small intestine: haemorrhagic
diathesis; diagnosis: history (fasting during high energy need for over 24 hours),
macroscopic (gross) examination.
- Pseudomembraneous covering of the duodenal wall: hexamitiasis; in cranes;
diagnosis: wet mounts, cytology and histology.
- Thickened wall with or without blood in the lumen: enteritis; diagnosis: wet
mount and cytology; parasitology; microbiology. Beware: in psittacines very
rarely coccidia, often ascaridia; in small passerines rarely worms, often coccidia
spp.
- Haemorrhagic contents: lead intoxication, clostridium infection,
pseudomonas infection, Giardia spp.; diagnosis: lead in gizzard; lead analysis
liver and kidneys; cytology, culture.
- Clear watery contents in small intestine with flabby wall: hexamitiasis; diagnosis:
fresh wet mount, cytology, histology.
- Yellow non-digested starch and broken seeds in small passerines: Cochlosoma
or Campylobacter spp.: diagnosis: fresh wet mount, cytology, selective culture.
- Enlarged caeca with pseudomembraneous to necropurulent content; typhlitis;
diagnosis: galliformes: histomoniasis ("blackhead"); diagnosis: cytology,
histology (often with liver lesions)
- Caeca with nodular lesions: parasitic typhlitis; pheasants: Heterakis isolonga;

diagnosis: worms and ova; histology
Cloaca
- Congested, swollen red mucosa: papilloma: diagnosis: histology.
Bursa
- Especially in young birds for detecting virus infections e.g. circovirus: diagnosis:
histology, PCR.
BEWARE: TRY TO ESTABLISH A RELATIONSHIP BETWEEN THE CLINICAL HISTORY

AND THE POST-MORTEM FINDINGS
10. Open the anterior part of the oesophagus from the beak, make a wet mount.
Remove the tongue and cut the salivary glands. Inspect the beak, choanae and
oesophagus.
Examples
- Tongue with yellow "abscesses" at the location of the salivary glands in
psittacines: metaplasia, due to vitamin A deficiency; diagnosis: wet mount,
diet history, histology.
- see also crop/intestines (eg. trichomoniasis, avipox, candidiasis).
- Chronic, necrotic lesions especially in commisures: tuberculosis: diagnosis:
cytology (acid fast stain), histology, culture.
11. Cut across the beak through the nostrils and sinuses.
Examples
- The presence of turbid mucus: sinusitis; diagnosis: wet mount, cytology, culture.
12. Inspect the joints, bones, bone marrow, brains.

Joints of the wings, legs and feet should be opened and examined. If exudate is
present, cytology should be done as well as a microbiological examination. White,
chalky deposits may represent urate deposition.
Bone marrow is most easily collected from the tibiotarsus for both cytology and
histology. In bone marrow tubercular lesions can be found; often visible on X-rays.
Ecchymoses within the calvarium are a common agonal change and do not imply
head trauma.
Examination of the nervous system and associated tissues is governed by the
presence or absence of neurological or ocular disease.
The brain may be removed by deskinning the head, making a sagittal incision through
the calvarium and removing the bony calvarium to expose the brain. When sampling
for histology, it is often better to leave the brain inside the skull, after opening it, and
immerse the whole head in formalin.
13. The muscles of the legs and the sciatic nerve running on the posterior surface of
the femur should be examined.
V. Final activities
1. Bacterial cultures are done from the liver and the rectum and all abnormal organs,
especially when bacteria are seen in the imprints!
The following media are selected: blood agar, selective Enterobacteriaceae agar
(brilliant-green-agar) and serum broth.
The intestinal contents are collected in tetrathionate broth as an enrichment medium
for Salmonella spp. When special microorganisms are expected (e.g. anaerobes,
Campylobacter spp.) contact the laboratory.
2. When a mycotic problem is suspected a Malt-agar, or other selective culture

medium, is selected as well.
3. The impression smears are allowed to dry, stained with 'HemacolorR' or "Diff
QuickR" and Stamp or Macchiavello (for Chlamydia), and examined by microscope
with objective 100x in immersion oil.
The slide for an IFT for Chlamydia is fixed in cold acetone (freezer -20°C) and sent to
the laboratory.
4. POSITIVE cytology for Chlamydia requires sampling for IFT or PCR.
5. Examine the wet mounts of gut contents.
6. Samples collected for ancillary diagnostics should be packed, labelled and stored
properly, until shipment. See that each sample is provided with the essential
documentation .
7. Make a detailed report and use this to document the samples.

References
1. Graham, D. L. 1992. Checklist for necropsy of the pet bird and preparation and sub-
mission of necropsy specimens - A mnemonic aid for the busy avian practitioner. AAV
Introduction to Avian Medicine and Surgery, New Orleans, LA, USA, pp. 1-4.
2. Lowenstine, L. J. 1986. Necropsy procedures. In: Harrison, G. J., and L. R. Harrison
(eds). Clinical Avian Medicine and Surgery, P.A. Saunders, Philadelphia, Pennsylvania.
Pp. 298 - 309.
3. Latimer, S. L., and P. M. Rakich. 1994. Necropsy examination. In: Ritchie, B. W., G. J.
Harrison, and L. R. Harrison (eds). Avian Medicine: Principles and application. Wingers
Publishing, Inc. Lake Worth, FL, USA. pp. 355-379.
4. Dorrestein, G. M. 1996. Cytology. In: Beyon, P. H., N. A. Forbes, and N. H. Harcourt-
Brown (eds), Manual of Raptors, Pigeons and Waterfowl, BSAVA, London, UK. Pp. 55-
62
5. Dorrestein Gerry M. and Wit, Martine de (2005) Chapter 7. Clinical pathology and
necropsy. In: BSAVA Manual of Psittacine Birds. 2nd ed. N. Harcourt-Brown and Chitty J.
(eds). pp. 60-86.
6. Dorrestein, G. M. 1997. Diagnostic Necropsy and Pathology and Avian Cytology. In:
Altman, R. B., S. L. Clubb, G. M. Dorrestein, and K. Quesenberry (eds). Avian Medicine
and Surgery, WB Saunders, Philadelphia, Pennsylvania, USA. Pp. 158-169 and pp. 211-
222.
Necropsy report form and check list
The following checklist can be use during the post-mortem examination as well as writing the
necropsy report:
1. Bird species, weight, age/leg band number, sex, summarized history.

2. Date of necropsy, your name.
3. Macroscopic (gross) examination:
External examination
- general bodily condition: muscle mass: robust, well muscled, moderately muscled,
thin, emaciated, depot fat)
- feathers/integument/ectoparasites
- palpate skeleton
- body openings/oral cavity
Internal examination
- fat/subcutis/body wall
- body cavities (air sacs/pleura/peritoneum)
- (para)thyroids, thymus
- spleen (size)
- heart, aorta, other vessels
- liver, gall bladder, bile ducts
- reproductive system (gonads, repr. tract)
- urinary tract (kidneys, ureters) and adrenal glands.
- respiratory tract (nasal/sinus, choanal, larynx, trachea, syrinx, air sacs, lungs)
-
- digestive tract (beak, tongue, oropharynx, oesophagus, crop, proventriculus, gizzard,
duodenum and pancreas, small intestine, yolk sac, caeca, rectum (colorectum),
cloaca, bursa of Fabricius, vent)
- special senses (eyes, ears, nares)
- musculoskeletal system: muscles, skeleton (sternum, ribs, vertebrae, long bones),
bone marrow, joints
- brain, pituitary, spinal cord, meninges, peripheral nerves
Wet mounts (crop, rectum, etc.)

Cytology (liver, spleen, lung, rectum)
Chlamydiosis
TENTATIVE (DIFFERENTIAL) DIAGNOSIS
Ancillary diagnostics: bacteriology, mycology, virology, parasitology, toxicology, others
Tissue saved:
Tissues submitted for histopathology:
VIII. GUIDELINES FOR CLEANING AND DISINFECTION IN

ZOOLOGICAL GARDENS
Matti Kiupel, Robin Mecklem, Birgit Hunsinger and Rachel E. Marschang
Diagnostic Center for Population and Animal Health (MK) and Office of Radiological,
Chemical and Biological Safety (RM), Michigan State University, Michigan, USA
Institut für Umwelt- und Tierhygiene (REM, BH), Hohenheim University, Garbenstr. 30,
70599 Stuttgart, Germany
Introduction
Hygienic measures are a major component for prevention of infectious diseases in

zoological exhibitions. A clean environment is not only a key feature in disease control, but
also attractive to the public. Preventive health care consists of multiple components of equal
importance including food storage, preparation and handling, insect and vermin control and
cleaning and disinfection (Fowler, 1978).
The purpose of this chapter is to provide guidelines for cleaning and disinfection to the range
of people who would be involved in managing a disease emergency involving a zoo,
zoological garden, game park, circus or any other facility keeping exotic animals. For the
information in this chapter to be effective, it is important that the described cleaning and
disinfection methods are incorporated into the routine husbandry and emergency
procedures. The goal is to provide individual zoos with information that can be used to
develop their own specific cleaning and disinfection protocols. Each of the fact sheets in the
“Transmissible Disease Handbook“ contains disease specific information about prevention
and control of diseases in zoos and suggested disinfectants for animal housing. Therefore,
the purpose of this chapter is not to provide detailed information on specific disinfectants for
the diseases listed in this handbook, but rather to provide an overview of the guiding
principles of cleaning and disinfection in zoological gardens and to review the various
classes of chemical disinfectants.
Defining Disinfection
The term “disinfectant” is defined as “an agent that frees from infection, usually a chemical
agent but sometimes a physical one, such as x-rays or ultraviolet light, that destroys
diseases or other harmful microorganisms but may not kill bacterial spores” (Block, 2000).
Disinfectants are used on inanimate surfaces and are assumed to act rapidly and efficiently
to kill or inhibit growth of microorganisms. In the veterinary care environment, disinfection is
most effective for diseases that are not vector-borne, but are acquired by direct contact with
contaminated fluids or animal products (Quinn, 2000). Therefore it is important to recognize
that disinfectants play a critical, yet limited role in infection control. Insect and vermin control
are equally important in preventing infectious diseases, especially for the control of insect-
VIII. Guidelines for Cleaning and Disinfection in Zoological Gardens
borne diseases that present particular challenges in zoos. Techniques available to minimize
insect-borne disease spread include the use of anthelmintics and insecticides through
direct application on at-risk species or through the spraying of housing of susceptible
animals. The assistance of epidemiologists and entomologists should be sought to establish
insect traps within the zoo for insect identification and to identify potential areas of a high-risk
exposure.
For situations where resistant microorganisms are present, or animals may be susceptible to
infection due to their health status or the invasiveness of the procedure to be performed, a
sterilization method should be used for treatment of contaminated items or surfaces.
Sterilization is achieved when all living microorganisms and bacterial endospores have been
destroyed. Common methods of sterilization include, dry heat, moist heat, and exposure to
specific chemical compounds. For dry heat sterilization, surfaces must be exposed to 160 C
to 170 C for periods of 2 to 4 hours (Heinsohn et al., 1995). Sterilization by steam may be
accomplished by exposing contaminated surfaces to moist heat at 121 C for at least 15
minutes (Quinn, 2000) This will not be sufficient for certain heat resistant organisms such as
some thermophilic spores and prions. Chemical sterilization may be suitable in situations
where items to be sterilized cannot withstand the temperatures and physical conditions
required for dry heat or steam sterilization. Chemical sterilants may be used in the form of a
vapor or gas, such as formaldehyde or ethylene oxide, or as an immersion liquid such as
glutaraldehyde. These chemical compounds and the requirements for their proper use will
be discussed in a later section of this chapter.
The efficacy of a certain disinfection or sterilization protocol will depend on a number of

factors. The nature of the surface that is to be disinfected plays an important role. An uneven
surface will be more difficult to disinfect than an even one. Dirt will interfere with the
disinfection. Another important factor are the pathogens present. Some pathogens are more
difficult to inactivate than others. The most difficult to inactivate are bacterial spores and
prions. For disinfection, non-enveloped viruses can also be a challenge.
Role of Cleaning and Disinfection in Infection Control
Having an effective cleaning and disinfection plan is a crucial step in every biosecurity
program. Such a program should be instituted for every new building and facility and should
be revised after the occurrence of an infectious disease and prior to the introduction of new
animals. The main purpose of a cleaning and disinfection program is to reduce the number
of pathogens (disease-causing agents) in the environment and thereby to reduce the
potential for diseases to occur.
The first step in an effective disease control plan is an exact identification of the routes how
infectious agents may enter zoological gardens (inputs), and how they may spread
throughout the zoo to other facilities and outside the zoo to become a threat to farming
operations or possibly humans. Inputs into and outputs from zoological institutions may vary
depending on the type of the facility.
Animal inputs may include: animals introduced from other facilities within the same
institution, animals from institutions, either from within Europe, or imported from another
continent, animals confiscated by customs/quarantine officers, sick or injured animals
brought in by members of the public, free-ranging animals, which may be either native
(rats, mice, birds), or feral (including cats and dogs), and animals imported from farming
operations. Feed inputs include dry processed preparation (concentrates, hay, pellets, seed
etc.) and wet feed, including fresh fruit, fish, meat, vegetables and pasture silage. Biological
specimens confiscated by customs and quarantine officers are sometimes brought to zoos
for identification. Semen and embryos may be imported for breeding purposes. Various
vehicles move into the facility and may be contaminated. Other materials entering the facility
include materials used during the importation of the animals (hay, sawdust, crates etc.).
Personnel entering the premises for normal work purposes may have contact with other
animals (pets/farm animals) outside of work hours. Local and international visitors pass
through the premises on a daily basis and may introduce disease.
Animals may leave the facilities for a number of reasons including: exchange and sale of
animals, relocation of animals to other facilities within and outside the institution, use of
animals in public relations exercises, i.e. animals taken to shows, shopping centers, schools
and television stations, animals taken home by staff for hand rearing, free-ranging of
native and feral animals, including cats and dogs that may have had contact with
animals/animal waste. Waste materials that have to be removed from the premises may
include hay and composted feces, effluent, some of which has secondary treatment, and a
small portion with tertiary treatment, waste-water. Biological specimens and feces are sent to
laboratories for testing and biological specimens are sometimes also sent to museums,
veterinary schools etc. In some institutions, offal and carcasses are taken off the property for
disposal or may be sent to veterinary schools for necropsy. Vehicles, crates and packing
material used in the transportation of animals are all regularly moved off the premises.
Possible outputs related to people include personnel who have been in contact with
animals and waste products (contaminated clothing and footwear), and visitors who may
have been in contact with animals.
Due to the public role of zoological gardens, it will be impossible to clean and disinfect all
inputs and outputs. In particular, visitors and the media would not except the imposition of
cleaning and disinfection programs onto them. Therefore a strict cleaning and disinfection
program of the animal premises incorporated into daily routines becomes even more
important. In addition, it is useful to identify premises according to their exposure to
infectious agents to select the proper cleaning and disinfection regimes and to possibly limit
the access to such premises. Areas (could be all or part of a facility) in which an infectious
disease exists, is believed to exist, or which may harbor the infective disease agent should
be classified as an infected premise (IP). Dangerous contact premises (DCP) are premises
containing animals with no clinical signs that were exposed to an infectious disease and
therefore will be subjected to disease control measures. Suspect premises (SP) are areas
containing animals that show no clinical signs, but may have been exposed to an infectious
disease through possible contact with infected animals or facilities, people, equipment,
semen or embryos, or animals with evident disease symptoms, but no confirmed diagnosis.
Using this classification in combination with specific cleaning and disinfection programs and
other measures, such as insect and vermin control, as well as treatment, vaccination and
quarantine of infected and exposed animals or animals at high-risk for infections, will help to
control and to eliminate an infectious disease after an outbreak has occurred. In some
cases, it may also be necessary to consider culling certain animals that have been exposed
to disease. Disposal of animal carcasses in the case of a disease outbreak should also be
incorporated into these plans.
Cleaning before Disinfection
The first step in any cleaning and disinfection program is cleaning. Cleaning is the removal
of organic material (i.e., feces, urine, blood, bedding, food, dust etc.). Disease agents are
often protected by such materials and can survive the disinfection process. Therefore,
thorough cleaning of a building prior to the disinfection is required. The cleaning process can
include a dry cleaning and a wet cleaning step. Dry cleaning physically removes the organic
material before the actual wet cleaning. Wet cleaning, as the name implies, involves the use
of water. There are 4 basic steps in the wet cleaning process: soaking, washing, rinsing, and
drying. The use of detergents will often benefit the wet cleaning process. However, it is more
important to have pressure washers with the proper pressure (500-800 psi) to ensure that all
organic materials are removed from the facilities. The final step for ensuring proper cleaning
is to dry the wet areas of the building quickly. If the building is not dried properly, the excess
moisture can result in bacteria multiplying to higher levels than prior to cleaning. It is vital to
make sure the cleaning procedure is done properly, as an improper cleaning can actually
increase the load of infectious agents! If done properly, a good cleaning can remove 90% of
all pathogens (Fotheringham, 1995). Special care must be taken when cleaning facilities that
are contaminated with suspected zoonotic or highly contagious animal pathogens. All
personnel should wear protective clothing, footwear and if necessary face masks, goggles,
and headwear. After the cleaning of facilities, proper care must be taken of the protective
clothing and equipment that has been used for cleaning and disposable products should be
disposed as biohazards. Organic material removed during cleaning may also contain
pathogens and should be treated accordingly.
The last step in a cleaning and disinfection program is the actual disinfection process that
will further reduce pathogens in the facilities. Disinfection is especially useful in reducing
infection risks in young animal nursery facilities, and in routine cleaning operations of animal
quarters and feeding utensils. To maximize the effectiveness of a cleaning and disinfection
program, it is crucial to modify such a program based on the suspected pathogens that
should be eliminated or reduced. In addition, specific disinfectants may be selected for
certain known microbial contaminants following an infectious disease outbreak. It is also
important to remember that many disinfectants can be toxic to the animals, or may be
caustic or corrosive. Animals must usually be excluded from facilities being disinfected. After
a suitable environmental exposure time to such disinfectants premises should be rinsed
thoroughly, before animals are allowed to return.
Levels of Disinfection
Disinfectants are tested for their bacteriocidal, tuberculocidal, sporicidal, fungicidal, virucidal
(against enveloped and non-enveloped viruses), and antiparasitic (against eggs and
coccidia) effects. They can generally be categorized into 3 groups based on their ability to
inactivate certain microorganisms. High-level disinfectants are effective against bacterial
endospores under specific conditions. Intermediate-level disinfectants include those
products that can inactive tubercle bacilli, but do not kill bacterial spores. Such products are
commonly marketed by manufacturers as “tuberculocidal”. Finally, normal (“low-level”)
disinfectants include products that kill vegetative bacteria and fungi but are not reliable for
the destruction of bacterial endospores, tubercle bacilli or small non-enveloped viruses
within a practical period of time (Boothe, 2000).
When selecting a disinfectant for use on items that will come in contact with patients during
animal care procedures, the invasiveness of the procedure and composition of the medical
device must be considered. A system for classifying such devices was developed by
Spaulding in the 1970s. This system has been recognized and referenced since that time by
many human healthcare organizations like the Association for Professional in Infection
Control and Epidemiology (Rutala, 1996). Although this system was developed for human
healthcare, there are notable similarities to procedures and devices used in veterinary care
environments. These principles may be adapted for disinfectant selection purposes in
zoological gardens and other exotic animal containments.
The Spaulding system categorizes devices into 3 groups based on the risk of infection
involved with their use: critical, semicritical and noncritical. Critical devices are those that
bear a substantial risk of acquiring infection if contaminated with microorganisms at the time
of use. Devices that are introduced directly into the body such as needles, scalpels,
catheters and implants fall into this category. These items must be sterilized in between
animal use (Favero and Bond, 2000). When choosing a sterilization method, consideration
must be given to the impact of the sterilization process on the integrity of the device.
Semicritical devices are those items that come in contact with mucous membranes but do
not normally penetrate the skin. Examples of these devices include endoscopes,
bronchoscopes, and urinary catheters. Sterilization of semicritical devices is desirable, but
high-level disinfection is a minimum requirement (Favero and Bond, 2000). High-level
disinfectants may also act as cold liquid sterilants under specific conditions of use.
Therefore, some products may be appropriate for both critical and semicritical devices.
Product versatility should be one of the considerations for disinfectant selection. Non-critical
devices are items that only come in contact with an animal’s intact skin, and therefore bear
the lowest relative risk of disease transmission. Stethoscopes, otoscopes, electrodes, and
common animal restraining tools are a few examples of non-critical devices. Proper
treatment of this category of devices in between animals will depend on the nature and
degree of contamination sustained during use. Where contamination is minimal, simple
scrubbing with detergent and warm water may be acceptable to assure safety. However, the
use of low- or intermediate-level disinfectants may be appropriate to assure that disinfection
has been accomplished (Favero and Bond, 2000).
Factors that Impact Efficacy of Disinfection
Effective disinfection can only be achieved if the following interrelated factors are taken into
account:
Nature of the item or surface to be disinfected
Items with smooth, non-porous surfaces are the easiest to disinfect because they can be
effectively cleaned of organic debris and this will allow for ample surface contact with the
disinfectant applied. As a rule of thumb, the “rougher” the surface (i.e. crevices, damaged
finish), the more difficult cleaning and subsequent disinfection will be. To account for this,
increased contact time of the disinfectant or a higher disinfectant concentration on these
surfaces may be beneficial (careful with increased concentrations: toxicity and corrosive or
caustic effects may also be increased).
Contact time and other factors
The surface contact time is the minimum required duration of exposure of the contaminated
surface to the disinfecting agent in order to achieve the level of disinfection that is claimed
by the manufacturer. Therefore, careful consideration should be given to the manufacturer’s
recommendations and conditions of use. These recommendations will also address other
factors that may impact efficacy such as pH, water hardness, performance in the presence
of organic debris and optimal temperatures for product use.
Presence of organic material
The presence of organic material on surfaces to be disinfected will generally compromise

disinfection. Any type of contamination will reduce the surface contact time of the applied
disinfectant and therefore its effectiveness. Blood, blood products, body fluids and feces
contain proteins that will bind and inactivate some disinfections or slow their action. This
reinforces the importance of cleaning surfaces prior to disinfection whenever possible. If
organic debris cannot be removed, an increased concentration of the disinfectant or contact
time may be warranted. Furthermore there are also products available that will be effective in
the presence of organic debris.
Number and type of microorganisms present
In general, items that have a higher level of microbial contamination will require a longer
exposure to chemical products to achieve disinfection. Level and type of microbial
contamination may be presumed by the nature of a procedure that a device was used for,
the health status of the animal, or other epidemiological factors associated with the
veterinary care environment.
Resistance of microorganisms
Microorganisms vary in their susceptibility to disinfectants. In general, obligate intracellular

bacteria, such as Mycoplasma, are the most susceptible to disinfection. Gram-positive and
Gram-negative bacteria, enveloped viruses and fungal spores are less susceptible, but are
not considered to be resistant to disinfection. Highly resistant microorganisms include non-
enveloped viruses and mycobacteria. Although these organisms are hearty, they are not as
resistant as bacterial endospores or protozoal oocysts and even more prions, which are
resistant to most disinfectants (Quinn and Markey, 2000). The level of disinfection should be
based on the most resistant microorganism posing a risk in a given situation.
Type and concentration of disinfectant
As previously noted, microorganisms vary in their overall resistance to disinfection. However,

resistance will also depend on the type of disinfectant used. Each class of chemical products
acts differently on the cells. It is important to assure that the mode of action for the
disinfectant is compatible for the type of cells to be destroyed. General modes of action for
commonly used disinfectant classes are addressed in a later section of this chapter.
Regarding concentration, with all other variables constant, the higher the concentration, the
greater the effectiveness of the disinfection and the shorter the contact time (Favero and
Bond, 2000). However, there are exceptions to this rule including alcohol and iodophors that
have an optimal range of concentration. While the concept of using concentrated
disinfectants to achieve faster, more effective disinfection may seem like an attractive time-
saver, caution should be exercised. Chemical disinfectants are often hazardous to human
and animal health and an increased concentration is likely to equate to increased chemical
exposure hazard to personnel, animals and the visiting puplic.
Factors for Selecting a Disinfectant
Because all disinfectants are unique in their chemical action and properties, it is unlikely that
one disinfectant will fit all needs. However, for veterinary care purposes, there are a number
of desirable qualities that have been identified and should be considered for product
selection. These include (Quinn and Markey, 2000):
o Wide antimicrobial range

o Absence of chemical hazards (i.e. toxicity, teratogenicity, carcinogenicity)
o Compatible with a wide range of chemicals
o Non-corrosive
o Active in the presence of organic debris
o Stable at ambient temperatures
o Long shelf-life
o Effective over wide range of temperatures
o Inexpensive and readily available
o Nonpolluting and biodegradable
In Europe, there are a number of organizations that test disinfectants for use in animal
husbandry. In Germany, the German Veterinary Medical Society (DVG) provides guidelines
for testing disinfectants and publishes a list of disinfectants that have been tested for use in
animal husbandry. The list also contains information on effectivity of the products against
different categories of pathogens, and suggested concentrations and incubation times.
Products in this list have also been tested under conditions meant to simulate “real life”
outside the laboratory, and include effectivity in a protein-contaminated environment and on
“rougher” porous surfaces. The European Committee for Standardization (CEN) is currently
establishing European guidelines for disinfectant testing in the veterinary field.
Overview of Categories of Chemical Disinfectants
There are a number of compounds that have specific use as disinfectants in the veterinary
care environment. An overview of the main classes of chemical disinfectants is given in this
section but periodic consultation to current literature is required as new products are
constantly under investigation and emerging infectious diseases continue to rise. For lists of
tested disinfectants see e.g. the DVG (German Veterinary Medical Society) for products
used for veterinary medicine or in the food industry or the DGHM (German Society for
Hygiene and Microbiology) for disinfection in human medicine and hospitals. At this time, no
general European lists are available.
Alcohols
Alcohols are inexpensive, relatively nontoxic, and colorless, and ethyl and isopropyl alcohol
are most widely used. They are considered to be intermediate level disinfectants that
inactivate organisms by denaturing proteins. While they are effective for destruction of
enveloped viruses and tubercle bacilli, they are less effective against non-enveloped viruses
(particularly isopropyl alcohol) and are not sporocidal (Maillard and Russell, 1997). A
concentration of 70% of ethyl alcohol is most effective. When used as a surface disinfectant,
rapid evaporation of these compounds makes sufficient contact time difficult to achieve.
Additionally, alcohols tend to harden and swell plastic tubing when used over time, and may
be absorbed by rubber products that can lead to irritation of the skin and mucous
membranes (Favero and Bond, 2000).
Aldehydes
Formaldehyde (available in an aqueous 37% solution) and glutaraldehyde are two common
examples of this class. Formalin in solutions of 3% - 8% is effective for intermediate to high
level disinfection. It is less effective than glutaraldehyde in the presence of organic matter.
Despite discussions on potential carcinogenicity, skin irritation, and irritating fumes,
aldehydes are common compounds in disinfectant preparations used in the veterinary field
(i.e. breeding, husbandry, transport and disposal of all animals).
Glutaraldehyde, which inactivates microorganisms by alkylation, is also an intermediate to

high level disinfectant. At 2% active ingredient, glutaraldehyde solutions are effective against
bacterial endospores. Although minimally affected by organic matter, product effectiveness
is influenced by a number of factors such as pH and temperature. Glutaraldehyde solutions
are most effective at alkaline pH and effectiveness increases with temperature (Quinn and
Markey, 2000). Glutaraldehyde solutions are noncorrosive and are typically used in
immersion baths for high level disinfection of semi-critical devices (i.e. endoscopes) that
cannot withstand steam or heat sterilization. Due to the toxic nature of this compound,
special considerations must be made for proper handling and storage of solutions.
Immersion baths should have tight-fitting lids and be placed in an exhaust hood to minimize
personnel exposure to vapors. Additionally, to eliminate skin and mucous membrane
exposure, splash goggles and appropriate chemical resistant gloves should be worn.
Aldehyds should not be used at temperatures below 10 °C.
Alkalis
Sodium hydroxide (NaOH), or lye is a caustic alkali that has a wide virucidal spectrum when
used at a 2 percent concentration (prepare by mixing 1/3 cup of NaOH pellets per gallon of
water – careful! hot). This is effective against most bacteria, and enveloped and non-
enveloped viruses, although somewhat higher concentrations may be necessary for some
viruses. This concentration is effective against virusesincluding the causes of Avian
Influenza, Rinderpest and Pest of Small Ruminants, Newcastle Disease, Malignant Catarrhal
Fever, Lumpy Skin Disease and Sheep and Goat Pox, African Horsesickness and
Bluetongue, Foot-and-Mouth Disease, and Swine Vesicular Disease. Sodium hydroxide
should not be used on wood. Sodium hydroxide is also one of the few compounds that can
be used for the inactivation of prions (in much higher concentrations) (Taylor, 2000). An
overview on deactivation of prions is given below. A pH above 12 is required to for the
inactivation of bacilli such as Mycobacterium bovis. Chemical exposure risks to personnel
and animals as well as surface incompatibility are factors for the limited use of such
products.
Ammonium hydroxide, a weak base, has a strong activity against coccidial oocytes
(Williams, 1997). The high pH of even low aqueous solutions and intense fumes require
protective clothing when using this alkali.
Sodium carbonate, containing 0.1% sodium silicate, has significant virucidal activity and is
commonly used for the decontamination of aircrafts.
Biguanides
Biguanides are a group of cationic compounds commonly used for hand washing and skin
preparation.
Chlorhexidine, a low-level disinfectant that is incompatible with anionic compounds, is the

most commonly used compound in this group. Its activity is pH dependent and it is
inactivated by organic debris (McDonnell and Russell, 1999). It is more active against gram-
positive than gram-negative bacteria and Pseudomonas spec. and Proteus spec. are
resistant against chlorhexidine (Widmer and Frei, 1999). Although it is active against
enveloped viruses, it can not be used against non-enveloped viruses, mycobacteria and
most fungal species, such as dermatophytes, i.e. Microsporum canis, are resistant (DeBoer
et al., 1995) Because chlorhexidine has a longer residual activity on teat skin than most
other disinfectants, it is widely used for mastitis control (Quinn and Markey, 2000).
Halogens
Chlorine Compounds
Chlorine and chlorine releasing compounds, such as hypochlorite, chlorine dioxide, sodium
dichloroisocyanurate, and chloramine-T, are used in some countries for disinfection of
surfaces, equipment, buildings and vehicles as well as disinfection of milking installations
and in the food industry.
Sodium hypochlorite (NaOCL or household bleach) solution is an intermediate-level

disinfectant when properly diluted. The mode of action for this solution is dependent on the
formation of undissociated hypochlorous acid that will oxidize peptide links and denature
proteins (Maris, 1995). Solutions diuted to final concentration must be prepared directly
before use and should not be stored for more than 1 day. At a concentration of 0.1%, this
compound is effective against microbial agents of diseases, including enveloped viruses and
Foot-and-Mouth Disease virus. It can be prepared at the time of use by adding
approximately 30 cc (ml) of household bleach to a gallon of water (or 1 gallon of bleach plus
50 gallons of water). As pH decreases microbicidal activity (as well as corrosivity) increases.
In concentrated form chlorine-based disinfectants are usually unstable, and affected by light
and heat. They can lose 50% of their concentration in a month when left in an open
container (Widmer and Frei, 1999). Additionally, the corrosivity of active solutions can be
damaging to surfaces and equipment and irritating to the skin and eyes. Bleach solutions are
readily inactivated by organic debris, limiting their effectiveness to situations where devices
or surfaces have been thoroughly cleaned prior to disinfection. In areas heavily
contaminated with secretions, excretions, and soil, there is a considerable organic demand
for available chlorine and disinfection should be repeated at least once. Under such
circumstances a 3% solution of sodium hypochlorite should probably be used (3 liters of
bleach to 2 liters of water). This concentration is effective against a variety of agents of viral
diseases including Avian Influenza, Rinderpest and Pest of Small Ruminants, Malignant
Catarrhal Fever, Lumpy Skin Disease and Sheep and Goat Pox, and African Horsesickness
and Bluetongue as well as other non-enveloped viruses. At concentrations used for water
treatment it is not effective against Cryptosporidium parvum (Fayer, 1995). Halogens rapidly
lose efficacy on rough surfaces such as concrete.
Iodine Compounds
Iodine compound disinfectants are more effective in the presence of organic matter than
chlorine compounds, and less chemically reactive. Inorganic iodine and iodophors,
compounds in which iodine has been complexed with polymers to sustain the release of free
iodine and to increase water solubility, have both been used for disinfection. However, the
instability of inorganic iodine in the environment, skin irritations, hypersensitivity reactions,
and intense staining of contaminated surface limit its use. At appropriate dilutions, iodophors
are bactericidal, mycobactericidal, sporicidal, fungicidal, and virucidal. They are also widely
used as teat dips (Saran, 1995). Because the activity of iodophors is greater at a low pH, the
compound should not be used in alkaline conditions or mixed with other compounds.
Certain acidified iodophor compounds that contain a generic formulation of polyethoxy-

substituted polypropoxy-ethane complex are extremely effective disinfectant compounds
with strong virucidal activity (Maillard and Russell, 1997). The active ingredient must provide
a minimum use concentration of 0.02% titratable iodine.
Organic and Anorganic Acids
Acids inhibit the growth of microorganisms and a number of compounds are commonly used
as preservatives in the food industry. Acetic acid has been used for the treatment of wounds
infected with Pseudomonas spec. (Lemarie and hosgood, 1995). The use of acids may be
warranted under certain circumstances in the animal care environment. Formic acid is the
most effective of these substances and should be used at a concentration of 4%. Citric acid,
formic acid, and strong mineral acids, such as phosphoric acid, are effective for inactivating
the Foot-and-Mouth Disease virus (Quinn and Markey, 2000). Two mineral acids,
hydrochloric acid and sulfuric acid, are sometimes used for cleaning and disinfection of
animal housing. A 2.5% concentration of hydrochloric acid can be used to inactivate
endospores of Bacillus anthracis on the skin and is also effective against Rotaviruses and
Vesicular Stomatitis virus (Maillard and Russell, 1997). However, the activity of these
compounds is highly pH dependent and these compounds are corrosive and hazardous to
workers and animals. To minimize chemical exposure risk to personnel, citric acid should be
used when possible.
Peroxygen Compounds
Strong oxidizing agents such as hydrogen peroxide and peracetic acid, have a broad
antimicrobial spectrum. Hydrogen peroxide is fast acting, nonpolluting, and decomposes to
oxygen and water. Because it is unstable in solution, stabilizers are usually added. It is
bactericidal, fungicidal and virucidal, but should not be used against Mycobacteria (Maillard
and Russel, 1997). It is quickly inactivated by protein contamination of treated surfaces.
Peracetic acid is a strong oxidizing agent and an even stronger disinfectant that has
algicidal, bactericidal, fungicidal, virucidal and sporicidal activity. Unfortunately it can corrode
most metals, rubber and has been suspected to act as a cocarcinogen (Sattar and
Springthorpe, 1999). Oxidising agents rapidly lose efficacy on rough surfaces such as
concrete.
Phenolic Compounds
Phenol is cited in the literature as being the original standard against which other
disinfectants were compared (Quinn and Markey, 2000). Due to its toxicity and unpleasant
odor, the use of phenol for disinfection purposes has been largely replaced by the use of
synthesized phenolic compounds. Examples include ortho-phenylphenol and ortho-benzyl-
para-chlorophenol. These compounds are a complex group of chemicals often found in
various combinations and concentrations in disinfectant products. The effectivity of phenolic
compounds increases with the length of the alkyl chains and number of halogen atoms in the
molecule, but this also increases the sensitivity of the compounds to organic material, lowers
their water solubility and increases their toxicity, so that these compounds are not frequently
found in disinfectants for veterinary medical use. These products are considered to be
intermediate to low level disinfectants. For veterinary care purposes, it is important to note
that these compounds generally leave a residue on surfaces that may increase chemical
exposure for personnel and animals. Additionally, pigs and cats are susceptible to the toxic
effects of these compounds (Quinn and Markey, 2000). Also available are substituted
phenolic compound, that have potent virucidal activity when prepared as a 1% solution of
stock disinfectant. These types of compounds are effective against Eimeria tenella (Williams,
1997) and the viruses of African Swine Fever, Avian Influenza, Hog Cholera, and Velogenic
Newcastle Disease, but not effective against the viruses of Foot-and-Mouth Disease and
Swine Vesicular Disease (Widmer and Frei, 1999).
Quaternary Ammonium Compounds (QACs)
Quaternary ammonium compounds (QACs) are cationic detergents that may be used for low
level disinfection, especially surface disinfection. Benzalkonium chloride and dodecyl
dimethyl ammonium chloride are common examples of this group. QACs are relatively non-
toxic making them appropriate for general cleaning purposes. However, QACs are
inactivated by organic debris, metal salts in water (i.e. hard water) and anionic detergents
(Heinsohn et al., 1995). Even some gram-negative bacteria, such as Pseudomonas spec.,
can survive disinfection with QAC and may grow in QAC solutions. Such limitations must be
considered when developing procedures for the use of the products in the animal care
environment. QACS are found in many disinfectant formulations used in the food industry.
Inactivation of prions
Because of the political impact transmissible spongiform encephalopathies have had in the
past few years and the public perception of these diseases, this paragraph has been
included. Prions are characterized by extreme resistance to conventional inactivation
procedures including irradiation, boiling, dry heat, and chemicals (formalin,
betapropiolactone, alcohols). While prion infectivity in purified samples is diminished by
prolonged digestion with proteases, results from boiling in sodium dodecyl sulfate and urea
are variable. Sterilization of rodent brain extracts with high titers of prions requires
autoclaving at 134 C at 3 bar for 1 h . Denaturing organic solvents such as phenol or
chaotropic reagents such as guanidine isothiocyanate or alkali such as NaOH can also be
used for sterilization. Prions are inactivated by 1 N NaOH (>1 h), 4.0 M guanidinium
hydrochloride or isocyanate, 2,5 % sodium hypochlorite (2% free chlorine concentration; >1
h), followed by heating in an autoclave at 134 C at 3 bar for >1 h (Anonymus, 2003). It is
recommended that dry waste be autoclaved at 134 C at 3 bar for at least 1 hour (depends
on the thickness of the material) or incinerated. Large volumes of infectious liquid waste
containing high titers of prions can be treated with 1 N NaOH (final concentration) before
autoclaving at 134 C at 3 bar for 1 hour. Disposable plasticware, which can be discarded as
a dry waste, is highly recommended. Because the paraformaldehyde vaporization procedure
does not diminish prion titers, biosafety cabinets must be decontaminated with 1 N NaOH,
followed by 1 N HCl, and rinsed with water. Although there is no evidence to suggest that
aerosol transmission occurs in the natural disease, it is prudent to avoid the generation of
aerosols or droplets during the manipulation of tissues or fluids and during the necropsy of
experimental animals. It is further strongly recommended that gloves be worn for activities
that provide the opportunity for skin contact with infectious tissues and fluids. Formaldehyde-
fixed and paraffin-embedded tissues, especially of the brain, remain infectious.
Considerations for Disinfectant Use
Disinfectant products, as well as range of applications vary widely. However, careful

consideration of manufacturer’s product information in conjunction with a basic
understanding of limitations to the disinfection process will go a long way in assuring that
disinfection is an effective part of an infection control program.
As previously addressed, selecting the proper disinfectant for specific needs is essential.
Before purchasing a product, request and review information from the manufacturer. Many
manufacturers have product sheets that will go into more detail than the information on the
product label and may even include efficacy studies that could impact the decision-making
process. Review the material safety data sheet (MSDS) for the product to determine the
nature of hazards associated with product use and specific product disposal requirements.
Remember that disinfectants are designed to destroy living cells. Therefore, all disinfectants
are likely to be hazardous in one way or another to personnel and to client animals if the
product has residual properties or is improperly used.
Before disinfecting surfaces or devices, it is essential to clean the surface first if at all
possible. Organic debris can inactivate many disinfectants. Additionally, feces and other
body excretions can shield pathogens from contact with the disinfectant thus hindering
disinfection.
When using a disinfectant, it is essential that the product be used in accordance with the
manufacturer’s instructions. Note that the manufacturer’s efficacy claims are based on its
prescribed dilution ratio, method of preparation, method of application, surface contact time
and shelf-life. To clarify, contact time is the minimal amount of time that a product must be in
contact with the surface to be disinfected in order to achieve the level of disinfection claimed
by the manufacturer. Shelf-life is the amount of time that a diluted product is actively
effective for use once prepared from a concentrated product.
There are a number of organizations in Europe that provide lists of disinfectants for use in
animal husbandry, human medicine, and the food industry. Examples are the German
Veterinary Medical Society (DVG), and the Germany Society for Hygiene and Microbioloy
(DGHM). These lists can be consulted for commercial disinfectant preparations that can be
used in certain situations and against different types of pathogens.
Under circumstances where it is critical to assure that disinfection practices are efficacious,
(i.e. infectious disease outbreaks, immunosuppressed animal housing), it may be beneficial
to perform a surface contamination test. There are several techniques described in the
literature that may be used for surface testing (Tamasi, 1995). The floor may be the surface
of choice for such a test because it is likely to receive the most contamination of the
environmental surfaces, and this surface often has irregularities that will make disinfection a
challenge.
In cases of disease outbreaks, disinfection procedures may be dictated by law and close
cooperation with veterinary authorities may be necessary.
Summary
Cleaning and disinfection are a major component of disease prevention and eradication in
every zoological exhibition. Despite the development of antimicrobial drugs and effective
vaccines, infectious diseases remain a major threat to animals kept in captivity as well as our
wildlife population. The proper selection and proper use of disinfectants requires extensive
knowledge not only about the effectiveness of specific disinfectant compounds, but also
about the infectious agent, the conditions under which the disinfectant will be used and the
side effects of each compound, including toxic, caustic and corrosive properties.
However, disinfection is only one aspect of an effective infectious disease control program in
any animal or human health environment. As described in this chapter proper sanitation
consists of multiple components in addition to cleaning and disinfection. Insect and vermin
control, and proper food storage, handling, preparation and distribution are equally important
for a healthy zoo environment. Furthermore, sanitation should be part of a preventive
veterinary medicine program that would further include training of personnel, proper nutrition
and feeding, routine surveillance and prophylactic medicine including vaccination,
quarantine of new and sick animals, proper holding facilities, correct disposal of waste
products and dead animals.
This chapter can only provide a brief overview of the guidelines of cleaning and disinfection.
Before using a disinfectant, the manufacturer’s label should always be studied. When
dealing with a specific disease problem, further information regarding disinfection can be
found in the disease specific fact sheets. Additionally, the listed references will provide more
detailed information.
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virus. Appl. Microbiol., 24: 115 - 122.
Tamasi, G. 1995. Testing disinfectants for efficacy. Rev. Sci. Tech. 14: 75 - 79.
Taylor, D. M. 2000. Inactivation of transmissible degenerative encephalopathy agents: a

review. Vet. J. 159: 10 - 17.
WHO/CDS/CSR/APH/2000.3, 1999. Report of a WHO consultation: WHO Infection Control

Guidelines for Transmissible Spongiform Encephalopathies, Geneva, Switzerland, March
23-26, Annex III.
Widmer, A. F. and R. Frei. 1999. Decontamination, disinfection and sterilization. In: Murray,
P. R., E. J. Barron, and M. A. Pfaller (eds). Manual of clinical microbiology. ASM Press,
Washington, DC. Pp. 138 – 164.
Williams, R. B. 1997. Laboratory tests of phenolic disinfectants as oocysticides against the

chicken coccidium Eimeria tenella. Vet. Rec. 141: 447 – 448.
Table 1: Effectiveness of classes of disinfectants against various pathogens

Class of Disinfectant
Organic and Peroxygen
Alcohols Aldehydes Alkalis Biguanides Halogens Phenols QAC
Anorganic Acids Compounds
Chlorine Iodine
Infectious Agent
Compounds Compounds
obligat intracellular
gram-positive
Bacteria gram-negative
bacilli
endospores
Fungi including spores
enveloped
Viruses
non-enveloped
Protozoa including oozysts
Prions
highly effective effective limited effectiveness not effective
This table provides only a general overview of the effectiveness of various classes of disinfectants. The effectiveness of a specific compound in each
of these classes may vary. For more detailed information please review the information provided in the text. Always consult the manufacturers label
for correct use and concentration of each compound.
The table is based on the following sources: Boothe, 1998; Bruins and Dyer, 1995; Jaroll, 1999; Jeffrey, 1995, Lemarie and Hosgood, 1995; Maillard and
Russell, 1997; McDonnell and Russell, 1999; Quinn and Markey, 1999 and 2000; Russell, 1996, 1998 and 1999; Sattar and Springthorpe, 1999;
Sringthorpe and Sattar, 1990; Taylor, 2000; Widmer and Frei, 1999; Williams, 1997.
IX. BLUETONGUE IN NON-DOMESTIC RUMINANTS: EXPERIENCES

GAINED IN EAZA ZOOS DURING THE 2007 & 2008 BTV8 AND
BTV1 EPIZOOTICS.
Stephanie Sanderson
Chester Zoo
Introduction
Until the introduction of Bluetongue virus serotype 8 (BTV8) in 2006, Bluetongue had not
been a significant disease problem in Europe. The ensuing epizootic and subsequent
incursion of BTV1 have caused widespread mortality and morbidity in livestock and been a
major focus for international disease control in the EU. This chapter provides information
pertaining to these European epizootics – focusing primarily on the clinical species
susceptibilities of non-domestic ungulates and their response to vaccination. The data
presented has been derived from the literature and two EAZWV endorsed Bluetongue web
surveys of EAZA zoos covering the 2007 and 2008 disease seasons.
General disease information on Bluetongue can be found in disease fact sheet 7.
BTV8 in Europe: an atypical virus strain
Bluetongue is an insect borne disease caused by an orbivirus and affecting mainly domestic
sheep breeds and occasionally cattle. It is transmitted by midges of a few select Culicoides
species and its global distribution is largely defined by suitable climatological factors for
these species. Bluetongue viruses have been found on all continents excepting Antarctica
but the disease is generally only endemic in the tropics and subtropics (34° S to 53°N OIE
terrestrial animal health code). (Hately 2009, MacLachlan 2009).
Until 2006, BTV was not considered to be a significant threat to Central and Northern
European livestock as the resident Palaearctic midge species were not competent vectors.
However the unexpected occurrence of an atypical BTV virus in Maastricht in 2006 has
challenged established thinking on the behaviour of this disease.
Key differences from other BTV serotypes and strains include:

 Ability to use Palaearctic midges as vectors (C.obsoletus and C.pulicaris)
 Significant morbidity and mortality in cattle as well as sheep
 Fairly frequent vertical transmission in pregnant ruminants – a rare event in other BTV
strains.
Other features of BTV epidemiology in temperate climates include a marked seasonality with
clinical cases occurring almost exclusively between July and December and with
recrudescence occurring the following summer. It is still unclear how the virus is maintained
from year to year.
The source of this BTV8 strain is still unknown. Phylogenetic studies indicate the likely origin
to be sub-Saharan Africa and it has been postulated that it could have been introduced either
by an infected animal originating from Africa, imported infected vectors or as an illegally
imported vaccine strain. (EFSA 2007)
IX. Bluetongue in non-domestic ruminants
Other BTV subtypes in Europe
A number of BT serotypes have been present in Southern Europe from 1900’s onwards. The
spread of different BT serotypes since 1998 is shown in figure 1. The distribution of
serotypes in May 09 is shown in figure 2. BTV1 has been seen on numerous occasions in
Southern Europe however it was not until 2008 that it spread northwards and is also now
capable of spread via Palaearctic midges. Cases of BTV6 and BTV11 were also reported in
the Netherlands and Belgium in 2008. These are postulated to have been introduced via
illegal imports of modified live vaccine containing these serotypes from Southern Africa. At
the time of writing it appears that these serotypes have not become established unlike BTV8
and BTV1. Increased surveillance has also uncovered a previously unknown orbivirus in
Toggenberg goats which may indeed be a new serotype of BTV. Its clinical significance is
currently unknown. (Chaignat et al. 2009)
Fig 1. Spread of
Bluetongue throughout
Europe since 1998.
Picture, Institute of Animal
Health, Pirbright
Fig 2.
Species susceptibility to clinical infection
It is generally accepted that all ruminant species and some camelids are likely to be capable
of supporting BTV infection. Clinical expression of the disease is however highly variable
between viral serotypes, species, breeds and indeed environmental conditions and individual
health status. Animals indigenous to endemic areas appear to be clinically resistant.
(Verwoerd & Erasmus, 2004).
The pathology and pathogenesis of the disease is reviewed in detail by MacLachlan et al.
and the clinical picture seen in domestic species is well documented. (MacLachlan et al.
2009). The classical clinical signs of fever, nasal discharge, dypsnea, cyanosis of the tongue,
oral lesions and ulcers, oedema of the head and neck, lameness and hyperaemia of the
coronary band are due to virus mediated vascular injury. These signs are most frequently
seen in sheep where mortality rates can be up to 30% or higher. Cattle rarely show clinical
disease (with the exception of the European BTV8 strain).(MacLachlan et al. 2009)
Little is published about clinical susceptibility of non-domestic species. In North America,

white-tailed deer (Odocoileus virginianus), prong-horn antelope (Antilocapra americana) and
desert bighorn sheep (Ovis canadensis) are known to develop severe disease similar to that
described in domestic sheep. (Verwoerd & Erasmus, 2004). Abortion and death has also
been seen in dogs injected with BT contaminated vaccine and in a European lynx fed
infected meat (EFSA 2007; Jauniaux et al. 2008, MacLachlan 2009). Natural, asymptomatic
infection of African carnivores has also been reported. With the incursion of BTV8 into
Europe, European Zoos were in a unique position to contribute to knowledge on species
susceptibility to disease as they hold a naïve individuals representing a wide taxonomic and
geographic spectrum.
A survey of all 313 EAZA zoos was undertaken in January 2008 to collate data on clinical
disease seen during the 2007 BTV season. 49 zoos had confirmed BTV8 cases within 20km
and could be classified as at risk of infection. These 49 zoos held over 1000 susceptible
individuals of 53 different species and 7 ruminant families indigenous to Europe, North and
South American, Africa and Asia. Clinical disease was seen in 62 individuals (6% of the at
risk population), spread between 13 zoos (27% of at risk collections). (Sanderson et al.
2008).
Mortality and morbidity rates and the clinical picture seen in each affected species is
summarised inTable 1 (Sanderson et al. 2008). Bovidae are the most susceptible family of
ruminants to clinical disease, with four species showing morbidity rates of greater than 20%
and mortality rates of greater than 10%. The average case fatality rate for the affected
Bovidae species was 69%. All the affected ruminant species in this study were indigenous to
Europe, Asia or South America. Clinical signs in these species and are consistent with those
recorded for BTV8 infection domestic livestock. (Elbers et al., 2007) and with those reported
in yak (Mauroy et al. 2008;). It is noteworthy that despite over 200 African ruminants of 20
species being held by zoos in at risk areas, none of these were reported to have shown
clinical signs of infection. This is consistent with observations in Africa that indigenous
antelope do not develop clinical disease (Verwoerd & Erasmus, 2004).
Data was also gathered by European Zoos on species susceptibility to BTV1. There is yet
insufficient data on BTV1 to draw any firm conclusions however experiences so far suggest a
similar clinical picture and species susceptibility to BTV8.
Table 1: Morbidity, mortality, case fatality and clinical signs reported in ruminant species by zoos situated within 20km of confirmed BTV8 outbreaks during the
period August 2006 - December 2007 (Sanderson et. al. 2008)
AFFECTED At risk individuals Clinically Morbidity Laboratory Mortality Case Fatality
* Deaths ** *** Clinical Signs Reported in Affected Animals.
SPECIES (BTV8 within 20km) affected Rate confirmation Rate Rate
Abs. No. Abs. No. % Abs. No. Abs. No. % (%sick that died)
AFFECTED
519 55 10.60 25 38 7.32 69.09
BOVIDAE
American bison Lethargy, fever, mouth ulcers, drooling, difficulty eating,
(Bison bison) 30 10 33.33 3 5 16.67 50.00 conjunctivitis, corneal oedema, lameness, inflammation
coronary band, sudden death.
European wisent Lethargy, fever, mouth ulcers, drooling, difficulty eating,
(Bison bonasus) 20 8 40.00 4 4 20.00 50.00 conjunctivitis, corneal oedema, respiratory difficulty, lameness,
inflammation coronary band, sudden death.
Yak Nasal discharge, conjunctivitis, corneal oedema, drooling,
(Bos grunniens) 35 6 17.14 6 6 17.14 100.00 difficulty eating, respiratory difficulty, lameness, inflammation
of coronary band, sudden death.
Blackbuck
17 2 11.76 2 2 11.76 100.00 Sudden death
(Antilope cervicapra)
Sheep/mouflon Lethargy, fever, swelling head and neck, mouth ulcers,
(Ovis aries) 101 6 5.94 5 2 1.98 33.33 drooling, difficulty eating, conjunctivitis, respiratory difficulty,
lameness, inflammation coronary band, sudden death.
Goat
208 2 0.96 2 2 0.96 100.00 Lameness
(Capra hircus)
Alpine ibex/Tur
34 2 5.88 2 2 5.88 100.00 Nasal discharge, sudden death.
(Capra ibex)
Siberian ibex
4 1 25.00 0 0 0.00 0.00 Swelling of head and neck.
(Capra sibirica)
Muskox
5 1 20.00 1 0 0.00 0.00 Lethargy, fever, conjunctivitis, abortion.
(Ovibos moschatus)
AFFECTED
83 5 6.02 5 2 2.41 40.00
CERVIDAE
Fallow deer Mouth ulcers, difficulty eating, drooling, lameness, sudden
43 2 4.65 2 2 4.65 100.00
(Dama dama) death
AFFECTED
40 2 5.00 2 2 5.00 100.00
CAMELIDAE
Bactrian camel
8 1 12.50 1 1 12.50 100.00 Sudden death
(Camelus bactrianus)
Alpaca
32 1 3.13 1 1 3.13 100.00 Sudden death
(Lama pacos)
*Morbidity rate= number clinically affected / number at risk; **Mortality rate = number that die/number at risk; ***Case fatality = number that die / number clinically affected. Note: Morbidity and mortality rates
for the 2006 BTV8 epidemic in domestic livestock were 20% and 5% for domestic sheep and 7% and 3% in domestic cattle (Elbers et al., 2006)
Control Strategies for BTV: legislative framework
Bluetongue is a disease of global importance. Its ability to cause death and debilitating
disease across international borders has led to its inclusion in the World Organization for
Animal Health (Office International des Epizooties) Terrestrial Animal Health Code which in
turn has implications for trade. In EU Member States, control and eradication provisions are
laid out in Council Directive 2000/75/EC. Measures include vector control, restriction to
movements of live ruminants from affected areas to non-infected regions where the vector is
present and the use of vaccines. Of these, vaccination is the mainstay of control in areas
where BTV has become established.
The OIE guidelines on movement controls, diagnostic methods and vaccine production can
be found at: http://oiebtnet.izs.it/btlabnet/.
Further information on EU control measures are laid out in

http://ec.europa.eu/food/animal/diseases/controlmeasures/bluetongue_en.htm
In addition, individual European Countries have their own legislation and detailed disease
control plans (e.g.
http://www.defra.gov.uk/animalh/diseases/notifiable/bluetongue/about/index.htm ).
Vaccination
Mass vaccination has been identified by the European Commission as the most efficient
veterinary measure in combating bluetongue. Mass emergency vaccination campaigns can
be used to achieve the following objectives (European Commission, 2008/655/EC, Savini et
al. 2007):
1. prevention of clinical disease
2. limiting regional spread of BT
3. allowing regional/country eradication
4. safe movement of animals between affected and free zones.
A variety of vaccines have been developed of three different types: modified live vaccines
(MLV), inactivated vaccines (either whole killed virus preparations or virus like particle
produced from recombinant bacluovirus) and recombinant vaccinia, capripoxvirus or
canarypox virus vectored vaccines. (EFSA 2007). Only vaccine types currently approved
under EC approved national disease programmes (MLV and killed whole virus preparations)
will be discussed further here.
MLV have been used for over 40 years in endemic bluetongue areas (Verwoerd D. W. and
Erasmus, B. J. 2004).They are quick to produce (8-10wks), highly immunogenic and can
confer long lasting protection after a single dose. Using live virus has significant
disadvantages as there is potential for under attenuation causing symptomatic disease, milk
drop and foetal pathology, and for infection of the vector population leading to local spread
and potential for reassortment with field strains leading to new serotypes. For these reasons,
inactivated vaccines are preferred even though they take longer (6-8mths) to develop, are
more costly and require regular boosters in order to maintain efficacy. (EFSA 2007; OIE
2008)
There is little or no cross protection between different serotypes of Bluetongue, hence
vaccines are produced specifically in response to circulating BTV serotypes and strains. All
of the vaccines currently available in the EU for the control of BTV1 and BTV8 are inactivated
vaccines using saponin and aluminium hydroxide as adjuvants. (Table 2)
Table 2: BTV 1 & 8 Vaccines licensed for use in 2008.

Manufacturer BTV8 Vaccine Trade Names BTV1 Vaccine Trade Names
Intervet Bovilis BTV8
CZ Veterinaria Bluevac 8 Bluevac 1
Fort Dodge Zulvac8 Bovis Zulvac1 Bovis
Zulvac8 Ovis Zulvac1 Ovis
Merial BTVPUR
AlSap 8
Virbac SYVAZUL 1
Safety
Trial and field experience has found these vaccines to be safe in domestic species (EMEA
2009, Gethman et al. 2009, Eschbaumer et al 2009). An overview of field experience
following administration of over 60million doses of BTV8 vaccine in 12 countries was
undertaken by the European Medicines Agency (EMEA 2009). Mass vaccination campaigns
often necessitate deviations from normal procedure. Large groups of animals are brought
together, less attention is paid to their individual health status, needle hygiene is less good
and government instructions may deviate from those of the manufacturers (eg minimum age
of vaccination, target species, duration of immunity). In addition, compensation schemes in
some countries may lead to over reporting of certain adverse reactions. Despite these
factors, adverse reactions were seen in less that 1 in 10,000 animal. Those recorded are
typical for other inactivated vaccines and include local reactions and non-severe general
reactions such as pyrexia (fever) and lethargy.
A survey of all 313 EAZA zoos was undertaken in February 2009 to collate data on
vaccination in non-domestic species. Over 2000 individuals of 57 species in 47 institutions in
9 European countries were vaccinated for BTV8 using 5 of the products on the market during
2008. Adverse reactions occurred at a rate of 0.5% with half of these being local reactions
and 40% being abortions. The slightly higher rate could well be due to the relatively small
sample size and also because the species studied are not used to handling and are likely to
have been more stressed than their domesticated counterparts. Nonetheless, the abortion
rate is still well below that considered acceptable for vaccines. (EAMEA 2009)
Efficacy
Vaccine efficacy can be assed both by response to virus challenge (both clinical and levels of
viraemia) and serological response induced by immunisation. (Savini et al. 2008). Whilst
experimental virus challenge under laboratory conditions provides the most accurate
measures of efficacy, are the mainstay of vaccine testing and are required for vaccine
licensing (European Parliament 2001), field experiences are also provide a useful data. The
licensed vaccines have been shown to be efficacious in domestic animals (Eschbauer et al.
2009, Gethmann et al. 2009 ) The 2008 European Zoo Survey (Sanderson et al. 2009) found
that of the 37 bovidae (cattle, sheep, goat and antelope sp) and girrafidae tested post
vaccination, 100% seroconverted post vaccination as did 87% of the 40 South American
camelids tested. Of the 9 cervidae (deer sp) represented only 50% seroconverted. No
vaccinated animals succumbed to clinical disease post vaccination, despite virus circulating
in the area. These data suggest that the inactivated BTV8 vaccines are efficacious in
bovidae, girrafidae and to a lesser extent camelids. The sample size in the cervidae is too
small to draw any firm conclusions and further work needs doing to evaluate efficacy in these
species.
Conclusions
BTV poses a significant risk of mortality and morbidity in naïve non-domestic ruminants. The
clinical picture is similar to that seen in domestic livestock with species indigenous to
temperate areas of Europe, Asia and the Americas being most severely affected. Species
indigenous to Africa, the putative source of BTV8, were clinically unaffected. This suggests
that there is a genetic resistance to particular BTV serotypes.
Inactivated BTV8 and BTV1 vaccines have been used in many European zoos both on a
voluntary basis as part of national control measures. Adverse reactions were rare and in line
with those seen in the domestic species for which they are licensed. Vaccination produced a
reliable immune response and no animals showed clinical evidence of infection post
immunisation despite the presence of circulating virus in the region. These vaccines would
appear to be safe in non-domestic ruminants and efficacious in the bovidae and camelidae.
Further work is required to evaluate their efficacy in cervidae.
Further work is underway within the zoo community to expand our knowledge on vaccine
efficacy and duration of immunity in non-domestic species. Data is also being collected on
species susceptibilities to other BTV serotypes as they appear.
References
Chaignat, V., Worwa, G., Scherrer, N., Hilbe, M., Ehrensperger, F., Batten, C., Cortyen, M.,
Hofmann, M., Thuer, B., (2009). Toggenburg Orbivirus, a new bluetongue virus: Initial
detection, first observations in field and experimental infection of goats and sheep. Veterinary
Microbiology 138, 11-19.
Elbers, A.R.W , K. Mintiens , C. Staubach , G. Gerbier , E. Meroc , H.M. Ekker , F.J.

Conraths , A.N. van der Spek , A. Backx (2007) Epidemiological analysis of the 2006
bluetongue virus serotype 8 epidemic in north-western Europe; Nature and severity of
disease in sheep and cattle. In EFSA scientific report: Epidemiological analysis of the 2006
bluetongue virus serotype 8 epidemic in north-western Europe
http://www.efsa.europa.eu/EFSA/1178620925100/efsa_locale-
1178620753812_Bluetongue.htm
EMEA CVMP.(2009)An overview of field safety data from the EU for bluetongue virus
vaccines serotype 8 emerging from the 2008 national vaccination campaigns. London:
EMEA/CVMP/652019/2008.
Eschbaumer, M., Hoffmann, B., Konig, P., Teifke, J.P., Gethmann, J.M., Conraths, F.J.,
Probst, C., Mettenleiter, T.C., Beer, M., (2009). Efficacy of three inactivated vaccines against
bluetongue virus serotype 8 in sheep. Vaccine 27, 4169-4175.
European Commission, 2008/655/EC: Commission Decision of 24 July 2008 approving the
emergency vaccination plans against bluetongue of certain Member States and fixing the
level of the Community's financial contribution for 2007 and 2008, Off J Eur Union L214/66
(August).
The European Parliament and the Council of the European Union (2001). Directive
2001/82/EC of the European Parliament and of the Council of 6 November 2001 on the
Community code relating to veterinary medicinal products. Off J Eur
Commun;L311/1(November).
European Food Standards Agency (EFSA) (2007) Report of the Scientific Panel on Animal
Health an Welfare (AHAW) on the EFSA Selfmandate on bluetongue origin and occurrence.
EFSA Journal 479,1-29; 480, 1-20
Gethmann, J., Huttner, K., Heyne, H., Probst, C., Ziller, M., Beer, M., Hoffmann, B.,
Mettenleiter, T.C., Conraths, F.J., (2009). Comparative safety study of three inactivated BTV-
8 vaccines in sheep and cattle under field conditions. Vaccine 27, 4118-4126.
Hateley, G., 2009. Bluetongue in northern Europe: the story so far. In Practice 31, 202-+.
Jauniaux, T.P., De Clercq, K.E., Cassart, D.E., Kennedy, S., Vandenbussche, F.E.,
Vandemeulebroucke, E.L., Vanbinst, T.M., Verheyden, B.I., Goris, N.E., Coignoul, F.L.,
(2008). Bluetongue in Eurasian lynx. Emerging Infectious Diseases 14, 1496-1498.
Mauroy, A., Guyot, H., De Clercq, K., Cassart, D., Thiry, E., Saegerman, C., (2008).
Bluetongue in captive yaks. Emerging Infectious Diseases 14, 675-676.
Maclachlan, N.J., Drew, C.P., Darpel, K.E., Worwa, G., (2009). The Pathology and
Pathogenesis of Bluetongue. Journal of Comparative Pathology 141, 1-16.
Office International des Epizooties (OIE). Bluetongue. In: Manual of diagnostic tests and
vaccines for terrestrial animals. Paris: OIE; 2008.
Sanderson, S., Garn, K., Kaandorp, J. (2008) Species Susceptibility to Bluetongue in

European Zoos during the Bluetongue Virus Subtype 8 (BTV 8) Epizootic Aug 2006-Dec
2007. Proc. European Association of Zoo and Wildlife Veterinarians.
Sanderson, S., Edwards, S.J., Setzkorn, C and Baylis M. (2009) Survey of Species
Susceptibility to Bleutongue virus and Bluetongue Vaccine Usage in European Zoos During
2008. Proc. International Conference on Diseases of Zoo and Wild Animals. 1. 2.
Savini, G., MacLaclalan, N.J., Sanchez-Vinaino, J.M., Zientara, S., (2008). Vaccines against
bluetongue in Europe. Comparative Immunology Microbiology and Infectious Diseases 31,
101-120.
Verwoerd D. W. and Erasmus, B. J. (2004) in: Infectious Diseases of Livestock. Eds Coetzer
J.A.W & Tustin R.C. Oxford University Press pp 1201-1220
X. TUBERCULOSIS IN ZOO SPECIES: DIAGNOSTIC UPDATE AND

MANAGEMENT ISSUES.
EAZWV Tuberculosis Working Group
1. Introduction
One person out of three is currently infected by tuberculosis over the world. More than 2
millions of people die from it every year. Recent raising of Multi Drug Resistance or even
Extreme Drug resistance, co-infection with HIV and incidence of non tuberculous
mycobacteria (NTM) makes the battle against tuberculosis more difficult: the disease is now
placed in the top 3 list by WHO, together with AIDS and Malaria.
Most of the mycobacteria from the ‘tuberculosis complex’ have the ability to infect wild
species, in whom the pathogenesis, receptivity and immune responses vary widely. Genetic
key factors (e.g. Interferon Gamma receptor or vitamin D genotypes) are obviously acting
towards these differences (SCHLUGER, 2005). Course of disease and occurence of latent
infection vs. open disease are variable among species, from extremely sensitive old world
monkeys to apparently resistant equids.
Table 1: Tuberculosis complex mycobacteria and their reported hosts.

Mycobacteria of the Major historical known Reported Wild and Zoo Host
Tuberculosis complex host or burden
M.tuberculosis Human, Non Human Elephant, NHP, Beisa Oryx,
Primates (NHP) Addax, Goats, Birds, Lowland
Tapir, Giraffes, Springboks,
mongoose, Rhinoceros
M.bovis Cattle (+buffalo, Bison) All ruminants, Badgers,
Possums, Meerkats, Big Cats,
Canids, Rodents, NHP, Wild
boars Elephants, Camelids,
Rhnoceros, Onager, Horse,
Birds
M.africanum Human Cattle, Swine, NHP
M.microti Vole, Camelids NW Monkeys, Big Cats
M.pinnipedii Pinnipeds Camel, Tapir, Big Cats
M.caprae Goat, Sheep, Swine Swine, Cattle Wild Boars, Red
& WT deer, Camel, Bison
M.canetii Human ?
« Dassie bacillus » Hyraxes Meerkats
X. Tuberculosis in Zoo Species
2. European regulation
National programs
Domestic species of cattle in zoos and safari parks should normally be subjected to routine
tuberculin testing as often as the indicated testing interval for the area in which the zoo is
located.
Zoo species are generally exempted from statutory TB testing and, in any case, there is no
recognized, approved screening test for TB in species other than bovines and deer.
Several texts are defining sanitary policy for tuberculosis within country members. The EU
policy mainly focuses on the eradication of bovine tuberculosis and is based on two
fundamental principles:
1/ The Member States are primarily responsible for the eradication of bovine tuberculosis
and may receive community financial support for the eradication program
2/ Eradication of bovine tuberculosis in the EU must be the final target and the Member
States must consider eradication as the defined aim.
Hence, most of the EU regulations apply only to M.bovis- sometimes M.tuberculosis -

screening.
Table 2: European legislation concerning animal tuberculosis.

LEGISLATION RELATED TO THE ERADICATION OF BOVINE TUBERCULOSIS
Council Directive 64/432/EEC of 26 June 1964 on animal health

problems affecting intra-Community trade in bovine animals and swine
Council Directive 77/391/EEC of 17 May 1977 introducing Community
measures for the eradication of brucellosis, tuberculosis and leucosis in
cattle
Council Directive 78/52/EEC of 13 December 1977 establishing the
Community criteria for national plans for the accelerated eradication of
brucellosis, tuberculosis and enzootic leukosis in cattle
Commission Decision 2003/467/EC of 23 June 2003 establishing the
official tuberculosis, brucellosis, and enzootic-leukosis-free status of
certain Member States as regards bovine herds
Commission Decision 2003/849/EC of 28 November 2003 approving
the programmes for the eradication and monitoring of animal diseases
and for the prevention of zoonoses presented by the Member States for
the year 2004
Council Decision 90/424/EEC of 26 June 1990 on expenditure in the
veterinary field
Council Decision 90/638/EEC of 27 November 1990 laying down
Community criteria for the eradication and monitoring of certain animal
diseases
Based on the Commission Decision 1999/467/EC of 15 July 1999 seven states of the
European Union were classified as free of bovine tuberculosis: Denmark, Germany,
Luxembourg, the Netherlands, Austria, Finland, and Sweden. In 2004 Belgium, the Czech
Republic, and France were added to this group and on 4th March 2005 an eleventh country,
Slovakia was added. Switzerland also has the status “free of bovine tuberculosis”.
BALAI application within Europe
The BALAI directive 92/65 requires that all ruminants traded between institutions must come
from officially free herds, as it’s mentioned in TRACES health certificates.
Moreover, to be BALAI approved, an institution must be free of “bovine tuberculosis”, as

listed into the Annex A of the directive, for at least three years, or “tuberculosis” (this term
includes all mycobacteria of the TB complex) for primates, felidae and ruminants, if the
member state has a control monitoring progam. During a transfer between two approved
bodies, Tb testing is not required by the BALAI directive, but many member states (UK,
Sweden,…) added TB test as an additional requirement when importing ungulate, whatever
institutions it is coming from.
Given these requirements, a lot of zoo mammals are still being exchanged in national and
international transfers without any serious individual testing.
3. Quick overview of current diagnostic methods
Standard screening tools like skin testing and sputum smears have limited application in
wildlife species, especially when prevalence is low. Inversely, investigations of cell-mediated
immunity through an vitro assay of gamma interferon have numerous advantages (good
sensitivity), as long as technical limits are known and can be improved. Furthermore, new
tools based on the investigation of humoral immunity seem very promising for the detection
of antibody directed against certain immunogenic mycobacterial antigens in a wide range of
species. All these methods are currently evaluated in field studies, despite difficulties to
ensure rigorous validation.
Thus, diagnostic methods are hardly homogenic and never validated for any zoo species.
Bearing in mind that validation will not easily be possible, zoo actors must be aware of actual
tests available.
Limits of unspecific diagnosis
Clinical signs of TB are rarely seen prior to death in zoo animals (LYASCHENKO & AL, 2006,
MONTALI & AL, 2001). If cough and dyspnea are noticed, this is always in a very late and
irreversible stage of the pulmonary form of disease. The most frequent sign noticed among
all mammals is chronic weight loss.
Imagery techniques can help, especially laparoscopy followed by biopsy of suspect

granulomas or tissue. A CT scan is useful to detect lesions in non-palpable lymph nodes. On
the other hand, though X-rays are a regular step in human diagnostics, it is not practically
useful in zoo veterinary medicine: the size of animals may prevent X-R use, references
images are often missing, skills required to interpret pictures are often out of “average zoo
vet” range and, on top of that, a lot of species do not show any calcified lesions.
Limits of direct examination
Culture stands as the “gold standard” method, but requires at least 2 to 6 weeks delay before
a test result can be obtained, even with recent fast techniques (VARGAS & al, 2005) which are
still not validated in veterinary medicine. Hence, faster testing still relies on microscopic
examination and mycobacterium DNA amplification methods.
The threshold number of bacilli needed to obtain a positive microscopic exam (Ziehl Neelsen
and other stains) is around 104 bacilli / ml, which is a rather important and infectious value. In
a culture, it is possible to detect bacilli loads superior to 101-102 / ml. As a comparison, less
than 10 colony forming units (CFU) of M.tuberculosis per ml is enough to infect a
cynomolgus macaque (LIN & al, 2006).
Many biological samples contain very few bacilli (highly calcified lesions, intermittent
shedding in biological fluids like bronchial secretions or milk). As an example, 15 to 20% of
active pulmonary forms are not even confirmed by any culture in human being (FRIEDEN & al,
2003). Therefore, only positive culture findings provide evidence of disease, whereas
negative culture results may not rule out infection in exposed/suspected animals.
Detection of DNA material in biologic samples have been used already in various zoo
species: elephant trunk wash (MIKOTA & AL), broncho-alveolar lavage (FLYN & al, 2003),
gastric lavage,.. Amplification methods (PCR) are now well developed and help specify the
mycobacteria with the use of selected probes. However, some of these biological samples
are likely to host many other bacteria that are impairing PCR efficacy. Within the last years,
very sensitive and specific PCR became available, but it still can’t be used as a broad
screening tool as described in table 3.
Table 3: Example of PCR sensitivity in human sputum with 5% prevalence. From (VEZIRIS,
pers. com.). With those given sensitivity and specificity, a positive PCR result is meaning an
even probability (49%)of TB infected or free animal, mainly because of the low prevalence.
The lower the prevalence is, the lower the PPV turns out.
PCR Disease No Disease
Positive predictive value
Se=72% (Active TB) (Latent or no TB)
PPV=3.6/(3.6+3.8)
Sp= 96%
=49%
Prevalence 5% 5 95
PCR + 3.6 3.8 Negative predictive value
PCR - 1.4 91.2 NPV=91.2/(91.2+1.4)
=98%
When there is enough DNA (rich sample or culture), molecular typing should be performed.
Spoligotyping or other methods (VNTR,..) allow to identify strains of mycobacteria and these
techniques can be a very useful tool to track the epidemiological circuit of the TB. In recent
years, several tuberculosis strains were spoligotyped and their fingerprint can be compared
to other circulating strains. Whenever a tuberculosis mycobacterium is discovered, this kind
of typing should always be performed, or at least samples must be kept frozen (-25°C or -
80°C) until further typing is available.
Cell mediated immunity (CMI) exploration
Memory of T cells regarding previous contact with Mycobacterium can be assessed by

presenting selected antigen(s) to them, either in vivo (skin test or “Mantoux” test) or in vitro
(Lymphoblastic Transformation Test –LTT- or Interferon Gamma tests).
Skin testing is far from being validated for zoo species, as there are great variations between
tegument and dermal structure within species. The test relies on local inflammatory cell
recruitment, which can be low or absent for many reasons: tegument cellular organization,
immunosuppressive status, superficial temperature, … Skin test sensibility is often poor: e.g
70 to 90% in human, 50 to 90% in zoo hooftstock (COUSINS AND FLORISSON, 2005).
Specificity depends on antigen (tuberculin) injected intradermally but could also be low
because of species-specific features (e.g. orangutans) or co-infection by other non
tuberculous mycobacteria (e.g. avium complex), leading to false positive results.
Materials and methods of application of the skin test vary between countries and vets:
standardized quality of tuberculin is still missing within EEC, reading methods also vary
between vets with a lot of “distant” appreciations of TST local reaction. Whenever possible, a
close exam, palpation and caliper measurement of tegument is strongly recommended in
order to avoid false negative results.
In vitro tests of the cellular immunity rely on (re)stimulation of T lymphocytes memory. Thus,
first compulsory step is to keep cells alive until they reach the lab. Blood sample should
reach lab for stimulation no later then 8-10h after collection and should be kept at ambient
temperature until there. Particular care must be paid to homogenic and full mixing of blood
and anticoagluant (heparine) at collection.
Sometimes part of experimental study, LTT is not a current option to a zoo vet because of its
limited reproducibility and the use of radio-elements. Gamma interferon (IFNg) tests stand as
a good option as they are already available and marketed for cattle, non human primates,
deer and humans. These tests are performed in two steps, for a total run of 48h minimum.
The first step is to incubate T cells with selected mycobacterial antigen (usually bovine and
avium ppd), leading them to produce IFNg when they were previously in contact with the
same antigen.. The second step is to reveal the amount of IFNg produced through an ELISA
or an ELISPOT (more sensible). This step can be delayed as soon as stimulation wells have
been frozen.
Commercial kits are available, designed for cattle (BOVIGAM®), primates (PRIMAGAM®),
deer (CERVIGAM®, but production is discontinued at this time) and human (QUANTIFERON
GOLD®). Recent studies show that these tests can be used in some exotic species : for
example a cattle-designed test will detect INFg of a large range of exotic Bovidae, but also
some animals standing outside of this family (e.g. Girafidae). On the other hand, other
artiodacyl species just don’t do well with the usual positive control antigens, or are not
detected by ELISA (RIQUELME, 2009). This also counts for the primate-referred test: although
a list of “validated” species is provided on its leaflet, field studies showed contrasted results
(LÉCU, 2008, RIQUELME, 2009).
In order to overcome the problem of specificity in ELISA detection of INFg, in-house modified
tests can be created. One solution is the detection of mRNA coding for INFg, which seems to
have broad nucleotid sequences, shared by a lot of different mammal species. This can only
be achieved by experienced laboratories. Another solution is to design a specific interferon
antibody, as is currently being developed in elephants and rhinoceros.
Whatever kind of cellular exploration is chosen, the duration of the cell-mediated immunity is
rather unknown. All studies concerning longest periods of time (VERVENNE & AL, 2004) and
work on BCG vaccination protection length suggest that stimulation tends to go back to a
baseline level, which remains unknown beyond a year.
Humoral immunity exploration
Serodiagniosis of tuberculosis suffered from “bad reputation” because early trials were
assessing antibody against broad mycobacterial antigens, showing a very low specificity in
these tests. During the course of an infection, Th1 activity (CMI pathway) is thought to be
initially greater than Th2 (humoral route) in order to control and confine infection. An
inversion of this Th1/Th2 balance control is often associated with a relapse or with active
disease (DOHERTY & ROOK, 2006). Thus, seeking for antibodies maybe of little help to screen
for latent infected animals, but is becoming more relevant to detect and monitor more active,
“sick” and shedding individuals. It has been clearly noticed also in human (ABEBE & al, 2007)
that some antibody rise occurs during the shift from latent to active disease, so that a
prognostic value may be added to certain serological results
Some mycobacterial antigens elicit humoral response in a wide range of species. Most
relevant antigens have been selected to design ELISA and Rapid Lateral Flow Technology
test, with good results in a lot of various species (elephants, primates, tapirs, camels,
deer,…). However, the panels of immunostimulant antigen will remain directly linked to the
type of mycobacterium involved, host species, timeframe of disease and also individual
condition. Some commercial tests validated for Asian and African elephants (ElephantTB
STATPAK®, DPP® VetTB) or primates (PrimaTB STATPAK®) also look promising in non
target species (GREENWALD ET AL. 2009; LYASHCHENKO ET AL. 2008).
Sensibility (Se) and specificity (Sp) of these serological assays are encouraging. However,
Se and Sp values are issued from descriptive studies performed on either very sensitive
species (i.e prone to start disease soon after infection) or under very clear enzootic
conditions, maybe jeopardizing the real Se and Sp. Moreover, serological profiles of animals
in long-term quiescent infection status are still remaining poorly known.
Repeated testing seems to be the only way to adapt with these actual limitations, as titers is
thought to arise when relapse is about to happen. As zoonotic risk increases a lot within this
shifting time, detection of early serological change may be relevant to trigger a deeper
screening (direct exam) and preventive measures towards staff and public.
4. Current recommendations
Within some species, current captive population shows more than 10% of infected herds. To
draw a parallel, for a country to keep the TB-free status regarding bovine tuberculosis, the
OIE requires that the percentage of herds confirmed infected with M. bovis has not exceeded
0.1% per year for 3 consecutive years.
All the zoo community should make an effort, even if available tools are still – and will likely
remain - unvalidated. Crossing the three exploration paths (direct, CMI and serology) is a
good way to partially get rid of some of their respective limitations.
The aim of detecting TB in zoo species has two levels: the first one is to protect staff
(keepers, vet) and public from zoonotic contamination. Few actual tools (serology, PCR) are
aimed at this predictive purpose, focusing on the active (=excretion) phase of the disease.
The second level is the census of latently infected individuals and their monitoring. For this
purpose, CMI testing can then help a lot, as long as their sensibility / specificity limits are
taken into account.
Table 4: Testing categories available. A positive test from one of these boxes should always
initiate a test from another box.
Any repeated positive outcome in the immunological investigationary tests should trigger
exploration of the direct exam category, in order to determine the shedding status of the
animal. If direct exam is positive, measures should be taken to avoid contamination of staff,
surrounding animals and premises.
“Euthanasia or treatment” is a choice that should be considered first with veterinary officials,
occupational physician, all related actors and then according to global captive population
status and dynamics (EEP, TAG, …). Depending on TB extension analysis, treatment may
be an alternative to euthanasia, to prevent infected animals from shedding mycobacteria.
Treating an animal for TB implies following strict rules of drug administration,
pharmacokinetic check, observance and excretion follow-up, that are very hard to deal with.
Zoos who engage in treatment must stick to these rules and require permission for treatment
from the official authorities. It must be borne in mind that failure in the treatment could lead to
emergence of resistant strain as it was already noticed (LYASHCHENKO ET AL, 2006).
The effect of a successful treatment is to bring the animal back to a latent stage, which
means that reactivation will always be possible in any stages of its life when treatment is
discontinued. Thus, treated animals should be monitored closely for the rest of their life. It
could be recommended that treated animals remain in the premises where they have been
treated or are transferred to an institution with at least the same level of monitoring abilities.
An updated database –working group leaded- is under progress, in order to gather and share
information on tests, categorized by zoo species. The feedback of test results is of primary
concern, as well as follow-up of suspect populations and fingerprinting of circulating strains.
Zoo vets, TAG & EEP advisor must arouse this interest within their relative animal & human
population. It’s not acceptable anymore to exchange animals without any medical history
including TB.
Table 5: Current TB risk and monitoring levels, and methods of diagnostic available for
relevant selected species in zoo. Refer to Annex 3 for more specific recommendations when
existing.
Species Risk - Recommended methods
(G): see TB Recommended monitoring
guidelines level
in Annex 3
Bovidae Depends of status of  Skin test: 0.1ml of bovine PPD, 1mg/ml or
surrounding animals, and at least 2000 UI/test injected intradermally
above all, contact with feral into a shaved cervical area. Reading at 24,
animals. 48 and 72h includes palpation and
Maintenance monitoring = measuring the skin thickness with an
testing on occasions: appropriate caliper. Caudal tail fold less
exchange, anesthesia – if appropriate to wild species.
purpose doesn’t interfere  Bovigam® : blood should be tested within
with TST (infection, use of AI 8h after sampling. Refer to literature for
drugs,…) adjusting TST and elicit booster effect.
 Serological test
Primates Old world monkeys are  Skin test with 0.1ml bovine ppd (at least
highly susceptible to 2000 IU/test, 1000 to 10000 times greater
(G) infection, new world than human) into eyelid or abdomen skin.
monkeys are fairly resistant Reading at 24, 48 and 72h. Old
but able to develop TB. mammalian tuberculin may be used to
OIE recommends quarantine increase sensibility but it decreases
on recently imported specificity
primates with 2 to 3 skin test  Primagam® : whole blood or white cells

at a 2-week intervals. should be stimulated 8h max. after
sampling. Refer to literature for validated
or successful species.
 RapidTest (PrimaTB STATPAK®) on
serum
Carnivores Not a standard test to  Comparative cervical test(CCT) maybe
perform except when there is used
a risk of exposure (Tb cases  Serological assays looks promising (ELISA
in zoo or fed with suspect and RapidTest)
carcass)  In-house interferon test could be used
(badgers), see references.
Sealions Some South American  Skin test with 0.1ml bovine ppd in cervical
sealions (O.byronia) infected area, up the shoulder.
(G) with M.pinnipedii. Likely to  RapidTest (ElephantTB STATPAK®) :
come from imported animals. prefer serum to whole blood
Transmission to other  ELISA on serum
species of pinnipeds (seals,  Stains, PCR and culture of any nasal
Californian sealion) and discharge
terrestrial mammals
occurred.
Test should be required for
any trade or as soon as
animals are trained enough.
Camelids As bovids  Skint test as indicated for bovids but one
has to be aware that the percentage of
false positive and false negative is much
higher than in bovids
 Serologic tests (ELISA, ElephantTB STAT
PAK)
Tapirs Malayan tapirs are most at  TST or Comparative skin test can be
(G) risk with a not yet defined performed with 5000 IU=0.1ml bovine ppd
proportion of captive tuberculine and 2000 IU avian ppd
population infected. tuberculine injected in the inguinal area.
Non specific reactions are not uncommon
 Serological test showed good results
Elephant Infected Asian elephants are  Serological tests show the best sensibility
(G) still detected. and specificity : ELISA, ElephantTB
STATPAK® and DPP®VetTB
Deers Clusters of infected feral  Comparative Cervical Test with avian and
deer remain in Europe. bovine ppd (M.avium infection is not rare in
cervids)
 Serologic tests (ELISA, CerivdTB STAT
PAK®, DPP®VetTB)
Rhinoceros M.tuberculosis infection from  Caudal Fold skin test
infected human reported in  Gastric lavage with culture and PCR
black rhino  Serologic tests (ELISA, CerivdTB STAT
M.bovis infected white and PAK®, DPP®VetTB)
black rhino reported in zoos.
Annex 1
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Annex 2
List of National Laboratory regarding Mycobacteria diagnostic.
VLA Weybridge
New Haw, Addlestone, Surrey KT15 3NB
UNITED KINGDOM
Tel: (44.1932) 34.11.11 Fax: (44.1932) 34.70.46
Glyn Hewinson
Email: r.g.hewinson@vla.defra.gsi.gov.uk
AFSSA Alfort
Unité Zoonoses Bactériennes
23 avenue du Général de Gaulle, 94706 Maisons-Alfort Cedex
FRANCE
Tel: (33 (0)1) 49.77.13.00 Fax: (33 (0)1) 49.77.13.44
María Laura Boschiroli-Cara
Email: ml.boschiroli@afssa.fr
Veterinary Research Institute

Hudcova 70, 62132 Brno
CZECH (Rep.)
Tel: (420.5) 33.33.16.01 Fax: (420.5) 33.33.12.29
I. Pavlik
Email: pavlik@vri.cz
VISAVET
Laboratorio de vigilancia veterinaria, Facultad de Veterinaria, Universidad Complutense de
Madrid
Avda. Puerta de Hierro, s/n. Ciudad Universitaria
28040. Madrid
SPAIN
J.M. Vizcaino
Email: jmvizcaino@visavet.ucm.es
Community reference laboratory for bovine tuberculosis from July 2008 until July
2013.
Friedrich-Loeffler-Institute
Federal Research Institute for Animal Health
Suedufer 10
D-17493 Greifswald - Insel Riems
GERMANY
Phone +49 38351 7-0
Fax +49 38351 7-219
Dr Irmgard Moser
Email : irmgard.moser@fli.bund.de
Central Veterinary Institute - Lelystad

Edelhertweg 15
8203 AA Lelystad
THE NETHERLANDS
Phone: +31 320 238159
Fax: +31 320 238050
Douwe Bakker
Email : d.bakker@wur.nl
National Veterinary Institute (SVA)

Department of Bacteriology
Section of Mycoplasma and Mycobacteria Diagnosis
S-Uppsala
SWEDEN
Göran BÖLSKE
E-mail: goran.bolske@sva.se
Institut National des Recherches Veterinaires (INRV)

Laboratory of Tuberculosis
B-Brussel
BELGIUM
Jacques Godfroid
E-mail: jagod@var.fgov.be
Laboratorio Nacional de Investigaçao Veterinaria

Ministerio de Agricultura, do Desenvolvimento Rural e das Pescas
Estrada de Benfica 701
1500 Lisboa
PORTUGAL
Tel. 351 1 7115200
Fax. 351 1 7115383
Alicia Amado
Email : alice.amado@lniv.min-agricultura.pt
Department of Bacteriology
National Veterinary and Food Research Institute
Hämeentie 57
Helsinki
FINLAND
Tel. 358 9 393 18 26
Fax. 358 9 393 18 11
Sinikka Pelkonnen
Email : sinikka.pelkonen@eela.fi
Sections of Bacteriology, Virology and Serology, and Pathology

National Veterinary Institute
8156 Dep
N-0033, Oslo
NORWAY
Tel. 47 22 96 46 75
Fax. 47 22 60 09 81
Oeivind Oedegaard
Email : oivind.odegaard@vetinst.no
Institute of Veterinary Research of Thesaloniki

80, 26th October Street
Thessaloniki
GREECE
Tel. 30 31 55 20 28
Fax. 30 31 55 40 22
Zoi Dimareli-Malli
Email : vetresi@the.forthnet.gr
EAZWV Working Group members

Annex 3
Tuberculosis guidelines for some species
Malayan Tapirs (Tapirus indicus), from M.Hoyer 2009.
South American Sea Lions (Otaria byronia) issued from the 1st Sealion TB Meeting,
Duisburg, Sept. 2009. Contact
Elephants (Elephas maximus)

W.Shaftenaar, vet advisor of Elephant EEP
26 June 2009
These recommendations for the control of tuberculosis in captive Asian en African elephants
are aimed to prevent the spread of Mycobacterium spp. that can cause tuberculosis in
mammals. The recommendations are based on the document “TB testing in captive
elephants
in the EEP, 23 July 2008”, (see annex to this document and are reflecting the current
possibilities for testing within Europe. The document will be updated when new relevant
developments become available.
The interpretation of the available diagnostic tests is under constant evaluation and the panel
of experts involved in TB-testing in elephants in recent years will be consulted when
questions arise.
Glossery
Antibody test (serum or plasma): ELISA. At present, the Central Veterinary Institute
Lelystad is the only institute in Europe running this ELISA on a routine base.
Antigens used: M.bovis, MPB70 and M.paratuberculosis.
Address:
Central Veterinary Institute,
DSU
Edelhertweg 15,
8219 PH Lelystad,
the Netherlands
Elephant TB STAT-PAK Assay: Also known as ERT. Test to be performed by a qualified zoo
veterinarian or veterinary institute.
The test is available through the following website: www.zootest.com
Culture of suspected material to be sent to: National Veterinary Laboratory
Tuberculin to be obtained from: National Veterinary Institute
Trunk wash for culture and/or PCR See definition in the annex
How to act when an elephant has to be transported from or to a zoo that participates
in the elephant EEP?
Before the transport of an elephant to or from an institute that participates in the elephant-
EEP, the veterinary advisor to the elephant TAG must be notified. Together with the institute
that is sending the animal as well as the receiving institute, the veterinary advisor will
propose the measures to be taken in order to reduce the risk of transmission of tuberculosis.
In case of disagreement, the studbook coordinator will be informed and the EAZA veterinary
committee will be consulted if the parties involved do not resolve these issues. The advice of
the veterinary advisor is always subject to the (inter-)national veterinary legislation applicable
in the countries involved at the time of shipment. The veterinary advisor will base his advice
on the pre-transport screening and specific test results as described below.
Pre-transport screening
A report about the history of the animal should be made, including the following data:
• Species / ID / date of birth / location of birth
• Locations where the animal has been kept during its entire life, including dates of entry
in any new location
• Has the animal previously been suspected of tuberculosis or treated for this disease?
• Has there been any known direct or indirect contact with confirmed or suspected TB-
cases in herd mates or other mammalian species, including humans?
• List of data when blood was sampled, tested and stored at below -20°C
• Test results of any TB-test performed in the animal in the past
• Does the animal show clinical signs that are suggestive for tuberculosis?
Specific tests
The animal(s) that will be transported must be tested 4-6 weeks prior to transport for the
presence of specific antibodies using the following tests: ELISA’s and Elephant TB STAT-
PAK Assay (ERT). Serum or heparin plasma of the elephant(s) must be shipped on dry ice to
the Central Veterinary Institute (CVI) in Lelystad to be tested in the ELISA’s that are routinely
used for elephants. The Elephant TB STAT-PAK Assay will also be performed at the CVI-
Lelystad. Costs for these tests will be covered by the sender of the samples.
On the same day when the blood sample is taken, a comparative skin test will be done: 2000
IU of ppd avian tuberculin* will be injected intracutaneously in a thin part of the skin on the
base of the backside of the ear. At the same time 3000-5000 IU of ppd bovine tuberculin* will
be injected intracutaneously on the contra-lateral ear at a similar location. If only one ear can
be used, both injections should be given at a minimum distance of 10 cm from each other.
* Standardization of the quality of the tuberculin is of outmost importance. In case of any
doubt, the tuberculin can be provided by the veterinary advisor for the elephant TAG.
The results of all tests and the anamnesis data will be evaluated by the zoo vet and the
veterinary advisor of the elephant-TAG. The latter will write his advice to the EEP studbook-
coordinator.
What to do when an elephant is suspected of tuberculosis?
An elephant can become suspected of tuberculosis for different reasons. The anamnesis
may contain certain data that raise suspicion or one or more tests may give a positive result.
When this is the case, the veterinary advisor will consult international experts in the field of
tuberculosis and may propose additional tests, which may include: repeated antibody tests
(ELISA, ERT), additional intradermal tuberculin test, trunk wash and other tests available at
that particular moment). Attempts will also be made to send a serum sample to the USA to
perform a Multi Antigen Print Immuno Assay. It is the responsibility of the zoo veterinarian to
consult the local veterinary authorities about the situation.
Willem Schaftenaar, DVM

Veterinary advisor to the elephant TAG
Rotterdam zoo
PO box 532
3000 AM Rotterdam, the Netherlands
Tel: +31 10 4431 485
Fax: +31 10 4431 414
E-mail: W.Schaftenaar@Rotterdamzoo.nl
TB testing in captive elephants in the EEP

23 July 2008
TB testing in elephants is a concern for all elephant keeping institutions. In Europe, no

validated tests for elephants, approved by EU-authorities or any other European country, are
available at this time. However, veterinary authorities often request elephants to be tested on
TB when they are moved to other countries. Zoos that receive elephants must be well aware
of the risk of the import of TB in their collection. This makes it very important to build up a
well documented history of elephant herds, including regular testing and monitoring other
animals and personnel.
In the EAZWV-recommendations for approval of zoos according to the EU Directive 92/65,

the trunk wash procedure for regular testing on tuberculosis (TB) has been discussed. In the
final recommendations, a more general text was chosen (see point C.5.h.): “Specific
guidelines for the systematic testing of specific animal species may be developed and
recommended by the Infectious Diseases Working Group of EAZWV”. This means, that at
this time, no official guidelines for TB-testing of elephants are described under the BALAI-
directive.
However, this does not mean that zoos keeping elephants should not prepare themselves to
establish a routine testing protocol. The risk of introducing TB in a zoo through elephants is
quite realistic as we can see from cases in the past decade both in the USA as well as in
Europe. This document does not touch the issue of introduction of TB through other animal
species and human contact, but should leave no doubts that a complete TB-surveillance is
the only way to reduce the risks of TB in a zoo.
Every national or regional government may have its own interpretation about how to deal
with suspected TB-cases. Much depends on the relationship between the zoo veterinarian
and the local veterinary authorities. Generally it helps if the zoo has a clear policy regarding
its health surveillance system. There is one approach that should not be practiced: the
“sleeping dog policy”. Zoos that make all efforts to be certified according to the EU Directive
92/65 should have the intrinsic desire to stay free of infectious diseases like TB. This
document describes methods that can be used to collect information about the TB-status of
individual elephants and an elephant herd. One should be aware that the European situation
can not be compared to the situation in the USA, where one federal government determines
guidelines for the entire country. Legislation in the EU and other non-EU-member states is
far from uniform. Quality of PPD’s even within the EU differs significantly between the
member states. This makes it impossible to write a guideline with an official status. This
document can only help to convince institutions that keep elephants to use any means
available to minimize the risk of contracting and spreading tuberculosis in their animals and
personnel. Finally it may help decision making in case of a planned elephant transfer; the
reliability of a “TB- history report” depends a priori of the amount of data collected over the
years, not only of a single test taken “on the day of transport”.
What are the diagnostic possibilities on live elephants?
As there are no tests that provide 100% accuracy, it is of outmost importance that each
serum or plasma sample obtained from elephants should always be banked at below -20°C.
This can be of great value for the interpretation of future test results and the evaluation of the
TB-status in an individual elephant as well as the herd status. To test elephants for carrying
TB-organisms, a very limited range of tests can be used, of which none has been validated
for elephants:
1. Immunologic tests
None of these tests are 100% conclusive, but positive results should be followed by further
investigations :
a. (Comparative) intradermal tuberculin test. In most countries, this test is only validated
for cattle, but it is used in a large range of other species. In elephants it may have
some diagnostic value, but results should be interpreted with great care. Bovine and
avian tuberculins are injected intradermally in an area where the skin is thin, like the
ear base area. The skin reaction is measured after 3, 4 and 5 days (swelling,
temperature, sensibility). This test may provide information about the cellular immune
response of the animal as a reaction on previous contact of the animal with
Mycobacterium sp. A positive response on bovine tuberculin should be followed by
further investigations.
b. Humoral antibodies:
ELISA: some laboratories run an ELISA for the detection of antibodies. There is no
official ELISA recognized in Europe for use in exotic mammals. The Central Veterinary
Institute in Lelystad (the Netherlands) is the only official institute that runs these
ELISA’s on a routine base for zoos. The antigens used at this CVI are M.bovis, MPB70
and M.paratuberculosis. Positive results should be followed by further investigations.
The Elephant TB STAT-PAK Assay is officially recognized in the USA and can be
obtained in Europe too (www.zootest.com) However, the test has no official status in
Europe at this moment.
c. Gamma interferon test: not yet available for elephants. This test is currently under
development at the Department of Infectious diseases and Immunology of the
Veterinary Faculty in Utrecht, the Netherlands. A positive test result should be followed
by further investigations.
2. Culture of Mycobacterium sp.
For several years the method of choice for sample collection has been the trunk wash*. If
positive, this is a conclusive test: you know that you have an animal that is excreting the
Mycobacterium sp. Action should be undertaken immediately, regardless the consequences
it may have for the status of the zoo. No zoo can allow its personnel to work with an animal
which is a serious risk for its employees without taking protective measures nor to expose
other animals to this disease. If the culture is negative, the animal may still be a carrier of
Mycobacterium sp. and may excrete the pathogens in low quantities. It may take several
months before you get the result back from the lab; in the Netherlands the Central
Veterinary Institute in Lelystad declares a culture to be negative for M. bovis-complex only
after 4 months. A two phase TB diagnostic method combining the culture method and the
PCR technique may shorten this period. However the PCR is especially useful, when the
samples are ZN positive, otherwise many cycles are needed to get a signal and this will
result in a certain numbers of false positive reactions in negative samples.
NB: In recent years the sensitivity of the trunk wash method has been proven to be extremely
low. If you are dealing with a TB-positive animal, the risk of spreading the Mycobacteria by
using this method is a great concern! In the view of the authors this method is not
recommended unless performed by qualified persons that are aware of the risks and low
sensitivity of the test.
* Trunk wash is an active manipulation at the elephant trunk which can be performed in free
and protected contact systems in non immobilized elephants after they are conditioned for
this procedure. The principle is that a sterile saline solution (approx. 100 ml) is injected in
each nostril of the trunk. The nostrils must be kept closed by an elephant keeper and the
trunk has to be lifted actively by the elephant or passively by the keeper so that the solution
is running up to the basis of the trunk. The mixture of the solution and trunk mucus is
collected in sterile plastic bags by active blowing of the elephant through its trunk or by
lowering the level of the nostrils. The sample must be sealed and stored at 4°C for immediate
culture or stored at –20°C if culture is to be performed at a later stage. Trunk wash under
non-contact situation requires a full anesthesia of the elephant and a portable fluid pump and
sucking system which allows the operation under sterile condition. The external pump and
sucking system will be connected to a sterile PVC tube (1 cm diameter) with a length of
approx. 2 meter. The amount of sterile solution and the collection bag are like described
before.
Every zoo should work on a TB-free status, also in elephants. This can be accomplished by
regular testing, especially when new animals are being brought in frequently. We propose to
test:
a. Every elephants at least opportunistically whenever blood can be collected, by

ELISA’s (Central Veterinary Laboratory-Lelystad, using antigens derived from M.bovis,
MPB70 and M. paratuberculosis) and the Elephant TB STAT-PAK Assay. Training of
the elephants should include blood collection;
b. New animals by ELISA’s (Central Veterinary Laboratory-Lelystad, using antigens
derived from M.bovis, MPB70 and M. paratuberculosis) and Elephant TB STAT-PAK
Assay) and skin test;
c. All suspect animals and contact animals by all immunological tests available. It is
advisable to repeat the antibody tests 6 weeks after the intradermal tests. Potential
carriers of TB may possibly react by an increase in the production of antibodies as
measured by ELISA’s or (if the first sample was negative) by the Elephant TB STAT-
PAK Assay. One has to be aware that this phenomenon may not occur in elephants,
though it has been seen in a few individual cases. As in other tests described here, it
may contribute to obtain more information about an unknown TB/health status.
What to do when an elephant has died or will be euthanized?
An intense search for lesions of tuberculosis is encouraged in all elephant necropsies. This
should include all elephants that die or are euthanized for other reasons even though TB is
not suspected. Be advised that elephant TB is likely to be caused by Mycobacterium
tuberculosis and M. bovis which are contagious to humans. Therefore be prepared with
proper protective apparel, and contain any suspicious organs or lesions as soon as possible.
Ideally, elephants should be tuberculin tested 3 days prior to euthanasia and bled for
serology (ELISA and Elephant TB STAT-PAK) at the time of euthanasia. Serum from
elephants that die naturally should be harvested from post mortem blood for serological
assays.
All elephants undergoing necropsies should have a careful examination of the tonsillar
regions and submandibular lymph nodes for tuberculous appearing lesions. Take any nodes
that appear caseous or granulomatous for culture (freeze or ultrafreeze), and fixation (in
buffered 10% formalin). In addition, search thoracic organs carefully for early stages of TB as
follows: after removal of the lungs and trachea, locate the bronchial nodes at the junction of
the bronchi from the trachea. Use clean or sterile instruments to section the nodes. Freeze
half of the lymph node and submit for TB culture (even if no lesions are evident). Submit
sections in formalin for histopathology. Carefully palpate the lobes of both lungs from the
apices to the caudal borders to detect any firm (nodular size) lesions. Due to the missing
pleural elephants intrathoracal handling is required for the lung removal during necropsy.
Please, wear a mouth and nose protector for this procedure. Take sections of any suspicious
lesions. Open the trachea and look for nodules or plaques and process as above. Regional
thoracic and tracheal lymph nodes should also be examined and processed accordingly.
Split the trunk from the tip to its insertion and take samples of any plaques, nodules or
suspicious areas for TB diagnosis as above. Look for and collect possible extra-thoracic TB
lesions, particularly if there is evidence of advanced pulmonary TB.
XI. AVIAN INFLUENZA
J.D.W. Philippa
WAZA/EAZA/EAZWV
1. VACCINATION OF NON-DOMESTIC AVIAN SPECIES AGAINST

HIGHLY PATHOGENIC AVIAN INFLUENZA (HPAI) VIRUSES
2. WAZA/EAZA/EAZWV RECOMMENDATIONS ON AVIAN
INFLUENZA
1. Vaccination Of Non-Domestic Avian Species Against Highly

Pathogenic Avian Influenza (HPAI) Viruses
ADAPTED FROM: “VACCINATION OF NON-DOMESTIC ANIMALS AGAINST EMERGING INFECTIOUS DISEASES”
- PHD THESIS ERASMUSMC/ROTTERDAM ZOO, 2007
J.D.W. Philippa
Introduction
Avian influenza viruses (AIV) are type A influenza viruses and belong to the
Orthomyxoviridae family. They can be classified according to the antigenicity of its surface
proteins haemagglutinin (H) and neuraminidase (N). Currently 16H (H1-16) and 9N (N1-9)
subtypes have been described in avian species (Fouchier et al., 2005). Furthermore the
subtypes can be classified on the basis of their pathogenicity in chickens after intravenous
inoculation.
Highly pathogenic avian influenza (HPAI, formerly termed fowl plague), an acute generalised
disease in which mortality in chickens may be as high as 100%, is restricted to subtypes H5
and H7, although most viruses of these subtypes have low pathogenicity, and do not cause
HPAI. Low pathogenic avian influenza (LPAI) virus strains cause more variable morbidity and
mortality (ranging from sub-clinical to fatal) but are generally associated in poultry with a
mild, primarily respiratory disease with loss of egg production (Capua & Alexander, 2004), or
mild enteric disease in non-domestic birds. In certain cases (in poultry flocks) the LPAI virus
phenotype (of subtype H5 or H7) may mutate into the HPAI virus phenotype by the
introduction of basic amino acid residues (arginine or lysine) at the cleavage site of the
precursor haemagglutinin (HAO) (Banks et al., 2001), which facilitates systemic virus
XI. Avian Influenza
replication. H5 and H7 subtypes with an amino acid sequence at the HA0 cleavage site
comparable to those that have been observed in virulent AI viruses are considered HPAI
viruses, even when mortality in chickens is low (Office International d'Epizooties., 2004).
However, the two forms of avian influenza (HPAI and LPAI) are distinctly different and should
be regarded as such.
Avian influenza viruses have a worldwide distribution and are infectious to all avian species
(commercial, domestic and wild), with variable morbidity per virus isolate and species.
Aquatic avian species, mainly those of the taxonomic orders Anseriformes and
Charadriiformes are considered the main natural reservoir of all avian influenza viruses,
including the LPAI ancestral viruses of HPAI strains (Munster et al., 2005; Munster et al.,
2007). Waterfowl were generally considered resistant to infection with HPAI virus until 2002.
However, in 2002 an outbreak of HPAI H5N1 virus occurred in wild migratory avian species
and resident waterfowl (Sturm-Ramirez et al., 2004). Since then, this particular HPAI virus
subtype has made an unprecedented spread from South East Asia throughout Asia and into
Europe and Africa, with morbidity and mortality not only in domestic poultry, but in more than
130 non-domestic avian species from various taxonomic orders: Anseriformes,
Charadriiformes, Ciconiiformes, Columbiformes, Falconiformes, Galliformes, Gruiformes,
Passeriformes, Pelecaniformes, Phoenicopteriformes, Strigiformes, Struthioniformes,
Psittaciformes, and Podicipediformes (USGS National Wildlife Health Center, 2008).
Additionally, this virus strain has has caused mortality in a large number of mammalian
species, and has caused 403 human cases with 254 deaths to date (27 January 2009)
(World Health Organisation, 2009).
Documented outbreaks of Asian lineage H5N1 HPAI virus in captive non-domestic birds
have been limited to 6 cases: Penfold Park, Hong Kong, (People’s Republic of China, 2002),
Kowloon Park, Hong Kong (People’s Republic of China, 2002), Phnom Tamao wildlife rescue
centre (Cambodia, 2004), Ragunan Zoo, Jakarta (Indonesia, 2005), Dresden Zoo (Germany,
2006) and Islamabad Zoo (Pakistan, 2007). Large felids with H5N1 infection have been
reported in Suphanburi Zoo (Thailand, 2003), and Sri Racha tiger zoo (Thailand, 2004). To
curtail these outbreaks, a combination of increased bio-security measures (isolation and
quarantine of infected animals, disinfection of the area), feeding of cooked poultry only,
treatment of infected animals in quarantine areas, selective culling, extensive surveillance of
migratory and captive birds and vaccination were used.
Vaccination
Vaccination is a useful means of reducing the horizontal spread of AIV in poultry (Capua et
al., 2004; van der Goot et al., 2005) (Ellis et al., 2004). Vaccination protects against disease
and mortality, but does not always prevent infection and virus spread. However, the dose
required for infection is much higher, and vaccinated birds shed far less field virus after
infection than unvaccinated birds (Brugh et al., 1979; Karunakaran et al., 1987).
Protective antibodies produced in response to infection or vaccination, are directed against

the H and N surface proteins. Vaccine-induced antibody responses are species-, dose-, and
vaccine strain-dependent, e.g. the antibody responses upon AIV vaccination are generally
higher in chickens than in other poultry species (Higgins, 1996). Published minimum serum
antibody titres measured by HI test in vaccinated chickens that correlate with protection after
challenge with HPAI virus are 1:10 (Swayne et al., 2006), or 1:16 (Ellis et al., 2004; Tian et
al., 2005). However, domestic ducks with very low or undetectable antibody titres post
vaccination have been shown to be protected from HPAI virus challenge (Middleton et al.,
XI. Avian Influenza
2006; Webster et al., 2006). Duration of protection from HPAI virus challenge may vary
between species: chickens for up to 40 weeks after one dose of vaccine, domestic ducks for
more than 52 weeks after 2 doses, while domestic geese which received 3 doses were
protected for 34 weeks (Tian et al., 2005).
The degree of homology of the H protein will largely affect the level of cross-protection and
therefore efficacy of the vaccine (Swayne et al., 2000). A so-called Differentiation of Infected
from Vaccinated Animal (DIVA) strategy, with a heterologous vaccine (using the same H
subtype as the field virus, but a different N subtype), is recommended to differentiate
between vaccinated and field-virus infected animals (Capua et al., 2003). However, in
housing systems where birds are not housed permanently indoors (e.g., in zoos), contact
with free-ranging birds can result in LPAI virus infections that go by unnoticed, but which may
interfere with the DIVA principle.
In the European Union routine vaccination of poultry against avian influenza viruses is
currently not practised as this would interfere with stamping-out policies and international
trade agreements. Instead, eradication measures during an outbreak in poultry include (long-
term) confinement, large-scale culling and safe disposal of carcasses of all poultry on the
infected farm, and, depending on the poultry density in the area and the epidemiological
situation, pre-emptive culling of poultry on neighbouring farms and emergency vaccinations
(Directive 92/94/EEC). Since 2003, more than 300 million birds have been culled to eradicate
HPAI outbreaks.
Vaccination in European Zoos
The standard measures used to prevent and eradicate HPAI virus outbreaks in poultry (long-
term confinement and large scale culling) would be detrimental to the welfare, conservation
status and breeding programmes of zoo birds, which often are irreplaceable, valuable and
endangered avian species (IUCN Red list, http://www.iucnredlist.org/). Directive 2005/94/EC
foresees a derogation from culling of birds provided the birds can be brought inside and are
subjected to virus detection tests (after the last death/positive finding, 2 tests at an interval of
21 days have to be performed according to the diagnostic manual Decision 2006/437/EC).
However, most zoos do not have the capability to suitably confine their entire bird collections
for extended time, and many species would not be able to adjust to confinement and
increased stress with subsequent welfare problems and increased exposure to pathogens
resulting in disease (e.g. aspergillosis and bumblefoot) (McMillian & Petrak, 1989; Redig,
2000; Harcourt-Brown, 1996).
Instead of confinement, vaccination of zoo birds against HPAI virus was allowed as an
additional preventive measure (while reducing confinement measures) in Belgian, Dutch and
German zoos during an outbreak of HPAI H7N7 virus in poultry in 2003 (Decision
2003/291/EC). Similarly, in 2005, Decision 2005/744/EC allowed vaccination in European
zoos against the encroaching H5N1 subtype. These campaigns were subject to rigorous
surveillance and control requirements.
Vaccination against HPAI H7N7 in zoos
During the HPAI H7N7 outbreak in poultry in 2003, birds in Dutch zoos were vaccinated
twice with six weeks interval using a whole inactivated oil-adjuvanted vaccine, based on
influenza virus A/chicken/Italy/473/99 (H7N1), with high homology to the field strain HPAI
H7N7 A/chicken/Netherlands/1/03 (97.4% nucleotide and 98.7 % amino acid sequence
XI. Avian Influenza
identity). This resulted in the induction of antibody titres ≥ 40 (used as a correlate of

protection in this study) in 81.5% of the vaccinated birds, with an overall GMT of 190. Birds of
the taxonomic orders Anseriformes, Galliformes and Phoenicopteriformes showed higher
GMT, and larger percentages developed a serum HI antibody titre ≥ 40 than those of the
other orders. Furthermore, a decrease in antibody response with an increase in body weight
> 1.5 kg was shown. The high agreement between post vaccination antibody titres
determined by serum HI test (using the vaccine strain), and VN titres (using the field strain),
was used as a further measure of immunogenicity. The broad efficacy demonstrated in a
large variety of taxonomic orders illustrated the benefits of vaccination as an additional
preventive measure against HPAI virus infection (Philippa et al., 2005).
In 2005, the Dutch zoos were the first to implement Decision 2005/744/EC to provide
protection against the encroaching HPAI H5N1 subtype. Birds were vaccinated with an
inactivated adjuvanted H5N2 vaccine with vaccine doses adapted to mean body weight per
species, using data collected during the H7N1 vaccination campaign. The vaccine strain
(A/duck/Pottsdam/1402/86) had a homology of 90% to the HA gene of the H5N1 field strain
(A/turkey/Turkey/1/05) on the basis of nucleotide sequence (1530 base pairs, including basic
cleavage site), and 92.4% on the basis of amino acids. Vaccination was safe, and proved
immunogenic throughout the range of species tested, with some variations between and
within taxonomic orders. After booster vaccination the overall homologous GMT to the
vaccine strain, measured in 334 birds, was 190 (95% CI:152–236), and 80.5% of vaccinated
birds developed a titre of ≥ 40. Titres to the HPAI H5N1 virus followed a similar trend, but
were lower (GMT: 61 (95% CI: 49–76); 61%≥ 40) (Philippa et al., 2007).
The breadth of the immune response was further demonstrated by measuring antibody titres
against prototype strains of four antigenic clades of currently circulating H5N1 viruses.
Antigenic distances to the prototype strains were determined using antigenic cartography
(Smith et al., 2004). Antigenic cartography uses the antigenic properties of influenza viruses
combination with epidemiological and genetic data, and is used to select virus strains for use
as human pre-pandemic (H5N1) vaccine candidates (World Health Organisation (WHO),
2006). Influenza vaccines whose haemagglutinins are antigenically similar to circulating
strains provide the highest level of protection from infection in humans (Subbarao, 1999).
The birds clustered in two groups based on the breadth of antibody responses. Group 1
(Anseriformes, Galliformes, Phoenicopteriformes, Psittaciformes and Struthioniformes)
showed a very broad response to vaccination, with predicted protection against future strains
up to 12 antigenic units from the current vaccine. Group 2 (Ciconiiformes, Gruiformes,
Pelecaniformes and Sphenisciformes) had low HI antibody titres against the prototype strain
of the most antigenically distant clade (A/Indonesia/5/05).
In 2006, a working group of Animal health and Welfare experts was established by the
European Food Safety Authority (EFSA)(European Food Safety Authority (EFSA), 2007), to
provide a scientific assessment of the preventive vaccination against avian influenza of H5
and H7 subtypes carried out in zoos in Member States (MS). The total number of birds
vaccinated, as reported by 12 MS, was 44721. Individual data from 4718 birds (374 species
from 19 taxonomic orders) were submitted. Not all of these could be used for every
evaluation: pre-vaccination titres could be evaluated for 3039 birds; titres after first
vaccination were evaluated for 1429 birds, and post-second vaccination titres for 2296 birds.
XI. Avian Influenza
Differences in vaccination schedules, doses and routes, differences in methodology and

antigens used in the HI tests between laboratories (due to the absence of international
reference standards, and the absence of inter-laboratory standardisation of methodology),
the use of different vaccines1 in different taxonomic orders and the sometimes incomplete
reporting of results, limited the evaluation of some of the data provided by EU MS. Cut-off
points varied with laboratory, and titres considered a measure of adequate immune response
were 8, 16, 32, 40, and 64. Most countries used dilution series starting at 4 or 8, therefore
results were evaluated for titres 16 and 32 [documented surrogate markers for protection in
chickens (Ellis et al., 2004; Office International d'Epizooties., 2004; Swayne et al., 2006; Tian
et al., 2005)], and undetectable titres were regarded as 4 for calculation of GMT. In the
absence of (and unfeasibility of obtaining) vaccination/challenge data in often endangered
zoo bird species, the evaluation had to be based on extrapolation of serological data from
poultry and limited other bird species.
The H5 vaccines registered for poultry in the EU showed differences in efficacy, measured
as serum HI antibodies induced by two doses of vaccine (Table 1.). Three of the five
vaccines evaluated induced relatively high GMT and high percentage seroconversion in the
vast majority of vaccinated birds. The HI titres induced by vaccination showed marked
differences between and within taxonomic orders. Both routes of vaccination (i.m. and s.c.)
were effective in inducing HI serum antibody responses, and for most avian species the
poultry dose was suitable. In some larger species higher doses adjusted to body weight,
induced higher serum antibody titres. (e.g. for ostriches a 10-fold increase of the poultry dose
(10 x 0.25ml). However, extremely high doses at a single site of injection (e.g. vaccination of
ostriches with 10 ml of vaccine) appeared to have a negative effect on the induction of serum
antibody titres, and induced local adverse reactions.
There were indications that one vaccination was sufficient to induce high serum antibody
titres in at least two taxonomic orders of birds (Anseriformes and Galliformes). However, a
second vaccine administration ensured seroconversion in the majority of birds of most
species. Limited data indicated that antibody titres persisted in several species for six months
after vaccination. Adverse effects and mortality associated with vaccination were low and
were mainly attributable to handling stress or trauma. Differences in adverse effects reported
from different zoos highlight the importance of proper skilled handling.
Longevity of antibody titres
One year after vaccination with the H5N2 vaccine, birds in Dutch zoos were revaccinated
with the same vaccine. Antibody titres one year after the initial two vaccinations and the
effect of one booster vaccination at this time were evaluated. In Rotterdam Zoo, 72
previously vaccinated birds could be evaluated for the effect of one booster vaccination
(Philippa et al, in press). For 44 birds, serum samples were available from 4 weeks after the
initial two vaccinations the previous year, at the time of revaccination, and 4 weeks later.
Birds which had been vaccinated with the H7 vaccine two years prior to the H5N2
revaccination were additionally tested for the presence of H7-specific antibodies.
Vaccine A: H5N9 (A/turkey/Wisconsin/68).

Vaccine B: H5N2 (A/duck/Pottsdam/1402/86).
Vaccine C: H5N2 (A/chicken/Mexico/232/94/CPA)
Vaccine D: H5N9 (A/chickenk/Italy/22A/98).
Vaccine E: H5N9 (A/chicken/ltaly/22A/H5N9/1998).
XI. Avian Influenza
Serum antibody titres of the birds tested in Rotterdam Zoo had clearly decreased in one year
time: while 80% of birds had a positive titre (≥ 8) and 68% a high positive titre (≥ 32) after 2
vaccinations, these figures were 61% and 30% respectively one year later. Four weeks after
re-vaccination these figures increased to 93% and 77% respectively. Although a larger
percentage of these 44 birds had a serum HI antibody titre ≥ 32 after re-vaccination, the
GMT was lower than GMT after 2 vaccine doses one year before (88 vs 66).
Birds from 4 out of 8 taxonomic orders did not have a GMT > 5 one year after vaccination,
and only one order (Phoenicopteriformes had a GMT > 40. Four weeks after one
revaccination 6/8 taxonomic orders tested had a GMT > 40.
GMTs had decreased even further two years after vaccination, as was shown by the H7
specific serum HI antibody titres. As these birds were not revaccinated with an H7 vaccine,
the effect of revaccination two years after the initial vaccinations is not known.
Conclusions
Bio-security measures remain the first line of protection of zoo birds against the introduction
of AI viruses and should be implemented in zoos. These bio-security measures should
include strict hygiene and quarantine measures, but should also exclude the possibility of
introducing AI viruses through feed animals such as day old chicks, other poultry or their
products. Continuous clinical monitoring of captive and wild birds in zoos should be
practiced, for early detection of introduced viruses by wild birds, domestic birds, or their
products. Strict bio-security measures will also reduce the risk of subsequent infection of wild
birds from zoo birds. Wild birds have been documented to be susceptible to HPAI virus
infection, and could potentially play a role in the spread of HPAI virus, although the majority
of avian influenza viruses detected in free-ranging birds have been LPAI viruses. If bio-
security measures cannot sufficiently protect zoo birds from exposure to HPAI viruses
coming from wild birds (based on an overall risk assessment which includes welfare aspects)
vaccination with vaccines against HPAI of H5 and H7 subtypes authorised for use in poultry
should be used to protect these zoo birds. In designing AI vaccination programmes and
schedules for zoo birds, recent data on wild bird migration and prevalence of AI viruses
should be taken into account. Vaccination against AI viruses of the H5 and H7 subtypes with
current inactivated oil-adjuvanted poultry vaccines is safe and, in most taxonomic orders of
zoo birds, effective in terms of inducing HI serum antibody titres. AI vaccines should be
administrated in a way that elicits high HI antibody titres in the vast majority of the zoo birds
vaccinated, i.e., by adjusting dose to average body weight. Although there are indications
that one vaccination might suffice for some species, a second vaccine dose ensures high
titres in the vast majority of species. Unless it is demonstrated that one vaccine
administration is sufficient, two administrations are recommended. The H5 and H7 vaccines
currently registered for poultry in the EU show differences in the performance in terms of HI
response in zoo birds after two doses. There appears to be no difference in route of
vaccination (s.c. or i.m.), so route can vary depending on the bird species to be vaccinated.
In order to maintain high titres in the captive populations in zoological collections, annual re-
vaccination seems to be required, as antibody titres decrease significantly in most taxonomic
orders, and high titres are seen after a single annual booster dose.
Mortality and adverse effects were low in all zoos evaluated in EU MS, and mainly attributed
to handling stress and trauma. Zoos can, and should therefore try to minimise these losses in
the execution of HPAI vaccination programmes. To minimise indirect losses due to
XI. Avian Influenza
decreased breeding results, AI vaccination during breeding seasons should be avoided

whenever possible. Mortality due to catching and handling stress can be reduced by handling
the birds less. Once the efficacy of a vaccination protocol has been validated for certain
species using certain vaccines, measurement of post-vaccination HI serum antibody titres
should no longer be mandatory by the EU. These birds will then only have to be handled for
vaccination, and not 4 weeks later. Further research should be carried out to establish
effective vaccination schedules, route, and dose regimen in different zoo bird species. This
may, amongst others, lead to a reduction in the number of booster vaccinations needed in
certain species. Novel generation vaccines which may be administered in the form of an
aerosol (as is used in vaccination of poultry against Newcastle disease virus) may prove to
be useful in non-domestic species, and would eliminate the need for handling the birds.
The vaccination campaigns against HPAI virus have focused on protecting birds in zoological
collections. However, a large number of mammalian species, including tigers and leopards,
have also been documented with HPAI virus infection with recent H5N1 subtypes. There is
currently no commercial vaccine available to protect mammals from HPAI H5N1 virus
infection. A recombinant fowlpox-vectored vaccine expressing the H5 gene has been shown
to produce high antibody titres against heterologous H5N1 virus antigen in cats after booster
vaccination (Karaca et al., 2005), and may prove to be useful in prophylactic vaccination
programs of mammals in the future. Until then, these animals have to be protected by
excluding the introduction of AIV through raw poultry used as feed.
The broad vaccine efficacy in the different avian taxonomic orders illustrates that vaccination
against avian influenza is a useful tool for the protection of non-domestic avian species in
zoos, which allows for an alleviation of confinement measures – and is therefore beneficial to
the health and welfare of these birds. However, increased bio-security measures in
combination with virological monitoring remain imperative in combating outbreaks of HPAI.
XI. Avian Influenza
References
Banks,J., Speidel,E.S., Moore,E., Plowright,L., Piccirillo,A., Capua,I., Cordioli,P., Fioretti,A.,
Alexander,D.J., 2001. Changes in the haemagglutinin and the neuraminidase genes prior to
the emergence of highly pathogenic H7N1 avian influenza viruses in Italy. Arch. Virol.
146:963-973.
Brugh,M., Beard,C.W., Stone,H.D., 1979. Immunization of chickens and turkeys against
avian influenza with monovalent and polyvalent oil emulsion vaccines. Am. J. Vet. Res.
40:165-169.
Capua,I., Alexander,D.J., 2004. Avian influenza: recent developments. Avian Pathol. 33:393-
404.
Capua,I., Terregino,C., Cattoli,G., Mutinelli,F., Rodriguez,J.F., 2003. Development of a DIVA
(Differentiating Infected from Vaccinated Animals) strategy using a vaccine containing a
heterologous neuraminidase for the control of avian influenza. Avian Pathol. 32:47-55.
Capua,I., Terregino,C., Cattoli,G., Toffan,A., 2004. Increased resistance of vaccinated
turkeys to experimental infection with an H7N3 low-pathogenicity avian influenza virus. Avian
Pathol. 33:158-163.
Ellis,T.M., Leung,C.Y., Chow,M.K., Bissett,L.A., Wong,W., Guan,Y., Malik Peiris,J.S., 2004.
Vaccination of chickens against H5N1 avian influenza in the face of an outbreak interrupts
virus transmission. Avian Pathol. 33:405-412.
European Food Safety Authority (EFSA). Opinion of the Scientific Panel AHAW related with
the vaccination against avian influenza of H5 and H7 subtypes as a preventive measure
carried out in Member States in birds kept in zoos under Community approved programmes.
2007.
Fouchier,R.A., Munster,V., Wallensten,A., Bestebroer,T.M., Herfst,S., Smith,D.,
Rimmelzwaan,G.F., Olsen,B., Osterhaus,A.D., 2005. Characterization of a novel influenza A
virus hemagglutinin subtype (H16) obtained from black-headed gulls. J. Virol. 79:2814-2822.
Harcourt-Brown,N.H. 1996. Bumblefoot. Pages 126-131 in Samour,J.H. editor. Avian
Medicine. Harcourt Publishers Ltd, London.
Higgins,D.A. 1996. Comparative immunology of avian species. Pages 149-205 in
Davison,T.F., Payne,L.N., Morris,T.R. editors. Poultry Immunology. Carfax publishing co.,
Abingdon.
Karaca,K., Swayne,D.E., Grosenbaugh,D., Bublot,M., Robles,A., Spackman,E., Nordgren,R.,
2005. Immunogenicity of fowlpox virus expressing the avian influenza virus H5 gene
(TROVAC AIV-H5) in cats. Clin. Diagn. Lab Immunol. 12:1340-1342.
Karunakaran,D., Newman,J.A., Halvorson,D.A., Abraham,A., 1987. Evaluation of inactivated
influenza vaccines in market turkeys. Avian Dis. 31:498-503.
McMillian,M., Petrak,M., 1989. Retrospective study of aspergillosis in pet birds. J. Avian Med
Surg211-215.
Middleton,D., Bingham,J., Selleck,P., Lowther,S., Gleeson,L., Lehrbach,P., Robinson,S.,
Rodenberg,J., Kumar,M., Andrew,M., 2006. Efficacy of inactivated vaccines against H5N1
avian influenza infection in ducks. Virology.
XI. Avian Influenza
Munster,V.J., Baas,C., Lexmond,P., Waldenstrom,J., Wallensten,A., Fransson,T.,

Rimmelzwaan,G.F., Beyer,W.E., Schutten,M., Olsen,B., Osterhaus,A.D., Fouchier,R.A.,
2007. Spatial, temporal, and species variation in prevalence of influenza A viruses in wild
migratory birds. PLoS. Pathog. 3:e61.
Munster,V.J., Wallensten,A., Baas,C., Rimmelzwaan,G.F., Schutten,M., Olsen,B.,
Osterhaus,A.D.M.E., Fouchier,R.A.M., 2005. Mallards and highly pathogenic avian influenza
ancestral viruses, northern europe. Emerg. Infect. Dis. 11:1545-1551.
Office International d'Epizooties. 2004. Avian Influenza.OIE Manual of Diagnostic Tests and
Vaccines for Terrestrial Animals.
Philippa,J., Baas,C., Beyer,W., Bestebroer,T., Fouchier,R., Smith,D., Schaftenaar,W.,
Osterhaus,A., 2007. Vaccination against highly pathogenic avian influenza H5N1 virus in
zoos using an adjuvanted inactivated H5N2 vaccine. Vaccine 25:3800-3808.
Philippa,J.D.W., Munster,V.J., Bolhuis,H., Bestebroer,T.M., Schaftenaar,W., Beyer,W.E.,
Fouchier,R.A.M., Kuiken,T., Osterhaus,A.D.M.E., 2005. Highly pathogenic avian influenza
(H7N7): Vaccination of zoo birds and transmission to non-poultry species. Vaccine 23:5743-
5750.
Redig,P.T. 2000. Aspergillosis. Pages 275-287 in Samour,J. editor. Avian Medicine. Mosby,
Philadelphia.
Smith,D.J., Lapedes,A.S., de Jong,J.C., Bestebroer,T.M., Rimmelzwaan,G.F.,
Osterhaus,A.D., Fouchier,R.A., 2004. Mapping the antigenic and genetic evolution of
influenza virus. Science 305:371-376.
Sturm-Ramirez,K.M., Ellis,T., Bousfield,B., Bissett,L., Dyrting,K., Rehg,J.E., Poon,L.,
Guan,Y., Peiris,M., Webster,R.G., 2004. Reemerging H5N1 influenza viruses in Hong Kong
in 2002 are highly pathogenic to ducks. J. Virol. 78:4892-4901.
Subbarao,K., 1999. Influenza vaccines: present and future. Adv. Virus Res. 54:349-373.
Swayne,D.E., Lee,C.W., Spackman,E., 2006. Inactivated North American and European
H5N2 avian influenza virus vaccines protect chickens from Asian H5N1 high pathogenicity
avian influenza virus. Avian Pathol. 35:141-146.
Swayne,D.E., Perdue,M.L., Beck,J.R., Garcia,M., Suarez,D.L., 2000. Vaccines protect
chickens against H5 highly pathogenic avian influenza in the face of genetic changes in field
viruses over multiple years. Vet. Microbiol. 74:165-172.
Tian,G., Zhang,S., Li,Y., Bu,Z., Liu,P., Zhou,J., Li,C., Shi,J., Yu,K., Chen,H., 2005. Protective
efficacy in chickens, geese and ducks of an H5N1-inactivated vaccine developed by reverse
genetics
6. Virology 341:153-162.
USGS National Wildlife Health Center. Referenced reports of highly pathogenic avian
influenza H5N1 in wildlife and domestic animals. USGS . 2008. 31-3-2009.
van der Goot,J.A., Koch,G., de Jong,M.C., van,B.M., 2005. Quantification of the effect of
vaccination on transmission of avian influenza (H7N7) in chickens. Proc. Natl. Acad. Sci. U.
S. A.
Webster,R.G., Webby,R.J., Hoffmann,E., Rodenberg,J., Kumar,M., Chu,H.J., Seiler,P.,
Krauss,S., Songserm,T., 2006. The immunogenicity and efficacy against H5N1 challenge of
reverse genetics-derived H5N3 influenza vaccine in ducks and chickens. Virology 351:303-
311.
XI. Avian Influenza
World Health Organisation. Cumulative Number of Confirmed Human Cases of Avian

Influenza A/(H5N1) Reported to WHO. World Health Organisation . 2009.
World Health Organisation (WHO). Antigenic and genetic characteristics of H5N1 viruses
and candidate H5N1 vaccine viruses developed for potential use as pre-pandemic vaccines.
2006.
XI. Avian Influenza
2. WAZA/EAZA/EAZWV Recommendations on Avian Influenza
Background
Highly pathogenic avian influenza (HPAI) is an extremely contagious viral disease of

domestic birds that can affect all species of birds. It is also known to have infected a range
of wild and domestic species of mammals (including civets, ferret, beech marten, domestic
cat, clouded leopard, leopard, tiger) and humans, and has experimentally been transmitted to
mice, rats, rabbits and crab-eating macaques. The disease is caused by the Influenza A
virus (Family Orthomyxoviridae), which can be divided into subtypes on the basis of distinct
haemagglutinin antigens (H1–H16) and neuraminidase antigens (N1–N9). It is a notifiable
disease under the Terrestrial Animal Health Code of the International Organisation for
Animal Health (OIE), thus notifiable by law in the 167 Member Countries of the OIE. The
Code defines notifiable avian influenza as an infection of poultry (i.e. not of wild birds!)
caused by any influenza A virus of the H5 or H7 subtypes or by any AI virus with an
intravenous pathogenicity index (IVPI) greater than 1.2 (or as an alternative at least 75%
mortality). Notifiable avian influenza can be divided into highly pathogenic and low
pathogenic notifiable avian influenza. Highly pathogenic avian influenza (HPAI) may result in
a mortality rate of 100% within a poultry flock. These viruses have been restricted to
subtypes H5 and H7, although not all viruses of these subtypes cause HPAI. All other viruses
cause a much milder low pathogenicity avian influenza (LPAI) consisting primarily of mild
respiratory disease and egg production problems in laying birds, or being completely
inapparent.
From 1959 to 1998, only 24 distinct outbreaks of HPAI from domestic poultry have been
reported. The total number of birds involved in all outbreaks over this 40-year period was
approximately 23 million. But the years 1999-2004 have witnessed six outbreaks that
resulted in a considerable socio-economic impact (e.g. 1999/2000 in Italy, 2003 in the
Netherlands and in Germany) and the unprecedented outbreak currently affecting several
Asian countries. The USA, Canada, Chile and some African countries have also recently
experienced HPAI disease outbreaks. From 1999-2004 over 200 million domestic birds
have been affected by the disease. With the exception of some countries, where the disease
in domestic poultry is not yet under control, a rigorous stamping out policy was applied by
national authorities. This strategy included in general the rapid culling of infected poultry and
those suspected of being infected together with the implementation of movement restrictions
for live poultry and poultry products, increased monitoring and biosecurity measures.
HPAI is considered to have been spread between countries by a number of different known
vectors, including through the movement of avian livestock and bird by-products, legal and
illegal trade in birds, equipment associated with these respective industries, movement of
people, and migration of waterbirds.
The genetic pool for AI viruses is primarily in the aquatic birds responsible for the
perpetuation of these viruses in nature. It still is a matter of debate whether HPAI viruses are
normally present in wild bird populations, or whether they arise only as a result of mutation
after H5 or H7 LPAI viruses have been introduced to poultry from wild birds. The unusual
situation of endemicity which is present in some Asian countries suggests a spill over of
HPAI of the H5N1 subtype to the wild bird population. This situation has never occurred in
XI. Avian Influenza
the past, and therefore the consequences of this epidemiological situation are unpredictable.
Until now, the limited number of known human infections with H5N1 virus has been through
close contact with, or possibly by consumption of, infected poultry and none through contact
with wild birds. If however the virus were to change its characteristics and to spread between
humans, the result could be a pandemic with millions of victims. However, nobody can
predict whether such changes will happen.
Although, traditionally, legislation of most countries did not address avian influenza in wild
birds, veterinary services, noting the pandemic potential of the H5N1 strain, have started
monitoring the disease in wild birds and implementing protection measures also in the case
of the presence of the virus in other species than farmed poultry. In Europe the legal basis
for the new approach is Council Directive 2005/94/EC of 20 December 2005 on Community
measures for the control of avian influenza, which repealed the previous Directive
92/40/EEC.
Current approaches to control the disease may refer to both HPAI and LPAI infections
and include: surveillance of bird species other than domestic poultry; early detection of
possible spread of avian influenza to mammals; notification of surveillance measures and
outbreaks; emergency measures applicable to holdings where outbreaks are suspected;
killing of all poultry and other captive birds, disposal of carcases and eggs, and disinfection of
the premises under official supervision, in holdings where outbreaks are confirmed;
establishment of protection, surveillance and further restricted zones around affected
holdings.
Routine vaccination, although technically possible and effective, continues to be prohibited

in many countries. Such countries may, however, have provisions for emergency
vaccinations, grant certain derogations, or introduce preventive vaccinations on the basis of
a risk assessment.
Recognising that avian influenza has always occurred, and will continue to occur, in wild
birds (although the incidence is usually very low), that WHO, FAO and OIE have concluded
that attempts to eliminate HPAI in wild bird populations through lethal responses such as
culling are not feasible and may exacerbate the problem by causing further dispersion of
infected birds, that it is technically impossible to keep wild birds out of zoological institutions,
and that zoological institutions need to keep birds of wild species in order to play their
important role in conservation communication, education and public awareness (as stressed
by resolutions of the Conferences of the Parties of the Ramsar Convention and Convention
on Migratory Species), legislation tends to be somewhat more flexible with regard to
zoos than legislation relating to other highly contagious diseases, such as foot-and-mouth
disease, African swine fever and so on. It may thus be possible for a zoo to be granted
certain derogations (e.g. to be allowed to vaccinate preventively, or not have to kill the
entire collection in the case of an outbreak at or near the zoo) under specified conditions.
These conditions may include, among other things, restrictions of the transfer of vaccinated
birds to other holdings.
XI. Avian Influenza
References
Convention on Migratory Species (2005). Res. 8.27 Migratory Species and Highly
Pathogenic Avian Influenza, adopted at Eighth Meeting of the Conference of the Parties to
CMS (COP 8), 20 - 25 November 2005, UNEP Headquarters Gigiri, Nairobi.
European Association of Zoo and Wildlife Veterinarians (2004) Transmissible Diseases

Handbook, 2nd edition. Houten NL.
European Food Safety Authority – AHAW (2005). Scientific Report - Animal health and
welfare aspects of Avian Influenza. Annex to the EFSA Journal (2005) 266, 1-21.
European Union (2005) Commission Decision 2005/731/EC of 17 October 2005 laying down
additional requirements for the surveillance of avian influenza in wild birds. OJ L 27/93 of
20.10.2005
European Union (2006) Council Directive 2005/94/EC of 20 December 2005 on Community

measures for the control of avian influenza and repealing Directive 92/40/EEC. OJ L10/16 of
14.1.2006
OIE (2005) Terrestrial Animal Health Code. 14th edition. Paris.

XI. Avian Influenza
WAZA Council Resolution on Avian Influenza (11th April 2006)

RECOGNIZING the potential risk of transmission of Highly Pathogenic Avian Influenza
(HPAI) from wild birds, in particular waterfowl, to birds kept in zoological institutions;
MINDFUL that the zoos’ role in environmental education may require to exhibit birds under
conditions, contact with wild birds cannot effectively be excluded;
CONCERNED about the impact an outbreak of HPAI in or near a zoological institution could
have on the well-being of the birds in the collection, the functioning of conservation breeding
programmes and the economy of the institution affected;
AWARE that legislation to control HPAI may provide for derogations or special conditions
applicable to zoological institutions;
CONVINCED that, in spite of the limited knowledge about the efficacy and safety of current
vaccines in non-domesticated species, appropriate preventive vaccination has more
advantages than disadvantages, and that vaccination with an inactivated vaccine should be
applied where allowed and appropriate to protect zoo birds likely to enter into contact with
possibly HPAI infected wild birds;
THE WORLD ASSOCIATION OF ZOOS AND AQUARIUMS RECOMMENDS:
ASSOCIATION MEMBERS should

 maintain a regular dialogue with the national veterinary services of their respective
countries with a view of providing input into the legislation process and exploring the
possibility of regulations being applied to zoological institutions that will take into
account the special situation under which these institutions have to operate2;
 inform their constituencies of current regulations and any existing derogations for
zoological institutions;
 advise their constituencies to head for common approaches and to coordinate such
approaches as appropriate.
INSTITUTION MEMBERS should

 assume that all bird species are susceptible to HPAI and not only those that may be
addressed by current legislation;
 maintain a regular dialogue with the veterinary authority responsible for supervising
the institution;
 implement, and have approved by the competent veterinary authority, a health
surveillance plan covering the entire collection;
 apply strict quarantine measures when introducing birds into the collection, even in
the event of these birds not being imported from a third country;
 explore the possibility of the institution being subdivided into different compartments
for the purpose of animal health measures;
 apply biosecurity measures as appropriate within the institution;
 take special precaution to avoid the spreading of any infection from one compartment
to another, in case the institution has been subdivided;
2
EAZA should, in collaboration with EAZWV act accordingly with regard to the European Commission.
XI. Avian Influenza
 develop and have agreed by the competent veterinary authority contingency plans to
be implemented in the event of a HPAI outbreak in or in the neighbourhood of the
institution;
 explore the possibility and assess the advantages and disadvantages of vaccinating
those birds in their collection that are likely to enter into close contact with wild birds,
in particular waterfowl.
XI. VACCINATION OF NON-DOMESTIC AVIAN SPECIES AGAINST

HIGHLY PATHOGENIC AVIAN INFLUENZA (HPAI) VIRUSES
ADAPTED FROM: “VACCINATION OF NON-DOMESTIC ANIMALS AGAINST EMERGING INFECTIOUS DISEASES”

- PHD THESIS ERASMUSMC/ROTTERDAM ZOO, 2007
J.D.W. Philippa
Introduction
Avian influenza viruses (AIV) are type A influenza viruses and belong to the
Orthomyxoviridae family. They can be classified according to the antigenicity of its surface
proteins haemagglutinin (H) and neuraminidase (N). Currently 16H (H1-16) and 9N (N1-9)
subtypes have been described in avian species (Fouchier et al., 2005). Furthermore the
subtypes can be classified on the basis of their pathogenicity in chickens after intravenous
inoculation.
Highly pathogenic avian influenza (HPAI, formerly termed fowl plague), an acute generalised
disease in which mortality in chickens may be as high as 100%, is restricted to subtypes H5
and H7, although most viruses of these subtypes have low pathogenicity, and do not cause
HPAI. Low pathogenic avian influenza (LPAI) virus strains cause more variable morbidity and
mortality (ranging from sub-clinical to fatal) but are generally associated in poultry with a
mild, primarily respiratory disease with loss of egg production (Capua & Alexander, 2004), or
mild enteric disease in non-domestic birds. In certain cases (in poultry flocks) the LPAI virus
phenotype (of subtype H5 or H7) may mutate into the HPAI virus phenotype by the
introduction of basic amino acid residues (arginine or lysine) at the cleavage site of the
precursor haemagglutinin (HAO) (Banks et al., 2001), which facilitates systemic virus
replication. H5 and H7 subtypes with an amino acid sequence at the HA0 cleavage site
comparable to those that have been observed in virulent AI viruses are considered HPAI
viruses, even when mortality in chickens is low (Office International d'Epizooties., 2004).
However, the two forms of avian influenza (HPAI and LPAI) are distinctly different and should
be regarded as such.
Avian influenza viruses have a worldwide distribution and are infectious to all avian species
(commercial, domestic and wild), with variable morbidity per virus isolate and species.
Aquatic avian species, mainly those of the taxonomic orders Anseriformes and
Charadriiformes are considered the main natural reservoir of all avian influenza viruses,
including the LPAI ancestral viruses of HPAI strains (Munster et al., 2005; Munster et al.,
2007). Waterfowl were generally considered resistant to infection with HPAI virus until 2002.
However, in 2002 an outbreak of HPAI H5N1 virus occurred in wild migratory avian species
and resident waterfowl (Sturm-Ramirez et al., 2004). Since then, this particular HPAI virus
subtype has made an unprecedented spread from South East Asia throughout Asia and into
Europe and Africa, with morbidity and mortality not only in domestic poultry, but in more than
XI. Vaccination of Non-domestic Avian Species
130 non-domestic avian species from various taxonomic orders: Anseriformes,

Charadriiformes, Ciconiiformes, Columbiformes, Falconiformes, Galliformes, Gruiformes,
Passeriformes, Pelecaniformes, Phoenicopteriformes, Strigiformes, Struthioniformes,
Psittaciformes, and Podicipediformes (USGS National Wildlife Health Center, 2008).
Additionally, this virus strain has has caused mortality in a large number of mammalian
species, and has caused 403 human cases with 254 deaths to date (27 January 2009)
(World Health Organisation, 2009).
Documented outbreaks of Asian lineage H5N1 HPAI virus in captive non-domestic birds
have been limited to 6 cases: Penfold Park, Hong Kong, (People’s Republic of China, 2002),
Kowloon Park, Hong Kong (People’s Republic of China, 2002), Phnom Tamao wildlife rescue
centre (Cambodia, 2004), Ragunan Zoo, Jakarta (Indonesia, 2005), Dresden Zoo (Germany,
2006) and Islamabad Zoo (Pakistan, 2007). Large felids with H5N1 infection have been
reported in Suphanburi Zoo (Thailand, 2003), and Sri Racha tiger zoo (Thailand, 2004). To
curtail these outbreaks, a combination of increased bio-security measures (isolation and
quarantine of infected animals, disinfection of the area), feeding of cooked poultry only,
treatment of infected animals in quarantine areas, selective culling, extensive surveillance of
migratory and captive birds and vaccination were used.
Vaccination
Vaccination is a useful means of reducing the horizontal spread of AIV in poultry (Capua et
al., 2004; van der Goot et al., 2005) (Ellis et al., 2004). Vaccination protects against disease
and mortality, but does not always prevent infection and virus spread. However, the dose
required for infection is much higher, and vaccinated birds shed far less field virus after
infection than unvaccinated birds (Brugh et al., 1979; Karunakaran et al., 1987).
Protective antibodies produced in response to infection or vaccination, are directed against

the H and N surface proteins. Vaccine-induced antibody responses are species-, dose-, and
vaccine strain-dependent, e.g. the antibody responses upon AIV vaccination are generally
higher in chickens than in other poultry species (Higgins, 1996). Published minimum serum
antibody titres measured by HI test in vaccinated chickens that correlate with protection after
challenge with HPAI virus are 1:10 (Swayne et al., 2006), or 1:16 (Ellis et al., 2004; Tian et
al., 2005). However, domestic ducks with very low or undetectable antibody titres post
vaccination have been shown to be protected from HPAI virus challenge (Middleton et al.,
2006; Webster et al., 2006). Duration of protection from HPAI virus challenge may vary
between species: chickens for up to 40 weeks after one dose of vaccine, domestic ducks for
more than 52 weeks after 2 doses, while domestic geese which received 3 doses were
protected for 34 weeks (Tian et al., 2005).
The degree of homology of the H protein will largely affect the level of cross-protection and
therefore efficacy of the vaccine (Swayne et al., 2000). A so-called Differentiation of Infected
from Vaccinated Animal (DIVA) strategy, with a heterologous vaccine (using the same H
subtype as the field virus, but a different N subtype), is recommended to differentiate
between vaccinated and field-virus infected animals (Capua et al., 2003). However, in
housing systems where birds are not housed permanently indoors (e.g., in zoos), contact
with free-ranging birds can result in LPAI virus infections that go by unnoticed, but which may
interfere with the DIVA principle.
In the European Union routine vaccination of poultry against avian influenza viruses is
currently not practised as this would interfere with stamping-out policies and international
trade agreements. Instead, eradication measures during an outbreak in poultry include (long-
term) confinement, large-scale culling and safe disposal of carcasses of all poultry on the
infected farm, and, depending on the poultry density in the area and the epidemiological
situation, pre-emptive culling of poultry on neighbouring farms and emergency vaccinations
(Directive 92/94/EEC). Since 2003, more than 300 million birds have been culled to eradicate
HPAI outbreaks.
Vaccination in European Zoos
The standard measures used to prevent and eradicate HPAI virus outbreaks in poultry (long-
term confinement and large scale culling) would be detrimental to the welfare, conservation
status and breeding programmes of zoo birds, which often are irreplaceable, valuable and
endangered avian species (IUCN Red list, http://www.iucnredlist.org/). Directive 2005/94/EC
foresees a derogation from culling of birds provided the birds can be brought inside and are
subjected to virus detection tests (after the last death/positive finding, 2 tests at an interval of
21 days have to be performed according to the diagnostic manual Decision 2006/437/EC).
However, most zoos do not have the capability to suitably confine their entire bird collections
for extended time, and many species would not be able to adjust to confinement and
increased stress with subsequent welfare problems and increased exposure to pathogens
resulting in disease (e.g. aspergillosis and bumblefoot) (McMillian & Petrak, 1989; Redig,
2000; Harcourt-Brown, 1996).
Instead of confinement, vaccination of zoo birds against HPAI virus was allowed as an
additional preventive measure (while reducing confinement measures) in Belgian, Dutch and
German zoos during an outbreak of HPAI H7N7 virus in poultry in 2003 (Decision
2003/291/EC). Similarly, in 2005, Decision 2005/744/EC allowed vaccination in European
zoos against the encroaching H5N1 subtype. These campaigns were subject to rigorous
surveillance and control requirements.
During the HPAI H7N7 outbreak in poultry in 2003, birds in Dutch zoos were vaccinated
twice with six weeks interval using a whole inactivated oil-adjuvanted vaccine, based on
influenza virus A/chicken/Italy/473/99 (H7N1), with high homology to the field strain HPAI
H7N7 A/chicken/Netherlands/1/03 (97.4% nucleotide and 98.7 % amino acid sequence
identity). This resulted in the induction of antibody titres ≥ 40 (used as a correlate of
protection in this study) in 81.5% of the vaccinated birds, with an overall GMT of 190. Birds of
the taxonomic orders Anseriformes, Galliformes and Phoenicopteriformes showed higher
GMT, and larger percentages developed a serum HI antibody titre ≥ 40 than those of the
other orders. Furthermore, a decrease in antibody response with an increase in body weight
> 1.5 kg was shown. The high agreement between post vaccination antibody titres
determined by serum HI test (using the vaccine strain), and VN titres (using the field strain),
was used as a further measure of immunogenicity. The broad efficacy demonstrated in a
large variety of taxonomic orders illustrated the benefits of vaccination as an additional
preventive measure against HPAI virus infection (Philippa et al., 2005).
In 2005, the Dutch zoos were the first to implement Decision 2005/744/EC to provide
protection against the encroaching HPAI H5N1 subtype. Birds were vaccinated with an
inactivated adjuvanted H5N2 vaccine with vaccine doses adapted to mean body weight per
species, using data collected during the H7N1 vaccination campaign. The vaccine strain
(A/duck/Pottsdam/1402/86) had a homology of 90% to the HA gene of the H5N1 field strain
(A/turkey/Turkey/1/05) on the basis of nucleotide sequence (1530 base pairs, including basic
cleavage site), and 92.4% on the basis of amino acids. Vaccination was safe, and proved
immunogenic throughout the range of species tested, with some variations between and
within taxonomic orders. After booster vaccination the overall homologous GMT to the
vaccine strain, measured in 334 birds, was 190 (95% CI:152–236), and 80.5% of vaccinated
birds developed a titre of ≥ 40. Titres to the HPAI H5N1 virus followed a similar trend, but
were lower (GMT: 61 (95% CI: 49–76); 61%≥ 40) (Philippa et al., 2007).
The breadth of the immune response was further demonstrated by measuring antibody titres
against prototype strains of four antigenic clades of currently circulating H5N1 viruses.
Antigenic distances to the prototype strains were determined using antigenic cartography
(Smith et al., 2004). Antigenic cartography uses the antigenic properties of influenza viruses
combination with epidemiological and genetic data, and is used to select virus strains for use
as human pre-pandemic (H5N1) vaccine candidates (World Health Organisation (WHO),
2006). Influenza vaccines whose haemagglutinins are antigenically similar to circulating
strains provide the highest level of protection from infection in humans (Subbarao, 1999).
The birds clustered in two groups based on the breadth of antibody responses. Group 1
(Anseriformes, Galliformes, Phoenicopteriformes, Psittaciformes and Struthioniformes)
showed a very broad response to vaccination, with predicted protection against future strains
up to 12 antigenic units from the current vaccine. Group 2 (Ciconiiformes, Gruiformes,
Pelecaniformes and Sphenisciformes) had low HI antibody titres against the prototype strain
of the most antigenically distant clade (A/Indonesia/5/05).
In 2006, a working group of Animal health and Welfare experts was established by the
European Food Safety Authority (EFSA)(European Food Safety Authority (EFSA), 2007), to
provide a scientific assessment of the preventive vaccination against avian influenza of H5
and H7 subtypes carried out in zoos in Member States (MS). The total number of birds
vaccinated, as reported by 12 MS, was 44721. Individual data from 4718 birds (374 species
from 19 taxonomic orders) were submitted. Not all of these could be used for every
evaluation: pre-vaccination titres could be evaluated for 3039 birds; titres after first
vaccination were evaluated for 1429 birds, and post-second vaccination titres for 2296 birds.
Differences in vaccination schedules, doses and routes, differences in methodology and

antigens used in the HI tests between laboratories (due to the absence of international
reference standards, and the absence of inter-laboratory standardisation of methodology),
the use of different vaccines1 in different taxonomic orders and the sometimes incomplete
reporting of results, limited the evaluation of some of the data provided by EU MS. Cut-off
points varied with laboratory, and titres considered a measure of adequate immune response
were 8, 16, 32, 40, and 64. Most countries used dilution series starting at 4 or 8, therefore
results were evaluated for titres 16 and 32 [documented surrogate markers for protection in
chickens (Ellis et al., 2004; Office International d'Epizooties., 2004; Swayne et al., 2006; Tian
et al., 2005)], and undetectable titres were regarded as 4 for calculation of GMT. In the
absence of (and unfeasibility of obtaining) vaccination/challenge data in often endangered
Vaccine A: H5N9 (A/turkey/Wisconsin/68).

Vaccine B: H5N2 (A/duck/Pottsdam/1402/86).
Vaccine C: H5N2 (A/chicken/Mexico/232/94/CPA)
Vaccine D: H5N9 (A/chickenk/Italy/22A/98).
Vaccine E: H5N9 (A/chicken/ltaly/22A/H5N9/1998).
zoo bird species, the evaluation had to be based on extrapolation of serological data from
poultry and limited other bird species.
The H5 vaccines registered for poultry in the EU showed differences in efficacy, measured
as serum HI antibodies induced by two doses of vaccine (Table 1.). Three of the five
vaccines evaluated induced relatively high GMT and high percentage seroconversion in the
vast majority of vaccinated birds. The HI titres induced by vaccination showed marked
differences between and within taxonomic orders. Both routes of vaccination (i.m. and s.c.)
were effective in inducing HI serum antibody responses, and for most avian species the
poultry dose was suitable. In some larger species higher doses adjusted to body weight,
induced higher serum antibody titres. (e.g. for ostriches a 10-fold increase of the poultry dose
(10 x 0.25ml). However, extremely high doses at a single site of injection (e.g. vaccination of
ostriches with 10 ml of vaccine) appeared to have a negative effect on the induction of serum
antibody titres, and induced local adverse reactions.
There were indications that one vaccination was sufficient to induce high serum antibody
titres in at least two taxonomic orders of birds (Anseriformes and Galliformes). However, a
second vaccine administration ensured seroconversion in the majority of birds of most
species. Limited data indicated that antibody titres persisted in several species for six months
after vaccination. Adverse effects and mortality associated with vaccination were low and
were mainly attributable to handling stress or trauma. Differences in adverse effects reported
from different zoos highlight the importance of proper skilled handling.
Longevity of antibody titres
One year after vaccination with the H5N2 vaccine, birds in Dutch zoos were revaccinated
with the same vaccine. Antibody titres one year after the initial two vaccinations and the
effect of one booster vaccination at this time were evaluated. In Rotterdam Zoo, 72
previously vaccinated birds could be evaluated for the effect of one booster vaccination
(Philippa et al, in press). For 44 birds, serum samples were available from 4 weeks after the
initial two vaccinations the previous year, at the time of revaccination, and 4 weeks later.
Birds which had been vaccinated with the H7 vaccine two years prior to the H5N2
revaccination were additionally tested for the presence of H7-specific antibodies.
Serum antibody titres of the birds tested in Rotterdam Zoo had clearly decreased in one year
time: while 80% of birds had a positive titre (≥ 8) and 68% a high positive titre (≥ 32) after 2
vaccinations, these figures were 61% and 30% respectively one year later. Four weeks after
re-vaccination these figures increased to 93% and 77% respectively. Although a larger
percentage of these 44 birds had a serum HI antibody titre ≥ 32 after re-vaccination, the
GMT was lower than GMT after 2 vaccine doses one year before (88 vs 66).
Birds from 4 out of 8 taxonomic orders did not have a GMT > 5 one year after vaccination,
and only one order (Phoenicopteriformes had a GMT > 40. Four weeks after one
revaccination 6/8 taxonomic orders tested had a GMT > 40.
GMTs had decreased even further two years after vaccination, as was shown by the H7
specific serum HI antibody titres. As these birds were not revaccinated with an H7 vaccine,
the effect of revaccination two years after the initial vaccinations is not known.
Conclusions
Bio-security measures remain the first line of protection of zoo birds against the introduction
of AI viruses and should be implemented in zoos. These bio-security measures should
include strict hygiene and quarantine measures, but should also exclude the possibility of
introducing AI viruses through feed animals such as day old chicks, other poultry or their
products. Continuous clinical monitoring of captive and wild birds in zoos should be
practiced, for early detection of introduced viruses by wild birds, domestic birds, or their
products. Strict bio-security measures will also reduce the risk of subsequent infection of wild
birds from zoo birds. Wild birds have been documented to be susceptible to HPAI virus
infection, and could potentially play a role in the spread of HPAI virus, although the majority
of avian influenza viruses detected in free-ranging birds have been LPAI viruses. If bio-
security measures cannot sufficiently protect zoo birds from exposure to HPAI viruses
coming from wild birds (based on an overall risk assessment which includes welfare aspects)
vaccination with vaccines against HPAI of H5 and H7 subtypes authorised for use in poultry
should be used to protect these zoo birds. In designing AI vaccination programmes and
schedules for zoo birds, recent data on wild bird migration and prevalence of AI viruses
should be taken into account. Vaccination against AI viruses of the H5 and H7 subtypes with
current inactivated oil-adjuvanted poultry vaccines is safe and, in most taxonomic orders of
zoo birds, effective in terms of inducing HI serum antibody titres. AI vaccines should be
administrated in a way that elicits high HI antibody titres in the vast majority of the zoo birds
vaccinated, i.e., by adjusting dose to average body weight. Although there are indications
that one vaccination might suffice for some species, a second vaccine dose ensures high
titres in the vast majority of species. Unless it is demonstrated that one vaccine
administration is sufficient, two administrations are recommended. The H5 and H7 vaccines
currently registered for poultry in the EU show differences in the performance in terms of HI
response in zoo birds after two doses. There appears to be no difference in route of
vaccination (s.c. or i.m.), so route can vary depending on the bird species to be vaccinated.
In order to maintain high titres in the captive populations in zoological collections, annual re-
vaccination seems to be required, as antibody titres decrease significantly in most taxonomic
orders, and high titres are seen after a single annual booster dose.
Mortality and adverse effects were low in all zoos evaluated in EU MS, and mainly attributed
to handling stress and trauma. Zoos can, and should therefore try to minimise these losses in
the execution of HPAI vaccination programmes. To minimise indirect losses due to
decreased breeding results, AI vaccination during breeding seasons should be avoided
whenever possible. Mortality due to catching and handling stress can be reduced by handling
the birds less. Once the efficacy of a vaccination protocol has been validated for certain
species using certain vaccines, measurement of post-vaccination HI serum antibody titres
should no longer be mandatory by the EU. These birds will then only have to be handled for
vaccination, and not 4 weeks later. Further research should be carried out to establish
effective vaccination schedules, route, and dose regimen in different zoo bird species. This
may, amongst others, lead to a reduction in the number of booster vaccinations needed in
certain species. Novel generation vaccines which may be administered in the form of an
aerosol (as is used in vaccination of poultry against Newcastle disease virus) may prove to
be useful in non-domestic species, and would eliminate the need for handling the birds.
The vaccination campaigns against HPAI virus have focused on protecting birds in zoological
collections. However, a large number of mammalian species, including tigers and leopards,
have also been documented with HPAI virus infection with recent H5N1 subtypes. There is
currently no commercial vaccine available to protect mammals from HPAI H5N1 virus
infection. A recombinant fowlpox-vectored vaccine expressing the H5 gene has been shown
to produce high antibody titres against heterologous H5N1 virus antigen in cats after booster
vaccination (Karaca et al., 2005), and may prove to be useful in prophylactic vaccination
programs of mammals in the future. Until then, these animals have to be protected by
excluding the introduction of AIV through raw poultry used as feed.
The broad vaccine efficacy in the different avian taxonomic orders illustrates that vaccination
against avian influenza is a useful tool for the protection of non-domestic avian species in
zoos, which allows for an alleviation of confinement measures – and is therefore beneficial to
the health and welfare of these birds. However, increased bio-security measures in
combination with virological monitoring remain imperative in combating outbreaks of HPAI.
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XII. VACCINATION OF NON-DOMESTIC CARNIVORES: A REVIEW
Joost Philippa, DVM
Institute of Virology, Erasmus MC, Rotterdam, The Netherlands
Since the introduction of vaccinia by Jenner 200 years ago, vaccines have been the first line
of defense in controlling infectious diseases in man and domestic animals. Large scale
domestic dog vaccination programmes against canine distemper virus (CDV) and canine
adenovirus (CAV) became possible in the late 1940’s, when egg-adapted vaccines became
available on a commercial basis, followed by tissue culture adapted vaccines in the late
1950’s (Piercy 1961). Before this time these devastating diseases had to be controlled
through quarantine and vigilance in capturing feral domestic animals (Dolensek et al. 1977).
Historically there have always been two major types of vaccines based on the living state of
the antigens. Modified live (MLV) vaccines use attenuated pathogens which reproduce in the
vaccinate, thereby elliciting an immunologic response without causing disease (a “controlled”
infection). The other major type of vaccine uses antigens that are non-living or inert – killed
vaccine (KV). These vaccinations are preferred when safety information is not available, as
they do not replicate and are therefore incapable of causing an infection. The proces of
inactivation however may be damaging enough to modify immunogenicity, usually resulting
in an immune response that is shorter in duration, narrower in antigenic spectrum, weaker in
cell-mediated and mucosal immune responses, and possibly less effective in totally
preventing viral entry (Murphy et al. 1999).
Recent advances in immunology, molecular biology and biochemistry have allowed the
construction of subunit vaccines based on viral or bactererial recombinants, peptides, or
plasmid vectors, which may lead to safer, more efficacious vaccines that can also be used in
exotic species.
Vaccines cannot be absolutely guaranteed to provide protection against disease. The

principal objective of vaccination is to mimic the protective immune response induced by
natural infection, ie to elicit a high titre of neutralising antibodies of the appropriate class, IgG
and/or IgA, directed against the relevant epitopes on the virion (Murphy et al. 1999).
Immunity induced by vaccination or infection in domestic animals has been evaluated mainly
by measuring the levels of serum antibodies. The immunologic response that provides
protection against infectious agents involves a cellular and a humoral response. For certain
infections (e.g. CDV, CAV, canine parvovirus (CPV), feline panleukopenia virus (FPV) or
Borrelia burghdorferi) the humoral responses, although not the only mechanism involved,
tend to correlate with level of protection from clinical disease, and therefore may be a useful
indicator of the immune status. Other agents (eg, Bordetella bronchiseptica, canine
coronavirus (CCoV), feline enteric coronavirus (FCoV), canine parainfluenzavirus (CPIV) and
Chlamydia psittaci) all replicate and cause damage on mucosal surfaces, and might require a
mucosal immune response for protection. As a consequence, serum antibody titres do not
necessarily correlate with protection (Pfizer 1998). A high concentration of antibodies in an
XII. Vaccination of Non-domestic Carnivores
animal implies that the animal is probably protected but also could indicate that it may not be
possible to stimulate an additional immune response in that animal. Cattle vaccinated with
baculovirus-expressed haemaglutinin (H) and fusion (F) proteins of Rinderpest virus (RPV)
were not protected against disease caused by challenge exposure despite having detectable
antibody titres (Bassiri et al. 1993). For some diseases, caused by persistent intracellular
pathogens (viral diseases or intracellular bacteria) the neutralising antibodies and
complement may play a less important role than the cellular immunity. Jones et al. (1993,
1997) demonstrated a lack of detectable antibody titre to CDV in ferrets and to peste des
petites ruminants (PPRV) in goats after vaccination with a RPV recombinant vaccine based
on fowlpox expressing the F and H genes. When these animals were challenged they
survived, suggesting that protection against clinical disease may be cell mediated rather than
humoral. A comparable study by Fisher et al (2003) using a DNA vaccine showed clear
protection after challenge with CDV, with only limited virus neutralising antibody titres. High
antibody concentrations do serve to inhibit the spread of virus between cells and thus
promote host resistance (Tizard & Ni 1998). Infection with feline rhinotracheitis virus, as all
herpesviruses, requires local and cell-mediated immunity, and one study suggests that there
is no correlation with protective immunity and antibody titre (Johnson & Povey 1985). Later
studies (eg Scott & Geissinger 1999, Dawson et al. 2001) use antibody titres as indicators of
protective effect. In general it can be stated that theoretically, cell-mediated immunity is the
most effective arm of the immune system in controlling, if not eliminating latent/persistent
infections such as those caused by herpesviruses and retroviruses (Murphy et al. 1999).
The usefulness of antibody titres to measure immunity is thus limited to only a few disease
agents, and in order to obtain a more complete view of the immunologic status of an animal
one therefore needs to look at the humoral and cellular responses. One should also keep in
mind that an animal that has mounted an immune response after vaccination will possess
memory T and B cells, which will remain for years after the antibody titre has declined. These
memory cells rapidly differentiate during a subsequent infection into effector cells that can
eliminate an infection before clinical signs appear, although the exact mechanism
responsible for this longevity is unknown (Ahmed & Gray 1996). One can only know if the
measured level of immunity is protective by challenging the vaccinated animal with the
pathogen.
One of the principal causes of vaccination failures in domestic dogs is maternal antibody
interference. MLV vaccines differ in their ability to evade antibodies, and may sometimes be
prevented from inducing an immunologic response in the vaccinate when the antibody level
is high. The duration of passive immunity is directly correlated with the metabolic size of the
animal. Therefore immunoglobulins will persist longer in a larger animal (Armstrong et al.
1942). The “window of vulnerability” during which the pup is vulnerable to infection with
virulent virus, but unresponsive to attenuated vaccine virus (Pollock and Carmichael 1990),
has been shown to range from 2-5 weeks for parvovirus infection in domestic dogs, but
varies between pathogens and vaccinate species. Females close to parturition may be
hyperimmunised with an inactivated parvovirus vaccine, so that high levels of maternal
antibody delay the window of vulnerability until the offspring are older and better able to
withstand the effects of parvovirus infection.
The recent debate in veterinary medicine concerning issues related to vaccine efficacy and
safety as well as duration of immunity induced by the currently available vaccines (Smith et
al. 1995, Schultz et al. 1998, Kruth & Ellis 1998, Tizard & Ni 1998, McCaw et al. 1998,
Gumley et al. 1999, Hustead et al. 1999, Twark et al. 2000) has resulted in the need for more
objective and scientific data and an increase in research in domestic animals.
In non-domestic animals however, there have been only few controlled studies of vaccination
(Heerden et al. 1980, Halbrooks et al. 1981, Behlert et al. 1981, Barker et al. 1983, Montali et
al. 1983, Green et al. 1984, Bush et al. 1985, Hoover et al. 1985, Paul-Murphy et al. 1985,
Briggs et al. 1986, Tham et al. 1987, Follmann et al. 1988, Spencer et al. 1991, 1992,
Goodrich et al. 1994, Harrenstien et al. 1995, Schubert et al. 1995, Williams et al. 1996,
Henke et al. 1997, Kadoi et al. 1998, Bingham et al. 1999, Harthorn et al. 1999, Pare et al.
1999, Wimsatt et al. 1999, 2003, Maack et al. 2000, Blasco et al. 2001, Federhoff et al. 2001,
Lambot et al. 2001, Maia et al. 2001, van Heerden et al. 1998, 2002). These are usually
restricted to measuring the (humoral) immune response and extrapolating the data from
those known in domestic animals, as subjecting (endangered) zoo animals to challenge
infections is generally not an option.
Challenge studies in dogs have shown a range of reported protective titres – these may differ
due to the variety of techniques and standards used. In humans there is a general
standardisation of assay methods to measure antibody titres. Non-standardisation of
serologic tests makes comparisons between laboratories of questionable use (Luff et al.
1987):
Table 1: Reported protective titres in domestic dogs:

Virus Protective Reference
titre
CAV 30 Cole et al. 1998
CDV >2 Gillespie et al. 1965; Ackerman & Siebel 1974.
20 Gillespie et al. 1958, 1972;Gorham et al. 1966; Prydie 1966;
Krakowka et al. 1978; Cooper et al. 1991.
24 Jones et al. 1997.
30 Gillespie 1966.
>50 Olson et al. 1988.
96 Mc Caw et al. 1998.
100 Appel 1969; Krakowka et al. 1975, Montali et al. 1983; Carmichael
et al. 1999.
CPV 80 Olson et al. 1988; Carmichael et al. 1983, 1994, 1997; Appel et al.
1979; Pollock and Carmichael 1982; Meunier et al. 1985.
RV 20 Bunn et al. 1984
Twark et al. (2000) report that CDV titres above 5 are indicative of an adequate antibody
response in domestic dogs, although level of protection is unknown, and recommend CDV
revaccination when titres are below 32. Carmichael et al. (1983) demonstrated that dogs with
a CPV titre below 100 were not protected. Titres of 200-800 were protective in some dogs,
titres above 1600 appeared to be protective in all dogs.
There is still no general consensus on how often domestic animals need to be revaccinated -
for most vaccines there is little information on the duration of immunity. It is, however,
recognised that protective immunity to CDV following MLV vaccination is of long duration,
perhaps even lifelong. For other viruses or components of combination vaccines this duration
may not be of such long duration.
Vaccines used in domestic animals are approved for use in specific animals under specific
conditions, and any other use is therefore extra-label. Most of the vaccines are not approved
for non-domestic species, therefore there is always a potential liability to such use (Bittle
1993). MLV vaccines have been designed to be minimally virulent, while retaining maximal
immunogenicity in their domestic counterparts. When used in other species or delivered by
another route the residual virulence may cause disease (Tizard et al. 1990). It is not unusual
to observe side-effects such as elevated temperature, swelling, and irritation at the site of
injection, or systemic anaphylactic reactions like hyperaemia, hypersalivation, or vomiting
(Greenacre 2003) that may in some cases be severe (Karesh et al. 1983).
Several viruses induce a supression of the immune system, and it is known that some
attenuated virus strains may still be able to cause immunosuppression, e.g. MLV CPV
(Krakowka et al 1982). Sometimes the individual vaccine strains are not detectably
immunosuppressive, but when combined in a combination vaccine they may induce a
suppression of blood lymphocyte counts (Phillips et al. 1989a). Enhanced virulence of canine
distemper virus produced in canine cell-cultures has been reported when used in
combination with CAV-1 and live CCV vaccines (Carmichael et al. 1983, Martin et al. 1985,
Wilson et al. 1986), and may lead to encephalitis.
Table 2: There are many examples of vaccine-induced disease:

Virus Species Reference
CAV Domestic dog (Canis familiaris) Appel et al. 1978.
Maned wolf(Chrysocyon brachyurus) Thomas-Baker et al. 1985.
CDV Domestic ferret (Mustela putorius furo) AVMA 1966; Wimsatt et al. 2001.
Kinkajous (Potus flavus) Kazacos et al. 1981.
Red panda (Ailurus fulgens) Bush et al. 1976; Itakura et al. 1979.
Black-footed ferrets (Mustela nigripes) Carpenter et al. 1976; Pearson et al. 1977.
African wild dogs (Lycaon pictus): Mc Cormick et al. 1983; Montali et al. 1983;
Durchfeld et al. 1990.
Maned wolf Thomas-Baker et al. 1985.
Bush dog (Speothos venaticus) Mc Innes et al. 1992.
European mink (Mustela lutreola) Sutherland-Smith et al. 1997.
Ek-Kommonen et al. 2003
Gray fox (Urocyon cinereoargenteus) Halbrooks et al. 1981.
Fennec fox (Fennecus zerda) Mehren et al. 1984.
RV Domestic dog Pedersen et al. 1978; Whetstone et al. 1984.
Domestic cat Erlewein et al. 1981.
Skunk (Mephitis mephitis) Debbie et al. 1979.
Raccoon (Procyon lotor) 1978.
FeLV Cheetah Briggs et al. 1986.
FHV Domestic cats North et al. 1978
Pallas cat Wallach & Boever 1983
FPV Felidae: Behlert et al. 1981
Cheetah (Acinonyx jubatus) Crawshaw et al. 1996
Preventive medicine is especially important in the management of non-domestic animals for

several reasons: the ability to mask or hide illness or distress as a means of survival means
that by the time signs of disease are exhibited, the underlying disease condition may have
advanced to a critical stage. Another reason is that the use of anaesthesia is required to
perform a proper physical examination. Active immunisation is only one of the factors (eg
nutrition, parasite control, hygiene) associated with preventive medicine. Recommendations
for use in exotic mammals are generally based on tradition, anecdotal/personal experiences
or taken from limited precise, published data. This has led to a plethora of differing opinions
and therefore the use of many different protocols in zoological collections. In general,
inactivated viral or bacterial vaccines are preferred for use in exotic animals. The type, serial
number, and source of product should be recorded in the medical records (Joslin et al.
1990). Use of polyvalent vaccines containing unnecessary antigen (one the species is not
susceptible to) should be avoided where possible. Animals with active clinical illness should
not be vaccinated. In the event of a viral disease outbreak in an animal collection, all
susceptible species should be vaccinated immediately and boostered 10-14 days later,
regardless of age and last time of immunization (Phillips 1989). Some drugs, such as
tetracycline, chloramphenicol, dapsone, clindamycin, griseofulvin, nalidixic acid and
sulphamethoxypyradizine have been associated with an inadequate response to vaccination
(Kruth – Ellis 1998). Vaccination should also be avoided in animals undergoing glucocorticoid
therapy, although challenge studies have been performed which show that “immuno-
suppressive” doses given at the time of vaccination do not significantly affect the level of
post-vaccinial immunity to canine distemper or rabies (Dhein et al. 1986). When using
remote delivery systems one must be sure that a full dose has been delivered. Syringe darts
may rebound quickly on impact and fail to deliver the dose required to elicit a satisfactory
immune response (Aiello 1998). Any vaccination programme should also take the current
local prevalence of the pathogen into account, upon which the decision can be made if
vaccination is warranted.
Table 3: Disease susceptibility

CAV CDV FPV FeLV FHV FCaV Rabies Lepto Toxo
Canidae + + - - - - + + +
Felidae - + ++ + + + + + ++
Ursidae + + + - - - + + +
Procyonidae - + + - - - + + +
Mustelidae - + + - - - + + +
Viverridae - + + - - - + + ?
Hyanidae + + + - - - + + ?
Pinnipedia + + - - - - + + +
Canine distemper virus (CDV)
All families of the order Carnivora are susceptible to CDV, and it is among the most
significant infections of many species. Christensen (1963) recommended vaccination against
CDV in susceptible zoo animals, with the addition that vaccination of young animals at an
earlier stage than in dogs is preferred. CDV vaccination is recommended in all members of
the Canidae, Procyonidae and Mustelidae by all authors. There is no mention of vaccination
of felids against CDV in literature until several outbreaks occurred among large cats in zoos
and the wild (Fix et al. 1989, Appel et al. 1994, Munson et al. 1995, Wood et al. 1995,
Roelke-Parker et al. 1996, Meehan et al. 1998, Cameron et al. 1998, Miller & Anderson
2000). Following this, the vaccination of large cats is mentioned as being possible, but not
recommended – unless in high risk situations - as risk of exposure is generally low, and
vaccination carries some risk (Junge et al. 1995, Kennedy-Stosskopf 1996, Aiello 1998,
Miller & Anderson 2000, Woodford 2001, www.5tigers.org). The susceptibility of members of
the Ursidae and Hyaenidae to canine distemper virus is deemed questionable by some
authors, and therefore not recommended by these authors (Fraser 1991, Aiello 1998).
Although clinical disease as a result of CDV infection is rare in ursids (Poston & England
1992), serologic surveys have shown the presence of CDV specific antibodies (Munson et al.
1995, Marsilio 1997, Dunbar et al. 1998, Maack et al. 2000). Clinical disease and presence
of CDV specific antibodies has been documented in spotted hyaenas (Crocuta crocuta)
(Montali et al. 1987, Alexander et al. 1995, Haas et al. 1996, Harrison et al. 2002) and a palm
civet (Paguma larvata) (Machida et al. 1992), therefore vaccination is recommended in these
species by other authors (Fowler 1986, Phillips 1989, Miller 1989, Burroughs 1992, Junge
1995, Woodford 2001). Miller & Anderson (2000) recommend vaccination of Viverridae, do
not recommend vaccination of Ursidae, and omit Hyanidae from their article. In general,
when vaccination is recommended, the same regime is used in the different families. Due to
the possibility of immunosuppressive effects of multivalent vaccines, it is recommended that
the distemper vaccine be given separately at a reasonable interval from the other
components (Montali et al. 1994)
A problem faced in the prophylaxis of distemper in exotic carnivores is the variation between
and within species in their reaction to MLV vaccines, with possible lethal consequences.
There are two distinct types of MLV CDV vaccine, one produced in avian cells and the other
in canine kidney cells, and neither is safe for use in all potential target species. Chicken
embryo-adapted live virus distemper vaccines attenuated for ferrets have protected them
from challenge with an infective dose of virulent virus within 48 hours after vaccination (Baker
et al. 1952). Fromm D (Solvay Animal Health Inc.) appeared to be safe and efficacious for
use in maned wolves, bush dogs and fennec foxes (Montali et al. 1983). But avian cell
vaccines have caused disease in mink, ferrets and foxes (Sutherland-Smith et al. 1997,
Carpenter et al. 1976, Henke et al. 1997). Fervac-D, also of avian origin caused fatal disease
in 1 of 8 red pandas (Montali et al. 1994). Vaccine of canine origin has been responsible for
vaccine induced distemper in a large number of species (see table ). Non-domestic canine
pups can be vaccinated with a MLV measles vaccine (Klös & Lang 1982, Frankenhuis &
Visee 1985) - measles and canine distemper virus are antigenically closely related, and the
measles virus is not neutralised by the maternal antibodies in 6 week old puppies of
domestic dogs (Appel et al. 1984). The second and all subsequent vaccinations should be
with a MLV CDV vaccine. Until 1983 the use of MLV is mentioned without warning (Klös &
Lang 1982, Wallach & Boever 1983, Miller 1989). After this KV are recommended for use in
exotic species (Fowler 1986, Gabrisch & Zwart 1987, Franke et al. 1989, Fraser 1991, Aiello
1998) even though the efficacy of KV has been questioned (Appel et al. 1984, Sikarski et al.
1991). Currently there are no killed CDV vaccines commercially available, because there is
no demand for its use in domestic dogs, and the market for zoo animals is too small (Appel &
Montali 1994). Between the different commercially available MLV vaccines there is a clear
difference in vaccine efficacy, as has been demonstrated in domestic dogs (Appel et al.
1987, Rikula et al. 1996, Kommonen et al. 1997). Considering the high incidence of vaccine
induced disease, Miller & Anderson (2000) mention that in some cases strict isolation may be
preferable to vaccination.
The large range of (highly susceptible) host species in zoos for which vaccination is
recommended underpins the need for the production of a safe and efficacious vaccine. An
experimental subunit vaccine incorporating the CDV F and H proteins into immuno
stimulating complexes (ISCOM) has been developed for use in dogs and seals (de Vries et
al. 1988, Visser et al. 1992), and proved to be capable of producing humoral and cellular
immunity. Although the immunity achieved was not sterile (infection of the upper respiratory
tract occurred), the vaccinated seals were protected from a potentially lethal challenge with
phocid distemper virus (Visser et al. 1992). The ISCOM vaccine has since been used
experimentally in several European zoos (Schaftenaar pers. comm), and its potential use in
non-domestic felids has been proposed in special cases (Kennedy-Stoskopf 1996). An
experimental adjuvanted killed CD vaccine produced by M.J.G. Appel has been used in red
pandas and giant pandas in several zoos. The vaccine appeared to be safe and effacacious,
but produced low titres with inadequate durability, requiring booster vaccinations two to three
times annually (Montali et al. 1994). This vaccine is no longer produced. In Germany a small
amount of inactivated vaccine is produced for use in zoos (Geyer and Matern pers. comm.
2001).
In 1997 a recombinant virus vectored CDV vaccine was introduced (Stephenson et al. 1997)
and tested for its safety and efficacy along with MLV components (Pardo et al. 1997).
Following vaccination experiments in mice using a vaccine expressing the H and F protein in
mice, a vaccine containing the H, F and nucleocapsid (N) constructs produced highly
encouraging results in domestic dogs after challenge with virulent virus, although the
mechanism of protection was not clear (Cherpillod et al. 2000). Recently a monovalent
canarypox-vectored vaccine expressing the H and F surface antigens of CDV has become
commercially available in the US, and its efficacy and safety in domestic ferrets has been
demonstrated (Wimsatt et al. 2001). In black-footed ferrets (Mustela nigripes) x Siberian
polecat (Mustela eversmanni) hybrids the use of this vaccine has produced a good immune
response (Williams & Montali 1998), and has since been used and evaluated in a large
number of exotic species (Montali pers. comm.). This vaccine, Purevax (Merial) is registered
for use in domestic ferrets, but its off-label use in all susceptible species in zoos is
recommended by the American Association for Zoo Veterinarians (AAZV) and Woodford
(2001). In the European Union its use is not permitted. The main advantage of avipox-
vectored vaccines is their safety, the foreign genes in the vector are expressed, inducing
protective cellular and humoral immunity in the absence of the complete virus, and therefore
eliminating the possibility of infection. Members of the Avipox genus are distinguished by
their host restriction to avian species, eliminating the potential for dissemination of the vector
within the vaccinate and therefore the spread of the vector to nonvaccinated contacts or the
environment (Paoletti 1996). Virus replication is blocked at a late stage of morphogenesis in
mammalian cells, importantly leaving the synthesis of viral proteins unimpaired (Sutter &
Moss 1992). For unknown reasons, canarypox virus appears to be superior to fowlpox virus
in the induction of immune responses in mammals (Moss 1996). Recent research has shown
that replication-competent CAV-2 recombinant vaccines expressing the H and F antigens of
CDV triggered both a significant seroconversion and protective immunity in puppies born to
CDV and CAV-2 immune dams, thereby overcoming the passive immunity (Fischer 2002).
CDV Vaccination regime recommended in domestic dogs:
 Initial at 6 weeks, repeat every 2 weeks until 12 weeks. Booster annually with MLV
vaccine
(Appel et al. 1999, Carmichael 1999, Dodds et al. 1999).
Table 4:
CDV vaccination Regimes recommended for use in non-domestic species
Species Vaccine Regime Booster Reference
All MLV Initial at 3-6 weeks, at 9 weeks biannually Klös & Lang 1982
susceptible combination vaccine. Single
vaccination after 12 weeks
Mustelidae MLV 10 weeks * Klös & Lang 1982
All KV or Initial at 5-6 weeks repeat every 2 annually Wallach & Boever
susceptible MLV weeks until 15 weeks 1985
Mustelidae KV for Initial at 8 weeks, repeat after 2-3 * Wallach & Boever
blackfoot weeks 1985
& initial
Procyonidae KV Initial at 6-8 weeks, repeat every 2-3 annually Wallach & Boever
weeks until 14 weeks 1985
All Initial at 8 weeks, repeat after 2-3 annually in affected Frankenhuis &
susceptible weeks areas, otherwise every Visee 1985
2-3 years
All KV Initial distemper/measles at 6-8 annually Burroughs 1992
susceptible (unavaila weeks, at 12-14 weeks combination
ble) vaccine.
Procyonidae Initial at 8 weeks, repeat at 12 and 16 * Paré et al. 1999
or 18 weeks of age
All Single dose after weaning im, monthly annually Fraser 1991,
susceptible booster up to 4 months Aiello 1998
All Initial at 6-8 weeks, repeat every 2-3 annually Phillips 1989,
susceptible weeks with a total of 3 vaccinations, Cubas et al. 1996
in special cases (ie early weaning, ill
juveniles, high probability of exposure
to disease) extended to 4 or 5.
All ISCOM 8, 11, 14 weeks of age annually Blijdorp
susceptible or KV
(unavaila
ble)
Parvovirus infections
Infections with Feline Panleukopeniavirus (FPV) have been reported in captive felids since
the 1930’s (Hindle & Findlay 1932, Goss et al. 1942), and have since been reported in a
large number of feline species. All members of the Canidae, Felidae, Viverridae, Mustelidae
(except for the domestic ferret, Parrish et al. 1987), Procyonidae and Ursidae are susceptible
and vaccination is recommended. Although there is some difference of opinion on the
susceptibility of Hyaenidae, vaccination is recommended (Junge 1995). Vaccination regimes
reported by these authors are as those for the exotic cats. In 1947 a new viral gastroenteritis
was observed in farmed mink (Mustela vison) in Canada (Schofield 1949). That virus, closely
related biochemically and serologically to FPV, was later named mink viral enteritis, and
currently probably occurs wherever mink are farmed (Pollock and Larsen 1990). In 1978 a
virus infecting canine species emerged with clinical similarities to FPV infection in cats (Appel
1979). This virus, referred to as canine parvovirus type 2 (CPV-2) is closely antigenically and
pathogenically related to FPV. Members of the Canidae, Mustelidae, Viverridae and
Procyonidae are considered to be susceptible, but the virus has not got the ability to replicate
in Felidae. In 1979 and around 1984 new antigenic types of CPV (CPV-2a and CPV-2b)
emerged with an increased host range including both domestic and large cats (Steinel et al.
2000), although the large cats appear to have a higher susceptibility for these virus types
(Steinel et al. 2001). For all susceptible species vaccination is recommended (Fraser 1991,
Aiello 1998, Steinel et al. 2001). Other authors recommend vaccination of canidae only
(Wallach & Boever 1985, Cubas et al. 1996). Canidae may be vaccinated with a FPV
vaccine, and Felidae may be vaccinated with CPV vaccine due to the close antigenic
relationship (Pollock & Carmichael 1983). In general, when vaccinating non-domestic
species, the vaccine selected should be based on the similarity of the hosts (e.g. CPV in
coyotes) or the known or probable virus susceptibility of the host to be vaccinated (e.g. FPV
vaccine in raccoons) (Barker and Parrish 2001). It is recommended to vaccinate members of
the Canidae with CPV-2 vaccines, and considering the high incidence of infections with CPV-
2a and CPV-2b in large cats an inactivated vaccine containing these types rather than CPV-2
would be desirable, but is not yet commercially available.
There is debate over vaccine dose in relation to size of species, and the use of KV and MLV
vaccines. Povey and Davis (1977) suggest that it is probably unnecessary to increase the
dose or antigen mass when using KV in large species, due to the antigenic efficiency of
parvoviruses and the use of adjuvants. Increased or double doses have been recommended
for the vaccination of large cats >50 kg, which will subsequently develop higher antibody
titres (Fowler 1977, Wallach - Boever 1983, Fraser 1991, Aiello 1998). In other studies
antibody titre does not appear to be dose dependent (Wack 1991), large felids do not require
larger doses than the 1ml dose used in domestic cats to develop protective titres (Bush et al.
1981). MLV vaccines probably overcome the consideration of animal size in relation to dose
of vaccine (Povey and Davis 1977), although the increased potency may result in decreased
safety. Some MLV vaccines reported to be safe for one species may be insufficiently
attenuated for use in another species, and have caused vaccine-induced disease (Klös &
Lang 1982, Wallach & Boever 1983, Frankenhuis & Visee 1985, Appel et al., Fraser 1991,
Aiello 1998, Woodford 2001). Christensen (1963) reported abortive cases of depression and
diarrhoea with recovery after 1 or 2 days’ symptoms following the introduction of systematic
FPV vaccination. Visee (pers. comm. 1974 & 1975) reported clinical panleukopenia in 3
vaccinated Siberian tigers and 1 leopard (panthera pardus), of which 2 fatal. Therefore the
use of KV is generally recommended in exotic species, although the production of antibodies
is not as effective and the duration of immunity is shorter. KV FPV vaccine used in bush dogs
(Speothos venaticus) did not protect them from infection – protective titres were not reached
before 23 weeks of age during which period they were susceptible to infection (Janssen et al.
1982). Fowler (1977) reports that MLV vaccines have been used in different wild felid
species, and are apparently safe. Many authors after this recommend the use of the highly
antigenic KV vaccine (Klös & Lang 1982, Wallach & Boever 1983, Appel et al., Fraser 1991,
Aiello 1999), or at least KV for the initial vaccinations which can then be boostered with MLV
(Frankenhuis & Visee 1985, Fowler 1986, Gabrisch & Zwart 1987). Phillips (1989)
recommends the use of KV without components for the feline respiratory viruses in
mustelidea, procyonidae and viverridae. MLV vaccine should never be used in in pregnant
felidae, foetal infection results in cerebellar hypoplasia with clinical ataxia in the kitten
(Fowler 1986).
Many different regimes have been tried, in which the timing of the initial vaccination is one of
the main variables. Interference with primary immunisation by maternal antibodies is the
commonest cause of “vaccine failure” in both domestic (Greene 1990) and non-domestic
carnivores (Janssen et. al. 1982; Wack et al. 1993). Vissee (pers. com. 1975) reorted that
after a change in the vaccination regime (6, 12, 26 weeks to 12, 16, 20 weeks) no clinical
signs were reported following MLV vaccination, suggesting that maternal antibodies interfere
with vaccination during the first 3 months. No controlled studies on the half-life of maternal
antibodies to FPV in non-domestic species have been performed, and it should not be
directly extrapolated from the 9.5 days determined in domestic cats (Scott 1970). Therefore
the vaccination recommendations for non-domestic species are the same as those used in
domestic species during maternal immunity.
Combination vaccines containing MLV CDV, CAV-2, CPI-3, and FPV/CPV have been used in
wild canidae without adverse effects, but with variable results, especially among non-canine
species. Burroughs (1992) recommends its use in canidae, viverridae and hyaenidae. A
multivalent KV vaccine (Fel-O-Vax PCT, Fort Dodge Lab Inc.) provides good antibody titres
to the three major infectious viral diseases of domestic cats (panleukopenia, rhinotracheitis
and calicivirus). The European Endangered Species Program (EEP) recommends 1 ml of
Fel-O-Vax be used for boosters in adults; juveniles should be vaccinated at 8, 12, and 16
weeks, repeated at 6 months, and then given annual boosters. This is the same schedule
used in domestic cats. Another regime recommended is repeated vaccinations with 2 week
intervals for 3 injections or until 16 weeks of age (Woodford 2001). When cheetah cubs are
vaccinated every 4 weeks from 8-16 weeks they may not develop and maintain protective
antibody titres during their first year of life. When these cubs are vaccinated every 2 weeks
from 8-16 weeks will develop protective titres. A booster may need to be given at the age of
40 weeks to insure that titres are maintained during the first year of life. There is much
individual variation – boosters every 3 months may be warranted in high risk situations
(Wack et al. 1993) - but a single annual vaccination may be adequate to maintain protective
antibody titres (Wack 1991).
FPV vaccination recommendation in domestic cats:
 8, 12, 16 weeks, booster at 6 months, then annually (Fel-O-Vax, Fort Dodge)
Table 5:
FPV vaccination Regimes recommended for use in non-domestic species
All MLV 6, 12, 26 weeks Visee pers
susceptible comm 1974
All MLV or KV 12, 16, 20 weeks Visee pers
susceptible comm 1975
All KV or MLV 4, 8, 12, 16 weeks annually when using Fowler 1977
susceptible KV
All 4, 8, 16 weeks (bi-)annually Klös & Lang
susceptible 1982
All 6-8 weeks, repeat every 2-3 weeks annually Wallach &
susceptible until 14 weeks of age Boever 1983
All 1-2 doses KV, then after 3 weeks annually in affected Frankenhuis &
susceptible MLV. MLV at 8, 11 weeks. areas, otherwise every Visee 1985
3 years
All KV 2, 4, 8, 12, 16 weeks (colostrum annually (KV or MLV) Fowler 1986
susceptible deprived). 8, 12, 16 weeks (naturally
weaned)
All 3 x KV with 2 weeks interval, then 2 x (bi-)annually Gabrisch &
susceptible MLV with 4 weeks interval (colostrum Zwart 1987
deprived), 3 x MLV with 3-4 weeks
interval starting at 7-8 weeks.
Skunks Initial at 8-10 weeks, repeat after 3 Gabrisch &
weeks Zwart 1987
All pregnant females should be boosted annually Sacramento zoo

susceptible during gestation 1990
Felidae KV, Initial KV at 3 months, MLV annually
(unavailabl combination vaccine at 4 months
e)
All Initial at weaning and repeated at annually Junge 1995
susceptible least twice with 2 week intervals
All 8 and 12 weeks annually Meltzer et al.
susceptible 1996
All 2 x with 2 weeks interval 6-12 months Fraser 1991,
susceptible Aiello 1998
All KV 8, 10, 12, 14, 16 weeks, repeat at 6 annually Miller &
susceptible months Anderson 2000
All KV 8, 11, 14 weeks annually Blijdorp
susceptible
Table 6:
CPV vaccination Regimes recommended for use in non-domestic species
All Initial at 8-12 weeks, 2nd vaccination 3 annually, every 6 Wallach &
susceptible weeks later months in endemic Boever 1985
areas
All As in domestic dogs Frankenhuis &
susceptible Visee, 1985
All MLV Initial at 3 months annually Burroughs 1992
susceptible
All Initial at 8 weeks, repeat 3-4 times with Cubas et al.
susceptible 2-3 week intervals 1996
All 8, 11, 14 weeks annually Blijdorp
susceptible
Infectious canine hepatitis / Canine Adenovirus-1 (CAV-1)
Canidae, Ursidae and Mustelidae are susceptible to canine adenoviruses (Mann et al. 1980,
Whetstone et al. 1988). Annual vaccination of Ursidae is recommended by some authors
(Sacramento zoo 1990, Fraser 1991, Aiello 1998), depending on risk of exposure (Fowler
1986, Appel 1987) or not at all (Wallach & Boever 1983, Frankenhuis & Visee 1985, Junge et
al. 1995, Miller & Anderson 2000). Burroughs (1992) recommends vaccination of Canidae
and Hyaenidae. KV has been recommended for use in genera other than Canidae, as MLV
may prove virulent (Klös & Lang 1982, Appel 1987). Currently there is no KV commercially
available. The diluent portion of Adenomune-7 however, contains a killed CAV-2 antigen that
may be used in highly susceptible species like maned wolves (Woodford 2001). Commercial
combination vaccines that include CDV and MLV CAV-1 or CAV-2 are generally used. CAV-
1 is the causative agent of of infectious canine hepatitis, CAV-2 is the causative agent of
respiratory disease. The two viruses are closely antigenically related, and vaccination with
CAV-2 provides cross-protection against CAV-1 without causing adverse postvaccinial
reactions like corneal opacity, common in domestic dogs after vaccination with MLV CAV-1,
and reported in maned wolves by Thomas-Baker et al. (1985). Although clinically dramatic,
the oedema usually resolves after a few days without consequence. Another reason was the
diminition of postvaccinial encephalitis which was noted after subtitution of CAV-2 for CAV-1
in combination vaccines (Carmichael 1999).
Table 7:
CAV vaccination Regimes recommended for use in non-domestic species
All Initial at 3-6 weeks bi-annually Klös & Lang
susceptible 1982
All MLV or KV Initial at 11-12 weeks, repeat annually Wallach &
susceptible after 2-3 weeks Boever 1983
All As in domestic dogs Frankenhuis &
susceptible Visee 1985
All MLV (caution) As in domestic dogs annually or prior to Appel 1987

susceptible KV possible exposure
All CAV-2 MLV Initial at weaning, monthly annually Fraser 1991,
susceptible booster up to 4 months of age Aiello 1998
Alls MLV Initial at 3 months annually Burroughs 1992
usceptible combination
Herpesvirus infections
All smaller exotic Felidae are susceptible to feline herpesvirus (FHV), larger Felidae have no
or mild symptoms. Vaccinations currently available are KV or MLV, but KV is recommended.
Vaccines commercially available (eg Fel-O-Vax) are usually in combination with other agents
(eg FPV, FCaV, Chlamydia). Humoral titres are usually short-lived and boosters every 3
months may be required in high-risk situations (Wack et al. 1993). Klös and Lang (1982)
recommended vaccination of Mustelidae and Viverridae, all other authors regard Felidae as
the only susceptible animals.
Fatal phocine herpesvirus type 1 (PhHV-1) infections have been reported in young and
immunocompromised harbour seals in rehabilitation centres (Osterhaus et al. 1985, Borst et
al. 1986, Gulland et al. 1997, Harder et al. 1997). An experimental sub-unit vaccine using the
gB protein of PhHV-1 has been produced that proved to be safe and provided protective
immunogenicity after challenge infection in domestic cats. Humoral and cellular
immunogenicity was produced in harbour seals (Martina et al. 2001; 2003).
Table 8:
FHV vaccination Regimes recommended for use in non-domestic species
All Initial at 9 weeks, repeat after 3 after 6 months Klös & Lang 1982
susceptible weeks. Adult cat twice with 2-3
weeks interval
Felidae MLV Initial at 8 weeks, repeat every 3-4 annually, in high Wallach & Boever
weeks until 14 weeks of age incident areas every 6 1983
months
Felidae As in domestic cats Frankenhuis &
Visee 1985
Felidae MLV Naturally weaned: Initial at 7-8 annually, endangered Gabrisch & Zwart
weeks, repeat 3 times with 3-4 cats and cheetahs 1987
weeks interval. Not naturally every 6-9 months.
weaned: Initial at 7-8 weeks, repeat With
2 times with 3-4 weeks interval. KV booster, larger
cats need 2-5 x dose
Felidae MLV Pregnant female should be boosted annually Sacramento Zoo
1990
Felidae KV or MLV Single dose at weaning. Monthly annually Fraser 1991, Aiello
combinatio intervals until 4 months of age 1998
n
All combinatio Repeat at 2 week intervals for 3 annually Woodford 2001
susceptible n KV injections or 16 weeks of age
All KV 8, 11, 14 weeks annually Blijdorp
susceptible
Feline Leukemia virus (FeLV)
Has been reported in exotic felids, and efficacy of a subunit vaccine has been demonstrated
(Briggs et al. 1986, Citino ety al. 1988, Pettan et al. 1992). It is recommended to serologically
test all Felidae for exposure. In 1986 vaccination with the then recently developed Leucocell
vaccine was recommended by Gabrisch & Zwart. Due to the low prevalence in exotic cats,
and the interference with serologic antibody screening, vaccination is not generally done
(Citino et al. 1988, Phillips 1989, Junge et al. 1995, Kennedy-Stosskopf 1996), but may be
done when there is close contact with feral cats (Miller & Anderson 2000).
Leptospirosis
Canidae, Procyonidae, Ursidae, Mustelidae, and Pinnipedia are susceptible (Shotts 1981). A
recent study in Rio de Janeiro Zoo, Brazil revealed an antibody prevalence of 37.7% of all
animals tested – carnivores and non-carnivores - belonging to 10 families, out of which
seroprevalence was most common in Canidae and Procyonidae (Lilenbaum et al. 2002).
Raccoons, opossums, and rodents can act as reservoirs and concurrently transmit infection
to zoo animals. Vaccination is highly serovar specific: the carnivores should be vaccinated
with bacterins that contain immunogens against Leptospira interrogans serovar canicola and
icterohaemorrhagiae. Vaccinations may influence the immune response in young animals -
vaccination of pups younger than 9-10 weeks of age is not recommended (Appel et al. 1999).
Vaccination does not necessarily prevent shedding of the organism (Aiello 1998).
Table 9:
Leptospirosis vaccination Regimes recommended for use in non-domestic species
Canidae Initial at 12 weeks, repeat after 2-3 weeks annually Wallach & Boever 1983
Procyonidae Initial at 12-14 weeks annually Wallach & Boever 1985
/ Mustelidae
All As in domestic dogs Frankenhuis & Visee
susceptible 1985
Ursidae Initial at 8 weeks, repeat at 12 and 16 weeks of annually Sacramento zoo 1990
age
All annually Junge et al. 1995
susceptible
All 1-2 ml dose im or sc at 6-8 weeks, repeat after bi-annual Fraser 1991, Aiello
susceptible 2 weeks 1998
Canidae/ bi-annual Woodford 2001
Procyonidae
Rabies
All mammal species are susceptible. Previously rabies vaccination was recommended in all
circumstances (Klös & Lang 1982, Wallach & Boever 1983, Fowler 1986, Appel 1987). More
recently recommendations are to vaccinate depending on location, risk of exposure, or
possible outbreak (Fraser 1991, Junge et al. 1995, Aiello 1998). In areas where the
incidence of rabies in local wildlife (skunks, raccoons, foxes) is high, vaccination is
recommended. Local veterinary authorities should be conctacted regarding the legal aspects
of extra-label vaccination, as some areas may have restrictions. Klös & Lang (1982)
recommended use of MLV, except for younger animals. All other authors agree that non-
domestic animals should be vaccinated with KV only. MLV vaccines have not been available
commercially after rare occasions where the vaccine caused rabies-like disease in dogs. The
efficacy of KV vaccines were questioned by Fowler (1986), but a KV vaccine (Imrab, Pitman
Moore) has proven to be efficacious and safe, and been approved for use in domestic ferrets
(Rupprecht et al. 1990). A great reduction in wildlife rabies has been accomplished by oral
immunization (see below).
Table 10:
RV vaccination Regimes recommended for use in non-domestic species
All KV Initial at 4-6 months annually Wallach & Boever
susceptible 1983; Junge et al.
1995
Felidae KV Initial at 6 months annually Wallach & Boever
1983
All KV Initial at 6 months annually Sacramento zoo 1990
susceptible
All KV Initial at 3-4 months annually Fraser 1991, Aiello
susceptible 1998
All Initial at 3-6 months tri-annually, during Burroughs 1992

susceptible outbreak more
often
Toxoplasmosis
Many species of zoo mammals (but also birds) are highly susceptible to Toxoplasma Gondii
infections, especially New World monkeys and marsupials, although members of the Felidae
family are the definitive host, and the only animals that pass the oocyst in the faeces. This
protozoan parasite has been reported in several species of non-domestic felidae, of which
the Pallas cat appears to be highly susceptible (Dubey et al. 1988, Dorny et al. 1989, Ocholi
et al. 1989, Swanson et al. 1999, Silva et al. 2001). There are a number of different vaccines,
of which New Zealand S48 vaccine containing live tachyzoites, proved to be capable of
inducing acute, fatal toxoplasmosis and is therefore considered to be unsuitable for use in
macropods (Lynch et al. 1993). An alternate study suggested that oral vaccination with
Hammondia Hammondi oocysts – a closely related non-pathogenic protozoan - may offer
partial protection to the clinical effects, but does not prevent infection by T. gondii (Reddacliff
et al. 1993). Recently an experimental recombinant FHV vaccine expressing the ROP2
antigen of T.gondii has been developed and proven efficacious in domestic cats (Mishima et
al. 2002), but this is not yet commercially available.
Vaccination of free-ranging wildlife
Should free-ranging wildlife be vaccinated? It is an interference of the natural selection, and

therefore a topic under discussion. Re-introduced or translocated animals will not have been
challenged under natural conditions with the local pathogens when young (and maternal
immunity is still present), and therefore need to be vaccinated to obtain a level of immunity
against these pathogens. Recently IUCN guidelines for the reintroduction of captive animals
into the wild have been published (Woodford 2001). Pre-release vaccination of animals to be
rehabilitated or translocated should be considered by the veterinarian after evaluating the
immunological status, and the risk of infection upon release into the destined area.
When vaccinating wildlife it is of utmost importance to consider the fact that MLV vaccines
may not be sufficiently attenuated for exotic species, and that vaccine induced disease or
shedding of virulent virus may occur, thereby infecting free-living populations. Another
problem faced is the difficulty to booster under field conditions, so that the level of immunity
may not be sufficient. It is therefore recommended to complete the vaccination regime before
release when possible. When this vaccination is carried out during the “preparation stage”,
sufficient time is allowed to develop the required immunity and detect possible adverse
effects.
Following the phocine distemper (PDV) epidemic of 1988 (Osterhaus et al. 1988, Osterhaus
& Vedder 1988, Kennedy et al. 1988) and the development of an experimental ISCOM
(Osterhaus et al. 1989, Visser et al. 1992), all rehabilitated seals from the rehabilitation and
research centre Pieterburen have been vaccinated before release. The duration of protective
immunogenicity following this vaccination is unknown, and is intended to last for the duration
of stay in the rehabilitation centre. The discussion of whether to start vaccinating the wild
population flared up during the recent PDV epidemic of 2002, but was not considered a
viable option (Trilateral seal expert meeting 2002, DEFRA 2002). Vaccination of seals with a
MLV vaccine is contraindicated (Kennedy et al. 2001).
Vaccination of endangered species to infectious diseases may aid in the survival of these
species. African wild dog (Visee et al. 2001) and black-footed ferret (Williams & Thorne
1999) conservation projects have been severely affected by CDV outbreaks, and much work
is done on the production of a safe and efficacious vaccine (Montali et al. 1998, Visee 2001,
van de Bildt et al. 2002).
Vaccination of a wild population of Mediterranean monk seals (Monachus monachus) was

considered during a morbillivirus epizootic in the mediterranean in 1992 (Osterhaus et al.
1992). During the PDV outbreak among Northern European Harbour seals in the summer of
2002, vaccination was also considered in the UK, but not deemed feasible (DEFRA 2002).
The zoonotic and economic aspects of rabies infection have resulted in prophylactic
immunization of domestic dogs and the eradication of canine rabies in several countries
(Bögel 1982). Following this achievement attention was focused on free-ranging vector
species, which were much more difficult to vaccinate. The development of a MLV vaccine
which vaccinated foxes by the oral route (Baer 1971) was a major step in the right direction,
which proved its value when an advancing epidemic was stopped by the vaccination zone
(Steck et al. 1982). This vaccine has since been replaced by a vaccinia recombinant vaccine
(Pastoret 1997), which has proven to be efficacious, and safe for the target species, the fox,
as well as for numerous non-target species (Black et al 1993). To be effective the vaccine
must be brought into contact with oral and /or pharyngeal mucosa in a sufficient number of
target animals via bait. Factors affecting uptake: size, texture, shape of bait and vaccine
container, as well as physical and chemical characteristics. One should take into account bait
density, distribution method (manual, aerial or both), sequence and frequency of bait
distribution, season, selection of specific baiting areas, strategies for expansion of baiting
areas and the overall duration of vaccination campaigns (Rupprecht et al. 2001). Research is
being conducted on the development of ideal baits and baiting systems for different species
(Steelman et al 1998, Knobel et al 2002, Linhart et al 2002).
Oral vaccination may be used in the eradication of other diseases of wildlife in the future.
Wimsatt (1999) reported potential oral efficacy of an experimental canarypox vectored
recombinant CDV vaccine in a preliminary study, and this oral efficacy was demonstrated in
Siberian polecats (Wimsatt 2003) but more research is needed to evaluate its efficacy and
safety in other species.
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Spencer, J.A., Burroughs, R. 1992. Antibody response to canine distemper vaccination in
African wild dogs. Journal of Wildlife Diseases 28(3):443-4.
Spencer, J.A. 1991. Antibody response to modified-live canine adenovirus vaccine in African
hunting dogs (Lycao pictus). Zentralblatt fur Veterinarmedizin 38(6):477-9.
Spencer, J.A., Burroughs, R. 1992. Decline in maternal immunity and antibody response to
vaccine in captive cheetah (Acinonyx jubatus) cubs. Journal of Wildlife Diseases 28(1):102-
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Steck, F., Wandeler, A., Bichsel, P., Capt, S., Schneider, L. 1982. Oral immunization of foxes
against rabies. A field study. Zentralblatt Veterinaermedizin 29(5):372-396.
Steelman HG, Henke SE, Moore GM. 1998. Gray fox response to baits and attractants for
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Steinel, A., Munson, L., van Vuuren, M., Truyen, U. 2000. Genetic characterization of feline
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Sutter, G. Moss, B. 1992. Nonreplicating vaccinia vector efficiently expresses recombinant
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Tham, K.M., Studdert, M.J., 1987. Antibody and cell-mediated response to feline
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Thomas-Baker, B. 1985. Vaccination induced distemper in maned wolves, vaccination
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XIII. REFERENCE LABORATORIES FOR ANIMAL DISEASES

This chapter includes listings of OIE and European Union reference laboratories for animal
diseases. Updated versions of these lists can be found at:
http://www.oie.int/eng/oie/organisation/en_listeLR.htm
and
http://ec.europa.eu/food/animal/diseases/laboratories/index_en.htm#list
The World Health Organization (WHO) also has Collaborating Centers for many diseases relevant
to zoo and wild animals. An up to date database of these centers can be searched at:
http://www.who.int/whocc/Default.aspx
Community Reference Laboratories
African horse sickness

Laboratorio de sanidad y producción animal
Ministerio de Agricultura, Pesca y Alimentación
28110 Algete, Madrid
Spain
African swine fever

Centro de Investigación en Sanidad Animal
Ctra. De Algete a El Casar, Valdeolmos
28130, Madrid
Spain
Avian Influenza
Veterinary Laboratories Agency
New Haw, Weybridge
Surrey KT 15 3NB
United Kingdom
Bivalve molluscs diseases

The Ifremer Laboratory
B.P. 133
17390 La tremblade
France
Bluetongue
Institute for Animal Health
Pirbright Laboratory
Pirbright, Woking
Surrey GU24 ONF
United Kingdom
Bovine tuberculosis
VISAVET
Laboratorio de vigilancia veterinaria, Facultad de Veterinaria, Universidad Complutense de Madrid
XIII. Reference Laboratories for Animal Diseases
Avda. Puerta de Hierro, s/n. Ciudad Universitaria

28040. Madrid
Spain
Brucellosis
AFSSA, Nancy
Laboratoire d’études sur la rage et la pathologie des animaux sauvages
Domaine de Pixérécourt, BP 9
F-54220 Malzéville
France
Classical swine fever

Institut für Virologie der Tierarztlichen Hochschule Hanover
Bischofscholer Damm 15
D-3000 Hannover 1
Germany
Crustacean diseases
Centre for Environment, Fisheries & Aquaculture Science (Cefas)
Weymouth Laboratory
The Nothe, Barrack Road, Weymouth
Dorset DT4 8UB
United Kingdom
Equine diseases other than African Horse Sickness

Agence Française de Sécurité Sanitaire des aliments (AFSSA)
Laboratoire d'études et de recherches en pathologie animale et zoonoses
23, avenue du Général de Gaulle
F-94706 MAISONS-ALFORT Cedex
France
Fish diseases
State Serum Laboratory
Hangovej 2
8200-Aarhus
Denmark
Foot and mouth disease

Institut für Virologieder Tierarztlichen Hochschule Hanover
Bischofscholer Damm 15
D-3000 Hannover 1
Germany
Newcastle disease
Veterinary Laboratories Agency
New Haw, Weybridge
Surrey KT 15 3NB
United Kingdom
Rabies
AFSSA, Nancy
Laboratoire d’études sur la rage et la pathologie des animaux sauvages
Domaine de Pixérécourt, BP 9
F-54220 Malzéville
France
Swine vesicular disease

Pirbright Laboratory
Pirbright, Woking
Surrey GU24 ONF
United Kingdom
Zootechnics (bovine breeding)

INTERBULL Centre
Department of Animal Breeding and Genetics Swedish University of Agricultural Sciences
Box: 7023;
S-750 07 Uppsala
Sweden
Organisation Mondiale de la Santé Animale

World Organisation for Animal Health
Organización Mundial de Sanidad Animal
OIE Reference Experts and Laboratories in Europe
African horse sickness
 Dr G.H. Gerdes
Onderstepoort Veterinary Institute
Private Bag X05, Onderstepoort 0110
SOUTH AFRICA
Tel: (27.12) 529.91.14 Fax: (27.12) 529.94.18
Email: oneillm@arc.agric.za
 Dr Concepción Gómez-Tejedor Ortiz (1)

Laboratorio Central de Veterinaria de Algete
Carretera de Algete, km 8, 28110 Algete, (Madrid)
SPAIN
Tel: (34.91) 347.92.82/92.77 Fax: (34.91) 347.82.96
Email: cgomezte@mapa.es
 Prof. P.S. Mellor

Institute for Animal Health, Pirbright Laboratory
Ash Road, Pirbright, Woking, Surrey GU24 ONF
UNITED KINGDOM
Tel: (44.1483) 23.24.41 Fax: (44.1483) 23.24.48
Email: philip.mellor@bbsrc.ac.uk
 Dr J.M. Sánchez-Vizcaíno (2)

Facultad de Veterinaria, Laboratorio de Vigilancia Sanitaria (VISAVET), HCV Planta sótano,
Universidad Complutense
Avda. Puerta de Hierro s/n, 28040 Madrid
SPAIN
Tel: (34.91) 394.40.82 Fax: (34.91) 394.39.08
African swine fever
 Dr J.M. Sánchez-Vizcaíno (2)

Facultad de Veterinaria, Laboratorio de Vigilancia Sanitaria (VISAVET), HCV Planta sótano,
Universidad Complutense
Avda. Puerta de Hierro s/n, 28040 Madrid
SPAIN
Tel: (34.91) 394.40.82 Fax: (34.91) 394.39.08
 Dr Chris Oura
UNITED KINGDOM
Tel: (44.1483) 23.24.41 Fax: (44.1483) 23.24.48
Email: chris.oura@bbsrc.ac.uk
 Dr Baratang Alison Lubisi

Diagnostic Virology, Molecular Epidemiology and Diagnostics Programme, Onderstepoort
Veterinary Institute, Agricultural Research Council
SOUTH AFRICA
Tel: (27.12) 529.95.85 Fax: (27.12) 529.95.95
Email: Lubisia@arc.agric.za
American foulbrood of honey bees

 Dr Adriana M. Alippi
Laboratorio de Loque Americana de la Unidad de Bacteriología del Centro de Investigaciones en
Fitopatología (CIDEFI)
calle 60 y 119 s/n c.c. 31, 1900 La Plata
ARGENTINA
Tel: (+54-221) 423.67.48 ext. 423 Fax: (+54-221) 425.23.46
Email: adrianaalippi@gmail.com
Email: alippi@biol.unlp.edu.ar
Anthrax
 Dr G. Harvey
National Veterinary Services Laboratories
P.O. Box 844, Ames, IA 50010
UNITED STATES OF AMERICA
Tel: (1.515) 663.75.65 Fax: (1.515) 663.75.69
Email: ginger.r.harvey@aphis.usda.gov
 Dr B. Golsteyn-Thomas
Canadian Food Inspection Agency, Lethbridge Laboratory
P.O. Box 640, Township Road 9-1, Lethbridge, Alberta T1J 3Z4
CANADA
Tel: (1.403) 382.55.51 Fax: (1.403) 381.12.02
Email: betty.golsteyn-Thomas@inspection.gc.ca
Antimicrobial resistance
 Dr Chris Teale
VLA Weybridge
UNITED KINGDOM
Tel: (44-1743) 46.76.21 Fax: (44-1743) 44.10.60
Email: c.teale@vla.defra.gsi.gov.uk
Aujeszky's disease
 Dr S.L. Swenson
P.O. Box 844, Ames, IA 50010
Tel: (1.515) 663.75.51 Fax: (1.515) 663.73.48

Email: sabrina.l.swenson@aphis.usda.gov
 Dr P. Vannier
AFSSA Ploufragan, Laboratoire d'études et de recherches avicoles et porcines, UR Station de
pathologie porcine
Zoopôle Beaucemaine-Les Croix, BP 53, 22440 Ploufragan
FRANCE
Tel: (33 (0)2) 96.01.62.22 Fax: (33 (0)2) 96.01.62.23
Email: p.vannier@afssa.fr
 Dr A.T.J. Bianchi
Central Veterinary Institute of Wageningen UR
P.O. Box 2004, 8203 AA Lelystad
THE NETHERLANDS
Tel: (31.320) 23.88.00 Fax: (31.320) 23.86.68
Email: andre.bianchi@wur.nl
Avian chlamydiosis
 Dr Konrad Sachse
Friederich-Loeffer Institute, Institute of Molecular Pathogenesis
Naumburger Str. 96a, 07743 Jena
GERMANY
Tel: (49.3641) 80.43.34 Fax: (49.3641) 80.42.28
Email: konrad.sachse@fli.bund.de
Avian mycoplasmosis (Mycoplasma gallisepticum, M. synoviae)

 Dr N. Ferguson-Noel
The University of Georgia, Poultry Diagnostic and Research Centrer
953 College Station Rd, Athens, Georgia 30602-4875
Tel: (1.706) 542.30.86 Fax: (1.706) 542.56.30
Email: naolaf@uga.edu
Avian tuberculosis
 Dr I. Pavlik
CZECH (Rep.)
Tel: (420.5) 33.33.16.01 Fax: (420.5) 33.33.12.29
Bacterial kidney disease (Renibacterium salmoninarum)

 Dr James R. Winton
Western Fisheries Research Center
6505 N.E. 65th Street, Seattle, Washington 98115
Tel: (1.206) 526.65.87 Fax: (1.206) 526.66.54
Email: jim_winton@usgs.gov
Bee diseases
 Monsieur Jean-Paul Faucon
AFSSA Sophia Antipolis, Unité Pathologie de l'abeille, Laboratoire de Pathologie des Petits
Ruminants et des Abeilles
105 route des Chappes, BP 111, 06902 Sophia Antipolis
FRANCE
Tel: (33 (0)4) 92.94.37.00 Fax: (33 (0)4) 92.94.37.01
Email: jp.faucon@sophia.afssa.fr
 Dr W. Ritter
Chemisches und Veterinäruntersuchungsamt Freiburg
P.O.B. 100462, 79123 Freiburg
GERMANY
Tel: (49.761) 150.21.75 Fax: (49.761) 150.22.99
Email: wolfgang.ritter@cvuafr.bwl.de
Bluetongue
 Dr G.H. Gerdes
SOUTH AFRICA
Tel: (27.12) 529.91.14 Fax: (27.12) 529.94.18
 Dr E.N. Ostlund
Diagnostic Virology Laboratory, National Veterinary Services Laboratories
P.O. Box 844, Ames, IA 50010
Tel: (1.515) 663.75.51 Fax: (1.515) 663.73.48
Email: eileen.n.ostlund@aphis.usda.gov
 Dr Giovanni Savini
Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise 'G. Caporale'
Via Campo Boario, 64100 Teramo
ITALY
Tel: (39.0861) 33.24.40 Fax: (39.0861) 33.22.51
Email: g.savini@izs.it
 Dr Peter Daniels
CSIRO, Australian Animal Health Laboratory (AAHL)
5 Portarlington Road, Private Bag 24, Geelong 3220, Victoria
AUSTRALIA
Tel: (61.3) 52.27.50.00 Fax: (61.3) 52.27.55.55
Email: peter.daniels@csiro.au
 Prof. P.S. Mellor

UNITED KINGDOM
Tel: (44.1483) 23.24.41 Fax: (44.1483) 23.24.48
Email: philip.mellor@bbsrc.ac.uk
Bovine babesiosis
 Prof. Ikuo Igarashi
National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary
Medicine
Inada-cho Nishi 2-13, Obihiro, Hokkaido 080-8555
JAPAN
Tel: (81.155) 49.56.42 Fax: (81.155) 49.56.43
Email: igarcpmi@obihiro.ac.jp
Bovine genital campylobacteriosis

 Dr Jaap Wagenaar
Animal Sciences Group (ASG), Division of Infectious Diseases
P.O. Box 65, 8200 AB Lelystad
THE NETHERLANDS
Tel: (31.320) 23.81.57 Fax: (31.320) 23.89.61
Email: j.wagenaar@uu.nl
Faculty of Veterinary Medicine (FVM), Department of Infectious Diseases and Immunology
P.O. Box 80.165, 3508 TD Utrecht
THE NETHERLANDS
Tel: (31.30) 253.12.42 Fax: (31.30) 253.31.99
Bovine spongiform encephalopathy
 Dr Marion Simmons
VLA Weybridge
UNITED KINGDOM
Tel: (44.1932) 35.75.64 Fax: (44.1932) 35.78.05
Email: TSEeucrl@vla.defra.gsi.gov.uk
Web: http://www.defra.gov.uk/corporate/vla/science/science-tse-rl-web.htm
 Prof. Andreas Zurbriggen

Institute of Animal Neurology, University of Bern
Bremgartenstrasse 109A, 3012 Bern
SWITZERLAND
Tel: (41.31) 631.25.09 Fax: (41.31) 631.25.38
Email: andreas.zurbriggen@itn.unibe.ch
 Dr Stefanie Czub
Canadian Food Inspection Agency, Lethbridge Laboratory
Township Road 9-1, Post Office Box 640, Lethbridge, Alberta
CANADA
Tel: (1.403) 382.55.49 Fax: (1.403) 382.55.83
Email: czubs@inspection.gc.ca
 Dr Takashi Yokoyama
Prion Diseases Research Unit, National Institute of Animal Health, National Agricultural Research
Organization
3-1-5 Kannondai, Tsukuba, Ibaraki 305-0856
JAPAN
Tel: (81.298) 38.77.40 Fax: (81.298) 38.83.32
Email: tyoko@affrc.go.jp
 Dr Francisco Javier Blanco Viera

Laboratorio Nacional de Referencia (LNR) para Encefalopatías Espongiformes Transmisible
animales, Institutos de Patobiología y Virología, Centro de Investigaciones en Ciencias
Veterinarias y Agronómicas (CICV), Instituto Nacional de Tecnología Agropecuaria (INTA)
Castelar, Casilla de Correo 77, 1708 Morón, Pcia. de Buenos Aires
ARGENTINA
Tel: (54.11) 46.21.12.89 Fax: (54.11) 46.21.17.43
Email: jviera@cnia.inta.gov.ar
Email: gpinto@cnia.inta.gov.ar
Bovine tuberculosis
 Dr Bernardo Alonso
Gerencia de Laboratorios (GELAB) del Servicio Nacional de Sanidad y Calidad, Agroalimentaria
(SENASA)
Av. Alexander Fleming 1653, 1640 Martinez - Pcia de Buenos Aires
ARGENTINA
Tel: (54.11) 48.36.00.36 Fax: (54.11) 48.36.00.36
Email: balonso@senasa.gov.ar
Email: dilab@inea.com.ar
 Mme María Laura Boschiroli-Cara

AFSSA Alfort, Unité Zoonoses Bactériennes, Laboratoire d'études et de recherches en pathologie
animale et zoonoses
FRANCE
Tel: (33 (0)1) 49.77.13.00 Fax: (33 (0)1) 49.77.13.44
 Dr Debby V. Cousins
Australian Reference Laboratory for Bovine Tuberculosis, Agriculture Western Australia
Locked Bag N° 4, Bentley Delivery Centre, Bentley WA 6983
AUSTRALIA
Tel: (61.8) 93.68.34.51 Fax: (61.8) 94.74.18.81
Email: dcousins@agric.wa.gov.au
 Prof. Glyn Hewinson

VLA Weybridge
UNITED KINGDOM
Tel: (44.1932) 34.11.11 Fax: (44.1932) 34.70.46
Email: r.g.hewinson@vla.defra.gsi.gov.uk
Bovine viral diarrhoea

 Dr D. Deregt (1)
Canadian Food Inspection Agency, Animal Diseases Research Institute
P.O. Box 640, Lethbridge, Alberta T1J 3Z4
CANADA
Tel: (1.403) 382.55.00 Fax: (1.403) 381.12.02
Email: deregtd@inspection.gc.ca
 Dr T.W. Drew
Head of Virology Department, VLA Weybridge
Woodham Lane, New Haw, Addlestone, Surrey KT15 3NB
UNITED KINGDOM
Tel: (44.1932) 35.76.37 Fax: (44.1932) 35.72.39

Email: t.w.drew@vla.defra.gsi.gov.uk
 Dr Peter Kirkland
Elizabeth Macarthur Agriculture Institute (EMAI), Virology Laboratory
PMB 8, Camden NSW 2570
AUSTRALIA
Tel: (61-2) 46.40.63.31 Fax: (61-2) 46.40.64.29
Email: peter.kirkland@dpi.nsw.gov.au
Brucellosis (Brucella melitensis)

 Dr Henrich Neubauer
Federal Research Centre for Virus Diseases of Animals (BFAV), Institute of Bacterial Infections and
Zoonoses
GERMANY
Tel: (49.3641) 80.42.00 Fax: (49.3641) 80.42.28
Email: heinrich.neubauer@fli.bund.de
 Dr Ana Maria Nicola

Gerencia de Laboratorios (GELAB), Servicio Nacional de Sanidad y Calidad Agroalimentaria
(SENASA)
Av. Alexander Fleming 1653, 1640 Martínez, Pcia. de Buenos Aires
ARGENTINA
Tel: (54.11) 48.36.19.92 Fax: (54.11) 48.36.19.92
Email: anicola@senasa.gov.ar
 Dr J.A. Stack
VLA Weybridge
UNITED KINGDOM
Tel: (44.1932) 35.76.10. Fax: (44.1932) 35.72.16
Email: j.a.stack@vla.defra.gsi.gov.uk
 Dr B. Garin-Bastuji
AFSSA Alfort, Unité Zoonoses Bactériennes, Lab. OIE/FAO de référence pour la brucellose
animale, Laboratoire d'études et de recherches en pathologie animale et zoonoses
FRANCE
Tel: (33 (0)1) 49.77.13.00 Fax: (33 (0)1) 49.77.13.44
Email: b.garin-bastuji@afssa.fr
 Dr K. Nielsen
P.O. Box 11300, Station H, Nepean, Ontario K2H 8P9
CANADA
Tel: (1.613) 228.66.98 ext. 48.04 Fax: (1.613) 228.66.69
Email: nielsenk@inspection.gc.ca
 Dr Massimo Scacchia
CESME, Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise 'G. Caporale', National
Centre for Exotic Diseases
ITALY
Tel: (390.861) 33.24.05 Fax: (390.861) 33.22.51
Email: m.scacchia@izs.it
 Dr Menachem Banai
Kimron Veterinary Institute
Department of Bacteriology, P.O. Box 12, Beit Dagan 50250
ISRAEL
Tel: (972.3) 968 16 98 Fax: (972.3) 968 17 53
Email: menachemba@moag.gov.il
Brucellosis (Brucella suis)

Zoonoses
GERMANY
Tel: (49.3641) 80.42.00 Fax: (49.3641) 80.42.28

(SENASA)
ARGENTINA
Tel: (54.11) 48.36.19.92 Fax: (54.11) 48.36.19.92
 Dr J.A. Stack
VLA Weybridge
UNITED KINGDOM
Tel: (44.1932) 35.76.10. Fax: (44.1932) 35.72.16
FRANCE
Tel: (33 (0)1) 49.77.13.00 Fax: (33 (0)1) 49.77.13.44
 Dr K. Nielsen
CANADA
Tel: (1.613) 228.66.98 ext. 48.04 Fax: (1.613) 228.66.69
ITALY
Tel: (390.861) 33.24.05 Fax: (390.861) 33.22.51
Brucellosis Brucella abortus

Zoonoses
GERMANY
Tel: (49.3641) 80.42.00 Fax: (49.3641) 80.42.28

(SENASA)
ARGENTINA
Tel: (54.11) 48.36.19.92 Fax: (54.11) 48.36.19.92
 Dr J.A. Stack
VLA Weybridge
UNITED KINGDOM
Tel: (44.1932) 35.76.10. Fax: (44.1932) 35.72.16
FRANCE
Tel: (33 (0)1) 49.77.13.00 Fax: (33 (0)1) 49.77.13.44
 Dr K. Nielsen
CANADA
Tel: (1.613) 228.66.98 ext. 48.04 Fax: (1.613) 228.66.69
ITALY
Tel: (390.861) 33.24.05 Fax: (390.861) 33.22.51
ISRAEL
Tel: (972.3) 968 16 98 Fax: (972.3) 968 17 53
 Dr Suk-chan Jung
Zoonosis Laboratory, Bacteriology and Parasitology Division, National Veterinary Research and
Quarantine Service (NVRQS), Ministry of Food, Agriculture, Forestry, and Fisheries (MIFAFF)
480 Anyang 6-dong, Manan-gu, Anyang-si, Kyunggi-do
CORÉE (RÉP. DE)
Tel: (82.31) 467.17.65 Fax: (82.31) 467.17.78
Email: jungsc@nvrqs.go.kr
Camelpox
 Professor Ulrich Wernery
Central Veterinary Research Laboratory
P.O. Box 597, Dubai
UNITED ARAB EMIRATES
Tel: (971.4) 337.51.65 Fax: (971.4) 336.86.38
Email: cvrl@cvrl.ae
Campylobacteriosis
Animal Sciences Group (ASG), Division of Infectious Diseases
P.O. Box 65, 8200 AB Lelystad
THE NETHERLANDS
Tel: (31.320) 23.81.57 Fax: (31.320) 23.89.61
Faculty of Veterinary Medicine (FVM), Department of Infectious Diseases and Immunology
P.O. Box 80.165, 3508 TD Utrecht
THE NETHERLANDS
Tel: (31.30) 253.12.42 Fax: (31.30) 253.31.99
Caprine arthritis/encephalitis
 Dr Stephen Valas
Laboratoire d'étude et de recherches caprines
60 rue du Pied de Fond, B.P. 3081, 79000 Niort
FRANCE
Tel: (33 (0)5 49.79.61.28 Fax: (33 (0)5 49.79.42.19
Email: s.valas@niort.afssa.fr
 Dr D.P. Knowles, Jr
Animal Diseases Research Unit,USDA, ARS, Washington State University
Pullman, Washington 99164-7030
Tel: (1.509) 335.60.22 Fax: (1.509) 335.83.28
Email: dknowles@vetmed.wsu.edu
Channel catfish virus disease

 Dr Larry A. Hanson
College of Veterinary Medicine, Fish Diagnostic Laboratory, Mississippi State University
P.O. Box 6100, Spring Street, Mississippi 39762
Tel: (1.662) 325.12.02 Fax: (1.662) 325.10.31
Email: hanson@cvm.msstate.edu
Chronic wasting disease

 Dr Aru Balachandran
Canadian Food Inspection Agency, Ottawa Laboratory
3851 Fallowfield Road, P.O. Box 11300, Station H, Nepean, Ontario K2H 8P9
CANADA
Tel: (1.613) 228.66.98 ext. 4854 Fax: (1.613) 228.61.03
Email: balachandrana@inspection.gc.ca
Classical swine fever
 Dr John Pasick
Canadian Food Inspection Agency, National Centre for Foreign Animal Disease
1015 Arlington Street, Winnipeg, Manitoba R3E 3M4
CANADA
Tel: (1.204) 789.20.13 Fax: (1.204) 789.20.38
Email: jpasick@inspection.gc.ca
 Dr Shunji Yamada
National Institute of Animal Health
6-20-1 Josui-honcho, Kodaira, Tokyo, 187-0022
JAPAN
Tel: (81.42) 321.14.41 Fax: (81.42) 325.51.22
Email: musasabi@affrc.go.jp
 Prof. V. Moennig
Institute of Virology, Hannover Veterinary School
Bünteweg 17, 30559 Hannover
GERMANY
Tel: (49.511) 953.88.40 Fax: (49.511) 953.88.98
Email: volker.moennig@tiho-hannover.de
 Prof. Dr Z. Pejsak
National Veterinary Research Institute
Partyzantow str. 57, 24-100 Pulawy
POLAND
Tel: (48.81) 889.30.30 Fax: (48.81) 886.25.95
Email: zpejsak@piwet.pulawy.pl
 Dr T.W. Drew
UNITED KINGDOM
Tel: (44.1932) 35.76.37 Fax: (44.1932) 35.72.39
Contagious agalactia
 Dr Robin A.J. Nicholas
Mycoplasma Group, Department of Statutory and Exotic Bacterial Diseases, VLA Weybridge
UNITED KINGDOM
Tel: (44.1932) 34 11 11 Fax: (44.1932) 34 70 76
Email: r.a.j.nicholas@vla.defra.gsi.gov.uk
Contagious bovine pleuropneumonia
 Dr F. Poumarat (1)
AFSSA Lyon, Laboratoire de pathologie bovine
31 avenue Tony Garnier, BP 7033, 69342 Lyon Cedex 07
FRANCE
Tel: (33 (0)4) 78.72.65.43 Fax: (33 (0)4) 78.61.91.45
Email: f.poumarat@lyon.afssa.fr
 Dr Ana Rosa Pombo Botelho

Laboratório Nacional de Investigaçâo Veterinária (LNIV)
Estrada de Benfica 701, 1500 Lisboa
PORTUGAL
Tel: (351.21) 711.53.33/39/40 Fax: (351.21) 711.52.36
Email: ana.botelho@lniv.min-agricultura.pt
 Dr A. Pini
CESME, Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise 'G. Caporale'
ITALY
Tel: (39.0861) 33.24.81 Fax: (39.0861) 33.22.51
Email: a.pini@izs.it
 Dr F. Thiaucourt (2)
UMR15 CIRAD-INRA, Control of exotic and emerging animal diseases
Campus international de Baillarguet TA A-15/G, 34398 Montpellier Cedex 5
FRANCE
Tel: (33(0)4) 67.59.37.24 Fax: (33(0)4) 67.59.37.98
Email: françois.thiaucourt@cirad.fr
Contagious caprine pleuropneumonia

 Dr F. Thiaucourt (2)
UMR15 CIRAD-INRA, Control of exotic and emerging animal diseases
Campus international de Baillarguet TA A-15/G, 34398 Montpellier Cedex 5
FRANCE
Tel: (33(0)4) 67.59.37.24 Fax: (33(0)4) 67.59.37.98
Email: françois.thiaucourt@cirad.fr
Contagious equine metritis

 Dr Brenda Morningstar-Shaw
P.O. Box 844, Ames, IA 50010
Tel: (1.515) 663.75.65 Fax: (1.515) 663.75.69
Email: brenda.r.morningstar@aphis.usda.gov
 Mr Paul Todd
VLA Bury St Edmunds
Rougham Hill, Bury St Edmunds, Suffolk IP33 2RY
UNITED KINGDOM
Tel: (44-1284) 72.44.99 Fax: (44-1284) 72.45.00
Email: p.todd@vla.defra.gsi.gov.uk
 Dr Hendrik-Jan Roest
Central Veterinary Institute of Wageningen UR, Bacteriology Department

P.O. Box 65, 8203 AA Lelystad
THE NETHERLANDS
Tel: (31.320) 23.80.26 Fax: (31.320) 23.81.53
Email: hendrikjan.roest@wur.nl
Control of Veterinary Medicinal Products in Sub-Saharan Africa

 Dr Assiongbon Teko-Agbo
Ecole Inter-Etats de Science et Médecine Vétérinaire (EISMV)
BP 5077, Dakar
SENEGAL
Tel: (221) 33.865.10.08 Fax: (221) 33.825.42.83
Email: tekoagbo2001@yahoo.fr
Crayfish plague (Aphanomyces astaci)

 Dr Birgit Oidtmann
The Centre for Environment, Fisheries, and Aquaculture Science (CEFAS), Weymouth Laboratory
Barrack Road, The Nothe, Weymouth, Dorset, DT4 8UB
UNITED KINGDOM
Tel: (44.1305) 20.66.61 Fax: (44.1305) 20.66.01
Email: birgit.oidtmann@cefas.co.uk
 Dr S. Viljamaa-Dirks
Finnish Food Safety Authority
Evira Kuopio, Neulaniementie 4, FIN-70210 Kuopio
FINLANDE
Tel: (358) 207.72.49.62 Fax: (358) 207.72.49.70
Email: satu.viljamaa-dirks@evira.fi
Crimean Congo haemorrhagic fever

 Dr Michèle Bouloy
Unité de génétique moléculaire des Bunyavirus, Département de Virologie, Institut Pasteur
25 rue du Dr Roux 75724 Paris cedex 15
FRANCE
Tel: (33.1) 40.61.31.34 Fax: (33.1) 40.61.32.56
Email: E-mail: mbouloy@pasteur.fr
Dourine
 Prof. V.T. Zablotsky
All-Russian Research Institute for Experimental Veterinary Medicine (VIEV), Veterinary
Department, Laboratory of Equine Viral Diseases
24-1, Ryazanskiy prosp., 109428 Moscow
RUSSIA
Tel: (7.495) 785.84.27 Fax: (7.495) 970.03.69
Email: efzabegina@mtu-net.ru
Echinococcosis/hydatidosis
 Prof. Masao Kamiya
Laboratory of Environmental Zoology, Department of Biosphere and Environmental Sciences,
Faculty of Environmental Systems, Rakuno-Gakuen University
Midori-machi 582, Ebetsu 069-8501
JAPAN
Tel: (81.11) 388.49.09 Fax: (81.11) 388.49.09

Email: mkamiya@rakuno.ac.jp
Web: http://www.k3.dion.ne.jp/~fea/
 Dr P.S. Craig
Cestode Zoonoses Research Group, Biosciences Research Institute, University of Salford
Manchester M5 4WT
UNITED KINGDOM
Tel: (44.161) 295.54.88 Fax: (44.161) 295.52.15
Email: p.s.craig@salford.ac.uk
 Prof. A. Dakkak
Institut Agronomique et Vétérinaire Hassan II, Département de Parasitologie
BP 6202, Rabat-Instituts
MOROCCO
Tel: (212.37) 77.64.32 Fax: (212.37) 77.64.32
Email: a.dakkak@iav.ac.ma
Enteric septicaemia of catfish (Edwardsiella ictaluri)

 Dr Larry A. Hanson
College of Veterinary Medicine, Fish Diagnostic Laboratory, Mississippi State University
P.O. Box 6100, Spring Street, Mississippi 39762
Tel: (1.662) 325.12.02 Fax: (1.662) 325.10.31
Email: hanson@cvm.msstate.edu
Enzootic abortion of ewes (ovine chlamydiosis)

 Dr Konrad Sachse
Friederich-Loeffer Institute, Institute of Molecular Pathogenesis
GERMANY
Tel: (49.3641) 80.43.34 Fax: (49.3641) 80.42.28
Email: konrad.sachse@fli.bund.de
 Dr Nicole Borel
Institute for Veterinary Pathology (IVPZ), Vetsuisse Faculty, University of Zurich
Winterhurerstrasse 268, CH-8057 Zurich
SWITZERLAND
Tel: (41.44) 635.85.71 Fax: (41.44) 635.89.34
Email: apos@vetpath.uzh.ch
Email: n.borel@access.uzh.ch
Enzootic bovine leukosis

 Mr C. Venables
VLA Weybridge
UNITED KINGDOM
Tel: (44.1932) 34.11.11 Fax: (44.1932) 34.70.46
Email: c.venables@vla.defra.gsi.gov.uk
 Dr Thomas W. Vahlenkamp
Friedrich-Loeffler-Institute, Institut für Molekularbiologie
Südufer 10, 17493 Greifswald-Insel Riems
GERMANY
Tel: (49-38351) 7.172 Fax: (49-38351) 7.151
Email: thomas.vahlenkamp@fli.bund.de
 Dr Jacek Kuzmak
SPartyzantow str. 57, 24-100 Pulawy
POLAND
Tel: (48-81) 889.31.14 Fax: (48-81) 886.25.95
Email: jkuzmak@piwet.pulawy.pl
Epizootic haematopoietic necrosis

 Dr A. Hyatt (1)
Australian Animal Health Laboratory, CSIRO Livestock Industries
5 Portarlington Road, Private Bag 24( Ryrie Street), Geelong, Victoria 3220
AUSTRALIA
Tel: (61.3) 52.27.00.00 Fax: (61.3) 52.27.55.55
Email: alex.hyatt@csiro.au
 Dr Richard Whittington (2)

Chair Farm Animal Health, Faculty of Veterinary Science, University of Sydney
425 Werombi Road, Private Bag 3, Camden NSW 2570
AUSTRALIA
Tel: (61.2) 93.51.16.19 Fax: (61.2) 93.51.16.18
Email: r.whittington@usyd.edu.au
Epizootic ulcerative syndrome

 Dr S. Kanchanakhan
Aquatic Animal Health Research Institute (AAHRI), Department of Fisheries, Kasetsart University
Campus
Paholyothin Road, Chatuchak, Bangkok 10900
THAILAND
Tel: (66.2) 579.41.22 Fax: (66.2) 561.39.93
Email: kanchanakhan@yahoo.com
Equine encephalomyelitis (Eastern)

 Dr E.N. Ostlund
P.O. Box 844, Ames, IA 50010
Tel: (1.515) 663.75.51 Fax: (1.515) 663.73.48
Equine encephalomyelitis (Western)

 Dr E.N. Ostlund
P.O. Box 844, Ames, IA 50010
Tel: (1.515) 663.75.51 Fax: (1.515) 663.73.48
Equine infectious anaemia

 Dr E.N. Ostlund
P.O. Box 844, Ames, IA 50010
Tel: (1.515) 663.75.51 Fax: (1.515) 663.73.48
 Dr K. Murakami
Viral disease Section, National Institute of Animal Health
3-1-5 Kannondai, Tsukuba, Ibaraki 305-0856
JAPAN
Tel: (81.29) 838.78.41 Fax: (81.29) 838.79.07
Email: muraken@affrc.go.jp
Equine influenza
 Dr W. Eichhorn
Institute for Medical Microbiology, Infectious and Epidemic Diseases, Veterinary Faculty, Ludwig-
Maximilians-University
Veterinärstrasse 13, 80539 München
GERMANY
Tel: (49.89) 21.80.25.31 Fax: (49.89) 21.80.59.03
Email: werner.eichhorn@micro.vetmed.uni-muenchen.de
 Dr Jennifer A. Mumford
Cambridge Infectious Diseases Consortium, Department of Veterinary Medicine
Madingley Road, Cambridge CB3 0ES
UNITED KINGDOM
Tel: (44.1223) 76.49.64 Fax: (44.1223) 76.46.67
Email: jam80@cam.ac.uk
 Dr T.M. Chambers
Maxwell H. Gluck Equine Research Center, Dept of Veterinary Science, University of Kentucky
108 Gluck Equine Research Center, Lexington, Kentucky 40546-0099
Tel: (1.859) 257.47.57 Fax: (1.859) 257.85.42
Email: tmcham1@uky.edu
 Prof. Ann Cullinane

Irish Equine Centre
Johnstown, Naas, Co. Kildare
IRLANDE
Tel: (353.45) 86.62.66 Fax: (353.45) 86.62.73
Email: acullinane@equine-centre.ie
Equine piroplasmosis
 Prof. Ikuo Igarashi
Medicine
JAPAN
Tel: (81.155) 49.56.42 Fax: (81.155) 49.56.43
Email: igarcpmi@obihiro.ac.jp
Equine rhinopneumonitis
 Dr Jennifer A. Mumford
Cambridge Infectious Diseases Consortium, Department of Veterinary Medicine
Madingley Road, Cambridge CB3 0ES
UNITED KINGDOM
Tel: (44.1223) 76.49.64 Fax: (44.1223) 76.46.67
Email: jam80@cam.ac.uk
 To be decided
Maxwell H. Gluck Equine Research Center, Dept of Veterinary Science, University of Kentucky
Tel: (1.859) 257.47.57 ext. 81119 Fax: (1.859) 257.85.42
Email:
 Prof. Konstantin P. Yurov

All-Russian Research Institute for Experimental Veterinary Medicine (VIEV), Laboratory of Equine
Viral Diseases
24-1 Ryazanskiy prosp. 109428 Moscow
RUSSIA
Tel: (7.495) 995.88.63 Fax: (7.495) 970.03.69
Email: efzabegina@mtu-net.ru
Equine viral arteritis

 Dr Peter J. Timoney
Maxwell H. Gluck Equine Research Center, Chair and Director, Dept of Veterinary Science,
University of Kentucky,
Tel: (1.859) 257.47.57 ext. 81084 Fax: (1.859) 257.85.42
Email: ptimoney@uky.edu
 Dr Takashi Kondo
Epizootic Research Center, Equine Research Institute, The Japan Racing Association,
K1400-4, Shiba, Shimotsuke-shi, Tochigi 329-0412
JAPAN
Tel: (81.285) 44.00.90 Fax: (81.285) 40.10.64
Email: kondo@epizoo.equinst.go.jp
 Dr T.W. Drew
UNITED KINGDOM
Tel: (44.1932) 35.76.37 Fax: (44.1932) 35.72.39
Escherichia coli
 Dr John Morris Fairbrother
The Echerichia coli Laboratory (EcL)
3200 Sicotte Saint-Hyacinthe, Québec, J2S 7C6
CANADA
Tel: (1.450) 773.85.21 Fax: (1.450) 778.81.08
Email: john.morris.fairbrother@umontreal.ca
Foot and mouth disease
 Dr Jef. M. Hammond
Ash Road, Pirbright, Woking, Surrey GU24 0NF
UNITED KINGDOM
Tel: (44.1483) 23.12.11 Fax: (44.1483) 23.26.21
Email: jef.hammond@bbsrc.ac.uk
 Dr O.G. Matlho
Botswana Vaccine Institute, Department of Animal Health and Production
Broadhurst Industrial Site, Lejara Road, Private Bag 0031, Gaborone
BOTSWANA
Tel: (267) 391.27.11 Fax: (267) 395.67.98
Email: gmatlho@bvi.co.bw
Web: www.bvi.co.bw
 Dr Ingrid Bergmann
Centro Panamericano de Fiebre Aftosa OPS/OMS
Av. President Kennedy 7778, Sao Bento, Duque de Caxias, ZC 20054-40 Rio de Janeiro
BRAZIL
Tel: (55.21) 36.61.90.56 Fax: (55.21) 36.61.90.01
Email: ibergman@panaftosa.ops-oms.org
 Dr V.M. Zakharov
Federal Governmental Institute, Centre for Animal Health, FGI-ARRIAH
600900 Vladimir, Yur'evets
RUSSIA
Tel: (4922) 26.19.14/26.06.14/26.38.77 Fax: (4922) 26.19.14/26.06.14/26.38.77
Email: mail@arriah.ru
Web: http://www.arriah.ru
Web: http://www.arriah.ru/portal/en
 Dr Eduardo D. Maradei
Laboratorio de Fiebre Aftosa de la Dirección de Laboratorios y Control Técnico
Av. Sir. Alexander Fleming 1653, Martínez (1640), Buenos Aires
ARGENTINA
Tel: (+54-11) 48.36.19.95 Fax: (+54-11) 48.36.19.95
Email: dilab@senasa.gov.ar
Email: emaradei@senasa.gov.ar
 Dr R.M. Dwarka
Onderstepoort Veterinary Institute, Team leader: Exotic Animal Health, Exotic Diseases Division
SOUTH AFRICA
Tel: (27.12) 529.95.89 Fax: (27.12) 529.95.43
Email: DwarkaR@arc.agric.za
 Dr Wilai Linchongsubongkoch
Department of Livestock Development
Pakchong, Nakhon Ratchasima 30130
THAILAND
Tel: (66.44) 27.91.12 Fax: (66.44) 31.48.89
Email: wilaifmd@loxinfo.co.th
Email: rrl@dld.go.th
Glanders
Zoonoses
GERMANY
Tel: (49.3641) 80.42.00 Fax: (49.3641) 80.42.28
 Professor Ulrich Wernery

Central Veterinary Research Laboratory
P.O. Box 597, Dubai
UNITED ARAB EMIRATES
Tel: (971.4) 337.51.65 Fax: (971.4) 336.86.38
Email: cvrl@cvrl.ae
Gyrodactylosis (Gyrodactylus salaris)

 Dr T.A. Mo
National Veterinary Institute, Section for Parasitology
P.O. Box 750 Sentrum, 0106 Oslo
NORWAY
Tel: (47) 23.21.61.10
Email: tor-atle.mo@vetinst.no
Heartwater
 Dr Dominique Martinez
CIRAD-EMVT
Head of Research Unit, Control of exotic and emerging animal diseases, TA30/G Campus
international de Baillarguet, 34398 Montpellier Cedex 5
FRANCE
Tel: (33(0)4) 67.59.37.12 Fax: (33(0)4) 67.59.37.98
Email: dominique.martinez@cirad.fr
Hendra and Nipah virus diseases

 Dr P. Daniels
AUSTRALIA
Tel: (61.3) 52.27.50.00 Fax: (61.3) 52.27.55.55
Email: peter.daniels@csiro.au
Highly pathogenic avian influenza and low pathogenic avian influenza (poultry)
 Dr John Pasick
Canadian Food Inspection Agency, National Centre for Foreign Animal Disease
1015 Arlington Street, Winnipeg, Manitoba R3E 3M4
CANADA
Tel: (1.204) 789.20.13 Fax: (1.204) 789.20.38
Email: jpasick@inspection.gc.ca
 Dr Hualan Chen
National Avian Influenza Reference Laboratory, Animal Influenza Laboratory of the Ministry of
Agriculture, Harbin Veterinary Research Institute, CAAS

427 Maduan Street, Harbin 150001
CHINA (People's Republic of)
Tel: (86-451) 85.93.50.79 Fax: (86-451) 82.73.31.32
Email: hlchen1@yahoo.com
Email: hlchen@hvri.ac.cn
Web: http://hvri.ac.cn
 Dr Timm C. Harder
Federal Research Centre for Virus Diseases of Animals (BFAV), Institute of Diagnostic Virology
Boddenblick 5a, 17493 Greifswald - Insel Riems
GERMANY
Tel: (49.383) 51.71.52 Fax: (49.383) 51.72.75
Email: timm.harder@fli.bund.de
 Dr Ian Brown
VLA Weybridge
UNITED KINGDOM
Tel: (44.1932) 35.73.39 Fax: (44.1932) 35.72.39
Email: i.h.brown@vla.defra.gsi.gov.uk
 Dr Paul W. Selleck
AUSTRALIA
Tel: (61.3) 52.27.50.00 Fax: (61.3) 52.27.55.55
Email: paul.selleck@csiro.au
 Dr B. Panigrahy
P.O. Box 844, Ames, IA 50010
Tel: (1.515) 663.75.51 Fax: (1.515) 663.73.48
Email: brundaban.panigrahy@aphis.usda.gov
 Dr Ilaria Capua
Istituto Zooprofilattico Sperimentale delle Venezie, Laboratorio Virologia
Via Romea 14/A, 35020 Legnaro, Padova
ITALY
Tel: (39.049) 808.43.79 Fax: (39.049) 808.43.60
Email: icapua@izsvenezie.it
 Dr H. Kida
Graduate School of Veterinary Medicine, Hokkaido University, Department of Disease Control
Kita-18, Nishi-9, Kita-ku, Sapporo 060-0818
JAPAN
Tel: (81.11) 706.52.07 Fax: (81.11) 706.52.73
Email: kida@vetmed.hokudai.ac.jp
 Dr S.C. Dubey
High Security Animal Disease Laboratory, Indian Veterinary Research Institute, Indian Council of
Agricultural Research
Anand Nagar, Bhopal 462021, Madhya Pradesh
INDIA
Tel: (91.7552) 26.94.87 Fax: (91.7552) 26.94.87
Email: scd_11@yahoo.in
Infection with Batrachochytrium dendrobatidis

 Dr A. Hyatt (1)
AUSTRALIA
Tel: (61.3) 52.27.00.00 Fax: (61.3) 52.27.55.55

AUSTRALIA
Tel: (61.2) 93.51.16.19 Fax: (61.2) 93.51.16.18
Infection with Bonamia exitiosa

 Dr Isabelle Arzul
IFREMER, Laboratoire de Génétique et Pathologie
BP 133, 17390 La Tremblade
FRANCE
Tel: (33-5) 46.76.26.10 Fax: (33-5) 46.76.26.11
Email: isabelle.arzul@ifremer.fr
Infection with Bonamia ostreae

FRANCE
Tel: (33-5) 46.76.26.10 Fax: (33-5) 46.76.26.11
Infection with Haplosporidium nelsoni

 Dr E.M. Burreson
Director for Research and Advisory Services, Virginia Institute of Marine Science, College of
William and Mary
P.O. Box 1346, Gloucester Point, VA 23062
Tel: (1.804) 684.70.15 Fax: (1.804) 684.77.96
Email: gene@vims.edu
Infection with Marteilia refringens

FRANCE
Tel: (33-5) 46.76.26.10 Fax: (33-5) 46.76.26.11
Infection with Marteilia sydneyi

FRANCE
Tel: (33-5) 46.76.26.10 Fax: (33-5) 46.76.26.11
Infection with Mikrocytos mackini

 Dr S. Bower
Department of Fisheries and Oceans Pacific Biological Station
3190 Hammond Bay Road, Nanaimo, British Columbia V9T 6N7
CANADA
Tel: (1.250) 756.70.77 Fax: (1.250) 756.70.53
Email: bowers@dfo-mpo.gc.ca
Infection with Perkinsus marinus

William and Mary
Tel: (1.804) 684.70.15 Fax: (1.804) 684.77.96
Infection with Perkinsus olseni

William and Mary
Tel: (1.804) 684.70.15 Fax: (1.804) 684.77.96
Infection with ranavirus

 Dr A. Hyatt (1)
AUSTRALIA
Tel: (61.3) 52.27.00.00 Fax: (61.3) 52.27.55.55

AUSTRALIA
Tel: (61.2) 93.51.16.19 Fax: (61.2) 93.51.16.18
Infection with Xenohaliotis californiensis

 Prof. Carolyn Friedman
Friedman Shellfish Health Laboratory, School of Aquatic and Fishery Sciences, University of
Washington
Box 355020, Seattle, Washington 98195

Tel: (1.206) 543.95.19 Fax: (1.206) 616.86.89
Email: carolynf@u.washington.edu
Infectious bovine rhinotracheitis/infectious pustular vulvovaginitis

 Dr Martin Beer
National Referance Laboratory for Bovine herpesvirus type 1, Institute of Diagnostic Virology,
Federal Research Centre for Virus Diseases of Animals (BFAV)
Insel Riems, Boddenblick 5a, 17493 Greifswald - Insel Riems
GERMANY
Tel: (49) 383.517.223 Fax: (49) 383.517.275
Email: martin.beer@fli.bund.de
 Dr M. Banks
VLA Weybridge
UNITED KINGDOM
Tel: (44.1932) 34.11.11 Fax: (44.1932) 34.70.46
Email: m.banks@vla.defra.gsi.gov.uk
Infectious bursal disease (Gumboro disease)

 Dr Y.M. Saif
Food Animal Health Research Program, Ohio Agricultural Research and Development Center, The
Ohio State University
1680 Madison Avenue, Wooster, OH 44691-4096
Tel: (1.330) 263.37.43 Fax: (1.330) 263.36.77
Email: saif.1@osu.edu
 Dr N. Eterradossi
AFSSA Ploufragan, Unité de virologie, immunologie et parasitologie aviaires et cunicoles
BP 53, 22440 Ploufragan
FRANCE
Tel: (33 (0)2) 96.01.62.22 Fax: (33 (0)2) 96.01.62.63
Email: n.eterradossi@ploufragan.afssa.fr
Infectious haematopoietic necrosis

 Dr James R. Winton
Western Fisheries Research Center
6505 N.E. 65th Street, Seattle, Washington 98115
Tel: (1.206) 526.65.87 Fax: (1.206) 526.66.54
Email: jim_winton@usgs.gov
Infectious hypodermal and haematopoietic necrosis

 Prof. Donald V. Lightner
Aquaculture Pathology Laboratory, Department of Veterinary Science and Microbiology, University
of Arizona
Building 90, Room 202 Pharmacy/Microbiology, Tucson, AZ 85721
Tel: (1.520) 621.84.14 Fax: (1.520) 621.48.99
Email: dvl@u.arizona.edu
Infectious myonecrosis
of Arizona
Tel: (1.520) 621.84.14 Fax: (1.520) 621.48.99
Infectious salmon anaemia

 Dr F. Kibenge
Atlantic Veterinary College, Department of Pathology and Microbiology, Faculty of Veterinary
Medicine, University of Prince Edward Island
550 University Avenue, Charlottetown, Prince Edward Island, C1A 4P3
CANADA
Tel: (1.902) 566.09.67 Fax: (1.902) 566.08.51
Email: kibenge@upei.ca
 Dr B. Dannevig
P.O. Box 750, Sentrum., 0106 Oslo
NORWAY
Tel: (47.23) 21.64.04 Fax: (47.23) 21.63.01
Email: birgit.dannevig@vetinst.no
Koi herpesvirus disease

 Dr Motohiko Sano
National Research Institute of Aquaculture, Fisheries Research Agency
422-1 Nakatsuhamaura, Minami-ise, Mie 516-0193
JAPAN
Tel: (81.599) 66.18.30 Fax: (81.599) 66.19.62
Email: sanogen@affrc.go.jp
 Dr Keith Way
The Centre for Environment, Fisheries and Aquaculture Science (CEFAS), Weymouth Laboratory
UNITED KINGDOM
Tel: (44.1305) 20.66.39 Fax: (44.1305) 20.66.01
Email: keith.way@cefas.co.uk
Leptospirosis
 Dr R.A. Hartskeerl
Royal Tropical Institute, N.H. Swellengrebel Laboratory of Tropical Hygiene, Division of Health,
Department of Biomedical Research
Meibergdreef 39, 1105 AZ Amsterdam
THE NETHERLANDS
Tel: (31.20) 566.54.38 Fax: (31.20) 697.18.41
Email: r.hartskeerl@kit.nl
 Dr W.A. Ellis
Department of Agriculture, Veterinary Sciences Division
Stoney Road, Stormont, Belfast BT4 3SD, Northern Ireland
UNITED KINGDOM
Tel: (44.2890) 51.94.41 Fax: (44.2890) 52.57.55

Email: bill.ellis@afbini.gov.uk
 Dr L. Samartino (1)
Instituto de Bacteriología, Centro de Investigaciones en Ciencias Veterinarias (CICV), Instituto
Nacional de Tecnología Agropecuaria (INTA)
ARGENTINA
Tel: (54.11) 46.21.12.89 Fax: (54.11) 46.21.17.12
Email: lsanma@correo.inta.gov.ar
 Dr Gleyre T.D. de Mazzonelli (2)

(SENASA)
ARGENTINA
Tel: (54.11) 48.36.19.92 Fax: (54.11) 48.36.19.92
Email: dilab@senasa.gov.ar/gmazzone@senasa.gov.ar
 Dr Mark Wilson
P.O. Box 844, Ames, IA 50010
Tel: (1.515) 663.73.42 Fax: (1.515) 663.76.73
Email: mark.a.wilson@aphis.usda.gov
 Dr L.D. Smythe
WHO/FAO/OIE Collaborating Centre for Reference and Research on Leptospirosis, Western
Pacific Region, Queensland Health Scientific Services
39 Kessels Road, Coopers Plains, P.O. Box 594, Archerfield, Queensland 4108
AUSTRALIA
Tel: (61.7) 32.74.90.64 Fax: (61.7) 32.74.91.75
Email: lee_smythe@health.qld.gov.au
Lumpy skin disease
 Dr G.H. Gerdes
SOUTH AFRICA
Tel: (27.12) 529.91.14 Fax: (27.12) 529.94.18
 Dr Eeva Tuppurainen
UNITED KINGDOM
Tel: (44.1483) 23.24.41 Fax: (44.1483) 23.24.48
Email: eeva.tuppurainen@bbsrc.ac.uk
Maedi-visna
 Dr Stephen Valas
Laboratoire d'étude et de recherches caprines
60 rue du Pied de Fond, B.P. 3081, 79000 Niort
FRANCE
Tel: (33 (0)5 49.79.61.28 Fax: (33 (0)5 49.79.42.19
Email: s.valas@niort.afssa.fr
 Dr D.P. Knowles, Jr
Animal Diseases Research Unit,USDA, ARS, Washington State University
Pullman, Washington 99164-7030
Tel: (1.509) 335.60.22 Fax: (1.509) 335.83.28
Email: dknowles@vetmed.wsu.edu
Marek's disease
 Dr K. Venugopal
Institute for Animal Health, Compton Laboratory, Compton
Newbury, Berkshire RG20 7NN
UNITED KINGDOM
Tel: (44.1635) 57.84.11 Fax: (44.1635) 57.72.63
Email: venu.gopal@bbsrc.ac.uk
 Dr Aly M. Fadly
USDA, ARS, Avian Disease and Oncology Laboratory
33606 East Mount Hope Roas, East Lansing, Michigan 48823
Tel: (1.517) 337.68.29 Fax: (1.517) 337.67.76
Email: fadly@msu.edu
Newcastle disease
 Dr Christian Grund
Federal Research Centre for Virus Diseases of Animals (BFAV), Institute of Diagnostic Virology
Boddenblick 5a, 17493 Greifswald - Insel Riems
GERMANY
Tel: (49.383) 51.71.96 Fax: (49.383) 51.72.75
Email: christian.grund@fli.bund.de
 Dr Ian Brown
VLA Weybridge
UNITED KINGDOM
Tel: (44.1932) 35.73.39 Fax: (44.1932) 35.72.39
Email: i.h.brown@vla.defra.gsi.gov.uk
 Dr Paul W. Selleck
AUSTRALIA
Tel: (61.3) 52.27.50.00 Fax: (61.3) 52.27.55.55
Email: paul.selleck@csiro.au
 Dr B. Panigrahy
P.O. Box 844, Ames, IA 50010
Tel: (1.515) 663.75.51 Fax: (1.515) 663.73.48
Email: brundaban.panigrahy@aphis.usda.gov
 Dr Ilaria Capua
Istituto Zooprofilattico Sperimentale delle Venezie, Laboratorio Virologia
Via Romea 14/A, 35020 Legnaro, Padova
ITALY
Tel: (39.049) 808.43.79 Fax: (39.049) 808.43.60
Email: icapua@izsvenezie.it
New world screwworm (Cochliomyia hominivorax)

 Dr Agustin Sagel
COPEG Panama/US Commission for the Eradication and Prevention of NWS)
Apartado Postal 0816-07636
PANAMA
Tel: (507) 296.00.06 Fax: (507) 296.09.69
Email: asagel@copeg.org
Email: veter56@yahoo.com
Email: tinso24@hotmail.com
Oncorhynchus masou virus disease

 Dr M. Yoshimizu
Laboratory of Biotechnology and Microbiology, Graduate School of Fisheries Sciences, Hokkaido
University, Faculty of Fisheries, Microbiology Department
3-1-1 Minato-cho, Hakodate, Hokkaido 041-8611
JAPAN
Tel: (81.138) 40.88.10 Fax: (81.138) 40.88.10
Email: yosimizu@fish.hokudai.ac.jp
Ovine epididymitis (Brucella ovis)

Zoonoses
GERMANY
Tel: (49.3641) 80.42.00 Fax: (49.3641) 80.42.28

(SENASA)
ARGENTINA
Tel: (54.11) 48.36.19.92 Fax: (54.11) 48.36.19.92
 Dr J.A. Stack
VLA Weybridge
UNITED KINGDOM
Tel: (44.1932) 35.76.10. Fax: (44.1932) 35.72.16
FRANCE
Tel: (33 (0)1) 49.77.13.00 Fax: (33 (0)1) 49.77.13.44
 Dr K. Nielsen
CANADA
Tel: (1.613) 228.66.98 ext. 48.04 Fax: (1.613) 228.66.69
ITALY
Tel: (390.861) 33.24.05 Fax: (390.861) 33.22.51
ISRAEL
Tel: (972.3) 968 16 98 Fax: (972.3) 968 17 53
Paratuberculosis
 Dr Jacek Gwozdz
Johne's Disease Laboratory, Research and Development Division, Department of Primary
Industries
475 Mickleham Road, Attwood, Victoria 3049
AUSTRALIA
Tel: (61.3) 92.17.42.00 Fax: (61.3) 92.17.42.99
Email: jacek.gwozdz@dpi.vic.gov.au
 Dr Bernardo Alonso
Gerencia de Laboratorios (GELAB) del Servicio Nacional de Sanidad y Calidad, Agroalimentaria
(SENASA)
Av. Alexander Fleming 1653, 1640 Martinez - Pcia de Buenos Aires
ARGENTINA
Tel: (54.11) 48.36.00.36 Fax: (54.11) 48.36.00.36
Email: balonso@senasa.gov.ar
Email: dilab@inea.com.ar
 Dr I. Pavlik
CZECH (Rep.)
Tel: (420.5) 33.33.16.01 Fax: (420.5) 33.33.12.29
 Mme María Laura Boschiroli-Cara

AFSSA Alfort, Unité Zoonoses Bactériennes, Laboratoire d'études et de recherches en pathologie

animale et zoonoses
FRANCE
Tel: (33 (0)1) 49.77.13.00 Fax: (33 (0)1) 49.77.13.44
Peste des petits ruminants
 Prof. Tom Barrett

UNITED KINGDOM
Tel: (44.1483) 23.24.41 direct 213 10 09 Fax: (44.1483) 23.24.48
Email: tom.barrett@bbsrc.ac.uk
 Dr Geneviève Libeau
CIRAD-BIOS, Control of Exotic and Emerging Animal Diseases
Programme Santé animale, TA A-15/G Campus international de Baillarguet, 34398 Montpellier
Cedex 5
FRANCE
Tel: (33 (0)4) 67.59.37.98 Fax: (33 (0)4) 67.59.38.50
Email: genevieve.libeau@cirad.fr
Porcine reproductive and respiratory syndrome

 Dr Tomasz Stadejek
National Veterinary Research Institute, Department of Swine Diseases
Partyzantow Str. 57, 24-100 Pulawy
POLAND
Tel: (48-81) 889.30.99 Fax: (48-81) 886.25.95
Email: stadejek@piwet.pulawy.pl
Rabbit haemorrhagic disease

 Dr L. Capucci
Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna, 'B. Ubertini'
Via A. Bianchi n° 7/9, 25124 Brescia
ITALY
Tel: (39.030) 229.06.17 Fax: (39.030) 229.05.59
Email: lorenzo.capucci@izsler.it
Rabies
 Dr A. Wandeler
Centre of Expertise for Rabies, Animal Diseases Research Institute
CANADA
Tel: (1.613) 228.66.98 Fax: (1.613) 228.66.69
Email: alex.wandelera@inspection.gc.ca
 Dr J. Barrat
AFSSA-LERPAS, Laboratoire d'études sur la rage et la pathologie des animaux sauvages
Domaine de Pixérécourt, BP 9, 54220 Malzéville
FRANCE
Tel: (33 (0)3) 83.29.89.50 Fax: (33 (0)3) 83.29.89.58
Email: j.barrat@afssa.fr
 Mme F. Cliquet
AFSSA-LERPAS, Laboratoire d'études sur la rage et la pathologie des animaux sauvages
Domaine de Pixérécourt, BP 9, 54220 Malzéville
FRANCE
Tel: (33 (0)3) 83.29.89.50 Fax: (33 (0)3) 83.29.89.58
Email: f.cliquet@afssa.fr
 Dr T. Müller
Institute of Epidemiology, Friedrich-Loeffler Institut, Federal Research Institute for Animal Health
Seest. 55, 16868 Wustherhausen/Dosse
GERMANY
Tel: (49.33) 97.98.01.86 Fax: (49.33) 97.98.02.00
Email: thomas.mueller@wus.bfav.de
 Dr Claude Taurai Sabeta

Onderstepoort Veterinary Institute, Rabies Unit
SOUTH AFRICA
Tel: (27.12) 529.94.39 Fax: (27.12) 529.93.90
Email: sabetac@arc.agric.za
 Dr Anthony Fooks
Rabies and Wildlife Zoonoses Group, Virology Department, VLA Weybridge
UNITED KINGDOM
Tel: (44.1932) 35.78.40 Fax: (44.1932) 35.72.39
Email: t.fooks@vla.defra.gsi.gov.uk
Red sea bream iridoviral disease

 Dr K. Nakajima
National Research Institute of Fisheries Science, Fisheries Research Agency
2-12-4 Fukuura, Yokohama-shi, Kanagawa 236-8048
JAPAN
Tel: (81.45) 788.76.15 Fax: (81.45) 788.50.01
Email: rsiv-lab@fra.affrc.go.jp
Rift Valley fever
 Dr G.H. Gerdes
SOUTH AFRICA
Tel: (27.12) 529.91.14 Fax: (27.12) 529.94.18
 Dr Michèle Bouloy
Unité de génétique moléculaire des Bunyavirus, Département de Virologie, Institut Pasteur
25 rue du Dr Roux 75724 Paris cedex 15
FRANCE
Tel: (33.1) 40.61.31.34 Fax: (33.1) 40.61.32.56
Email: E-mail: mbouloy@pasteur.fr
Rinderpest
 Prof. Tom Barrett

UNITED KINGDOM
Tel: (44.1483) 23.24.41 direct 213 10 09 Fax: (44.1483) 23.24.48
Email: tom.barrett@bbsrc.ac.uk
 Dr Geneviève Libeau
CIRAD-BIOS, Control of Exotic and Emerging Animal Diseases
Programme Santé animale, TA A-15/G Campus international de Baillarguet, 34398 Montpellier
Cedex 5
FRANCE
Tel: (33 (0)4) 67.59.37.98 Fax: (33 (0)4) 67.59.38.50
Email: genevieve.libeau@cirad.fr
Salmonellosis
 Dr R.H. Davies
VLA Weybridge
UNITED KINGDOM
Tel: (44-1932) 35.73.61 Fax: (44-1932) 35.75.95
Email: r.h.davies@vla.defra.gsi.gov.uk
 Dr M. Hartung
Bundesinstitut für Risikobewertung (Federal Institute for Risk Assessment)
P.O. Box 330013, 14191 Berlin
GERMANY
Tel: (49.30) 84.12.22.12 Fax: (49.30) 84.12.29.52
Email: m.hartung@bfr.bund.de
Web: http://www.bfr.bund.de
 Dr Cornelius Poppe
Laboratory for Foodborne Zoonoses, Guelph Laboratory, Health Canada, Public Health Agency of
Canada
110 Stone Road West, Guelph, Ontario, N1G 3W4
CANADA
Tel: (1.519) 822.33.00 Fax: (1.519) 822.22.80
Email: cpoppe@sympatico.ca
 Dr Antonia Ricci
Istituto Zooprofilattico Sperimentale delle Venezie, National Reference LaboratoryL for Salmonella
Viale Dell'Università 10, 35020 Legnaro (PD)
ITALY
Tel: (39.049) 808.42.96 Fax: (39.049) 883.02.68
Email: aricci@izsvenezie.it
Scrapie
 Dr Marion Simmons
VLA Weybridge
UNITED KINGDOM
Tel: (44.1932) 35.75.64 Fax: (44.1932) 35.78.05
Email: TSEeucrl@vla.defra.gsi.gov.uk
Web: http://www.defra.gov.uk/corporate/vla/science/science-tse-rl-web.htm
 Prof. Andreas Zurbriggen

Institute of Animal Neurology, University of Bern
Bremgartenstrasse 109A, 3012 Bern
SWITZERLAND
Tel: (41.31) 631.25.09 Fax: (41.31) 631.25.38
Email: andreas.zurbriggen@itn.unibe.ch
 Dr Aru Balachandran
Canadian Food Inspection Agency, Ottawa Laboratory
CANADA
Tel: (1.613) 228.66.98 ext. 4854 Fax: (1.613) 228.61.03
Email: balachandrana@inspection.gc.ca
 Dr Francisco Javier Blanco Viera

Laboratorio Nacional de Referencia (LNR) para Encefalopatías Espongiformes Transmisible
animales, Institutos de Patobiología y Virología, Centro de Investigaciones en Ciencias
Veterinarias y Agronómicas (CICV), Instituto Nacional de Tecnología Agropecuaria (INTA)
ARGENTINA
Tel: (54.11) 46.21.12.89 Fax: (54.11) 46.21.17.43
Email: jviera@cnia.inta.gov.ar
Email: gpinto@cnia.inta.gov.ar
Sheep pox and goat pox
 Dr H.R. Varshovi
RAZI Vaccine and Serum Research Institute
P.O. Box 31975/148, Hessarak, Karadj, Teheran
IRAN
Tel: (98.21) 311.79.08 Fax: (98.261) 455.31.94
Email: int@rvsri.com
Email: hr_varshovi@yahoo.com
 Dr Eeva Tuppurainen
UNITED KINGDOM
Tel: (44.1483) 23.24.41 Fax: (44.1483) 23.24.48
Email: eeva.tuppurainen@bbsrc.ac.uk
Spherical baculovirosis (Penaeus monodon-type baculovirus)

of Arizona
Tel: (1.520) 621.84.14 Fax: (1.520) 621.48.99
 Dr Grace Lo
Department and Institute of Zoology, National Taiwan University
1, Sec. Roosevelt Road, Taipei
CHINESE TAIPEI
Tel: (886.2) 23.63.35.62 Fax: (886.2) 23.63.81.79
Email: gracelow@ntu.edu.tw
Spring viraemia of carp

 Dr Peter Dixon
The Centre for Environment, Fisheries and Aquaculture Science (CEFAS), Weymouth Laboratory
UNITED KINGDOM
Tel: (44.1305) 20.66.42 Fax: (44.1305) 20.66.01
Email: peter.dixon@cefas.co.uk
Surra (Trypanosoma evansi)

 Dr Filip Claes
Institute of Tropical Medicine Antwerp, Department of Parasitology
Nationalestraat 155, B-2000 Antwerpen
BELGIUM
Tel: (32.3) 247.65.34 Fax: (32.3) 247.63.73
Email: fclaes@itg.be
 Prof. Noboru Inoue

Medicine
JAPAN
Tel: (81.155) 49.56.47 Fax: (81.155) 49.56.43
Email: ircpmi@obihiro.ac.jp
Swine vesicular disease
 Dr Jef. M. Hammond
Ash Road, Pirbright, Woking, Surrey GU24 0NF
UNITED KINGDOM
Tel: (44.1483) 23.12.11 Fax: (44.1483) 23.26.21
Email: jef.hammond@bbsrc.ac.uk
 Dr E. Brocchi
Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna 'B. Ubertini'
Via A. Bianchi n° 9, 25124 Brescia
ITALY
Tel: (39.030) 229.03.10 Fax: (39.030) 229.03.69
Email: emiliana.brocchi@bs.izs.it
Taura syndrome
of Arizona
Tel: (1.520) 621.84.14 Fax: (1.520) 621.48.99

Tetrahedral baculovirosis (Baculovirus penaei)

of Arizona
Tel: (1.520) 621.84.14 Fax: (1.520) 621.48.99
Theileriosis
 Dr Dirk Geysen
Department of Animal Health, Institute of Tropical Medicine
Nationalestraat 155, 2000 Antwerpen
BELGIQUE
Tel: (32.3) 247.62.66 Fax: (32.3) 247.62.68
Email: dgeysen@itg.be
Transmissible gastroenteritis
 Dr Linda J. Saif
Food Animal Health Research Program, Ohio Agricultural Research and Development Center, The
Ohio State University
1680 Madison Avenue, Wooster, OH 44691-4096
Tel: (1.330) 263.37.44 Fax: (1.330) 263.36.77
Email: saif.2@osu.edu
Trichinellosis
 Dr E. Pozio
Istituto Superiore di Sanita, Laboratorio di Parasitoligia
Viale Regina Elena 299, 00161 Roma
ITALY
Tel: (39.06) 49.90.23.04 Fax: (39.06) 49.90.35.61
Email: edoardo.pozio@iss.it
 Dr A. Gajadhar
Canadian Food Inspection Agency, Centre for Animal Parasitology
116 Veterinary Road, Saskatoon, Saskatchewan S7N 2R3
CANADA
Tel: (1.306) 975.53.44 Fax: (1.306) 975.57.11
Email: agajadhar@inspection.gc.ca
Trypanosomosis (tsetse-transmitted)
 Dr M. Desquesnes
CIRAD-EMVT
Programme Santé animale, TA30/G Campus international de Baillarguet, 34398 Montpellier Cedex
5
FRANCE
Tel: (33(0)4) 67.59.37.24 Fax: (33(0)4) 67.59.37.98
Email: marc.desquesnes@cirad.fr
Tularemia
 Dr T. Mörner
National Veterinary Institute, Department of Wildlife
751 89 Uppsala
SWEDEN
Tel: (46.18) 67.40.00 Fax: (46.18) 67.46.90
Email: torsten.morner@sva.se
Turkey rhinotracheitis
 Dr N. Eterradossi
AFSSA Ploufragan, Unité de virologie, immunologie et parasitologie aviaires et cunicoles
BP 53, 22440 Ploufragan
FRANCE
Tel: (33 (0)2) 96.01.62.22 Fax: (33 (0)2) 96.01.62.63
Email: n.eterradossi@ploufragan.afssa.fr
Venezuelan equine encephalomyelitis

 Dr E.N. Ostlund
P.O. Box 844, Ames, IA 50010
Tel: (1.515) 663.75.51 Fax: (1.515) 663.73.48
Vesicular stomatitis
 Dr Ingrid Bergmann
Centro Panamericano de Fiebre Aftosa OPS/OMS
Av. President Kennedy 7778, Sao Bento, Duque de Caxias, ZC 20054-40 Rio de Janeiro
BRAZIL
Tel: (55.21) 36.61.90.56 Fax: (55.21) 36.61.90.01
Email: ibergman@panaftosa.ops-oms.org
 Dr S.L. Swenson
P.O. Box 844, Ames, IA 50010
Tel: (1.515) 663.75.51 Fax: (1.515) 663.73.48
Email: sabrina.l.swenson@aphis.usda.gov
Viral encephalopathy and retinopathy

 Dr G. Bovo
Istituto Zooprofilattico Sperimentale delle Venezie, Dipartimento di Ittioviroligia,
Via Romea 14/A, 35020 Legnaro PD
ITALY
Tel: (39.049) 808.42.48 Fax: (39.049) 808.43.92
Email: gbovo@izsvenezie.it
 Dr T. Nakai
Hiroshima University, Graduate School of Biosphere Science, Laboratory of Fish Pathology
Higashi Hiroshima 739-8528
JAPAN
Tel: (81.82) 424.79.47 Fax: (81.82) 424.43.80

Email: nakaitt@hiroshima-u.ac.jp
Viral haemorrhagic septicaemia

 Dr Niels Jørgen Olesen
National Veterinary Institute, Technical University of Denmark
Hangøvej 2, 8200 Aarhus N
DENMARK
Tel: (45) 72.34.68.31 Fax: (45) 72.34.69.01
Email: njol@vet.dtu.dk
West Nile Fever

 Dr E.N. Ostlund
P.O. Box 844, Ames, IA 50010
Tel: (1.515) 663.75.51 Fax: (1.515) 663.73.48
White spot disease

of Arizona
Tel: (1.520) 621.84.14 Fax: (1.520) 621.48.99
 Dr Grace Lo
Department and Institute of Zoology, National Taiwan University
1, Sec. Roosevelt Road, Taipei
CHINESE TAIPEI
Tel: (886.2) 23.63.35.62 Fax: (886.2) 23.63.81.79
Email: gracelow@ntu.edu.tw
White tail disease

 Dr A. Sait Sahul Hameed
C. Abdul Hakeem College, Aquaculture Biotechnology Division, Department of Zoology
Melvisharam-632 509, Vellore Dt. Tamil Nadu
INDIA
Tel: (91.4172) 26.94.87 Fax: (91.4172) 26.94.87
Email: cah_sahul@hotmail.com
Yellow head disease

 Dr P. Walker
CSIRO, Aquaculture and Aquatic Animal Health (AAHL)
AUSTRALIA
Tel: (61.3) 52.27.54.65 Fax: (61.3) 52.27.55.55
Email: peter.walker@csiro.au
EAZWV Transmissible Disease Fact Sheet Sheet No.
DISEASE NAME
ANIMAL TRANS- CLINICAL FATAL TREATMENT PREVENTION

GROUP MISSION SIGNS DISEASE ? & CONTROL
AFFECTED
In houses
in zoos
Fact sheet compiled by Last update
Fact sheet reviewed by
Susceptible animal groups
Causative organism
Zoonotic potential
Distribution
Transmission
Incubation period
Clinical symptoms
Post mortem findings
Diagnosis
Material required for laboratory analysis
EU Reference Laboratory
OIE Reference Laboratories
Relevant diagnostic laboratories
Treatment
Prevention and control in zoos
Suggested disinfectant for housing facilities
Notification
Guarantees required under EU Legislation
Guarantees required by EAZA Zoos
1
EAZWV Transmissible Disease Fact Sheet Sheet No.
Measures required under the Animal Disease Surveillance Plan
Measures required for introducing animals from non-approved sources
Measures to be taken in case of disease outbreak or positive laboratory findings
Conditions for restoring disease-free status after an outbreak
Contacts for further information
References
2
INSTRUCTIONS FOR AUTHORS
1) If you plan to write a fact sheet or a general chapter on a certain topic, please INFORM THE
SECRETARIAT first. In this way we can avoid that two people work on the same
disease/subject at the same time.
2) ONE (OR MORE) AUTHOR(S) SHOULD BE LISTED on each fact sheet. This author will be
the reference/contact person responsible for redaction and corrections.
3) Each fact sheet has to be REVIEWED BY TWO PEOPLE. Reviewers are not necessarily
members of IDWG but should be specialists or at least have a minimal experience with the
disease concerned. Both reviewers should be listed on the concerned fact sheet. The author
has to find reviewers himself and should SUBMIT HIS FACT SHEETS TO THE
SECRETARIAT AFTER REVIEW.
4) Be short in your descriptions! Each fact sheet should NOT EXCEED 4 PAGES (including
references and laboratories!).
5) USE THE EMPTY FACT SHEET MODEL to write your text (write directly in the model). To
become the model in an electronic format, please contact the secretariat.
6) As a text format, use ARIAL standard, size 10.
7) Don't forget to send the fact sheets to the IDWG-secretary in an ELECTRONIC FORMAT
(email attachement or floppy disc).
8) NAME YOUR FILE according to the following model: "disease name" and "date of the text
version". Example: Shigellosis 24.08.01
9) REFERENCE LABORATORIES: The EU and the OIE established a list of reference

laboratories. National laboratories are the official labs recognized by the EU. For diseases
which don’t need to be notified, no lab is mentioned in the official lists. However, if there is
an important lab to recommend for the analysis, it can be mentioned under “Relevant
Laboratories”.
10) The 3 points "Measures required under the Animal Disease Surveillance Plan", "Measures
required for introducing animals from non-approved sources" and "Measures to be taken in
case of a disease outbreak or positive laboratory findings" are conditional to the adoption of
the revised Annex C of the Balai directive. Ask the secretariat for the actual situation before
submitting your fact sheet.
11) AVOID LISTING TOO MANY REFERENCES. It might be more helpful to suggest a few
good books for further readings.
12) List the LITERATURE according to the existing fact sheets. Basically, the presentation
follows the GUIDELINES OF THE JOURNAL OF ZOO AND WILD ANIMAL MEDICINE.
__________
May 2004
4th Edition, February 2010
Coordination of the work Jacques Kaandorp

& Editor of the Handbook
Co-editors Norin Chai

Ayla Bayens
Pictures (cover page) Nico Schoemaker
Authors In alphabetical order:
Gian Lorenzo d’Alterio

Luca Bacciarini
Hester van Bolhuis
Debra Bourne
Manfred Brack
J. Brandt
V. Briones
Norin Chaï
R. De Deken
Peter Dollinger
Gerry Dorrestein
Marein van der Hage
Jesus Fernandéz Morán
Scott Fitzgerald
Edmund Flach
Klaus Gunther Friedrich
Kai Frölich
Chris Furley
Fabricio Gamberale
S. Geerts
Manuel Garcia Hartmann
Ursula Höfle
Jacques Kaandorp
Marja Kik
Antoine Leclerc
Alexis Lécu
Matti Kiupel
Michael Lierz
Rachel Marschang
Marian Mensink
(continued on next page)

Authors (continuation)
Joost Philippa
Willem Schaftenaar
Stefanie Sanderson
Elvira Schettler
Gabrielle Stalder
Martin Straube
S. Téllez
Werner Tschirch
Francis Vercammen
Chris Walzer
Marno Wolters
Reviewers In alphabetical order:
R. Adone, J. Alunda, S. Bergmann, G. Bertoni, S. Bolin, S.

Blahak, M. Brack, J. Brandt, C. Buonavoglia, J. Caird, M.
Coosemans, T. Cornish, A. Cunningham, O. Czupalla, R. De
Deken, G. Dorrestein, P. Duff, C. Feliu, G. Ferrari, M. Fox,
C. Furley, D. Geysen, G. Goodman, A. Gröne, O. Haenen,
H. Hafez, J. Hatt, W. Jakob, T. Jauniaux, J. Kaandorp, E.
Kaleta, M. Kiupel, T. Kuiken, A. Lécu, J. Lewis, H. Li, N.
Majó i Masferrer, R. Marschang, B. Martina, R. McLean, T.
Mettenleiter, D. Miller, R. Montali, J. Mortelmans, B.
Neurohr, G. Paiba, T.Papp, F. Pasmans, P. Pasquali, M.
Popoff, A. Pospischil, C.Reid, L. Richman, W. Rietschel, W.
Schaftenaar, J. Sikarskie, J. Slingenbergh, G. Stevenson, G.
Strauß, P. Sutmöller, H. Thiel, U. Truyen, F. Vercammen, W.
Vosloo, T. Wahli, J. Wellehen, O. Werner, E. Williams, L.
Woods, P. Zwart

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Transmissible Diseases Handbook

II. GENERAL CONSIDERATIONS J. Kaandorp

III. EUROPEAN UNION J. Kaandorp

2. DATA ON EU MEMBER STATES

3. EU ANIMAL HEALTH STRATEGY AND ANIMAL

4. EU ANIMAL HEALTH LAW

5. EAZA POSITION STATEMENT ON THE ANIMAL

IV. LINKS J.Kaandorp

2. USEFUL (VETERINARY) WEBLINKS

V. ANIMAL HEALTH LEGISLATION IN EUROPE P. Dollinger

VI. RECOMMENDATIONS FOR THE APPLICATION OF EAZWV – IDWG – DEFRA –

VII. POST-MORTEM PROCEDURES G. Dorrestein et al.

VIII. GUIDELINES FOR CLEANING AND DISINFECTION IN M. Kiupel et al.

IX. BLUETONGUE IN NON-DOMESTIC RUMINANTS: S. Sanderson

X. TUBERCULOSIS IN ZOO SPECIES: DIAGNOSTIC EAZWV Tuberculosis Working

1. ELEPHANTS (ELEPHAS MAXIMUS) W. Shaftenaar

XI. AVIAN INFLUENZA

1. VACCINATION OF NON-DOMESTIC AVIAN J. Philippa

2. WAZA/EAZA/EAZWV RECOMMENDATIONS ON WAZA – EAZA – EAZWV

XII. VACCINATION OF NON-DOMESTIC CARNIVORES: A J. Philippa

XIII. LIST OF LABORATORIES

XIV. TEMPLATE FACT SHEET

XV. INSTRUCTIONS FOR AUTHORS

XVI. FACT SHEETS

II. GENERAL CONSIDERATIONS

III. EUROPEAN UNION

2. Data on the 27 EU Member States

3. The EU Animal Health Strategy (2007-2013) where “prevention is

4. The EU’s Animal Health Law

5. EAZA Position Statement on the Animal Health Strategy for the

This statement presents the position of the European

EAZA’s current status and general position;

What we would like to improve in the future;

6. The O.I.E. (World Organisation for Animal Health) (Organisation

Universitá di Pisa Facoltá di Medicina Veterinaria

Universidade do Porto – Instituto de Cièncias Biomédicas de Abel Salazar

2. Useful (Veterinary) Weblinks

European (Union) links

VI. RECOMMENDATIONS FOR THE APPLICATION OF ANNEX C TO

D. The added animals procedure

A. The term ‘animals’

1. ‘Animals’ means specimens of animal species other than those referred to in

Directive Article Species Comments

B. The approved veterinarian

1. In order to be granted official approval under Article 13 of the Directive, a zoo

C. The Annual Disease Surveillance Plan

1. The approved veterinarian has to draw up and implement an annual disease

D. The Added Animals Procedure

2. Animals from another approved establishment

3. Animals from a non-approved establishment in the same Member State

4. Animals from a non-approved establishment in another Member State

5. Animals from non-approved establishment to a non-approved establishment

6. Animals from third countries

E. Quarantine / Isolation requirements

3. General conditions - Structural requirements

4. General conditions - Management procedures

5. Additional requirements specifically for birds

6. Additional requirements specifically for ungulates

7. Additional requirements specifically for primates

Where certificates are required

V. ANIMAL HEALTH LEGISLATION IN EUROPE

VDZ / zooschweiz / EAZWV Office

As a result of the Uruguay round, an Agreement on the Application of Sanitary and