development motivated him to enroll for Ph.D study in the ... · 2. Siri Syarahan Inaugural KUT: 2...
Transcript of development motivated him to enroll for Ph.D study in the ... · 2. Siri Syarahan Inaugural KUT: 2...
Mohd. Effendy bin Abd. Wahid was born in Banda Hilir, Melaka on 26th. October 1966. He completed his secondary education at Sekolah Dato’ Seri Amar Di Raja, Muar, Johor. He was then pursuing for his Diploma in Animal Health and Production in 1984 at Universiti Pertanian Malaysia (UPM). After obtaining his diploma, he continued his study for Doctor in Veterinary Medicine and graduated in 1994. His passion for research and development motivated him to enroll for Ph.D study in the field of Veterinary Immuno-pathology in1995. He was awarded the Best PhD Thesis Award by the Faculty of Veterinary Medicine, UPM in 1998. Effendy started his academic journey at UPM Terengganu (UPMT) in November 1998 at the Faculty of Science and Professional Arts. He joined the Faculty of Science and Technology in 2001 before accepted a Post-doctorate position at Chonnam National University, Kwang-ju, South Korea by Korean Institute of Science and Technology Evaluation and Planning (KISTEP) in 2003.
Effendy was appointed as the Head of Student Welfare and Development Centre at Division of Student Affairs, KUSTEM in 2004. He was then given a task to establish the Institute of Marine Biotechnology at the end of 2005, and appointed as the founding Director of the Institute of Marine Biotechnology from 2006 to 2013. During the tenure, he faced many challenges until the Ministry of Higher Education approved it on February 26th 2008. Effendy was then appointed as the Assistant Vice Chancellor (Research and Innovation Affairs) in 2013, and the first Deputy Vice Chancellor (Research and Innovation) for UMT in 2014. Effendy has great interest in research and innovation. Until present, he has taught eight different courses and still teaching a course at the school. He started his first research on histopathology of uterine involution and side effects of Kacip Fatimah at a small laboratory at INOS in 1999.
Since then, he has supervised and graduated 12 PhD, 24 Master Science and 180 final year students and published 60 scientific papers in various journals, particularly in microbiology, immunology and biotechnology. His works with his colleagues were also disseminated through more than 110 seminars, conferences and symposiums. Apart from that, Effendy also involved in writing books and monograph, and reviewing research papers and thesis. He also acquired two full patent rights on mucosal vaccine against pneumonic pasteurellosis in small ruminants and nutritious health drinks from golden sea cucumber extracts, and others that are still pending. Throughout his research activities, he was recognized with 13 Special Research Awards, 16 medals at International Research Competitions and 24 medals at national research exhibitions and competitions. He was awarded Excellent Scientist Award by the Ministry of Higher Education in 2005 and the first recipient of National Intellectual Property Award 2006 (Individual Category). At the moment, Effendy focus his research on development of novel adjuvants for mucosal vaccines, establishing of seahorse sanctuary in Johor Darul Ta’zim and sustainable aquaculture management utilizing microalgae program with Japan International Collaboration Agency (JICA).
cover-syarahan-inaugural-prof-EFFENDYj.indd 1 5/12/16 4:41 PM
Mucosal Immunity:Animal Vaccination
and Disease Prophylaxis
INAUGURAL LECTUREUNIVERSITI MALAYSIA TERENGGANU
Penerbit UMTUniversiti Malaysia Terengganu
21030 Kuala Terengganu2016
Mohd Effendy Abd. Wahid
Mucosal Immunity:Animal Vaccination
and Disease Prophylaxis
INAUGURAL LECTUREUNIVERSITI MALAYSIA TERENGGANU
Mucosal Immunity:Animal Vaccination and Disease Prophylaxis
© 2016 All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical including photocopy, recording or any information storage and retrieval system, without permission in writing from Director, Penerbit UMT, Universiti Malaysia Terengganu, 21030 Kuala Terengganu, Terengganu, Malaysia.
Hak Cipta Terpelihara © 2016. Tidak dibenarkan mengeluar ulang mana-mana bahagian artikel, ilustrasi dan isi kandungan buku ini dalam apa juga bentuk dan dengan apa cara sekalipun sama ada secara elektronik, fotokopi, mekanik, rakaman, atau cara lain sebelum mendapat izin bertulis daripada Pengarah, Penerbit UMT, Universiti Malaysia Terengganu, 21030 Kuala Terengganu, Terengganu, Malaysia.
Published in Malaysia by / Diterbitkan oleh Penerbit UMT, Universiti Malaysia Terengganu, 21030 Kuala Terengganu, Terengganu, Malaysia.
http://www.umt.edu.my/penerbitumt E-mel: [email protected]
Perpustakaan Negara Malaysia Cataloguing-in-Publication Data
Mohd. Effendy Abd WahidMucosal Immunity : Animal Vaccination and Disease Prophylaxis / Mohd Effendy Abd. Wahid.Bibliography: page 71ISBN 978-967-0962-17-71. Livestock--Vaccination.2. Mucosal diseases in cattle.3. Mucous membrane--Immunology.4. Dental prophylaxis.5. Vaccination of animals.I. Title636.089
Set in Optima
Design: Penerbit UMTLayout: Penerbit UMT
Print by: Reka Cetak Sdn. Bhd.No. 14, Jalan Jemuju Empat 16/13D,Seksyen 16, 40200 Shah Alam,Selangor.
INAUGURAL LECTURE SERIES
1. KUT Inaugural Lecture Series: 1 (2000) Fisheries and the National Food Security: The Malaysian
Perspective Prof. Dr. Mohd. Azmi Ambak
2. Siri Syarahan Inaugural KUT: 2 (2000) Development of Ocean Modelling: The Malaysian Perspective Prof. Dr. Alejandro Livio Camerlengo
3. KUT Inaugural Lecture Series: 3 (2000) Into the Wonders of Surfactant Behaviour Prof. Dr. Hamdan Suhaimi
4. KUT Inaugural Lecture Series: 4 (2000) Food Chain in the Sea - Its Values, Challenges and Prospects Prof. Dr. Lokman Shamsudin
5. KUSTEM Inaugural Lecture Series: 5 (2002) The Fascinating World of Flukes Prof. Dr. Faizah Mohd. Shaharom
6. KUSTEM Inaugural Lecture Series: 6 (2002) The Width is Unreachable, the Travel is at the Speed of Light
Prof. Dr. Ismail Mohd
7. KUSTEM Inaugural Lecture Series: 7 (2004) Turtles in Trouble Prof. Dr. Chan Eng Heng
8. KUSTEM Inaugural Lecture Series: 8 (2004) Exploring the Interface: The Enigmatic Mangroves Prof. Dr. Mohd. Lokman Husain
9. KUSTEM Inaugural Lecture Series: 9 (2004) Metals in the Marine Environment Prof. Dr. Noor Azhar Mohamed Shazili
• vii
10. Siri Syarahan Inaugural KUSTEM: 10 (2004) Deria Pemikiran Matematik: Daripada Konkrit ke Niskala Prof. Dr. Abu Osman Md Tap
11. KUSTEM Inaugural Lecture Series: 11 (2007) The Quest for High Performance Computers
Prof. Dr. Md. Yazid Mohd Saman
12. UMT Inaugural Lecture Series: 12 (2008) Resizing Homo Sapiens Tan Sri Dato’ Dr. Ahmad Mustaffa Babjee
13. UMT Inaugural Lecture Series: 13 (2009) Corporate Governance in Malaysia: Towards Stronger Boards and Audit Committees Prof. Dr. Shamsul Nahar Abdullah
14. Siri Syarahan Inaugural UMT: 14 (2010) Realiti Sebuah Kehidupan dari Perspektif Ekonomi Alam Sekitar
dan Falsafah Islam Prof. Dr. Nik Hashim Nik Mustapha
15. UMT Inaugural Lecture Series: 15 (2010) Plant Parasitic Nematodes A Challenging Pest to Third National Agriculture Policy: Myth or Reality? Prof. Dr. Abdul Rahman Abdul Razak
16. Siri Syarahan Inaugural UMT: 16 (2010) Pembangunan Sumber Manusia: Salah Tafsiran Konsep dan
Strategi Luhur untuk Meningkatkan Prestasi Pekerja dan Pencapaian Organisasi
Prof. Dr. Ibrahim Mamat
17. Siri Syarahan Inaugural UMT: 17 (2010) Inovasi Kurikulum Sekolah Menengah: Kes Sekolah IMTIAZ Prof. Dr. Shukery Mohamed
viii •
18. Siri Syarahan Inaugural UMT: 18 (2013) Kepelbagaian Genetik Tanaman: Pemuliharaan dan Kepentingan dalam Bidang Pertanian Prof. Dr. Sayed Mohd Zain S. Hasan
19. UMT Inaugural Lecture Series: 19 (2014) Wastewater Treatment Technology: A Green Application in
Aquaculture Prof. Dr. Ir. Ahmad Jusoh
20. UMT Inaugural Lecture Series: 20 (2014) Cognitive Tools in Social Science Research Prof. Dr. Wan Salihin Wong Abdullah
21. Siri Syarahan Inaugural UMT: 21 (2016) EvolusidanBiogeografiMamaliaMalaysia Prof. Dato’ Dr. Mohd Tajuddin Abdullah
• ix
CONTENT
Acknowledgement xi
Abstract xiii
DOMESTICATION OF ANIMALS 1
DISEASE AND LIVESTOCK 9
VACCINATION STRATEGY IN CONTROLLING ANIMAL 13 DISEASE
THE SYSTEMIC IMMUNE SYSTEM 17
THE MUCOSAL IMMUNE SYSTEM 21
RESEARCH IN MUCOSAL VETERINARY VACCINE 25
CASE 1: PNEUMONIC MANNHEIMIOSIS 29
CASE 2: HAEMORHAGIC SEPTICAEMIA 41
ADVANTAGES OF MUSOCAL VACCINATION APPROACH 61
IMPROVING IMMUNOLOGICAL RESPONSE 65
CONCLUSION 69
References 71
ACKNOWLEDGEMENT
First and foremost, alhamdulillah and praise to Allah SWT for His
guidance and consent to whatever I acquired today. I seek for His
blessing and merciful towards my life and the life of my love ones. I
would like to take this opportunity to express my heartfelt gratitude
to my beloved wife, Dr. Shahnaz Ismail and my adorable children;
Ahmad Irfan Azfar, Nur Irdina Danisyah and Nur Amirah Aqilah for
theirsacrificesandunderstandingonthenatureofresponsibilities
of my work and interest. Their moral supports have been motivating
me to strive for excellence. There is no word or sentences that
could describe my feeling towards their love and patience. The
same sentiment goes to my parents, Hj. Abdul Wahid Ismail and
Hjh. Nyonya Yoon Abdullah for their love and encouragement
that brought me up as I am today. Without their encouragement,
guidance and love, I would not be able to achieve my dreams.
I would also like to thank my teachers, lecturers and mentors,
especially Prof. Dr. Mohd. Zamri Saad who had supervised
my final year project and alsomy PhD degree at the Faculty of
Veterinary Medicine, UPM. I am also indebted to Prof. Emeritus
Dato’ Dr. Mahyuddin Mohd. Dahan, Prof. Emeritus Dr. Sulaiman
Md. Yassin and Prof. Emeritus Dato’ Dr. Ibrahim Komoo for their
believe and trust on my capability to serve the university at various
departments level. Special dedication to all my postgraduate and
undergraduate students that put their trust in me on supervising
their research projects without hesitation. With their assistance and
diligence, our team has completed many research projects with
great achievements.
Thousands appreciation to my friends and colleagues in UMT
or outside the university that have shown their interest and shared
their knowledge in making my remarkable research journey. My
• xiii
gratitude also dedicated to the funding agencies; MOE, MOSTI,
JBiotechCorp, MARDI and UMT, without their kindness I would not
be able to complete all my research works, publishing my papers
injournals,presentingmyfindingsinconferencesandproceedings,
and funding my research assistants and graduate students along this
journey.
Last but not least, my sincere thanks dedicated to Prof. Dato’
Dr. Nor Aieni Mokhtar for her support towards the completion of
this manuscript.
xiv •
ABSTRACT
Animal protein is considered as very important diet in human and
animal. Although some people consume the protein from plant,
animal protein acquires different amino acids profiles that are
readily and rapidly absorbed within the body. Thus the demand is
still higher when compared to plant protein. However the supply
of animal protein can be affected by lack of good farm practices
and disease management. Disease control and prevention is very
important in livestock husbandry. With the emergence of new
pathogenic microorganisms, and increasing incidents of antibiotic
resistance, vaccination is the best prophylactic measure to contain
the infections. With the advancement of technology, different types
of vaccines have been developed. Most of the vaccines available
for livestock animals are delivered through parenteral route, which
includes intramuscular, intradermal, subcutaneous or intravenous
injection. The parenteral route will only induce systemic immune
responses after the reentering of the same antigen species into the
host’s body. Most of these vaccines were designed for the regime
of one dose of injection, without any guarantee of contact with
the same antigen again. If the animals did not encounter the same
antigen again, the level of antibodies will gradually subside due
to the inability of the immune system to sustain sufficiently high
antibody titers against the pathogen, which lead to the failure of the
vaccination program. Part of the failures of systemic vaccination
programs is due to the lack of understanding on the pathogenesis of
the disease, which leads to inappropriate choice of site and route of
administration of vaccine delivery.
The mucosal route has becoming a better alternative for
vaccination delivery system since most of the bacteria need to
adhere to the mucosal surface prior to the colonization and
• xv
disease development. Pneumonic pasteurellosis and haemorrhagic
septicaemia had been used as disease model in studying the potential
of mucosal immune responses toward protection against the disease.
Thecausativeantigensareknowntobepartofnormalfloraatthe
upper respiratory tract of ruminant, but become pathogenic when
the animals are in stress condition and compromised the infection.
The vaccines were delivered onto the mucosal membrane surfaces
through the intranasal route of delivery and induce immune
responses locally and systematically. They also induced immune
responses to the other effector sites that are connected through
the common mucosal-associated lymphoid tissues. The mucosal
route of delivery is suitable for herd health vaccination program
for livestock due to its rapid delivery, time saving, feasibility, and
reduced stress in animals as well as the technician without any
needs for special training. The mucosal membrane surfaces is also
suitable to induce innate immune responses in lower taxonomy
ranks of animal such as fish andmussels with short duration of
protection.Thesefindingsareimportantindevelopmentofprotocol
for other diseases in aquatic animals as well.
Other route of mucosal vaccine delivery are not studied
thoroughly. Our team in UMT has initiated study on the potential
of oral delivery vaccine, which also focus on the gut-associated
lymphoid tissue (GALT) that lining the mucosal surfaces of the
gastrointestinal tract as part of mucosal immune system. This type
of vaccine is not uncommon in birds and chicken vaccination. But
the approach is good for disease management in wildlife and other
straying animals that are not easily restrained during vaccination
program. Innate immunity equipped the mucosal defense system
and has been studied in an animal that has less developed immune
system. We have initiated a study of housekeeping genes that
are responsible to induce innate immune defense in order to
xvi •
understand their role in protection against bacterial colonization.
Themechanism,albeitnonantigen-specificbutasimportantasthe
firstlineofdefenseasitwillalsopreventtheadhesionofpathogen
on the mucosal surfaces thus prevent further pathogenic process.
Thus the new direction in facing newly emerging and re-emerging
disease is to capitalize on the studying the role of this housekeeping
in animal models with established immune system. It does not
requirespecific-antigenvaccinethatwouldneedlongerperiodof
time to produce.
There are huge tropical marine resources available for researcher
to be explored since the location of the university is facing directly
to the South China Ocean. As a start, a search for novel adjuvant
from marine resources has been initiated with those from other
disciplines. This is because most of adjuvants used in the vaccines
are toxic and harmful to human and animal tissues. Preliminary
findingsshowedthattheadjuvantishydrophilicinnaturewithout
harmful effects on tissue cultures and experimental animals. Instead
the adjuvant from marine resources is found to stimulate replication
of macrophages in tissue culture experiment. It also promotes
greaterimmuneresponsesinlabanimalandisreadyforfieldtrial.
• xvii
DOMESTICATION OF ANIMALS
All living things require protein in their diets. Proteins are regarded
as large molecules consisting of series of amino acids that are
important for the body to function properly. Most of the tissues
structures, enzymes, hormones and antibodies are made up of
proteins. Deficiencyof proteins in thebodywill lead to growth
disturbances, shrinkage of muscle tissues and poor immune system
responses that would lead to susceptibility to infections, apathy,
swollen belly and legs due to changes in hydrostatic pressures
which lead to edema and anemia. Lack of proteins will also cause
problem to the nervous system since some of these proteins act
as neurotransmitter, which play an important role in normal body
functions.
Proteins can be obtained from either plants or animals. For
instance, grain, vegetables and legumes are examples of protein
sources that consumed by strict vegetarian. An online survey
conducted by Harris Interactive® on behalf of The Vegetarian
Resource Group, USA in 2009 on 2,397 U.S. adults aged 18
years and older on vegetarian practitioner found out only 8% of
their respondents have never consumed meat protein in their diet
(www.vrg.org/nutshell/faq.htm#poll). On the other hand, majority
of this group of people; especially the semi-vegetarian dieters are
still depending on meat-type of protein in their routine diet. The
data also acknowledge that most of the people around the globe are
still subscribed to seafood, dairy products, eggs, ruminant and non-
ruminant meats for their protein sources.
Otherthanmeatandmilkanimals,fishhasgraduallybecoming
a significant protein source for human. While fishermen still
engagedwiththeseafishingactivities,reportsshowedthatfishing
2 • Mucosal Immunity: Animal Vaccination and Disease Prophylaxis
from the sea reached their maximum potential, where the majority
of the fish stocks being fully exploited. Therefore, aquaculture
farming showed an increasing trend in production whereby the
output from farmedfishandshellfish increasedsignificantly from
13 million tonnes in 1990 to 33 million tonnes in 1999. The inland
and marine aquaculture production now is steadily increased by
10% per year since 1999 (FAO, 2009). Majority of these products
are derived from farms outlets, or at bigger scales from the livestock
industry. The supply for animal proteins would not be possible
without the establishment of livestock farming as what we see today.
Withoutmodernfarming,itwouldbedifficultforhumantofulfill
their meat protein requirement, and for that reason we are indebted
to our forefathers for their efforts on animal domestication about
9,000 to 7,000 years Before Present (BP) ago.
According to history, most of the livestock species raised in the
modern farming were domesticated from the wild. The domestication
activity does bring a lot of advantages as well as disadvantages into
the way of life of the people involved in this industry. Although
scientists have different school of thoughts on the definition of
domestication,generally itcanbedefinedasaprocessbywhich
captive animals adapt to man and the environment he provides
(Price, 1999). The domestication process can be considered as
human intervention with expectation that the phenotype of nature
of these animals will be different from their wild counterparts. As
these domesticated animals need to adapt their new environment,
the changes will also involve at their genetic level over generations,
and make them less sensitive to environmental stimulation during
their lifetime (Price, 1984).
Domestication of Animals • 3
Figure 1: Old Egyptian hieroglyphic painting showing an early instance of a domesticated animal where cattle is being milked. From 1000 Fragen an die Natur, via The Metropolitan Museum of Art, Rogers Fund, 1948
Interestingly,thefirstanimalspeciesthathasbeendomesticated
wasnotafoodanimal.Itwasthewolvesorscientificallyknownas
Canis lupus,aneffectiveandefficientassistantforhuntersduringthe
Late Glacial around 17,000 to 15,000 years BP (Pionner-Capitan et
al., 2011). The wolves were domesticated for food animal hunting
activities,tofulfilltherequirementforproteins.Whenhuntingper
se could not meet the requirement, the domestication of wild food
animals was then started around 12,000 to 10,000 years BP (Figure
1); (Harris, 1996).
It is interesting to know that not every species of wild animals
can be domesticated for food. But the domestication activities of
new species are still continuing until today. The domestication of
wild food animals caused major changes in the way of life of human
society of different levels of society throughout the world ever since.
For instance, a process of changes known as Neolithisation, is a
slow process, that involved a drastic technology shift from hunting-
gathering to food production, based on cultivation and husbandry of
4 • Mucosal Immunity: Animal Vaccination and Disease Prophylaxis
the domesticated animals (Vigne, 2011). Table 1 shows the timeline
estimated for different wild food animals’ species domesticated by
human at different regions of the world.
Table 1: Estimated timeline of livestock domestication (Zeder, 2008)
Animal Species Region Date
Dog Undetermined ~14-30,000 BC?
Sheep Western Asia 8500 BC
Cat Fertile Crescent 8500 BC
Goats Western Asia 8000 BC
Pigs Western Asia 7000 BC
Cattle Eastern Sahara 7000 BC
Chicken Asia 6000 BC
Guinea pig Andes Mountains 5000 BC
Taurine Cattle Western Asia 6000 BC
Zebu Indus Valley 5000 BC
Llama and Alpaca Andes Mountains 4500 BC
Donkey Northeast Africa 4000 BC
Horse Kazakhstan 3600 BC
Silkworm China 3500 BC
Bactrian camel Southern Russia 3000 BC
Dromedary camel Saudi Arabia 3000 BC
Honey Bee Egypt 3000 BC
Banteng Thailand 3000 BC
Water buffalo Pakistan 2500 BC
Duck Western Asia 2500 BC
Yak Tibet 2500 BC
Goose Germany 1500 BC
Mongoose Egypt 1500 BC
Reindeer Siberia 1000 BC
Animal Species Region Date
Turkey Mexico 100 BC-AD 100
Dog Undetermined ~14-30,000 BC?
Sheep Western Asia 8500 BC
Cat Fertile Crescent 8500 BC
Goats Western Asia 8000 BC
Pigs Western Asia 7000 BC
Cattle Eastern Sahara 7000 BC
Chicken Asia 6000 BC
Guinea pig Andes Mountains 5000 BC
Taurine Cattle Western Asia 6000 BC
Zebu Indus Valley 5000 BC
Llama and Alpaca Andes Mountains 4500 BC
Donkey Northeast Africa 4000 BC
Horse Kazakhstan 3600 BC
Silkworm China 3500 BC
Bactrian camel Southern Russia 3000 BC
Dromedary camel Saudi Arabia 3000 BC
Honey Bee Egypt 3000 BC
Banteng Thailand 3000 BC
Water buffalo Pakistan 2500 BC
Duck Western Asia 2500 BC
Yak Tibet 2500 BC
Goose Germany 1500 BC
Mongoose Egypt 1500 BC
Reindeer Siberia 1000 BC
Turkey Mexico 100 BC-AD 100
Domestication of Animals • 5
6 • Mucosal Immunity: Animal Vaccination and Disease Prophylaxis
During the domestication process, animals were usually kept
incaptivity,or/andinopenconfinesystem.Unliketamingpetor
game animal, domestication involves more crucial changes in the
behavioral pattern, physiological and socialization of the animals.
Theseanimalsnormallywillbeselectedbasedonspecificdesired
phenotype of interest, for instance; big body size and faster weight
gain, high reproductive pattern, good temperament, fast maturation
(Mignon-Grasteau et al., 2005; Per Jensen, 2006) and disease
resistance.
In their natural wild environment, these animals are continually
exposed to threats such as hunger, predation, habitat destruction
and limited resources. Most of these animals are more aggressive,
stronger and resistance to disease. These behaviors’ gradually
changed as they were kept in the confine areas (Veissier et al.,
1994). Although the intention of captivity and isolation is to improve
breeding and minimize competition for food, water, mates and
personal space, they became stressful and showedmodifications
in behavioral development (Price and Wallach, 1990). When
the animals’ body is stressed, they will start to compromise with
pathogenic microorganism, thus prone to infection and diseases.
In captive environment, foods are provided at scheduled time,
while water is available ad libitum. Although captivity secured
these animals from predation, proper management system is
crucial for breeding and disease preventive programme. Therefore,
the domestication leads to food security, in term of meeting up
sufficient demand of basic protein requirements for human.
Domestication serves the purpose to harness the animals for work
and transportation, and also producing pets as companion animal
for human (Hansen and Damgaard, 1991). Although domestication
process can be an ample solution for food security issues, it brought
new challenges in the area of disease management.
The development of livestock industry and modern agriculture
farming also contribute to new research discipline i.e. economy,
agronomy, horticulture, breeding, nutrition, animal behavior,
microbiology and immunology.
“Only a handful of wild animal species have been successfully
bred to get along with humans. The reason, scientists say, is found
in their genes.”
- Evan Ratliff
Domestication of Animals • 7
DISEASE AND LIVESTOCK
There are different types of management systems in livestock
farming, such as free ranging system, intensive system, semi-
intensive or extensive system. Regardless whichever types of
livestock management systems practiced in the farm, the farmer
always concern about the potential pathogenic microbes that might
infect their herds. These pathogenic microbes are comprise by
different types of bacteria, viruses, protozoa, parasites or even prion.
The infection or disease would contribute to greater economic
losses due to reduction in production and also mortality if proper
preventive care were not practiced in the farm.
The animals survived from the disease can potentially become
a silent carrier, and harbor the pathogen to healthy animal during
the stress situation. In disease state, the infected animals would not
able to perform their normal physiological functions, thus leading to
poor performances i.e. low meat quality, reduced milk production,
abortion or unable to compete for food and water. Indirectly, their
situation could become worst due to chronic starvation caused by
prolong malnutrition. The predisposing factors such as chilling, poor
ventilation, overcrowding and malnutrition can deteriorate their
health condition and lead to stressful situation, which will make
them prone to infection.
The pathogens might derive from introduction of new batch
or individual animal into the herds, or from those that are just
recovered from a disease but still carry the pathogen. The infection
could also obtain from consuming contaminated food, water,
utensils or surgical equipment and sharing needle during delivery
of antibiotic. Apart from that, diseases can also disseminate by
airborne, which is more crucial when it involves dense populated
10 • Mucosal Immunity: Animal Vaccination and Disease Prophylaxis
farm, artificial insemination practice, and genetics hereditary or
through embryo transfer procedure. The disease could spread and
become infectious with the presence of infected rodents, birds or
flies as an intermediate carrier (Condy et al., 1985). The carrier
animal will not show any clinical signs but will harbor the pathogen
in their body. They will start shedding the pathogen from their body
only when they are in stress or at initial stage of infection, without
showing any obvious clinical sign.
It is imperative to treat the animal that are down with any
particular disease at this stage, and depends on the etiological agent;
proper disease management should be imposed as early as possible.
Antibiotic for instance, is the treatment of choice by the farmer to
terminate the course of disease. There are different types antibiotic
used to treat diseases that depends on whether the bacteria is gram-
positive or gram-negative types, while anti-viral is used to handle
DNA- or RNA-types of viral pathogen. But all these measures are
taken after the animals have been infected or in a disease state.
When dealing with certain contagious disease, for instance
brucellosis; the infected livestock usually will not be subjected to
any further treatment. They will be culled or destroyed through
mass killing and sometimes burning. Their milk products will be
banned from entering markets of many countries. This sanction
will cause market shocks when the supply suddenly drops and
disrupt international trade of livestock and their products. A good
classic example is the outbreak of rinderpest in 1890s that has
killed approximately 80% of cattle in southern Africa and caused
widespread starvation in the Horn of Africa. Rinderpest was then
reemerged in 1980s and killed estimated 100 million cattle in Africa
and West Asia.
It took a long time to eliminate the disease from the livestock
world map. The highly pathogenic avian influenza (HPAI) that
begins around 2003/2004 resulted in market shocks in a number of
countries that witnesses the loss of 250 to 300 million poultry from
the market (McLeod, 2009). Fortunately the recovery was rapid at
global market level and the effects on food security are limited. In
countries like Malaysia and Brazil, Foot and Mouth Disease (FMD)
has been a threat to cattle and buffaloes. The causative agent is a
virus known from the family of Picornaviridae.
Whenever the outbreaks occur, there will a ban from other
countries for our meat and by products from our livestock. When
Brazil was hit by FMD outbreaks in 2005, some parts of their
country lost exports market. They managed to compensate internally
and the export market shares bounce back (FAO, 2006). The best
feasible approach of livestock farming disease control is usually by
implementing specific preventivemeasure. Diseased animals are
always considered as liability to the operators and impose extra
management costs in term of time and manpower to control them
from spreading to other animals. They best way to prevent the
disease from infecting the animals are through proper vaccination
programme.
Disease and Livestock • 11
VACCINATION STRATEGY IN CONTROLLING ANIMAL DISEASE
It was evident that the science of vaccination was born with Edward
Jenner’s discovery of smallpox vaccine in the 1700s. It took another
150 years to understand the whole principles behind this new
science of vaccination. The discipline of vaccinology has fully
beenshaped in thepastfivedecades (Andre,2003). Oneof the
major achievements in this discipline was the global eradication
of smallpox endorsed by the World Health Assembly in 1980. On
contrary, vaccination approaches in animal are different than in
human.
Differ from human and companion animals, the veterinary
vaccination strategy would focus mainly on herd health approach.
This is simply because the animals are usually raised in a group
or herds, and it is often time and labour consuming to vaccinate
the herds by individual approach. Most of the time, veterinary
vaccination is focus on monitoring the herd health and welfare,
as well as to ensure sufficient food production and potection
of public health. The reasons are simply because vaccination is
a cost-effective way to deliver safe and sound food supply with
highefficiency,andatthesametimepreventthezoonoticdisease
transmission and foodborne infection to human (James, 2011).
Imagine how will the veterinarian execute a vaccination
programme in a farm with more than 2000 animals with individual
injection approach? If he decided to vaccinate the herds in batches,
the animals would become stressful due to restraining and handling
by the workers during the vaccination process. Another critical
issue is whether the farm have enough manpower to conduct the
whole programme since they need to deal with a huge number of
animals.Thefishfarmoperatorsalsofacethesameproblemwhere
14 • Mucosal Immunity: Animal Vaccination and Disease Prophylaxis
thefishesareusuallykeptindifferentpondsorhatcheriessincethey
can easily distress by any untoward distraction.
The form of herd immunity occurs when the vaccination of a
significant percentage in a flock (or herd) provides ameasure of
protection for individuals that have not developed immunity (John
and Samuel, 2000). The theory behind herd immunity proposes
that the chains of infection are likely to be disrupted when large
numbers of population are immune or less susceptible to the
disease. In other words, the higher the proportion percentage of
resistant individuals in a the herd, the lower the probability that a
susceptible individual will come down with the disease (History
and Epidemiology of Global Smallpox Eradication From the training
course titled “Smallpox: Disease, Prevention, and Intervention”. The
CDC and the World Health Organization. Slide 16-17).
The next consideration would be the type of vaccine to be
delivered; whether they are in the form of attenuated (live) vaccine,
live vector vaccine, killed (whole pathogen) vaccine, DNA-
based vaccine, conjugate vaccine, recombinant vaccine, subunit
vaccine etc. Should the vaccine be delivered intramuscularly,
subcutaneously, intradermal, orally etc.? Why is it important to know
thetypeofvaccinethatwearedealingwith?Whatisthesufficient
dosage and regime, method of delivery or whether a booster is
needed etc. for effective vaccination? The aspect of feasibility and
frugality are the factors taken into consideration when one has to
vaccinate large population of livestock animals. This would assist
them to evaluate whether it is more economic to cull the affected
batch in the population or offer a treatment in case of disease
outbreakinadjacentareas.Whataboutiftheherdisaflockofwild
free ranging animals? What if the vaccine or vaccination delivery
method did not provide good protection to the herd? What if the
recipient shows adverse effects following vaccination programme?
Most of the veterinary vaccines in the market employ the
systemic type of delivery. This type of vaccine uses parenteral
route of administration, either through injection into muscular
area, subcutaneous, intradermal, intravenous or intraperitoneally.
The aim of this type of vaccine is to induce immunity at systemic
level. This systemic immunity only stimulated when the antigen
successfullyovercometheso-calledthefirstlineofdefense.
Vaccination Strategy in Controlling Animal Disease • 15
THE SYSTEMIC IMMUNE SYSTEM
Thefirstlineofdefenseisreferringtonon-specificimmunesystem,
which refer to physical barrier against invasion of viruses, bacteria
and parasites (Figure 2). The main physical barrier is the epithelium
that line the external body surfaces, and mucous membranes that
line the digestive, respiratory and reproductive tracts, measures
about 400 square meters or as big as two single-tennis courts!
(Lauren, 2008). The epithelium and mucosal membranes arm with
structures like hairs that trap the dust and dirt, microscopic cilia that
line some mucous membranes and direct foreign particles out of the
body;gastricjuice,vaginalsecretionsandurinewithacidicfluids
with protective function; and tears, sweat and saliva that posses
some anti-bacterial properties.
Figure 2: The non-specific defenses of 1st and 2nd line of defenses, and specificdefensesofthe3rd line of defenses
18 • Mucosal Immunity: Animal Vaccination and Disease Prophylaxis
Whenthepathogenmanagetoovercomethefirstlineofdefense,
it will encounter the second line of defense (Bona and Bonilla,
1990) that includes defensive cells (mostly are phagocytic cells).
In the second line of defence, the host body deploys different types
ofmechanismtocombact the invader, for instance, inflammatory
reactions; fever mechanism; and antimicrobial substances (Tortora
et al., 2010). Among the cells that perform at second line of defense
are granulocytes (neutrophils, basophils and eosinophils) as well as
agranulocytes (monocytes, dendritic cells - derived from monocytes;
involve in phagocytosis and initiation of adaptive immune response,
lymphocytes such as Natural Killer (NK) cells, T cells that involve in
cell-mediated immunity and B cells that function as descendants of
B cells (plasma cells) that produce antibodies).
The defensive cells, which consist of leukocytes, will perform
phagocytic activities in supporting the adaptive immunity by
stimulating the T and B cells. These cells act in concert but
withoutproducinganyspecificantibodyresponse.Ifthepathogen
successfully defeated the second line of defense, the third line of
defensewillbeinitiatedwhichinvolvethespecificimmunesystem
that serve as a complex barrier to challenge the infection (Eales,
2003). The defense mechanism comes in concert with the second
lineofdefensebutwithspecificresponsethatwillleadtoprimary
antibody production following the identification of the pathogen
as non-self foreign molecules by the body (Campbell and Reece,
2002).
The immune system is activated whenever the non-self foreign
molecules overcome the second line of defense and invade the host
body. The system consists of a complex organization of organs and
cells that able to recognize the pathogen and induce an immune
response against it. The substance that are capable of inducing an
immune response is known as an antigen or an immunogen. The
antigen also includes their toxins or enzymes that are produced by the
pathogen itself. Then, certain cells known as the antigen-presenting
cells will trap the antigen and present it to the lymphocytes. The
lymphocytespossesreceptorsspecificforthatantigentobindtoit.
The binding process will activate the lymphocytes will secrete
variety of cytokines that promotes the growth and maturation of
other immune cells i.e. cytotoxic T lymphocytes. The cytokines
also act on B cells, which will then stimulating them to divide
and transform into antibody secreting cells. The non-self foreign
pathogen is either killed by the cytotoxic T cells or neutralized by
antibodies. The whole process of inducing the immune response is
regarded as immunization process.
The primary antibody response is less protective and does not
linger in the system for long period of time (Elgert, 1996). Generally,
the primary antibody response can be divided into four phases; the
firstoneisthelagphase,whichrefertothedurationoftimepassed
before the antibody can be detected in the serum. Normally the lag
phase will take about one to two weeks post-exposure. The duration
will also depends on the species being immunized, the antigen
involved and other factors as well (Coico et al., 2003). Second is
the log phase, which antibodies concentration in the serum rises in
logarithm manner. Then the stable phase, which is indicated when
the production and deterioration of antibodies are the same. The
declining phase starts when the concentration of antibodies in the
serum declines rapidly (Coico et al., 2003).
Although the production of antibodies may cease completely
within a few weeks, the immunized host will establish a long
lasting cellular memory cells, or immunological memory (Coico
et al., 2003; Elgert, 1996). The secondary antibody response due
to invasion of the same non-self foreign pathogen will triggered
instantaneous recognition and rapid responses, thus clearance of
The Systemic Immune System • 19
20 • Mucosal Immunity: Animal Vaccination and Disease Prophylaxis
the antigen effectively (Brigham, 2003). The log phase in secondary
antibody response takes shorter time and the antibodies can be
detected half the time required for primary antibody response. The
concentration of antibody is much more higher and measurable in
theserum.Moreover,theproductionofantibody,whichisspecific,
may continue for longer period of time at constant level in the serum
for months or even years later.
In systemic vaccination programme, the process is similar to
the third line of defense. But by the time, the antigen-antibody
responses develop; the damage has already been done to the host’s
tissue by the harmful substance released by the pathogen. On
contrary,themucosalimmunitymimicsthefirstlineofdefenseand
prevents the adhesion of the pathogen at the site of attachment from
the beginning.
THE MUCOSAL IMMUNE SYSTEM
Themucosalsurfacesprovidethefirstlineofdefenseandfunctionas
physical barriers that separate the hostile external environment from
the internal. As mentioned earlier, the hostile environment contains
diversity of microorganism with capability of colonizing and
invading the epithelial surfaces with their virulence mechanisms.
Some of these microorganisms are non-pathogenic in nature i.e.
commensal or normal flora, food- and airborne antigens but still
requireanappropriateresponsefromthehostnon-specificimmune
system to eliminate them (Aguilera et al., 2004). Apart from that,
the mucosal membrane also allows critical nutrients, oxygen and
other molecules to constantly across the barrier (Orga et al., 2001).
The daily routine tasks provide a great challenge to the mucosal
membrane in order to determine the potentially harmful from the
beneficialones(Orgaet al., 2001; Rosenthal and Gallichan, 1997).
The mucosal immune system is composed of the lymphoid
tissues that are associated with common mucosal surface (MALT or
mucosa-associated lymphoid tissue), which is recognized as the main
hub of the mucosal immune system that integrated through other
local lymphoid tissues, such as; NALT or nasopharinx-associated
lymphoid tissue (nostril area), BALT - bronchus-associated lymphoid
tissue (pulmonary or lung area), GALT - gut-associated lymphoid
tissue (gastrointestinal area), the mammary and salivary glands, as
well as the genitourinary organs (vagina) (Kelsall and Strober, 1996;
Greshwin et al., 1995).
The mucosal immune system has developed two layers
of adaptive non-inflammatory defense: (i) immune exclusion
provided mainly by the secretory antibodies to limit contact and
penetration of any microorganisms, and harmful pathogens; and
22 • Mucosal Immunity: Animal Vaccination and Disease Prophylaxis
(ii) immunosuppressive mechanisms to inhibit overreaction against
harmless luminal agents (Brandtzaeg, 2008). To assist the mucosal
immunesysteminconductingitstaskefficiently,themucosaltissues
are heavily populated with immune cells. The intestinal mucosal
lining, for instance, contains more lymphoid cells and produces
more antibodies than any other organs (McGhee et al., 1992).
Figure 3: Structure of the mucosal immune system comprises of mucosal inductive site and mucosa effector sites with their cells (Brandtzaeg et al., 2008). The structure of mucosal immune system shows it’s great potential as a route of vaccine delivery for herd health programme in livestock industry. Most of the pathogen will need to adhere to the mucosal sites priortothecolonizationandinvasionprocess. Theimmunityat thefirstcontact point at the mucosal site will provide better protection especially when mass vaccination is needed. Research on the effectiveness of the mucosal vaccination has been conducted to prove that the impact of immune responses can protect the animals from the infection and disease
The mucosal immune system is composed of the mucosal
effector site and the mucosal inductive site (Figure 3). Immune
responses in all effector tissues might be stimulated when one of
the mucosal inductive site is being immunized. Higher antibody
concentration might be detection at the site of exposure following
the stimulation (Orga and Karzon, 1969; Haneberg et al., 1994).
These inductive sites comprise secondary lymphoid tissue, where
naive immune cells are generated and, memory and effector cells
being produced (Brandtzaeg, 1998). The secondary lymphoid
tissues contain immune cells such as IgA class switching and
clonal expansion of B-cells, which occurs in response to antigen
specificT-cellactivation.Afteractivationand IgAclass switching,
T- and B-cells migrate from inductive sites to effector sites. Effector
sites are present in all mucosal tissues as disseminated lymphoid
tissue diffusely distributed throughout the lamina or substantia
propria in the gastrointestinal tracts (Yan et al., 2003). In effector
sites, secretory IgA or S-IgA (2 IgA molecules joined by a J-chain
and bound to secretory component, an epithelial cell membrane
receptor) is secreted across the mucosal epithelium (Pabst, 1987).
The Mucosal Immune System • 23
RESEARCH IN MUCOSAL VETERINARY VACCINE
Most of the veterinary vaccines in the market employ the systemic
type of delivery. This type of vaccine uses parenteral route of
administration, either through injection into muscular area,
subcutaneous, intradermal, intravenous or intraperitoneally. The
aim of this type of vaccine is to induce immunity at systemic level.
The systemic immune system is only stimulated when the antigen
had successfullyovercome the so-called thefirst lineof defense.
Apart from the parenteral route, vaccines can also be administered
through the mucosal lining routes, for instance nasal, oral, vaginal
and rectal (Montilla et al., 2004). Most of the vaccines in birds are
delivered through mucosal route, for instance intranasal drops for
Newcastle Disease Vaccine or through drinking water. The vaccine
usually contains attenuated virus, which replicates at the site of
inoculation at reduced strength.
It is important to learn how the pathogen infecting the host
in order to develop a vaccine and the dosage and regime for its
efficacy.Thisshouldbetheendinmindofeveryvaccineresearcher
since effectiveness of the vaccine will depend on how the immune
system responds to it. Apart from that, the strain of the pathogen
usedasthevaccineseedmustbespecificwiththeparticularstrain
that cause the disease. Most of the imported veterinary vaccines
did not work effectively in this country because they employed
different strains of antigen from the local host, thus raising the issues
ofcompatibilityandefficacy.Someoflocalvaccinesproducedin
the country did not work effectively due to the regime or the type of
excipient used as adjuvant in the vaccine formulation.
26 • Mucosal Immunity: Animal Vaccination and Disease Prophylaxis
In Malaysia, most of the livestock animals are raised either in
free range, semi intensive or intensive system. The intensive system
usually run by the big scale producers with proper management
system while the medium and small-scale producers handle semi
intensive system. These types of farms have their own herd health
system that comprises of debudding, dehorning, deworming,
vaccination and antibiotic treatment. Animals are monitored
with recorded tagging numbers individually and undergoing
proper management system. Since their management system
is organized with enough number of operators, disease usually
occurred seasonally i.e. during stress condition due to pregnancy
and eventually labor, transportation, parasitism, introduction of new
animals (Jasni et al., 1991; Zamri-Saad et al., 1991) or concurrent
diseases (Buddle et al., 1990; Zamri-Saad et al., 1994).
When vaccination programme is timely, the farm will need to
allocate sufficientmanpower to run the activity smoothly. For a
small farm with 2,000 heads of animals, 4 days should be allocated
to deliver an injectable vaccine. It is indeed time consuming and
cause stressful condition to the animals as well as the operators
that need to restrain the animals properly. There are side effects of
injectable type of vaccination such as temporary lameness due to
incidental nerves injury, abortion and loss of appetite. The rule of
thumb for injectable vaccination is one needle for one animal, but
the farmer usually will use the needle for more animals until the
needles are blunt. Apart from injuring the site of injection, there
is possibility to transfer infection from infected animal to others by
sharing needles. Some farmers purposely re-use the needles just
to reduce the operational cost. Other system practiced in Malaysia
and Southeast Asia countries is backyard husbandry. The animals,
particularly ruminant, such as sheep, goats, cattle and buffaloes
are let loose to stray from one place to another foraging for grass.
Theseanimalsrepresentsignificantnumbersoflivestockintermof
exposure to disease in endemic areas, or carrier for certain disease,
orevenhost forzoonoticpathogen. It isdifficult togetclose to
these animals especially when vaccination is required. Due to these
obstacles, vaccination programme becomes difficult and animals
are prone to diseases.
But injection approach is not the only way to deliver a vaccine.
The important thing is to look for suitable strategy for vaccination
programme.Thesolutionistofindthewaytodeliverthevaccine
moreeffectivelywithhighefficacyespeciallywheninvolvecrowds
of animals with different age groups and condition. Can we deliver
the vaccine through oral or intranasal route of administration?
Both are consider as mucosal immunity approach, which elicit the
local immunity or humoral type of antibody. To prove the ability
of inducing mucosal immunity system, few studies had been done
using white rats, goats, sheep and buffaloes as animal models and
few bacteria as pathogen model.
Research in Mucosal Veterinary Vaccine • 27
CASE 1: PNEUMONIC MANNHEIMIOSIS
Pneumonic mannheimiosis is one of the most common respiratory
diseases of small ruminant worldwide. The disease is cause either
by Mannheimia haemolytica type A or Pasteurella multocida types
A and D (Gilmour, 1980). In our country, 70% of the disease in
sheep and goats has been associated with M. haemolytica type
A (Jamaludin, 1993) and 30% of the bacterium isolated from
pneumonic lungswas identified asM. haemolytica serotype A2.
Vaccination is the best control measures for the disease, but the
protection provided by several commercial mannheimia vaccines
was uncertain (Wan Mohamed et al., 1988). Several factors have
been recognized as the caused for vaccination failures, for instance
the route of administration, the antigen and the adjuvant used in the
vaccine (Bahaman et al., 1991; Mosier, 1993).
The pathogen can be found in the upper respiratory tract of
healthy animals and considered as normal flora or commensal
bacteria. Isolations have also been made from nasopharynges,
tonsils and lungs of sheep and goats (Dungworth, 1985). The
bacterium isolated from nasal cavity of healthy sheep and goats
are said to be less pathogenic and will become virulent before it
invade the lungs during the immune-compromised condition. It
will eventually colonize the upper respiratory before lung lesions
developed (Gonzalez and Maheswaran, 1993). These virulence
factors include leukotoxin (LKT), lipopolysaccharide (LPS),
adhesins, capsule, outer membrane proteins, and various proteases.
The toxin is enhanced by lipopolysaccharide, which is associated
withthereleaseofproinflammatorycytokinesfromtheleukocytes
(Sutherland, 1985; Sutherland and Donachie, 1986). The toxin will
eventually produce effects such as neutrophil membranes rupture,
30 • Mucosal Immunity: Animal Vaccination and Disease Prophylaxis
production of numerous vesicles within the cytoplasm, pyknotic
nuclei and cell cytolysis (Singh et al., 2011; Berggren et al., 1981)
LPS is also known as endotoxin and is part of the outmost layer
of the outer membrane in gram-negative bacteria (Figure 4). LPS will
induceinflammatoryreactionsinlungsandaffectingthesurfactant
in the alveolus that will lead to reduction of surface tension at the
air-alveolar interface in the alveolus (Brogden et al., 1987). This
reaction might lead to the collapse of the alveolus structure, which
is irreversible in nature. The capsule of M. haemolytica also play
important role in inhibition of phagocytosis and intracellular killing
this enable it to establish itself in the lungs (Confer et al., 1990).
Figure 4: Location of lipopolysaccharides (LPS) in the gram negative bacteria. (Courtesy from John Wiley and Sons)
As been mentioned earlier, imported vaccines, which employ
the parenteral route of delivery, had been used to control the
disease in local sheep and goats with limited success rate (Wan
Mohamad et al., 1988; Chandrasekaran et al., 1993; Zamri-Saad et
al., 1993). The Veterinary Research Institute in Ipoh has formulated
an oil adjuvant vaccine with formalin-killed of whole organism of
local strain of M. haemolytica and P. multocida for intramuscular
injection but the vaccine did not shown significant success in
controlling the disease.
However, another experiment using the improved oil-adjuvant
vaccine incorporated with formalin-killed M. haemolytica A7 and P.
multocida types A and D showed better protection when challenged
against either M. haemolytica A2 or A7 (Zamri-Saad et al., 1993).
Unfortunately, due to the thick viscosity of the oil adjuvant, it was
verydifficulttodeliverthevaccinebyintramuscularinjectiontothe
big herds of animals. Furthermore, the adjuvant caused swelling
and painful at the site of injection, and lameness with uneventful
recovery approximately ten percent of vaccinated animals
(Jamaludin, 1993). The locally produced oil-adjuvant vaccine was
not popular among farmers (Jamaludin, 1993) thus resulted in high
incidence of the disease in many farms in Malaysia.
The study conducted by Jasni et al., (1990) involving six
farms over a 5-year period in Malaysia showed average monthly
percentage of deaths due to pneumonic pasteurellosis between
31.5% and 32.4% annually. It is also discovered that the highest
and lowest incidence of the disease correlated well with the highest
and lowest rainfall. The changes in temperatures during the season
changes have caused stressful condition to the animals. Improper
vaccination regime has also contributed to low serological antibody
titers in30% to80%of animals infive farms (Zamri-Saadet al.,
1993), due to the unpopularity of the vaccines among farmers
(Jamaludin, 1993).
M. haemolytica is part of the normal flora in the upper
respiratory tract of the animals and inhalation of large numbers
of the bacterium into the lungs will affect or initiate the infectious
Case 1: Pneumonic Mannheimiosis • 31
32 • Mucosal Immunity: Animal Vaccination and Disease Prophylaxis
process at the mucosal surfaces (McNeilly et al., 2008). Mucosal
immune system serves as an important component of an effective
immune response of the animal. Consequently, there is increasing
interest in the development of vaccines, which generate robust
mucosal immune responses. M. haemolytica uses the respiratory
route to cause the infection (Gilmour et al., 1991) which is armed
with lymphoid tissues of mucosal immunity (Kaltrieder, 1976). The
firstlineofcontactofthebacteriumandhostisalongthemucosal
membrane lining the epithelial of the respiratory tract, and the best
way to prevent the colonization is by stimulating the pulmonary
lymphoid tissue that will provide substantial local cell-mediated
and humoral immune responses (Kaltrieder, 1976).
Pulmonary lymphoid tissues arm the mucosal epithelial lining
with second line of defense should be the mucociliary clearance
mechanism fails to brush off the bacterium from upper and lower
respiratory tracts. The lymphoid tissues associated with the airways
of the lungs known as bronchus-associated lymphoid tissue (BALT)
has been described (Beinenstock et al., 1973; Anderson et al., 1986).
Good understanding of the pulmonary lymphoid tissues to stimulate
the local respiratory immunity is crucial. The lower respiratory tract,
which is the functioning part for oxygen-carbon dioxide exchanges,
is always in sterile condition. The wrong attempt in stimulating the
BALT directly by introducing any foreign materials in this sterile area
would cause pneumonia or bronchopneumonia that can be fatal to
the animal.
The best option was to induce the BALT indirectly through the
route of oral delivery, which is the gut-associated lymphoid tissue.
Thefirstattemptwasdonebyinoculatinganoraladministrationof
formalin-killed of M. haemolytica after determination of the dosage
at the strength 106 CFU/mL. To maintain the antibody response,
the second booster was delivered in similar method at two weeks
interval.Theresultsshowedthatsignificantnumberofnodulartype
of BALT (Table 2) in the treatment groups compared to the negative
control group (p<0.01) (Effendy et al., 1998). Although the size the
BALT was significantly increased, the increment in secreting-IgA
producedbytheBALTcellswerenotsignificant(Table3).
Table 2: The number of bronchus associated lymphoid tissue (BALT) and their average size in the lungs of goats following different treatments. a,b,c figureswithsamesuperscriptarestatisticallyinsignificant(p>0.01)
Group Nodular BALT Aggregate BALT
Number Size(μm2) Number Size(μm2)
1 17.3+2.5b 592.5+390.0e 11.3+1.5f 187.5+197.8g,h
2 2.33+3.2c 685.5+577.8e 10.7+8.6f 350.5+335.8h
Table 3: The effects of oral administration of formalin-killed Mannheimia haemolytica A2 on the total number of cells and the percentage of IgA-producing cells in BALT of goats. a,b,cfigureswithdifferentsuperscriptdiffersignificantly(p<0.01)
GroupAverage number of cells in BALT
Percentage of IgA-producing cells in BALT
1 1269±963c 12.05±2.36d
2 953±1014c 15.35±3.73d
Delivery of M. haemolytica A2 vaccine through oral route is
consideredtheeasiestmethodinanimal.Intheveryfirstexperiment
to study the response of mucosal-associated lymphoid tissues
(MALT) however, we found that oral route delivery was unable
to stimulate significant changes in the gut-associated lymphoid
tissue (GALT). But the results did show that the nodular type of
BALT was significantly increased following the oral inoculation,
which indicated promising result for mucosal immunity study.
Case 1: Pneumonic Mannheimiosis • 33
34 • Mucosal Immunity: Animal Vaccination and Disease Prophylaxis
The complexity of the digestive tract of ruminant actually is more
challenging compared to monogastric animal, and more studies is
needed to explore this new potential for ruminants. Interestingly,
the results also subscribe to the general believe that immunoblasts
in the lamina propria of gut will be transit to the lymphatic channel
to the blood circulation and seed to selected mucosal sites, mainly
canalsoinfluencetheirhomingproperties(JanHolmgrenandCecil
Czerkinsky, 2005).
The potential of intranasal cavity as a site for drug delivery has
been studied by few research groups (Aungst and Rogers 1988).
Pathogen-specific and other antibodies, particularly of the IgA
isotype, are found commonly in secretions of upper respiratory
tracts in health and diseased animals (Duncan Hannant, 2002).
The predominance of these antibodies is partly due to high rate of
IgA isotype switching in antibody secreting cells and their selective
localization and proliferation at mucosal effector sites (Husband et
al., 1999). Intranasal administration of live and dead M. haemolytica
A2 wasdoneonfivegroupsofgoats;Group1and2wereexposed
once to live and dead organism respectively, Group 3 with double
exposures of live organism at two weeks interval, Group 4 with
the same protocol with Group 3 but with formalin-killed organism
while group 5 remained as control untreated group.
The lungs tissues from each group were also collected into
Hank’s nutrient media for in-vitro colonization study. The results
showed that goats exposed to double intranasal spray of live or dead
M, haemolytica A2 at two weeks interval exhibited significantly
(p<0.05) larger size of nodular BALT (Table 4) (Effendy et al., 1998).
Table 4: Average size and number of bronchus-associated lymphoid tissue (BALT) in the lungs of goats following either a single intranasal exposure to live (Group 1) or dead Pasteurella haemolytica A2 (Group 2) or a double intranasal exposures to live (Group 3) or dead Pasteurella haemolytica A2 (Group 4) while Group 5 is control untreated. a,b values with same superscriptwithineachcolumndonotdiffersignificantly(p>0.05)
Group(n=20)
Average size of nodular BALT (mμ2)
Average size of aggregate BALT (mμ2)
Average number of BALT
1 1536+137a 392+50a 22+3.2a
2 1108+106a 284+10a 16+2.4a
3 3536+145b 311+28a 37+5.3b
4 4026+324b 336+51a 36+5.1b
5 1365+40a 385+88a 18+2.5a
The in vitro colonization study showed that despite the increment
secreting-IgA cell numbers were not significant, lung tissues of
treatment group showed less colonization of M. haemolytica during
the challenged (Table 4) (Effendy et al., 1998). After 4 to 6 hours
of colonization study, the lungs exposed to formalin-killed M.
haemolytica A2wasmarkedlyandsignificantly(p<0.01)lesssevere
colonization compared to others.
It is important to know whether the changes in sizes of BALT
alsorelateswith the levelofantibody in lung lavagefluidand in
the serum following the stimulation of mucosal immunity of the
respiratory tract by formalin-killed M. haemolytica A2. The same
experimental design was conducted to study the levels of IgM, IgA
and IgG using the same regime and dosage with formalin-killed M.
haemolytica A2.
Case 1: Pneumonic Mannheimiosis • 35
36 • Mucosal Immunity: Animal Vaccination and Disease Prophylaxis
1 2 4 6
Hours Post Inoculation
0
1
2
3
4
5C
olon
isat
ion
Scor
eGroup 1 Group 2 Group 3 Group 4 Group 5
Figure 5: The colonisation of Pasteurella haemolytica A2 in the lung tissues of goat with different treatments. Group 1 and 2 were intranasally exposed once to live or dead P. haemolytica A2 respectively while groups 3 and 4 were intranasally exposed twice to live or dead P. haemolytica A2 respectively
Following intranasal exposure to formalin-killed Pasteurella
haemolytica A2, the IgA level against Pasteurella haemolytica
A2inlunglavagefluidincreasedrapidlyandsignificantly(p<0.05)
asearlyas1weekpost-firstexposuretoformalin-killedPasteurella
haemolytica A2. The IgA levels remained significantly (p<0.01)
high compared to those of control-unexposed group throughout the
6-week study period, reaching a peak level at week 4 post-second
exposure (Figure 5). The IgM levels against Pasteurella haemolytica
A2 were slightly lower than those of IgA, and the increasing rate
was intermediate following intranasal exposure. The IgM reached
significantly(p<0.05)highlevelatweek1post-secondexposure
andremainedsignificantly(p<0.01)highforanotherweekbefore
thelevelsstartedtodeclinethereaftertoinsignificant(p>0.05)levels
compared to the control unexposed group.
B
1 2 3 4 5 60
0.1
0.2
0.3
0.4
0.5
0.6
0.7
IgA (control) IgA (exposed) IgM (control) IgM (exposed) (IgG (control) IgG (exposed)
Figure 6:Antibody responses in the lung lavage fluid of goats followingintranasal exposures to formalin-killed Mannheimia haemolytica A2
At the beginning of the study, IgG levels against Pasteurella
haemolytica A2werelowestinthelunglavagefluidamongthethree
immunoglobulins. Following intranasal exposure, the increasing
patternofIgGlevelswasgradualandinsignificant(p>0.05)initially
before they reached significantly (p<0.01) high levels toward the
end of the study period, at week 2 post-second exposure (week 4)
onward (Figure 6). The increased level of secreting-IgA prevented
further bacterial adhesion and colonization while the increased of
IgG level acts as opsonizing antibody in the lungs (Zamri-Saad et
al., 1999b).
The serum IgM and IgG responses were similar to those of
lunglavagefluid(Figure5.2).Therewasnoresponseshownbythe
IgM throughout the entire study period, except at week 4 after the
start of the trial or week 2 post-second exposure, which showed a
markedlyandsignificantly(p<0.01)highIgMlevelcomparedtothe
control unexposed. The high level, however, lasted only a week
Case 1: Pneumonic Mannheimiosis • 37
38 • Mucosal Immunity: Animal Vaccination and Disease Prophylaxis
(Figure 16). Similarly, the serum IgG levels in the exposed animals
increased gradually compared to the control unexposed to reached
significantly (p<0.01) high level atweek2 post-second exposure
before declined slightly the following week and increased again on
week 4 post-second exposure (Figure 5.2). The IgA levels, however,
remained similar to those of control unexposed. The differences in
the IgA levels between the exposed and unexposed animals were
insignificant(p>0.05)throughoutthestudyperiod(Figure7).
1 2 3 4 5 6
WEEKS POST-EXPOSURE
0
0.1
0.2
0.3
0.4
0.5
0.6
OP
TIC
AL
DE
NS
ITY
(OD
)
IgG (control) IgG (exposed) IgM (control) IgM (exposed)IgA (control) IgA (exposed)
Figure 7: The serum antibody responses following intranasal exposures of goats to formalin-killed Mannheimia haemolytica A2
Thefinalexperimentwastoconductaclinicaltrialinafarmto
determine whether the intranasal spray vaccine is able to protect the
sheep against pneumonic mannheimiosis. Two farms located at the
East Coast and West Coast of Peninsular Malaysia were selected for
the study. Prior to the start of the experiments, high mortality rates
at about 60% due to pneumonic pasteurellosis during the rainy
seasons from October to December in East Coast and March to May
in West Coast were observed in both farms respectively. At the end
of experiment, the mortality rates showed 52% reduction to only
1% to 3% annually. The 52% reduction of the annual death rate
indicatedgoosefficacyofthevaccine(Effendyet al., 1998).
Figure 8: Mortality rates before and after vaccination programmes in two farms
Under experimental conditions, the efficacyof the intranasal
mannheimia spray vaccine was between 95% to 98%, which
obtained within 2 weeks post-vaccination (Effendy et al., 1998a,b)
and lasted for a period of 12 weeks (Effendy et al., 1998c). The
vaccine also induced high level of IgAs in the lungs and nasal
mucosa, which prevented the colonization of the respiratory tracts
(Effendy et al., 1998a). The rainy season normaly will cause stress
to the animals and affects the host immune defence against the
disease. The field trials showed that rainy season did not affect
the protection provided by the mucosal route of vaccine delivery
and this is evidence by the reduced mortality rates during the rainy
seasons (Figure 8). Findings from this study have pioneered other
study on mucosal vaccination either through oral or intranasal
routes.
Case 1: Pneumonic Mannheimiosis • 39
40 • Mucosal Immunity: Animal Vaccination and Disease Prophylaxis
CASE 2: HAEMORHAGIC SEPTICAEMIA
Haemorhagic septicaemia is one of the diseases of cattle and
buffaloes caused by Pasteurella multocida B2 in many countries
in the world, particularly Asia. The pathogen is known to be host
specific and can be isolated from ruminant and non-ruminant
animals in endemic areas in East Coast of Malaysia (Effendy, 2004).
The disease can be induced by stressful condition, which will then
cause the outbreaks. In this country, outbreaks of the disease have
lead to annual loss of approximately RM2.4 million (Joseph, 1989).
Vaccination is the most feasible way to control the disease and has
been practiced since 1928 while research activities on control of
haemorrhagic septicaemia in Malaysia was carried out since 1960s
(Zamri-Saad, 2005). In 1978, Chandrasekaran and Yeap developed
an oil-adjuvant vaccine to control the disease but the outcomes
were uncertain. This is because the vaccine has thick viscosity and
difficulttodeliverwheninjectingtheanimalsindeepmuscles.This
leads to low vaccination coverage (Saharee and Salim, 1989) since
the farmers were not favored the type of vaccine. Between 1977 to
1987, approximately 42,000 deaths were recorded that amounted
to a loss of approximately RM38 million in Malaysia (Joseph, 1989).
ItisalsodifficulttocontrolthediseaseinMalaysiasincethedisease
is endemic in Malaysia and other countries too.
The same approach has been considered in formulating
a mucosal vaccine for oral and intranasal route of delivery. The
other form of vaccine, the lyophilized form of formalin-killed P.
multocida B2 was also studied. This also includes the study on the
suitable animal’s age to start the intranasal vaccination programme.
Goats have been used as model animals to study the disease since
they are susceptible to P. multocida infection (Sirous Sadeghian
42 • Mucosal Immunity: Animal Vaccination and Disease Prophylaxis
et al., 2011). They were divided into three groups; Group 1 was
remain as untreated control, Group 2 was subjected to intranasal
spray vaccine once of formalin-killed P. multocida B2 and Group
3 with twice intranasal spray of formalin-killed P. multocida B2 at
two weeks interval. The lungs of goats that were exposed once
and twice with intranasal inoculation of the crude vaccine did not
show any significant lesions (Figure 9). Results showed that the
size of BALT and numbers of lymphocytes were of similar pattern
when compared with previous study using M. haemolytica A2 (Siti-
Raudah et al., 2006).
The inoculation of formalin-killed P. multocida B2 through
intranasal route was able to induce BALT in the lungs (Siti-Raudah
et al., 2005a) (Figure 10). The levels of serum IgG showed increasing
patternbutwasnotsignificant (p>0.05) while thelevelofserum
IgAshowedsignificantlyincreased(p<0.05)inthegoatsinoculated
twice with intranasal spray of formalin-killed P. multocida B2 (Siti-
Raudah et al., 2005b) (Figure 11).
A
Figure 9: Gross appearance of lungs from animal inoculated with formalin-killed P. multocida B2 once (A) and twice at two weeks interval (B). Both lungs look normal without any lesions.
(i) (ii)
Case 2: Haemorhagic Septicaemia • 43
Figure 10: Histology of BALT in control untreated goats (left) and those inoculated intranasally with formalin-killed P. multocida B2 (right). Note that the size and numbers of BALT were enormous. H&E staining. x20
00.010.020.030.040.050.060.07
Day 1 Day 5 Day 10 Day 15 Day 19 Day 24 Day 29
Days
Op
tic
al D
en
sit
y
Control Once Twice
0
0.2
0.4
0.6
0.8
1
1.2
1.4
Day 1 Day 5 Day 10 Day 15 Day 19 Day 24 Day 29
Days
Op
tica
l D
ensi
ty
Control OnceTwice
Figure 11: The levels of serum IgG (i) and IgA (ii) showed increment after once and twice exposure towards intranasal administration of formalin-killed P. multocida B2
(i)
(ii)
44 • Mucosal Immunity: Animal Vaccination and Disease Prophylaxis
The intranasal spray vaccine of formalin-killed P. multocida
B2 that was delivered directly onto the membrane mucosae of the
nostril was able to induce both mucosal and systemic immunity
andgavebetterprotectionasshowninthepreviousfieldtrialofM.
heamolytica A2 vaccine. To increase the nostril membrane mucosal
surface area during the intranasal spray delivery, the lyophilized
form of live P. multocida B2 was prepared at three different dosages.
The final concentration was then adjusted to 1 x 109 CFU/mL.
Then three different doses of lyophilized vaccine were prepared
at 3.0 mg (Group 1), 5.0mg (Group 2) and 7.0mg (Group 3). All
the groups were exposed with twice intranasal inoculation at two
weeks interval. Results showed that lyophilized vaccine was able to
stimulate the BALT as shown in Figure 12 (Ooi et al., 2005). Results
also showed that the sizes of BALT and numbers of lymphocytes
were significantly higher in goats exposed twice at two weeks
interval with 2mg doses (Effendy and Zamri-Saad, 2007).
Figure 12: Histology appearance of lungs from control untreated animals with formalin-killed P. multocida B2 once (right). Note the nodular types of BALT with numerous lymphocytes.
First experiment in goats showed that 5mg and 7mg of
lyophilized dust contained 1 x 109 CFU/mL was able to stimulate
the BALT and lymphocytes numbers, as well as the of levels of
serum IgA and IgG (Figure 13) above the protective level (Ooi
et al., 2005). The levels of IgA and IgG in the broncho-alveolar
lavages(Figure14)werealsosignificantly(p<0.05)increased.The
lyophilized vaccine of P. multocida B2 also showed the ability to
stimulateGALTsignificantlyparticularlyattheduodenum,jejunum
and ileum associated lymphoid tissues (Figure 15, 16 and 17).
These are the aggregations of lymphoid tissue that were usually
found in the lowest portion of the small intestine, the ileum, in
humans; as such, they differentiate the ileum from the duodenum
and jejunum. The duodenum can be identified using Brunner’s
glands as the indicator. The jejunum has neither Brunner’s glands
nor Peyer’s Patches (PP). Because the lumen of the gastrointestinal
tract is exposed to the external environment, much of it is populated
with potentially pathogenic microorganisms. PP thus establishes
their importance in the immune surveillance of the intestinal lumen
and in facilitating the generation of the immune response within the
mucosa.
Case 2: Haemorhagic Septicaemia • 45
46 • Mucosal Immunity: Animal Vaccination and Disease Prophylaxis
Ig G Levels After Administration of Lyophilized Pasteurella multocida B:2 Crude
0
0.05
0.1
0.15
0.2
0.25
0.3
Pre-tre
tmen
tD3 D5
D10 D14 D20 D24 D28
Days
IgG
leve
l
Cut-off Value:ControlGroup 1Group 2Group 3
IgA Levels After Administration of Lyophilized
Pasteurella multocida B:2 Crude
00.020.040.060.08
0.10.120.140.16
Pre Day 3 Day 5 Day10
Day14
Day19
Day24
Day28
Day
IgA
Lav
el
ControlGroup 1Group 2Group 3Cut-off:
Figure 13: The response of serum IgG (i) and IgA (ii) following intranasal inoculation of 3.0mg (Group 1), 5.0mg (Group 2)and 7.0mg (Group 3) of lyophilized P. multocida B2. The highest level of serum IgG and IgA was noted in Group 3
(ii)
(i)
Control Group 1 Group 2 Group 3
0
0.05
0.1
0.15
0.2
0.25
Ig G
Lev
el
Group
Ig G Level in Lung Lavage:
First Administration: Second Administration:
Con
trol
Gro
up 1
Gro
up 2
Gro
up 3
00.020.040.060.080.1
0.120.140.160.18
Ig A
leve
l
Group
Ig A Level in Lung Lavage:
First Administration: Second Administration:
Figure 14: The response of IgG (i) and IgA (ii) in lung lavages following intranasal inoculation of 3.0mg (Group 1), 5.0mg (Group 2) and 7.0mg (Group 3) of lyophilized P. multocida B2. The highest level of serum IgG and IgA was noted in Group 3
Case 2: Haemorhagic Septicaemia • 47
(ii)
(i)
48 • Mucosal Immunity: Animal Vaccination and Disease Prophylaxis
Con
trol
Gro
up 1
Gro
up 2
Gro
up 3
First Administration:Second Administration:0
50001000015000
20000
25000
Number of Lymphocytes in Duodenum (Intraephithelial):
First Administration: Second Administration:
Figure 15: The response of GALT (duodenum) following intranasal inoculation of 3.0mg (Group 1), 5.0mg (Group 2) and 7.0mg (Group 3) of lyophilized P. multocida B2 (i). Note the size of intraepithelial lymph nodes and numbers of lymphocytes (ii). H&E staining. x20
(ii)
(i)
Control Group1
Group2
Group3
05000
1000015000200002500030000
35000
Number of Lymphocytes in Jejunum (Lamina propia):
First Administration: Second Administration:
Figure 16: The response of GALT (jejunum) following intranasal inoculation of 3.0mg (Group 1), 5.0mg (Group 2) and 7.0mg (Group 3) of lyophilized P. multocida B2 (i). Note the size of lamina propria and numbers of lymphocytes (ii). H&E staining. x20
Case 2: Haemorhagic Septicaemia • 49
(ii)
(i)
50 • Mucosal Immunity: Animal Vaccination and Disease Prophylaxis
Con
trol
Gro
up 1
Gro
up 2
Gro
up 3
First Administration:0
5000
10000
15000
20000
Number of Lymphocytes in Ileum (Intraephithelial):
FirstAdministration:
SecondAdministration:
Figure 17: The response of GALT (ileum) following intranasal inoculation of 3.0mg (Group 1), 5.0mg (Group 2)and 7.0mg (Group 3) of lyophilized P. multocida B2 (i). Note the size of intraepithelial lymph nodes and numbers of lymphocytes (ii). H&E staining. x20
(ii)
(i)
These results indicated that stimulation of the respiratory
mucosal immune system would also stimulate the GALT and the
systemic or humoral immunity as well.
But how soon could we start the mucosal vaccination strategy
to the newborn animals by intranasal route of administration?
Another research was conducted to study the normal morphology
development of the BALT and GALT in goats at the age of 1 month-,
3 month- and 5 month-old. In general farm practice, the newborn
kids will normally be weaned at the age of 90 days or three months
(Smith and Sherman, 1994). Mucosal tissues are continuously
exposed to both commensal and harmful antigens since birth, and
it is important to determine whether the mechanisms that activate
the appropriate immune responses to different types antigens are
fully organized and functioning prior to the delivery of mucosal
vaccine. The time to start vaccination in the newborn is crucial
since colostral antibodies will interfere with vaccinations at very
young ages and affect their immune status.
The study was done at a medium scale commercial farm in Ladang
Rakyat Terengganu involving 18 kids at the age of 1, 3 and 5 months
old (Eamy and Effendy, 2011). They were randomly selected and
divided equally into two groups; Group 1 was the control untreated
group while Group 2 was treated with formalin-killed P. multocida
B2 as antigen model. The results showed that the development of
IgM, IgA and IgG were inconsistent in control untreated animals. In
Case 2: Haemorhagic Septicaemia • 51
52 • Mucosal Immunity: Animal Vaccination and Disease Prophylaxis
general, the level of IgG was the highest while IgA was the lowest
immunoglobulin being secreted in lung lavages. In 1-month-old
kids, the level of IgM (P>0.05) was the highest immunoglobulin
secreted while IgG was the highest at the age 3 and 5 months old
(Figure 25) (Eamy and Effendy, 2009a).
Figure 18: Comparison on mean value of IgA, IgM and IgG antibody titres in normal untreated and vaccinated groups of goats age 1, 3 and 5 month. Note the switching activities of IgM to isotypes IgA and IgG.
In ruminants, the level of IgG was relatively high compared to
the level of IgA in the colostrum (Brambell, 1970). The level of
IgG at the age of 3 and 5 months old was high since the goats
werereceivingunspecificIgGinthemilkofthedam.Thefindings
were consistence with the level of IgA that was comparatively low
in the 1-, 3- and 5 months old goats. The highest level of IgM titre
showed that the newborns were continuously exposed to foreign
antigens after birth, that has elicited an early rise of antibodies in
their immune system.Thiseventuallywillbe followedbyaffinity
maturation, isotype switching and eventually the rise in antigen-
specific IgG and IgA antibodies (Marianne Boes, 2000). The
intranasal inoculation of formalin-killed P. multocida B2 at the
concentration of 1 x 108 CFU/mL has shown a reduction in the
levels of IgM in 1- and 3 months old kids indicating the possibility
of isotype switching to IgA and IgG (Figure 18).
The development of BALT and GALT is an important indicator
for mucosal immune system development in newborns. The
organized mucosa-associated lymphoid tissue (MALT) is one of the
subjects that have been extensively being investigated recently. The
MALT is widely distributed in the mucosal surfaces and considered
as one of the most important part of the mucosal immune system
(Liebler-Tenorio and Pabst, 2006). The findings showed that the
development of BALT in untreated kids groups was age-related.
As the animals’ grew older, more develop organized lymphoid
aggregates will develop and armed the bronchial areas (Po Po S
et al., 2005) (Figure 18 and 19). Findings in previous study have
showed that BALT is more organized in antigen challenged ovine
lungs (Joel and Chanana, 1985; Zamri-Saad et al., 1999b).
Case 2: Haemorhagic Septicaemia • 53
54 • Mucosal Immunity: Animal Vaccination and Disease Prophylaxis
Figure 19: Comparison of the size of nodular type of BALT in normal untreated and vaccinated animals at different age (i). Comparison of the number of lymphocytes between normal untreated and vaccinated group that occurred in nodular type of BALT in three different age groups of animals (ii)
(ii)
(i)
The intranasal inoculated P. multocida B2 has potentially
encountered multiple collections of lymphoid tissues while passing
from bronchioles proximally to hilar lymph nodes (Meyer, 2001).
Then, the lymphocytes may migrate into the lung lymphoid aggregates
via the high endothelium of post capillary venules, where it will be
located in the tissues for a period of time the systemic circulation.
TheoverlyinglargenodulesofBALTalsofoundtobeinfiltratedby
lymphocytes to form lymphoepithelium (Figure 19). Enlargement
of BALT and GALT structure showed the evidence of lymphocytes
migration into the submucosa epithelium prior to antigen exposure
tothemucosalsurface.Exposuretothespecificantigeninthisstudy
givesrisetothespecificdevelopmentoflymphocytesfortheIgM,
IgA and IgG immunoglobulin in the BALT structure.
Figure 20: (Left) Note that nodule (arrow) extending from the submucosa area with the cartilage (C) to the basement membrane of the mucosal epithelium around the lumen (L). (Right) Histological structure of nodular BALT observed in small bronchi and lymphoid tissue is located under the epithelium (arrow) and extending from the epithelium to tunica adventitia (TC). H&E staining. x20
When the foreign molecules intrude into the mucosal surface,
the evolved organized lymphoid tissue will facilitate their uptake.
This is known as the inductive sites which contains the B and T
lymphocytes where it is respond towards the antigen in the presence
of antigen-presenting cells (APC) by developing into effector and
C
L
C
L
C
Case 2: Haemorhagic Septicaemia • 55
56 • Mucosal Immunity: Animal Vaccination and Disease Prophylaxis
memoryBandTcells. Theseantigen specificBandTcellswill
next emigrate to the lymphatic drainage, circulate the bloodstream
and home to mucosal effector regions. In this mucosal effector
region,thisantigenspecificBandTcellswillresideandperform
their function by secreting antibody and cytokines to protect the
mucosal surface. The kid that being exposed to the P. multocida
B2organismhassuccessfullytriggeredtheantigenspecificBandT
cells to emigrate to the effector regions (Figure 19 and Figure 20).
Figure 21: (Left) Histology of bronchioles shows the infiltration oflymphocytes (arrow) forming the lymphoepitheliumwithpseudostratifiedcolumnar with cilia (C), goblet cells (G) and basal cells (B) H&E staining. x40. (Right) IgA immunolabeled lymphocytes prominently located in the bronchioles and alveolus area in the lung of animals at age 3 month old (arrow).
The specific lymphocytes stained brown with DAB substrate
has been suggested that it is not only B and T lymphocytes being
emigrates, but other cellular elements of the immune system being
circulated together with the lymphocytes into the effector region.
All these cells are essential to orchestrate the development and
secretionsofspecificantibody.
C
G
G
B
Figure 22: (Left) IgM immunolabeled lymphocytes prominently located in the bronchioles and alveolus of animals at age 3 month old (arrow). (Right) IgG immunolabeled lymphocytes prominently located in the bronchioles andalveolusinanimalsage5monthold(arrow).Thespecificlymphocytesstained brown with DAB substrate
The recirculation routine of these cellular elements are
contributed by the immune cellular systems including macrophages,
monocytes, eosinophils, neutrophils, basophils, mast cells and
dendriticcells (Pruett,2003). Therewasa significantdifferent in
the size of GALT between animals at age 1, 3 and 5 month old. The
GALT showed increment pattern in size of duodenum and jejunum,
but vice versa for ileum in relation to age. The size of ileal GALT
area in 1 month old of normal animals were larger than the size
observed in duodenum and jejunum. A strong interaction with the
antigensofadigestivetractatearlyagemayinfluencethenumberof
lymphocytes where they are exposed to the environmental antigens
(Lillehoj and Chung, 1992). In neonate, jejunal PPs and ileal PP
showed a similar morphology during their rapid growth, where they
start to develop distinctive features (Rothkotter and Pabst, 1989).
Peyer’s patches (or aggregated lymphoid nodules, or occasionally
PP for brevity) are organized lymphoid nodules, named after the
17th-century Swiss anatomist Johann Conrad Peyer.
Case 2: Haemorhagic Septicaemia • 57
58 • Mucosal Immunity: Animal Vaccination and Disease Prophylaxis
Figure 23: Development of GALT size in jejunum of normal animals was linear with the age of animals. Note that only animals at age 1 and 3 month oldwassignificantlydifferent(p<0.05)
Activated lymphocytes passes into the blood stream via the
thoracicductandtraveltothegutwheretheycarryouttheirfinal
effector functions. Eventually, plasma cells differentiated from the
B-cells, will excrete antibodies of type IgA back to the lumen. Even
though the size of GALT and lymphocyte number in this structure
is significantly difference, there might be unspecific antigenic
stimulation in the gastrointestinal tract due to the oral uptake of
antigenvia food.But afinding in this study is similarwithother
study where the GALT can be extensively developed in the ileum
compared to the duodenum and jejunum. The mucosal immune
system can be defined as one of the complex immunological
structure and lymphoid structure serves as the guard or barrier for
the pathogen entry.
Figure 24: Development of GALT size in duodenum and ileum of vaccinated animals was linear with the age of animals. Duodenum GALT wassignificantlydifferentbetweenage3and5monthold(p<0.05).WhileileumGALTwassignificantlydifferent(p<0.05)inallthreedifferentageofanimals
Findings in this study have showed the development of BALT and
GALTareaisage-relatedinruminantsandexposuretothespecific
antigen will give rise to the development of lymphocyte number
in this structure (Saw Po Po et al., 2004a; 2004b; 2004c). Animals
at age 3 month old showed a good response in immunoglobulin
development and also in the development of BALT and GALT
structure via vaccination. It is very crucial however, albeit the timing
of vaccination and selection of product or antigen, there is also
other external factor that should be considered for the protection
of animals against pathogens; the nutrition, care of the newborn,
sanitation and housing also playing an important factor to the health
and protection of the animals (Eamy and Effendy, 2009b;c).
There are still challenges for oral and intranasal immunization
in ruminant. Few studies showed that polysaccharides from marine
microalgae showed better immune response as adjuvant (Effendy
and Sharzehan, 2013; Effendy et al., 2011) in white rats. Many
studies using the polysaccharides from microalgae species showed
Case 2: Haemorhagic Septicaemia • 59
60 • Mucosal Immunity: Animal Vaccination and Disease Prophylaxis
their suitability for adjuvanted vaccine with better protection
against challenge pathogens. The next challenge is to develop the
polysaccharides as novel adjuvant and characterize their property
for future mucosal adjuvant study.
ADVANTAGES OF MUSOCAL VACCINATION APPROACH
There are many advantages for mucosal vaccination in livestock and
even better in wildlife disease management. Most of small holders
in Southeast Asia, including Malaysia practice the “let loose system”
in raising their animals. This kind of practice makes it difficult
for the owner, or the veterinarian in order to deliver an injection
for vaccination programme. The animals need to be properly
restrained before the injection can de done, and this will take
number of people to stop the animals from moving. The struggles
will eventually stress the animals especially of physiological burden
i.e. pregnant or diseased group.
The rule of thumb when delivering injection is to change to a
fresh needle to new animals in line. The reason is simply because
to prevent injury to the surrounding tissues where the injection will
take place due to the blunt needle. Replacement needle will also
prevent any possible transmission of disease from other animals.
But most of the farmers would prefer to use the same needles for
more animals since it will save them the cost. The used needles are
also not environmental friendly since they are not easily dispose
and improper management of these needles would lead to choking
upon incidental ingestion or other physical injuries either to the
animals or handlers.
The injection method focuses on systemic immune system
that capitalizing on humoral antibody production. The humoral
immunity only reacts with good impact only after the same antigen
re-enter the host’s body. In humoral immune system, B-lymphocytes
will be activated and differentiated to form plasma cells when the
system is exposed to antigenic determinants in lymphatic organs.
62 • Mucosal Immunity: Animal Vaccination and Disease Prophylaxis
These plasma cells are specialized and differentiated cells that are
synthesizedandsecretedantibodiesthatarespecifiedtoantigenthat
is exposed to them. The other activated B-lymphocytes will form
a memory cells that will be activated later to plasma cells rapidly
following the exposure of the same antigen. The production of the
specificantibodieswillrequirelongertime,andlargelydependson
the physiological status of the host.
While theprocessof antibodiesproduction require sufficient
time to maturation, the infected tissues and their surrounding
might have been injured by the virulence factors acquired by the
antigen. In case of respiratory disease such as Mannheimiosis, the
alveoli might collapse and leads to pneumonia and bronchitis that
is irreversible. This kind of lesions would limit the physiological
ability of the animals and eventually affect their productions.
Mucosal type of vaccination offers different approach of
inducing immune responses. When the vaccine seeds (formalin-
killed antigen) attempt to intrude the mucosal surface, the evolved
organized lymphoid tissues will facilitate their uptakes at the
inductive site. This inductive site contains the B- and T-lymphocytes
that respond towards the presence of the antigen in the APC by
developing into effector and memory B- and T-cells. These antigen-
specificB-andT-cellswillthenmigratetothelymphaticdrainage
and circulate in the blood circulation, and home to mucosal effector
region. In mucosal effector region, the antigen-specific B- and
T-cells will reside and perform their function by secreting antibody
and cytokines to protect mucosal surfaces.
The results suggested that animals that were exposed to P.
multocida B2antigenhassuccessfullytriggeredtheantigen-specific
B- and T-cells to emigrate to the effector site, either in the lungs or
GIT. Apart from B- and T-lymphocytes, other cellular elements such
as macrophages, monocytes, eosinophils, neutrophils, basophils,
mast cells and dendritic cells (Pruett, 2003) are also being circulated
into the effector region. This is because that these cells are essential
toorchestratethedevelopmentandsecretionsofspecificantibody.
These findings showed that induction of mucosal immune
system will concurrently stimulate the systemic immune system,
which happened to be one of the advantages in mucosal
vaccinationprogramme(EffendyandZamri-Saad,2007).Thefield
trialsconductedinthreedifferentfarmsshowedhighefficacyand
protection level of the vaccine locally and systematically. Apart
from that, the induction of immune responses in unvaccinated goats
that shared the same pen was also noticed. This suggested that
the vaccinated animals transferred part of the vaccine seed when
they sneezed and inhaled by the non-vaccinated animals. The
vaccination coverage became wider and protected more numbers
of animals.
Only one nostril needs to be sprayed in the form of droplet
nuclei at about 50 microns during the delivery of mucosal vaccine.
The animals will need only minimum restraining and fewer handlers
compared to injection vaccine. Thus, 2000 heads of goats took
onlyhalfadaytofinishwhencomparedtoinjectionmethodthat
required 4 days to be completed. Injection also caused temporary
lameness to 10% of the vaccinated animals and compromised the
normal physiological condition. The affected goats would not be
able to compete for feed without their strong feet.
Although the booster spray needs to be delivered 2 weeks later,
the antibodies produced both locally and systematically lasted for
longer time. The secretory IgA (sIgA) had also been detected on
the lungs mucosal surfaces by other researcher (Pilette et al., 2008).
The sIgA is the most abundant immunoglobulins in the mucosal
fluidsandcaninteractwithphagocyticcells.Besidethat,thesIgA
acquires multiple roles for defenses at mucosal surfaces (Lamm,
Advantages of Musocal Vaccination Approach • 63
64 • Mucosal Immunity: Animal Vaccination and Disease Prophylaxis
1997). They also give rise to the promotion to the entrapment of
antigens thus preventing direct contact with mucosal surfaces.
The presence of MALT was also evidence by the ability of the
intranasal mucosal vaccine to induce the development of GALT
particularlyinthejejunumandileumsignificantly.Thefindingsalso
showed that we could also induce immunity in protection against
gut-related disease antigen. The networks of MALT also enable the
induction of BALT to prevent respiratory disease through oral route of
vaccine delivery. Researcher now can study the appropriate regime
and dosage to induce immunity in local lymph nodes attached to
mucosal surfaces through the delivery oral vaccination. Wildlife
thataredifficulttocatchorrestraintcanbevaccinatedthroughoral
feedingsaccordingtothesefindings.
IMPROVING IMMUNOLOGICAL RESPONSE
The vaccine’s seed usually mixed with solution that is called
adjuvant. In immunology, adjuvant is incorporate to enhance
immunologicalresponses.Immunologicaladjuvantcanbedefined
as any substance or biomaterial that can increase, or enhance
antigen-specific immune responseswhenmix in specific vaccine
antigen (Shin Sasaki and Kenji Okuda, 2000). There are different
types of adjuvants available in the market, but most of this adjuvant
is toxic in nature. Some are thick in viscosity and making them
difficult to be delivered through injection method. In animal
vaccination, Freund adjuvant and oil adjuvant have been used for
systemic vaccination programme.
Is itnecessary tofind suitableadjuvant formucosalvaccine?
As we learned before, there are different mucosal route of vaccine
delivery such as intranasal, intra-vagina, intra-rectum or through
oral. Due to the complexity of the digestive system, especially in
ruminant, good adjuvant candidate that can protect the vaccine seed
before their arrived to gastrointestinal tract is futile. The adjuvant
must not be toxic in nature and can resist chemical reactions in
the stomach and intestinal tract. Oral type of vaccination is always
a good alternative for wildlife or straying animals where minimal
restraining procedure is needed. Although it is easy to deliver, the
effectiveness of the oral vaccine in ruminant animals is still under
research.
Recently we found a good candidate of adjuvant, extracted as
exopolysaccharides (EPS) from probiotic bacteria and algae. The
resource is ubiquitously found in aquatic environment and did
not show any toxic effects to cell lines. The EPS instead induced
proliferation of macrophage, a large phagocytic cell found in
66 • Mucosal Immunity: Animal Vaccination and Disease Prophylaxis
leukocytes that involve in the first line of defense. The adjuvant
encapsulated the vaccine seeds and sticky in nature. We are
going to test the effectiveness of the adjuvant through intranasal,
intramuscular and oral vaccination soon in different animal species.
The adjuvant might be suitable for human vaccination in future and
we expect that it would be ready in a couple of years. Our team also
developing other types of adjuvant such as palm oil extract, virus
like particles that is non-infectious and other marine biomaterials.
Fish is also a species that depends mostly on innate and
mucosal immune responses. It is hardly to induce acquired immune
responses although the structure of their immune system is very
similar to vertebrates (Uribe et al., 2011). Most of the smaller type
offishdependsonthenonspecificimmunitytoprotectthemselves
from pathogenic microorganism. The strategy for disease prevention
insmallerfishisusuallybyfocusingonimmunologicalparameters
such as growth inhibitors, lytic enzymes, the classic complement
pathways, the alternative and lectin pathway, agglutinins and
precipitins (opsonins and primary lectins) and antibacterial
peptides. Since antibiotics abuse has always a problem in
aquaculture practices (Eamy et al., 2010), the right approach is
to minimize physical and chemical parameter changes, or any
suppressive factors that would leave to stress condition and at the
same time formulating better food additives and immunostimulants
to enhance their immune responses. Our team has been studying
the Streptococcis infectioninculturedfishsystemwiththeaimto
produce oral vaccination against the disease but with uncertain
results. The successful colonization of the pathogen depends on
various combination factors (Effendy et al., 2009). Ascorbic acid
supplement formulations in the feed pallets have shown promising
effectsininducingnonspecificimmuneresponsesinClariashybrid
catfish(Phuonget al., 2007).
Our research is now expanding towards other aquatic species
such as the green mussel, Perna viridis. There is a need to study
their ability to produce mucosal protection by inducing the
innate immune system. Recently we found that the exposure
of green mussels towards non-lethal heat temperature (NLHT)
has successfully induced the expression of heat shock protein
70 (HSP70) and increased their tolerance towards lethal heat
temperature (LHT). Most of the mussels that expressed the protein
were also resistance against the colonization of Vibrio pathogen in
green mussel (Aleng et al., 2014, Effendy et al., 2014), but further
studiesneedtobedonetoconcludethefindings.Thisspeciesareof
the lower taxonomic rank animals that employ the innate immunity
as the main defense system. Due to their simple immune system
that only response to antigenic stimuli, the approach in disease
management are different. They acquire hemolymph cells that are
fundamental in their immunity, but it is not clear if other cells also
contribute to the innate responses towards pathogenic organism.
Animals that have simple defense mechanism might have
different types of innate immune system, which are not antigen-
specificinnature.Althoughinnateimmunesystemisnotantigen-
specific, it is important since it can induce expression of certain
housekeeping genes that plays important role in the prevention of
colonizationprocessoftheantigen.Thefindingsmightbeimportant
for the future since emerging diseases are rapidly occurred and
difficultyinproducingvaccineinshortperiodoftime.
Improving Immunological Response • 67
CONCLUSION
In the earlier section, more studies need to be done on determining
the suitability of different mucosal surfaces for vaccine delivery site,
thus preventing the initial contact of the other antigen to this site.
The mindset of the veterinary researcher in developing traditional
type of vaccine needs to be changed. This is partly due to the
potential of other mucosal membrane as effector sites for vaccine
route of delivery has not been fully explored. The oral vaccination,
which is well established in avian and human, could be the
answer in controlling endemic diseases of wildlife in future. It is
noteasytoproducehighefficacyvaccinesduetothediversityof
the pathogenic microorganism. The new vaccines need to undergo
several assays to determine their safety, specificity, effectiveness,
regimeanddosagepriortothefieldtrial.Somepathogensmight
undergo mutation during the preparation of the vaccines, which will
affecttheefficacyofthevaccinesagainstthemutatedpathogens.It
is imperative to understand that livestock vaccination approach is
onflockorherdhealthprogramme.Thus,thebestwaytoproduce
effectivemucosalvaccineswithhighefficacyandprotectionlevel
to assist the veterinarian in maintaining high antibody level towards
infectious diseases in their farm.
It is imperative to move our research from sub-disciplinary
based into the niche area of the university. This is in line with
the blue ocean strategy that enable us to work in our research
domain without the need to competing with others. The attempt
to get research grants from the government or other institutions and
agencies has become highly competitive. Young academic should
strategize their research and development programme by setting the
end in mind on the main issues and problem in the community,
70 • Mucosal Immunity: Animal Vaccination and Disease Prophylaxis
and positioning their knowledge and research skills accordingly.
Although we could not solve all the problems faced by the world,
we could offer better solution by working at multi-disciplinary
levels, and produce greater impact results through our collaboration
with academic from various disciplines.
REFERENCES
Abraham, E. P., & Chain, E. (1940),. An Enzyme from Bacteria Able
to Destroy Penicillin. Nature, 146: 837.
Aleng, N.A., Effendy, A.W.M. & Sung, Y.Y. (2014). Enhancement
of Heat Shock Protein 70 promotes thermotolerance in the
Asian Green Mussel, Perna viridis. International Conference on
Marine Science and Aquaculture. Universiti Malaysia Sabah,
Kota Kinabalu.
Anderson, M.L., Moore, P.F., Hyde, D.M. & Dungworth, D.L.
(1986). Bronchus Associated Lymphoid Tissue in the Lungs of
Cattle: Relationship to Age. Research in Veterinary Science, 41:
211-220.
Andre, F.E. (2003). Vaccinology: Past Achievements, Present Road-
blocks and Future Promises. Vaccine, 21: 593–595.
Aungst, B.J., Rogers, N.J.; (1988). Site Dependence of Absorption
Promoting Actions of Laureth-9, Na Salicylate, Na2EDTA, and
Aprotinin on Rectal, Nasal, and Buccal Insulin Delivery. Pharm.
Res, 5: 305–308.
A.W.M. Effendy, Pang, S.T. & Zahrah, S.A. (2009). In-vitro
colonization of Streptococcus agalactiae in Hybrid Tilapia Gills.
International Congress of Malaysian Society for Microbiology
(ICMSM 2009). “Current Issues for Sustainable Research and
Innovation in Microbial Biotechnology”. Parkroyal Hotel
Penang, Malaysia. 1 – 4 December 2009.
Bahaman, A.R., Nurlida, A.B., Sheikh-Omar, A.R. & M, Zamri-Saad.
(1991). Biotypes and Serotypes of Pasteurella haemolytica and
Their Importance in the Production of Vaccines for Pneumonic
Pasteurellosis in Sheep. J. Vet. Malaysia. 3: 33-35.
72 • Mucosal Immunity: Animal Vaccination and Disease Prophylaxis
Berggren, K.A., Baluyut C.S., Simonson, R.R., Bemrick, W.J. &
S.K. Maheswaran. (1981). Cytotoxic Effects of Pasteurella
haemolytica on Bovine Neutrophils. American Journal of
Veterinary Research, 42: 1383-1388.
Bienenstock, J., Johnston, N & Perey, D.Y.E. (1973). Brochial
Lymphoid Tissue. I. Morphological Characteristics. Laboratory
Investigation, 28: 686-692.
Black, J.G. (2013). Microbiology. International Student Version. (8th
Ed.). Singapore: John Wiley & Sons, Inc. Singapore Pte. Ltd.
Buddle, B.M.A., Cole, D.J.W., Pulford, H.D. & M.J. Ralston. (1990).
Experimental Respiratory Infection of Goats with Caprine
Herpesvirus and Pasteurella Haemolytica. N. Z. Vet. J., 38: 22-
27.
Brambell, F.W. (1970). The Transfer of Passive Immunity from Mother
to Young. In Frontiers of Biology (eds Neuberger, A., Tantum,
E.L.) 18: 201-233pp. Northern Holland, Amsterdam.
Brogden, D.A., Rimler, R.B., Cutlip, R.C. & H.D. Lehmkuhl. (1987).
Incubation of Pasteurella haemolytica Lipopolysaccharides
with Sheep Lung Surfactant. American Journal of Veterinary
Research, 47: 727-729.
Burton K.A. & M. W. Smith. (1977). Endocytosis and Immunoglobulin
Transport across the Small Intestine of the Newborn Pig. J.
Physiol., 270: 473-488.
Chain, E., Florey, H. W., Gardner, A. D., Heatley, N. G., Jennings,
M. A., Orr- Ewing, J., & Sanders, A. G. (2005). The Classic:
Penicillin as a Chemotherapeutic Agent. 1940. Clin. Orthop.
Relat. Res., 439: 23–26.
Confer, A. W., Panciera, R.J. Clinkenbeard, K.D. & D.A. Mossier.
(1990). Molecular Aspects of Virulence of Pasteurella
haemolytica. Canadian Journal of Veterinary Research
(Supplement), 54: S48-S52.
Duncan Hannant. (2002). Mucosal Immunology: Overview and
Potential in the Veterinary Species. Veterinary Immunology and
Immunopathology, 87: 265-267.
Dungworth, D.L. (1985). The Respiratory System. In: K.V.F. Jubb, P.C.
Kennedy and N. Palmer (eds), Pathology of Domestic Animals,
(Academic Press, London). 470-471.
Eamy, N.Y. & Effendy, A.W.M. (2011). Response of Bronchus-
Associated Lymphoid Tissue (BALT) in Goats at Three Different
Age Post-exposure with Pasteurella mutocida B;2, 3rd Malaysia
Association of Veterinary Pathology Conference, 13/05/2011,
15/05/2011, Pahang.
Eamy, N.Y., Effendy, A.W.M., Yii-Siang Hii & Elisa, C.S.Y. (2010).
Antibiotic Resistance in Isolated Bacteria Associated with
Cultured Abalone (Haliotis asinine). Annual Seminar on
Marine Science and Aquaculture: Indicators for Sustainability
of Fisheries and Aquaculture. Dewan Canselor UMS, Kota
Kinabalu, Sabah. 10 – 12 March 2010.
Eamy, N.Y. & Effendy, A.W.M. (2009a). Response of Bronchiole
Alveolar Lavage Immunoglobulin in 1,3 and 5 Month Old
Goats Upon Intranasal Exposure of Deactivated Pasteurella
multocida B2. International Congress of Malaysian Society for
Microbiology (ICMSM 2009). 1-4 December, Parkroyal Penang
Hotel, Malaysia.
References • 73
74 • Mucosal Immunity: Animal Vaccination and Disease Prophylaxis
Eamy, N.Y. & Effendy, A.W.M. (2009b). Development of Bronchiole
Alveolar-Lavage Immunoglobulin in 1, 3 and 5 Month Old
Goat. Faculty of Science and Technology Postgraduate Seminar
2009. 6 April, Universiti Malaysia Terengganu.
Eamy, N.Y. & Effendy, A.W.M (2009c). Response of Bronchus-
Associated Lymphoid Tissue (BALT) and Secreting Antibody in
Goats at Different Age Prior to Intranasal Exposure of Whole-
killed Pasteurella multocida B2. Postgraduate Seminar 2009, 10
June, Universiti Malaysia Terengganu.
Effendy A.W.M. (2004). Isolation,IdentificationandDNAAnalysis
of Pasteurella multocida from Ruminant and Non-ruminant of
Endemic Areas in Kuala Terengganu. KUSTEM Research Seminar
: Marine Biotechnology, Turtle Research and Conservation. May
31 – June 2, 2004. KUSTEM. pp 136-138
Effendy, A.W.M., Siti-Nurtahirah, J., Hussin, Z.M. & M. Zamri-Saad.
(2006). The Side Effects of Kacip Fatimah Extracts on Liver and
Kidney of White Rats. Journal of Sustainability Science and
Management, 1: 40-46.
Effendy, A.W.M. & M.A.K. Sharzehan. (2013). Response of Serum
IgG Upon Intranasal Inoculation of Exopolysaccharides-
Adjuvanted Vaccine of Pasteurella multocida B:2 in Wistar
rats. Conference Proceedings: Life Science and Biological
Engineering Conference 2013. Tokyo, Japan. March. page 3-4.
Effendy, A.W.M, Nadiah-Hanim M.N., Eamy N.Y. & Thirukanthan
C. (2011). Chlorella sp As Mucosal Vaccine Adjuvant against
Pasteurella multocida B:2. 8th Annual Seminar on Marine
Science and Aquaculture (SOMSA 2011). March 8-10, 2011.
Effendy, A.W.M., Pang, S.T. & Zahrah, S.A. (2008). In-vitro Challenge
of Streptococcus agalactiae towards Gill, Intestine and Nostril
of Fish. 17th EMSM Scientific Conference and 18th Annual
General Meeting, Shah Alam. 18th – 20th Dec 2008.
Effendy, A.W.M. & Zamri-Saad, M. (2007). Stimulation of Bronchus-
Associated Lymphoid Tissue against Pasteurella multocida B:2.
12th AITVM International Conference. Mont Pellier. France.
20-23 August 2007.
Effendy, A.W.M. & A.M. Siti Ruhaya. (2000). DNA Analysis of
Pasteurella haemolytica A2, A7, A9 and T Isolated from Goats
with Pneumonia. J. Vet. Malaysia (2000), 12(2): 97-100.
Effendy, A.W.M., Zamri-Saad, M., Maswati, M.A., Ismail, M.S. &
Jamil, S.M. (1998). Stimulation of the Bronchus Associated
Lymphoid Tissue of Goats and Its Effect on the In-vitro
Colonization of Pasteurella haemolytica. Veterinary Research
Communication, 22: 147-153.
Effendy, A.W.M., Zamri-Saad, M., Mohamad, M. & H. Omar. (1998).
Vaccination of Sheep Against Pneumonic Pasteurellosis Using a
New Spray Vaccine. Jurnal Veterinar Malaysia, 10: 17-20.
Effendy, A.W.M., Zamri-Saad, M., Puspa, R. & Rosiah, S. (1998).
Efficacy of Intranasal Administration of Formalin-killed
Pasteurella haemolytica A2 against Intratracheal Challenge
Exposure in Goats. Veterinary Record, 142: 428-431.
European Centre for Disease Prevention and Control/European
Medicines Agency Joint Working Group (ECDC/EMEA). (2009).
The Bacterial Challenge: Time to React. Available at www.
ecdc.europa.eu/en/publications/Publications/ 0909_TER_The_
Bacterial_Challenge_Time_to_React. pdf.
FAO. (2006). Impacts of Animal Disease Outbreaks on Livestock
Markets. Introductory Paper on Animal Disease Outbreaks
prepared for 21st Session of the Inter-Governmental Group on
Meat and Diary Products. Rome, Italy. 14 November 2006.
(available at
References • 75
76 • Mucosal Immunity: Animal Vaccination and Disease Prophylaxis
FAO. (2007). Food Outlook Global Market Analysis - Poultry Meat:
(available at http://www.thepoultrysite.com/articles/918/food-
outlook-global-market-analysis-poultry-meat on July 20th.,
2013 at10.20pm)
FAO. (2009). Livestock in the Balance. State of Food and Agriculture
2009. FAO, Rome.
FAO. (2011). World Livestock 2011 - Livestock in Food Security.
Rome, FAO.
George Christakos, Ricardo A. Olea, Marc L. Serre , Hwa-Lung Yu &
Lin-Lin Wang. (2005). Interdisciplinary Public Health Reasoning
and Epidemic Modelling: The Case of Black Death Hardcover.
Springer; 2005 edition (June 24, 2005).
Gilmour, N.J.L. (1980). Ovine Pasteurellosis. Vet. Q., 2: 191-198.
Gilmour, N.J.L., Angus, K.W. & J.S. Gilmour. (1991). Pasteurellosis.
In: Diseases of Sheep. Martin, W.B. and I.D. Aitken. (eds.).
BlackwellScientificPublication,London.pp133-139.
Gonzalez, C.T. & S.K. Maheswaran. (1993). The Role of Induced
Virulence Factors Produced by Pasteurella haemolytica in the
Pathogenesis of Bovine Pneumonic Pasteurellosis: Review and
hypotheses. Brit. Vet. J., 149: 183-193.
Hansen, S. W. & B. Damgaard. (1991). Effect of Environmental Stress
and Immobilization on Stress Physiological Variables in Farmed
Mink. Behav. Processes., 25 (1991): 191-204.
Harris, D.R. (1996). The Origins and Spread of Agriculture and
Pastoralism in Eurasia. Smithsonian Inst. Washington.
http://www.vrg.org/journal/vj2009issue4/2009_issue4_2009_poll.
php
Husband, A.J., Bao, S. & K.W. Beagley. (1999). Analysis of the
Mucosal Micro-environment: Factors Determining Successful
Responses to Mucosal Vaccines. Vet. Immunol. Immunopathol.,
72: 135-142.
Jamaludin, R. (1993). In: Pasteurellosis in Production Animals.
ACIAR PROCEEDINGS NO. 43, PP238-239.
James A. Roth. (2011). Veterinary Vaccines and Their Importance to
Animal Health and Public Health. Procedia in Vaccinology 5
(2011) 127 – 136.
Jan Holmgren & Cecil Czerkinsky. (2005). Mucosal Immunity and
Vaccines. NATURE Medicine Supplement. (11, 4, April 2005):
S45-S53.
Jasni, S., Zamri-Saad, M., Mutalib, A.R. & Sheikh-Omar, A.R.
(1991). Isolation of Pasteurella haemolytica from the Nasal
Cavity of Goats. British Veterinary Journal, 147: 352-355.
Jasni, S., Zamri-Saad, M., Kamal Hizat, A., Mutalib, A.R., Salim, N.
& Sheikh-Omar, A.R. (1990). Seasonal Occurrence of Caprine
Pneumonic Pasteurellosis in central Peninsular Malaysia. Jurnal
Veterinar Malaysia, 2: 147-148.
Joel, D.D. & A.D. Chanana. (1985). Pulmonary Immune Responses
in Sheep, in: Morris B., Miyasaka, M. (Eds), Immunology of the
Sheep, pp. 382-409, Roche, Switzerland.
John T.J. & R. Samuel. (2000). “Herd Immunity and Herd Effect:
NewInsightsandDefinitions”.Eur. J. Epidemiol, 16 (7): 601–6.
doi:10.1023/A:1007626510002. PMID 11078115
Joseph, P.G. (1989). Haemorrhagic Septicaemia in Peninsular
Malaysia . Kaj. Vet, 11:65 – 79.
Kaltreider, H.B. (1976). Expression of Immune Mechanisms in the
Lungs. Am. Rev. Resp. Dis, 113: 347-379.
References • 77
78 • Mucosal Immunity: Animal Vaccination and Disease Prophylaxis
Kauffmann, S.H.E., Sher, A. & R. Ahmed. (2002). Immunology of
Infectious Diseases. Washington DC: ASM Press.
Liebler-Tenorio, E.M. & R. Pabst. (2006). MALT Structure and
Function in Farm Animals. Vet. Res, 37: 257-280.
Lillehoj, H.S. & K.S. Chung. (1992) Postnatal Development of
T-lymphocyte Subpopulations in the Intestinal Intraepithelium
and Lamina Propria in Chickens. Vet Immunol Immunopathol,
31: 347-360.
Lombard, M., Pastoret, P.P. & A.M. Moulin. (2007). A Brief History of
Vaccines and Vaccination. Rev. Sci. Tech. Off. Int. Epiz., 2007,
26 (1): 29-48.
Marianne Boes. (2000). Role of Natural and Immune IgM Antibodies
in Immune Responses. Molecular Immunology, 37: 1141–1149.
McNeilly, T.N., McClure, S.J. & J.F. Huntley. (2008). Mucosal
Immunity in Sheep and Implications for Mucosal Vaccine
Development. Small Ruminant Research, 76 (2008) 83–91.
Meeusen, Els N.T., John Walker, Andrew Peters, Paul-Pierre Pastoret
& Gregers Jungersen. (2007). Current Status of Veterinary
Vaccines. Clinical Micobiology Reviews, 20(3): 489-510. 0893-
8512/07/$08.00+0 doi:10.1128/ CMR. 00005-07.
Meyer, K.C. (2001). Lung Immunology and Host Defense. Pulmonary
Biology. In E.E. Bittar (Ed.), Health and Disease. New York:
Springer.
Montilla, N.A., Blas, M.P., Santalla M. L. & J.M. Martín Villa. (2004).
Mucosal Immune System: A Brief Review. Inmunología. 23. (2
Abril-Junio 2004): 207-216.
Mosier, D. (1993). Prevention and Control of Pasteurellosis. In:
Pasteurellosis in Production Animals. ACIAR Proceedings. No
43. pp121-134.
Ooi K.W., Effendy A.W.M. & M. Zamri-Saad. (2005). The Role of
Lyophilized Crude of Pasteurella multocida B2 in Stimulation
of Bronchus-associated Lymphoid Tissue (BALT) in Goats.
Regional Symposium on Haemorrhagic Septicaemia, 1 – 2
December 2005, Palm Garden Hotel, Putrajaya. pp54-56
Oxford Dictionaries Online; website: http://www.oxforddictionaries.
com/.
Per Jensen. (2006). Domestication - From Behavior to Genes and
Back Again. Applied Animal Behaviour Science, 97: (2006)
3–15.
Per T Sangild. (2003). Uptake of Passive Immunity by the
Compromized Newborn Animal. Acta Veterinaria Scandinavica
2003, 44(Suppl 1): p23.
Phuong, D.N. Effendy, A.W.M. & A.B Abol-Munafi. (2007).
Non-specific Immune Responses Towards Ascorbic Acid
Aupplementation in Clarias hybrid catfish (Clarias gariepinus
x Clarias macrocephalus). Asian-Pacific Aquaculture 2007
“Prospering from Dynamic Growth”. Melia Hotel Hanoi.
Vietnam. 6-8 August 2007.
Po Po S., Zuki A.B.Z., M Zamri-Saad, Omar A.R. & A.W.M. Effendy.
(2005). Morphological Features of Bronchus-associated
Lymphoid Tissue in Lungs of Calves in Relation to Age. Regional
Symposium on Haemorrhagic Septicaemia, 1–2 December
2005, Palm Garden Hotel, Putrajaya. pp50-53.
Porter JR. (1976). “Antony van Leeuwenhoek: Tercentenary of His
Discovery of Bacteria”. Bacteriological Reviews, 40 (2): 260–9.
Ponnier-Capitan, M., Bemilli, C., Bodu, P., Celerier, G., Ferrie, J.G.,
Fosse, P., Garcia, M. & J.D. Vinge. (2011). New Evidence for
Upper Palaeolithic Small Domestic Dogs in South Western
Europe. J. Archael. Sci., 38 (9, September 2011): 2123–2140
References • 79
80 • Mucosal Immunity: Animal Vaccination and Disease Prophylaxis
Price, E.O. (1984). Behavioral Aspects of Animal Domestication. Q.
Rev. Biol., 59: 1–32.
Price, E.O. (1999). Behavioral Development in Animals Undergoing
Domestication. Applied Animal Behaviour Science, 65 (1999):
245–271.
Price, E.O. & S.J.R. Wallach. (1990). Physical Isolation of Hand-
reared Hereford Bull Increases Their Aggressiveness toward
Human. Appl. Anim. Behav. Sci., 27 (1990): 263-267.
Pruett, S.B. (2003). Stress and the Immune Cell. Pathophysiology,
9: 133-153.
Rothkotter, H.J. & R. Pabst. (1989). Lymphocyte Subsets in Jejuna
and Ileal Peyer’s Patches of Normal and Gnobiotic Minipigs.
Immunology, 67: 103-108.
Saharee AA, & Salim N (1993). Haemorrhagic Septicaemia Carriers
among Cattle and Buffalo in Malaysia. ACIAR Proceedings No.
42: 89-91.
Sandrine Mignon-Grasteau, Alain Boissy, Jacques Bouix, Jean-Michel
Faure, Andrew D. Fisher, Geoffrey N. Hinch, Per Jensen, Pierre
Le Neindre, Pierre Morme`de, Patrick Prunet, Marc Vandeputte
& Catherine Beaumont. (2005). Genetics of Adaptation and
Domestication in Livestock. Livestock Production Science. 93
(2005): 3 –14.
Simister, N.E., & Story, C.M. (1997). “Human Placental Fc Receptors
and the Transmission of Antibodies from Mother to Fetus.”
Journal of Reproductive Immunology, 37: 1-23.
Sirous Sadeghian, Mohammad Reza Mokhber Dezfouli, Gholam
Ali Kojouri, Taghi Taghipour Bazargani & Abbas Tavasoli.
(2011). Pasteurella Multocida Pneumonic Infection in Goat:
Hematological, biochemical, clinical and pathological studies.
Small Ruminant Research, 100 (2): 189 - 194.
Saw Po Po, A.B.Z. Zuki, M. Zamri-Saad, A. Rahman-Omar & A.W.M.
Effendy. (2004a). Macroscopic Evaluation of the Gut-associated
Lymphoid Tissues (GALT) of the Small Intestine of Three-month-
Old Calves. 11th. International Conference of the Association of
Institutions for Tropical Veterinary Medicine and 16th Veterinary
Association Malaysia Congress 23-27 August 2004, Sunway
Pyramid Convention Centre, Petaling Jaya: 336-37.
Saw Po Po, A.B.Z. Zuki, M. Zamri-Saad, A. Rahman-Omar & A.W.M.
Effendy. (2004b). Histological Evaluation of the Gut-associated
Lymphoid Tissues (GALT) of the Small Intestine of Three-month-
Old Calves. 11th. International Conference of the Association of
Institutions for Tropical Veterinary Medicine and 16th Veterinary
Association Malaysia Congress 23-27 August 2004, Sunway
Pyramid Convention Centre, Petaling Jaya: 384-85.
Saw Po Po, A.B.Z. Zuki, M. Zamri-Saad, A. Rahman-Omar & A.W.M.
Effendy. (2004c). Distribution and Morphological Study of the
Bronchus Associated Lymphoid Tissues (BALT) in Three Months
Old Calves. Journal of Animal and Veterinary Advances, 3 (9):
564-570.
Singh, K., Ritchey, J. W. & A. W. Confer. (2011). Mannheimia
haemolytica Bacterial– Host Interactions in Bovine Pneumonia.
Veterinary Pathology, 48 (2): 338-348.
References • 81
82 • Mucosal Immunity: Animal Vaccination and Disease Prophylaxis
Siti-Raudah S.A.K., A.W.M. Effendy, Zamri-Saad, M. & Zuki M.B.Z.
(2006). Lymphocyte aggregation of gastrointestinal-associated
lymphoid tissue (GALT) following administration of formalin-
killed Pasteurella multocida B:2. 3rd. Life Sciences Postgraduate
Conference. 1st. USM-Penang International Postgraduate
Convention. 24-27 May 2006. Universiti Sains Malaysia. Pp
101.
Siti-Raudah, S.A.K., Effendy A.W.M., Zamri-Saad, M. & Zuki M.B.Z.
(2005). Systemic Immune Responses in Goats Following
Intranasal Exposure of Formalin-killed P. multocida B:2. 2nd.
KUSTEM Annual Postgraduate Seminar; 27 November 2005.
FST KUSTEM, p45.
Siti-Raudah S.A.K., Effendy, A.W.M., Zamri-Saad M. & Zuki, M.B.Z.
(2005a). Responses of Bronchus-associated Lymphoid Tissue
(BALT) and Gut-associated Lymphoid Tissue (GALT) Following
Administration of Formalin-killed Pasteurella multocida B2.
Regional Symposium on Haemorrhagic Septicaemia, 1–2
December 2005, Palm Garden Hotel, Putrajaya. pp45-49.
Siti-Raudah S.A.K., Effendy A.W.M., Zamri-Saad M. & Zuki A.B.
(2005b). Stimulation of Bronchus-associated Lymphoid Tissue
(BALT) Following Intranasal Exposure of Formalin-killed
Pasteurella multocida B2. KUSTEM 4th Annual Seminar on
Sustainability Science and Management: Meeting Challenges
in Sustainable Agrotechnology. 2- 3 May 2005. pp79-81.
Smith, M. C., & D. M. Sherman. (1994). Goat Medicine. New York:
Lea & Febiger.
Stefan Riedel. (2005). Edward Jenner and the History of Smallpox
and Vaccination. Baylor University Medical Center Proceedings.
18 (1): 21–25.
Sutherland, A.D. (1985). Effects of Pasteurella haemolytica Cytotoxin
on Ovine Peripheral Blood Leukocytes and Lymphocytes
Obtained from Gastric Lymph. Veterinary Microbiology. 10:
431-438.
Sutherland, A.D. and W. Donachie. (1986). Cytotoxic Effect of
Serotypes of Pasteurella haemolytica on Sheep Bronchoalveolar
Macrophages. Veterinary Microbiology, 11: 331-336.
The Use of Conventional Immunologic Adjuvants in DNA Vaccine
Preparations, by Shin Sasaki & Kenji Okuda. In D.B. Lowrie and
R.G. Whalen (editors), DNA Vaccines: Methods and Protocols,
Humana Press, 2000. ISBN 978-0-89603-580-5.
Tortora, G.J., Funke, B.R. & L.C. Christine. (2013). Microbiology: An
Introduction. (11th ed.). Pearson.
Totaro, R. (2005). Suffering in paradise: The Bubonic Plaque in
English Literature from More to Milton. Pittsburgh. Duquesne
University Press. p26.
Ullmann, Agnes (August 2007). “Pasteur-Koch: Distinctive Ways
of Thinking about Infectious Diseases”. Microbe (American
Society for Microbiology). 2(8): 383–7. Retrieved December
12, 2007.
Uribe, C., Folch, H. Enriquez, R. & G. Moran. (2011). Innate and
Adaptive Immunity in Teleost Fish: A Review. Veterinarni
Medicina, 56: 486-503.
Vessier, I., Gesmier, V., Le Neindre, P., Gautier, J.Y. & G. Bertrand.
(1994). The Effects of Rearing in Individual Crates on Subsequent
Social Behavior of Veal Calves. Appl. Anim. Behav. Sci., 41
(1994): 199-210.
References • 83
84 • Mucosal Immunity: Animal Vaccination and Disease Prophylaxis
Vigne, J-D. (2011). The Origins of Animal Domestication and
Husbandry: A Major Change in History of Humanity and
Biosphere. C.R. Biologies, 334 (2011): 171-181.
Wan Mohamad, W.E., Mohamad, N. & A. Abdul Rahman. (1988).
Perspectives and Progress of Sheep Industry at Kumpulan
Guthrie. In: Proc. of symposium on sheep production in
Malaysia. pp98-108. Kuala Lumpur.
Willis N.J. (1997). Edward Jenner and the Eradication of Smallpox.
Scott Med, 42:118–121.
Winkelstein W. Jr. (1992). Not Just a Country Doctor: Edward Jenner,
Scientist. Epidemiol Rev, 14:1-15
Zamri-Saad, M. (2005). Attempts to Develop Vaccines against
Haenorrhagic Septicamie: A Review. Proceedings: Regional
Symposiumon Haemorrhagic Septicaemia. pp1-15.
Zamri-Saad, M., Effendy, A.W.M., Israf, D.A. & Azmi, M.L. (1999a).
Cellular and Humoral Response in the Respiratory Tract of
Goats Following Intranasal Stimulation Using Formalin-killed
Pasteurella haemolytica A2. Veterinary Microbiology, 65: 233-
240
Zamri-Saad, M., Maswati, M.A., Effendy, A.W.M. & Jasni, S.
(1999b). Changes in the Lungs of Goats with Acute Pneumonia
Following Experimental Challenge with Pasteurella haemolytica
and Pasteurella multocida. Jurnal Veterinar Malaysia, 11(2): 67-
70.
Zamri-Saad, M., Subramaniam, P., Sheikh-Omar, A.R., Sani, R.A.
& Rasedee, A. (1994). The Role of Concurrent Haemonchosis
in the Development of Pneumonic Pasteurellosis in Goats.
Veterinary Research Communications, 18: 119-122.
Zamri-Saad, M., Jasni, S., Nurida, A.B. & Sheikh-Omar, A.R. (1991).
Experimental Infection of Dexamethasone-treated Goats with
Pasteurella haemolytica A2. British Veterinary Journal, 147:
565-568.
Zeder MA. (2008). Domestication and Early Agriculture
in the Mediterranean Basin: Origins, Diffusion,
and Impact. Proceedings of the National Academy of
Sciences, 105(33): 11597-11604.
References • 85
Mohd. Effendy bin Abd. Wahid was born in Banda Hilir, Melaka on 26th. October 1966. He completed his secondary education at Sekolah Dato’ Seri Amar Di Raja, Muar, Johor. He was then pursuing for his Diploma in Animal Health and Production in 1984 at Universiti Pertanian Malaysia (UPM). After obtaining his diploma, he continued his study for Doctor in Veterinary Medicine and graduated in 1994. His passion for research and development motivated him to enroll for Ph.D study in the field of Veterinary Immuno-pathology in1995. He was awarded the Best PhD Thesis Award by the Faculty of Veterinary Medicine, UPM in 1998. Effendy started his academic journey at UPM Terengganu (UPMT) in November 1998 at the Faculty of Science and Professional Arts. He joined the Faculty of Science and Technology in 2001 before accepted a Post-doctorate position at Chonnam National University, Kwang-ju, South Korea by Korean Institute of Science and Technology Evaluation and Planning (KISTEP) in 2003.
Effendy was appointed as the Head of Student Welfare and Development Centre at Division of Student Affairs, KUSTEM in 2004. He was then given a task to establish the Institute of Marine Biotechnology at the end of 2005, and appointed as the founding Director of the Institute of Marine Biotechnology from 2006 to 2013. During the tenure, he faced many challenges until the Ministry of Higher Education approved it on February 26th 2008. Effendy was then appointed as the Assistant Vice Chancellor (Research and Innovation Affairs) in 2013, and the first Deputy Vice Chancellor (Research and Innovation) for UMT in 2014. Effendy has great interest in research and innovation. Until present, he has taught eight different courses and still teaching a course at the school. He started his first research on histopathology of uterine involution and side effects of Kacip Fatimah at a small laboratory at INOS in 1999.
Since then, he has supervised and graduated 12 PhD, 24 Master Science and 180 final year students and published 60 scientific papers in various journals, particularly in microbiology, immunology and biotechnology. His works with his colleagues were also disseminated through more than 110 seminars, conferences and symposiums. Apart from that, Effendy also involved in writing books and monograph, and reviewing research papers and thesis. He also acquired two full patent rights on mucosal vaccine against pneumonic pasteurellosis in small ruminants and nutritious health drinks from golden sea cucumber extracts, and others that are still pending. Throughout his research activities, he was recognized with 13 Special Research Awards, 16 medals at International Research Competitions and 24 medals at national research exhibitions and competitions. He was awarded Excellent Scientist Award by the Ministry of Higher Education in 2005 and the first recipient of National Intellectual Property Award 2006 (Individual Category). At the moment, Effendy focus his research on development of novel adjuvants for mucosal vaccines, establishing of seahorse sanctuary in Johor Darul Ta’zim and sustainable aquaculture management utilizing microalgae program with Japan International Collaboration Agency (JICA).
cover-syarahan-inaugural-prof-EFFENDYj.indd 1 5/12/16 4:41 PM