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


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



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


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


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


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


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


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


(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.


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,


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


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.



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.



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



(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 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


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

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0.8

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Day 1 Day 5 Day 10 Day 15 Day 19 Day 24 Day 29

Days

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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)


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.



Ig G Levels After Administration of Lyophilized Pasteurella multocida B:2 Crude

0

0.05

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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

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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

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Ig G

Lev

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Ig G Level in Lung Lavage:

First Administration: Second Administration:

Con

trol

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up 1

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up 2

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up 3

00.020.040.060.080.1

0.120.140.160.18

Ig A

leve

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Ig A Level in Lung Lavage:


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


(ii)

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up 1

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First Administration:Second Administration:0

50001000015000

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Number of Lymphocytes in Duodenum (Intraephithelial):


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):


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


(ii)

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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



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).



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



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.



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



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.


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


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


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,


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.

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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).

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