SAARC Regional Training - SAARC Agriculture Centre Diagnosis...SAARC Regional Training On Molecular Diagnosis and Laboratory Surveillance of PPR 21-26 July 2019 Editors Mohammed A

i

SAARC Regional Training

On

Molecular Diagnosis and Laboratory Surveillance of PPR

21-26 July 2019

Editors

Mohammed A Samad

Md. Abu Yousuf

Md. Nure Alam Siddiky

Md. Giasuddin

Nathu Ram Sarker

Ashis Kumar Samanta

S.M. Bokhtiar

Bangladesh Livestock Research Institute

Savar, Dhaka-1341

SAARC Agriculture Centre Dhaka, Bangladesh


ii

The SAARC Regional Training on “Molecular Diagnosis and Laboratory Surveillance of

PPR” is held at Bangladesh Livestock Research Institute, Savar, Dhaka-1341 during 21-

26 July 2019. The training is sponsored by SAARC Agriculture Centre, Dhaka,

Bangladesh

Editors

Dr. Mohammed Abdus Samad, SSO, AHRD, BLRI

Dr. Md. Abu Yousuf, SO, AHRD, BLRI

Dr. Md. Nure Alam Siddiky, Consultant, CAMR-ZD in BD, BLRI

Dr. Md. Giasuddin, Head, AHRD, BLRI

Dr. Nathu Ram Sarker, Director General, BLRI

Dr. Ashis Kumar Samanta, SPS, SAC

Dr. S. M. Bokhtiar, Director, SAC

Recommended citation

Samad, M.A., Yousuf, M.A., Siddiky, M.N.A., Giasuddin., M., Sarker, N.R., Samanata,

A.K., & Bokhtiar, S.M. eds. (2019). Training Manual on Molecular Diagnosis and

Laboratory Surveillance of PPR. Bangladesh Livestock Research Institute, Savar, Dhaka-

1341, P. 84

Published by


Savar, Dhaka 1341, Bangladesh

Tel: +88-02-7791670-2, 7791676, Fax: +88-02-7791675

Email: [email protected]

www.blri.gov.bd

ISBN: 978-984-34-7035-5

All rights reserved

This work is subjected to copy rights. All rights reserved by the publishers, whether

whole or part of the material thereof. The authors are solely responsible for the content of

the abstract papers compiled in this publication. The publisher/editors shall not be

responsible for the views, opinion and materials expressed by the authors.

Printed by

Nathudhara Printing Press

277/3 Elephant Road (1st Floor), Kataban, Dhaka

mailto:[email protected]


iii

Secretary Ministry of Fisheries and Livestock

Govt. of Bangladesh

Message

It gives me an immense pleasure that Bangladesh Livestock Research Institute is

organizing a regional training on “Molecular Diagnosis and Laboratory Surveillance of

Peste des petits ruminants (PPR)” from 21st to 26

th July 2019 under the aegis of SAARC

Agriculture Centre at BLRI, Savar, Dhaka-1341.

The role of livestock in livelihood, nutritional and food security of millions of

people live in SAARC region is very significant. Formulation of effective disease control

strategies is a daunting task for sustainable development of livestock sector in the region.

PPR is one of the important economic and devastating disease for small ruminants

prevalent in most of the SAARC countries. PPR can easily be transmitted from one

SAARC countries to another due to trans-boundary nature of the disease as well as

porous borders. Regional concerted and coordinated effort is prerequisite to eradicate

PPR in compliance with the global initiatives. Bangladesh has also been developed

national strategic action plan for the eradication of PPR following the guideline of PPR

global eradication campaign. Govt. of Bangladesh has taken different initiatives to

address the PPR eradication in align with global strategy. I believe BLRI has a very good

laboratory capacity as well as skilled human resources to organize the training very

successfully.

I am sure that the participants from SAARC countries would be exposed to many

conventional and new approaches employed for the precise molecular diagnosis of PPR.

Further, this training would also provide a common platform and networking to the

researchers from fellow SAARC countries to discuss strategic plan for the progressive control

of PPR to eradicate by 2030.

I wish this training program a great success.

(Md. Raisul Alam Mondal)


iv

Director General


Message

Bangladesh Livestock Research Institute (BLRI) a pioneer institute has been

entrusted to conduct Research & Development in the field of animal health, production

and different cross cutting issues related to livestock development in the country. BLRI is

hosting Regional Leading Diagnostic Laboratory (RLDL-PPR) for PPR under the aegis

of South Asian Association for Regional Cooperation (SAARC).

Livestock plays an important role in rural livelihood of subsistence farmers

dependent on the sector. The livestock sector in this region has been facing challenges

for transboundary, emerging and re-emerging threats of diseases. PPR is one of the

common notifiable disease for small ruminant prevalent in the region. BLRI has

developed progressive control pathway of PPR eradication model in accordance with

the guideline of OIE. Capacity building in the field of molecular diagnosis,

bioinformatics and surveillance of PPR will be of immensely useful for the participants

to containment disease in the region. There is need for continuous exchange of

knowledge & ideas and disease outbreak information among SAARC countries for early

detection and emergency preparedness of emerging, re-emerging and transboundary

diseases of the region.

It is a matter of pride and responsibility of the Institute to host the SAARC

regional training on ‘Molecular Diagnosis and Laboratory Surveillance of Peste des

petits ruminants” is held from 21st to 26

th July 2019. I hope the deliberations in the

training will be mutually beneficial among the participants as well as to the host

organizations. This training will create an avenue for BLRI to deliver our experience and

expertise in the field of molecular diagnosis of PPR among the participants which will

pave a better way to control the disease within the SAARC region.

I wish the participants a pleasant stay and I wish the training a great success.

(Dr. Nathu Ram Sarker)


v

Director

SAARC Agriculture Centre

Message

I am pleased to know that a training manual is published for the SAARC regional

training programme on "Molecular Diagnosis and Laboratory Surveillance of PPR"

jointly organized by SAARC Agriculture Centre (SAC) and Bangladesh Livestock

Research Institute.

SAARC Agriculture Centre (SAC), under the aegis of South Asian Association for

Regional Cooperation (SAARC) has been working for the promotion of agricultural

research & development as well as technology transfer through regional networks among

agricultural research/extension institutions and policy makers in the SAARC region.

BLRI is one of the specialized premier institute undertaking research and developmental

activities on animal health and production for the promotion of livestock sector. Livestock

is one of the important sector for food (milk, meat, egg) production, livelihood

improvement, employment generation and women empowerment. The livestock sector in

the region has been facing numerous challenges such as disease burden, scarcity of feeds

and fodder, poor quality genetic resources and so on. Peste des petits ruminants (PPR) is

endemic in all over the SAARC region with huge economic implications. SAARC has

developed a regional road map for the eradication of PPR by 2025. The regional training

on "Molecular Diagnosis and Laboratory Surveillance of PPR" would provide theoretical

as well as hands-on knowledge to the participants and exposure on different techniques and

technologies for molecular diagnosis and characterization of PPR. I believe the contents of

the manual is certainly the store of information related to advanced research and

development of PPR particularly molecular diagnosis. This manual is unique and store of

knowledge for anyone who is interested in pursuing research on PPR prevention and

control.

I wish all the grand success for this regional training programme and its endeavors.

(Dr. S. M. Bokhtiar)


vi

FOREWORD

Bangladesh Livestock Research Institute is organizing a training program on “Molecular

Diagnosis and Laboratory Surveillance of PPR” from 21st to 26

th July 2019, in

collaboration with SAARC Agriculture center, Dhaka, Bangladesh. The participants for the

training program are from Bangladesh, India, Nepal and Sri Lanka. The objective of the

training program is to impart the training to member nations in the domain of molecular

diagnosis and surveillance for PPR which is an important area need to be addressed to foster

the PPR global control strategy. The theme of the training program is appropriately chosen

by the SAARC Agriculture Center considering the urgent need to build capacity in order

to formulate PPR control and prevention strategies.

PPR is also known as goat plague which is increasing importance in Africa and Asia

wherever small ruminants form an important component of agricultural food production.

It threatens the food security and sustainable livelihood of farmers across the region. The

world organization for animal health (OIE) has identified PPR as a noticeable and

economically important trans-boundary viral disease of sheep and goats. The capacity

building in terms of laboratory and skilled human resources are still in meager and scanty

for PPR diagnosis and surveillance. The training encompasses theory followed by hands

on exposure on different novel techniques and technologies on diagnosis and sero-

surveillance. The resource speakers have been chosen very appropriately with their long

experience and devotion in the field of PPR. I hope the course contents of the training

would be immensely useful to the participants from SAARC countries with constructive

exchange of knowledge and experience.

I am sure that the present SAARC Agriculture Centre sponsored training program on

Molecular Diagnosis and Laboratory Surveillance of PPR would be quite useful for the

participants from the SAARC countries and this document will serve them as a reference

for carrying out various analytical procedures in their laboratory for molecular diagnosis

and surveillance. I would like to extent my sincere thanks to the SAARC Agriculture

Centre, Bangladesh for giving us the opportunity to organize hands on training on such

an important aspect. I would also like to thank Director General, Bangladesh Livestock

Research Institute for his support and guidance to conduct this training program

effectively.

(Mohammed A Samad, PhD)

Director

SAARC RLDL for PPR


vii

Contents

Message iii

Message iv

Message v

Foreword vi

PPR Eradication Strategy of Bangladesh 1

Making History: Eradicating Peste des petits ruminants –Saving Lives 7

and Livelihoods

Regional Roadmap on Progressive Control of Peste des petits ruminants 16

(PC PPR) for South Asian countries

BLRI Developed PPR Control Model 23

An Overview of General Guidelines for PPR Sample Collection, Preservation 26

and Genomic Analysis

Principles of PCR and Guidelines for Good Laboratory Practices 37

ELISA: An Essential Tool for Surveillance of PPR 42

National Animal Disease Referral Expert System (NADRES) 52

Laboratory Management with Biosafety and Biosecurity Practices 58

RT-PCR: The Technique for the Detection of PPR 60

Detection of PPRV Antibody in Sera by Competitive Elisa (cELISA) 67

Cell Culture for Virus Isolation and Identification 72

List of the Participants 81

List of the Resource Persons 83


1

Figure 1. District-wise goat population

distribution for the year 2017-18

PPR ERADICATION STRATEGY OF BANGLADESH

Mohammed A Samad* and Abu Sufian1

Director, SAARC Regional Leading Diagnostic Laboratory for PPR, Bangladesh Livestock

Research Institute, Savar, Dhaka-1341 1Department of Livestock Services, Khamarbar, Farmgate, Dhaka-1215


Livestock is an integral component of the complex farming system in Bangladesh as it

not only a source of meat protein but also a major source of farm power services as well

as employment. The livestock sub-sector provides full time employment for 20% of the

total population and part-time employment for another 50%. The GDP contribution of

this sub-sector has been a modest 1.54% annually in the 2017-18 fiscal year (DLS. 2018)

with the growth rate of livestock 3.40%. However, the sector’s actual contribution has

been consistently underestimated as the value added in draught power used in farm

operation, threshing, sugarcane and oilseed crushing, local transport, dung for cooking

fuel and manure for fertilization of crop fields were not taken into account. An estimate

of the uncounted sectoral contribution of livestock indicates a foregone value of three

times the amount of official GDP attributed to this sector (FAO 1990). Moreover,

livestock products, namely, leather and leather products, hides and skins are important

exportable items. Consequently, given versatile nature of the potential contribution

offered by the livestock sector including curbing of malnutrition prevalent in Bangladesh

also.

Bangladesh is blessed with small ruminant

(SR) population of 29.57 million (goats:

26.10 million and sheep: 3.47 millions). SR

usually kept by landless farmers in

marginal areas and are ranked second to

poultry in the livestock species ladder while

prioritizing the species kept by poor. Out of

the SR, goat represents 47.34% of the total

livestock populations in Bangladesh (DLS,

2017) and are normally reared by the rural

women community in the country. Thus

goat rearing plays an important role in

women empowerment. Nowadays

commercial goat farming

has become popular using intensive

farming system. Commonly available

breeds are Black Bengal goat, exotic breeds

such as the Jamunapari, Sirohi, Beetal and

crossbreds. Most recently, a few exotic



2

Figure 2. Spatial distribution of PPR

cases for the year 2017-18

breeds like Boer goat is being adapted as meat type goat in the country. However, Black

Bengal goat is the dominant (about 90%) breed in the country.

Sheep in Bangladesh, stands as third in position after cattle and goat population, and are

used primarily for meat production. Bangladesh possesses 3.40 million sheep at present

(DLS, 2017). Small and landless farmers rear about 38%, medium farmers 40% and large

farmers 22% of total sheep in Bangladesh. They are sparsely distributed throughout the

country but relatively higher concentration (about 32% of total sheep population) are

found in three different ecological zones like, Barind, Jamuna river basin and Coastal

areas, where farmers maintain larger commercial (meat) flocks. Sheep available in

Bangladesh are mostly indigenous non-descript type. Nonetheless, Garoles is one of the

native Bangladeshi sheep which are found in the extreme south-west of Bangladesh

adjacent to the Sundarbans forest in the coastal area and western districts of Bangladesh.

The coat is usually light brown in colour, with some animals having black spots on the

legs and the head region. Garole has an earlier puberty age, produces twin kids and has a

high resistance to internal parasites. They can survive better in saline water than other

sheep populations and the Black Bengal goat.

Peste des petits ruminants (PPR) is

considered as one of the major threats to

SR population in Bangladesh. The first

outbreak of PPR in Bangladesh occurred in

1993 in a bordering district, Meherpur,

south western part of the country. Since

then disease is continuing to occur and has

become endemic. The disease has been

described as the most important single

cause of morbidity and mortality in small

ruminant’s population in Bangladesh. As

per Upazila veterinary hospital based

secondary surveillance program 89,093

cases were identified, of which in Rajshahi

division ranked top position (69,899)

followed by Chattagram and Rangpur

(37,815) and Chattagram division (34,059)

shown in Figure 2. There is no reliable and

published data on the economic losses

incurred due to PPR in Bangladesh. A study

depicted, in 2010, there were 84,000

clinical cases reported, causing an

estimated loss of 1,842 million Taka (US$ 24.6 million). Since secondary surveillance

will not cover entire area of a sub-district (Upazila), for this reason, the actual number of

infected cases may not represent in the data as the cases of remote rural areas are not

included.


3

PPR is an acute, highly contagious, world organization for animal health (OIE) notifiable

and economically important transboundary viral disease of sheep and goats associated

with high morbidity and mortality and caused by PPR virus. The disease is capable of

destroying whole of the susceptible host population by infuriating epidemics and

panzootics, thus damaging economy, undermining food security and livelihood of poor

people of the society. PPR virus (PPRV) belongs to the family Paramyxoviridae, genus

Morbillivirus. The PPRV was first identified in Côte d’Ivoire in 1942 and for many

yea’rs it was considered as an African disease localized mainly in Western and Central

Africa. However, over the passage of time it became endemic across the Africa, Arabian

Peninsula, Middle East, Turkey, Pakistan, India, Bangladesh, Nepal, Tajikistan and

Kazakhstan in Central Asia, Mongolia and China. The PPR virus has been classified into

four distinct genotypes/ lineages (I, II, III, and IV). All four lineages are prevalent in

Africa while across Asia only lineage IV has been reported.

Seventeen Sustainable Development Goals (SDGs) of United Nations (UN) integrate the

three dimensions of sustainable development economic, social and environmental and are

targeted 2030 towards fulfilling the such goals, of which nine (9) goals are directly or

indirectly relate to livestock sectors. Goat rearing in Bangladesh has envisaged the SDGs

targets are- no poverty, zero hunger, gender equality, reduced overall inequality. But at

present small goat farmers in Bangladesh are facing massive economic losses due to PPR

as the disease still endemic in Bangladesh. It has been estimated that losses due to PPR

worth 24.6 million US$ annually that has a direct impact to fulfillment of Sustainable

Development Goals (SDGs) of United Nations. Thus, considering the fact, it is very

pragmatic to take immediate intervention to control and finally eradicate the disease by

2030. Since the disease is transboundary in nature, so regional approach to be optimized

to control the disease from country and the region as well.

Based on the country self-assessment using the PPR Monitoring and Assessment Tool

(PMAT), Bangladesh holds the position in Stage 2 of PPR, Global Control and

Eradication Strategy (GCES) and will continue up until 2020 towards reaching the Stage

3 in 2021, finally getting free status by 2025.

A national strategic plan (NSP) for the control and prevention of PPR and its subsequent

eradication by 2025 in Bangladesh has been framed in the light of Global Strategy for the

Control and Eradication of PPR. The methodology proposed in the NSP targets at

institutionalizing the efforts for the progressive control leading to the eradication of PPR

in Bangladesh. These activities will be carried out mainly through a donor funded

program, however, government is implementing the activities for PPR control and

prevention through own resources.

The control strategy consists of following main components:

PPR strategy and technical plans

Legal framework

Stakeholder awareness and engagement

Strengthening veterinary services


4

Support to the diagnostic and surveillance

Systems strengthening of laboratory and surveillance capacities

Epidemiology and laboratory networks

Measures toward PPR eradication

Demonstration of PPR freedom

Control of other small ruminant diseases (SRD) in support of PPR Eradication

Coordination, Management and Partnerships

Overall objective of NSP

To control and eradicate PPR from Bangladesh for poverty reduction, livelihoods, food

and nutrition security, resilience, market access and economic development.

Specific objectives of SP

a) To ensure an enabling environment for PPR eradication by improving policies,

awareness, legal framework and veterinary services.

b) To develop a robust diagnostic and surveillance system by improving capacity for

epidemiological assessment, surveillance, diagnosis and networking.

c) To ensure accessibility and availability of quality PPR vaccine and implement

vaccination program for achieving desired level of herd immunity.

d) To ensure in country and transboundary movement control to prevent the spread of

PPR virus and other SRDs.

e) To develop an effective coordination mechanism at national, regional and global

level by developing partnership and networking.

Expected outputs

Output -1: Enabling environment created for PPR Eradication by developing and

implementing National PPR strategy, guidelines, SOPs, awareness raising and improved

veterinary service delivery.

Output -2: A robust diagnostic and surveillance system developed by improving

capacity for epidemiological assessment, surveillance, diagnosis and networking.

Output -3: Accessibility and availability of quality PPR vaccine achieved and

vaccination programme undertaken for achieving desired level of herd immunity.

Output -4: In country and transboundary movement control of animal and animal

products to prevent the spread of PPR virus and other SRDs ensured.

Output - 5: An effective coordination mechanism at national, regional and global level

developed by ensuring partnership and networking.


5

Time bound logical framework

NSP description Verifiable indicators

of achievement

Time

Frame

Goal Improvement the socioeconomic conditions of small

ruminant’s producers by ensuring livelihood, poverty

reduction and achieving food security through progressive

control towards eradication of PPR in goat and sheep in

Bangladesh as compliance with SDGs target and obtain PPR free status from OIE

Significant reduction

of PPR incidence with

a positive

socioeconomic impact in the country.

2019-2015

Purpose Bangladesh will move towards eradication stage (Stage #3)

of GCES.

Report and document

have prepared and

submitted.

Output -1 Enabling environment created for PPR Eradication by

developing and implementing National PPR strategy,

guidelines, SOPs, awareness raising and improved veterinary service delivery.

SOPs and Guidelines

developed and

improved veterinary services developed.

2019-2020

Activities Develop and implement national strategic plan for the

control of PPR.

Develop a PPR vaccination and surveillance plan for

PPR and SRDs.

Prepare communication guidelines and materials for

raising awareness on PPR

Develop and Update standard operating

procedures/protocol for the control and eradication of

PPR.

Training of field vets, paraprofessionals and laboratory

staffs on surveillance, epidemiology and diagnosis of

PPR including TADs.

NSP developed

Communication

guidelines developed

Vaccination and

surveillance

strategy/plan developed

Relevant

manpower trained

Output -2 A robust diagnostic and surveillance system developed by

improving capacity for epidemiological assessment,

surveillance, diagnosis and networking.

A real-time

surveillance and robust

diagnostic system developed.

2020-2024

Activities Assess the epidemiological situation relating to PPR

disease distribution, risk factors for endemicity along the value chain and risk analysis.

Support to dedicated Community Animal Health

Workers for PPR Vaccination and primary health care.

Active surveillance, outbreak investigation and report

accordingly to be installed.

Sero-monitoring in immunized herd will be established.

Listing of wild life species along with surveillance of

susceptible species

Establish an Early Warning System (EWS) for TADs

on real time basis.

Strengthen PPR diagnosis capacities of regional

laboratories (FDILs), Central Disease Investigation Lab

(CDIL) and Regional Leading Diagnostic Laboratory.

PPR disease

epidemiology understood

Real-time

surveillance installed

Supported to all

laboratories for

PPR disease

diagnosis

Strengthened the

capacity of district

and upazila level laboratories


6

NSP description Verifiable indicators

of achievement

Time

Frame

for PPR (RLDL)

Capacity building of Upazila/ District Veterinary

Hospitals for collection, storing and Shipment of PPR samples.

Output 3 Accessibility and availability of quality PPR vaccine is

achieved and vaccination programme undertaken for achieving desired level of herd immunity.

Vaccine induced

desired level of herd immunity.

2020-2021

Activities Support to current vaccine production in terms of better

vaccine production technology and including quality

control in public and private sector.

Support to thermostable PPR vaccine production for

field use.

Strengthening of LRI in setting up regular testing of all batches of vaccines produced through standard SOPs.

Quality vaccine

production enhanced

and Quality control of

produced vaccine established.

Output 4 In country and transboundary movement control of

susceptible animal and animal products to prevent the spread of PPR virus and other SRDs is ensured.

TADs have prevented

though controlling of

animal movement by

border inspection.

2023-2025

Activity Support to animal quarantine station monitoring animal movement/transportation.

Border guard training on PPR transmission and control.

Animal Quarantine

station established and

Border guard

sensitized on PPR disease.

Output 5 An effective coordination mechanism at national, regional

and global level is developed by ensuring partnership

and networking.

A functional

Coordination

mechanism established

2019-2025

Establish a PPR Eradication Management Unit

(PEPMU) at DLS with accommodating necessary staffs.

A Technical Committee headed by DG DLS for the

periodical oversight of the programme and monitor the progress and keep the programme on right track.

A high level multi-sectoral Steering committee headed

by Secretary, Ministry of Fisheries and Livestock will

be formed drawing members from relevant public

sector agencies, academicians and stakeholders from the private sector and farming community

A functional Central

Coordination mechanism established

Monitoring and evaluation

The government, industry representatives, civil society and non-government

organizations will jointly evaluate the implementation of this strategy work plan every

year. They will jointly conduct annual evaluation of implementation of the strategic

action plan and identify an achieved level of each criteria. The evaluation results will be

presented publicly after approval from the government. For this activity, the PPR

Monitoring and Assessment Tool (PMAT) will also be used.


7

MAKING HISTORY: ERADICATING PESTE DES

PETITS RUMINANTS – SAVING LIVES AND

LIVELIHOODS

Bouna Diop Secretary, FAO/OIE PPR Global Secretariat, Viale delle Terme di Caracalla, 00153

Rome, Italy


Introduction

A global strategy to control and eradicate PPR was agreed at an international conference

hosted by FAO and OIE in April 2015 in Abidjan, Côte d’Ivoire. Drawing from their

experience in eradicating rinderpest, FAO and OIE have formed a joint global secretariat

to guide efforts to eradicate PPR worldwide by 2030; this timeframe coincides with the

2030 Agenda for Sustainable Development. This note provides an overview of the state

of play in implementing the global strategy and the challenges encountered.

Importance of small ruminants

Small ruminants – totaling 2.2 billion heads worldwide according to FAOSTAT - are the

primary livestock resource of 300 million poor rural families around the globe, including

subsistence farmers and landless villagers as well as pastoralists. For these households,

sheep and goats are a source of food and regular income, a means to capitalize savings,

and a safety net during times of hardship. Selling animals or their products provides the

necessary resources to access food, as well as educational and social services

Food products derived from sheep and goats are an essential part of the diet for many

people around the world and contribute to overcoming malnutrition. Sheep and goat milk

and meat are of high nutritional value and provide high-quality protein, vitamins and

minerals critical for cognitive development and physical strength, particularly for

children. Small ruminants are well adapted to arid and semi-arid environments, and are

kept in a variety of production systems throughout the world. These include pastoral

areas, where goats and sheep make a mixed flock. Households may totally depend on the

animals for survival, as crop production is almost absent in such arid or desert areas.

Small ruminants are mobile assets; pastoralists move with them in search for water and

new pasture, or in times of climatic stress and volatile security situations. In such pastoral

systems, meat and milk are key for food security and nutrition. Income from sales of live

animals and their products account for between 60 and 80 percent of total household

income. This money is essential for purchasing cereals and other household items,

covering social and financial obligations, paying for school, or dealing with doctors’ fees.

In most pastoralists’ cultures, women are in control of small ruminant operations and the

associated income flow. This favors gender balance and contributes to an equitable

allocation of earnings and animal-source foods within the household. Pastoralism is


8

dominant in some large regions in Africa (Sahel region, Afar in Ethiopia, Turkana in

Kenya, Somali region), the Middle East and Central and East Asia. In particular, in the

dry zone in the Sahel region, it is the only way of life.

Peste des petits ruminants

Peste des petits ruminants (PPR) is a highly contagious viral disease of domestic and wild

small ruminants first reported in 1942 in Côte d’Ivoire. The disease is caused by a

morbillivirus, Peste des petits ruminants virus (PPRV) belonging to the genus

Morbillivirus in the family Paramyxoviridae. PPR was first reported in Cote d’Ivoire,

West Africa. For some time, the disease was reported only from West Africa before

expanding through other regions in Africa (East, Central), the Middle and Near East, and

Asian countries extending from West Asia to China. PPR primarily affects sheep and

goats, although cattle, camels, buffaloes and some wild ruminant species can also be

infected, indicating spillover from domestic sheep and goats. Morbidity and mortality

rates in small ruminants vary, but can be as high as 100% and 90%, respectively in

previously unexposed flocks. PPRV also acts as a predisposing factor for secondary

bacterial infections which can contribute to high morbidity and mortality.

A PPR outbreak is an emergency due to its rapid spread and high animal mortality rate.

Fatal diseases of small ruminants, such as PPR, affect the already vulnerable livelihoods

and can decimate the savings of poor populations, in particular in pastoral areas. People

become desperate when they lose their assets. PPR outbreaks, and the desperation due to

the loss, can therefore trigger turmoil, migration, and volatile security situations.

Eradicating PPR will increase sustainability, alleviate poverty, improve the resilience of

poor pastoralists and their communities, enable them to better cope with other shocks and

threats, prevent forced migration and mitigate extremist trends.

Following the world-wide eradication of rinderpest in 2011, a global consensus was

reached on the need to eradicate PPR. The disease can be readily and cost-effectively

diagnosed and a reliable, inexpensive and high quality vaccine is available that confers

lifelong immunity to vaccinated animals after a single dose. The virus also has a

relatively short infectious phase and does not survive for long outside a host, making it an

ideal candidate for a concerted eradication effort. Controlling and eventually eradicating

PPR means fighting rural poverty, ensuring food security and nutrition, and strengthening

resilience and national economies. It will contribute significantly to achieving the

Sustainable Development Goals (SDGs), particularly SDG 1 (no poverty), SDG 2 (zero

hunger), but also SDGs 5 (gender equality) and 8 (decent work and economic growth).

PPR Global Control and Eradication Strategy

The PPR Global Control and Eradication Strategy (GCES) developed by FAO and OIE

was endorsed during an international conference on PPR held in Abidjan, Côte d’Ivoire,

in April 2015, with the vision of a PPR-free world by 2030. The PPR GCES promotes a


9

stepwise approach based on four stages (figure 1), which provide an overview of how the

programme will operate. These stages correspond to a combination of decreasing levels

of epidemiological risk and increasing levels of prevention and control, and comprise a

multi-stage, multi-country process involving assessment, control, eradication and

maintenance of PPRV free status. This ranges from stage 1 (where the epidemiological

situation is being assessed), to stage 4 (when the country can provide evidence that there

is no virus circulation either at a zonal or national level, and is ready to apply for the OIE

official PPR-free status). Control activities, including vaccination, are implemented in

stage 2 while stage 3 corresponds directly to PPR eradication. Of note, to enter stage 4

vaccination must be suspended in order to facilitate epidemiological monitoring of

disease. Implementation requires the concerted delivery of preparedness plans, capacity

building, improved stakeholder awareness and engagement; as well as the establishment

of appropriate legal frameworks.

Figure 1: The four Stages of the PPR GCES

Regardless of the stage in which a country initially places itself, it is imperative that

sufficient capacity is secured in 5 key areas so that the country can move, with

confidence, to the next stages of control and eradication. These five technical elements

are: i) provision of an adequate PPR diagnostic system, ii) development of a PPR

surveillance system, iii) implementation of a PPR prevention and control system, iv)

establishment of a legal framework system and v) ensuring adequate stakeholders’

involvement in the campaign. The PPR Monitoring and Assessment Tool (PMAT) is

being used to support countries in conducting self-assessments of their current stage. As

the implementation of the PPR GCES requires effective national Veterinary Services, it

provides capacity building using proven frameworks such as the Performance of

Veterinary Services (PVS) Pathway. The programme also promotes activities geared

towards reducing the prevalence of other prioritized small ruminant diseases. Finally, the

PPR GEP provides required technical assistance and coordination at regional and global

levels.

PPR Global Eradication Programme (GEP) 2017 - 2021

In October 2016, FAO and OIE launched the first five-year (2017 – 2021) of the PPR

GEP developed through an inclusive and peer-reviewed drafting process. The programme

aims to lay the foundations for and commence the PPR control and elimination effort in


10

infected countries by developing capacity; understanding the epidemiological situation

and defining appropriate implementation strategies to reduce the prevalence of PPR and

eventually eradicate the disease. For non-infected countries, the programme will assist in

developing capacity to demonstrate the absence of PPR virus and move towards OIE

official PPR free status recognition. The programme will also support countries to reduce

the prevalence of other prioritized small ruminant diseases and strengthen veterinary

systems. But the programme goes beyond disease eradication alone– it also aims to

improve national production models and help herders build the strongest, most resilient

livelihoods with their animal resources.

Overall coordination of the PPR GEP

To drive the PPR eradication effort on a global scale and effectively support countries in

fighting the disease, and building on the efforts of the FAO-OIE Global Framework of

the Progressive Control of Transboundary Animal Diseases (GF-TADs), FAO and OIE

established a Joint PPR Secretariat in March 2016 in FAO Headquarters. The Secretariat

is responsible for the overall coordination of the PPR GEP. The Secretariat is supported

by an Advisory Committee (AC) which provides strategic guidance and oversight on the

execution of the programme while also playing an important advocacy role with policy

makers, donors, national veterinary services and livestock owners. The Global Research

and Expertise Network (PPR GREN) was launched in April 2018 during a meeting

hosted by the Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture

in Vienna. PPR GREN is established as a forum for scientific and technical consultations

to foster a science-based and innovative debate on PPR.

What the PPR GEP is doing?

PPR infected-countries are found in nine regions throughout Africa, Asia, the Middle

East and Europe (Map 1). PPR Regional Roadmap Meetings have been organized in all

these regions. The first round of meeting provided the opportunity to present the PPR

GCES and its tools; carry out a first self-assessment of each country’s situation regarding

PPR and the capacity of its Veterinary Services to control the disease; and develop the

regional Roadmap for the region and obtain countries engagement for its implementation.

The meetings also served to identify other small ruminant diseases that could be

controlled together with PPR and set up the Regional Advisory Group (RAG) to oversee

the implementation of PPR control activities in the region. The regional roadmap

meetings are important to ensure continuous assessment and monitoring of the disease

situation, to discuss challenges faced on PPR GEP implementation and progress made

and to promote regional approaches because of the transboundary nature of the disease.

FAO and OIE have also developed partnerships with regional organizations, the African

Union – Inter African Bureau for Animal Resources (AU-IBAR), the African Union Pan

African Veterinary Vaccine Centre (AU-PANVAC) and Regional Economic


11

Communities such as AMU1, AOAD

2, ASEAN

3, ECCAS

4, ECO

5, ECOWAS

6, GCC

7,

IGAD8, SAARC

9, SADC

10 as well as with relevant civil society organizations.

FAO, OIE and partners are providing support to countries and regions to formulate their

PPR National and Regional Strategic Plans, which detail the steps for assessing,

controlling, and eradicating PPRV, and maintaining PPRV freedom, as well as the

financial resources required and committed by national and regional authorities to

implement the Plans. Eight out of the nine regions have formulated their respective

regional strategies, which now need to be endorsed by their constituencies. In addition,

68 infected countries have formulated their National Strategic Plans (NSP) in alignment

with the regional and global strategy. The formal endorsement of NSP by national

authorities and the integration of PPR into existing agriculture sector programmes and

activities are essential to make more national budgets available for the PPR eradication

programme.

Meetings of PPR vaccine manufacturers are held every two years since 2014. The 3rd

meeting was organized by FAO and OIE in Amman, Jordan in April 2019 in

collaboration with the Veterinary Services of Jordan and JOVAC. On December 2017,

the PPR Secretariat organized a workshop on thermotolerant PPR vaccines with the

funding support of GALVmed. The workshop reviewed the current research on

thermotolerant PPR vaccines, discussed the parameters for defining thermotolerance

Standard Operating Procedures (SOPs) developed by AU-PANVAC and explored

modalities for the development/production of thermotolerant PPR vaccines.

1 Arab Maghreb Union 2 Arab Organization for Agriculture Development 3 Association of Southeast Asian Nations 4 Economic Community of Central African States 5 Economic Cooperation Organization 6 Economic Community of West African States 7 Gulf Cooperation Council 8 Intergovernmental Authority for Development 9 South Asian Association for Regional Cooperation 10 Southern African Development Community


12

FAO and OIE organized in March 2019 in Rome a workshop on Controlling PPR at the

livestock/wildlife interface in collaboration with the Wildlife Conservation Society and

Royal veterinary College.

A Joint PPR Resource Mobilization and Marketing Strategy was developed which

includes a marketing narrative, an analysis of potential funding sources and a detailed

action plan. The marketing narrative is a human centered approach outlining that ending

PPR will greatly contribute to ending rural poverty, ensuring food security and to

strengthening resilience (SDG1 and SDG2). The subsequent market analysis identifies

potential resource partners at global, regional and national levels as well as strategic

alliances. Domestic resources from affected countries will represent a crucial funding

source. In September 2018, FAO, OIE and the European Union organized the Global

Conference ``Partnering and Investing for a PPR-free World’’ in Brussels which resulted

in a ministerial Declaration highlighting the need to fill a funding gap of USD$ 340

million.

PPR global situation

Fifty seven countries are recognized as free from PPR by OIE and one country on zonal

basis (Namibia) (May 2019). Seventhly nine countries engaged in the Regional

Roadmaps.

Figure 2: Global distribution pattern of PPR

PPR Stage 1 2 3 4

Number of countries 30 38 5 6


13

Challenges to the PPR GEP implementation

The implementation of the PPR GEP poses a series of challenges that need to be

addressed.

PPR vaccination campaigns conducted by most of the countries are not in line with

the PPR GCES, as not really based on epidemiology assessment, with an insufficient

number of vaccinated animals and an inappropriate PVE (Post-vaccination

evaluation). The coordination of control measures between neighboring countries is

not satisfactory and the regional or epizone approach is not taken into consideration.

A technical meeting to discuss about PPR vaccinations and epidemiological

assessment as well as PVE is scheduled to be held by end of 2019.

The PMAT, a companion tool to the PPR GCES, aiming to categorize countries

according to the four different stages of the PPR GCES and to provide PPR infected

countries guidance, milestones based on epidemiological, and activity-based

evidence is not used correctly. Training and capacity building at country level are

urgently needed on the use of PMAT, in a revised version easier to use, and PVE

Improving epidemiological understanding of PPRV: epidemiological research is

required to better understand PPR transmission dynamics, in particular its spread and

infectivity and the differing roles of wildlife and livestock species, production

systems, ecosystems and viral lineages in this process. The overall goal is to identify

critical control points, and optimal methods for intervention at these points, to

support effective management of eradication. One example is a need to support the

evaluation of R0 values, in relation to the various lineages of PPR and the various

ecosystems it affects. Along these lines evidence of a fully functional disease

reporting system (in order to properly quantify the effectiveness of vaccination) or a

prolonged period of field work in one or more infected countries may help to

generate some of the basic longitudinal data required for computer simulations. The

results of these epidemiological studies will likely be the main guide to decisions

about what levels of progressive control might practically be achieved, as well as the

feasibility or otherwise of timely eradication.

Infection of wildlife and other species: there are now convincing reports

demonstrating the ability of PPRV to cross the species barrier. Indeed, PPRV can

infect animal species other than small ruminants, with dromedaries, pigs and cattle

reportedly being identified with PPRV (Roger et al., 2000; Gopilo et al., 2005;

Munir, 2014). It is currently unclear whether these infections are relevant from an

epidemiological and eradication perspective; however, it is essential to fully

understand the role of wildlife in the spread and potential maintenance of PPRV in

the environment in order to be able to initiate successful control strategies. The

importance of this research was highlighted by a recent outbreak in Mongolia. In

December 2016 the disease was diagnosed in several wildlife populations in the East

of the country, including saiga antelope (Saiga tatarica mongolica), ibex (Capra

sibirica) and goitered gazelle (Gazella subguttorosa), with 50% mortality of the

10,000 highly endangered saiga population (Aguilar et al, 2018). Moving forward, an


14

important first step is to ensure that the currently available tests for sero-diagnosis of

PPRV are validated in serum samples from these animal species, e.g. camels, saiga

and ibex. With specific reference to the PPRV eradication campaign, and as

mentioned above, the significance of these infections as a whole should be carefully

evaluated, as they may not significantly affect the ultimate success of the programme.

Applying movement controls: the control of animal movement, including the

imposition of quarantines and other sanitary measures, are integral to most infectious

disease control and eradication programmes. However, strict movement control can

be counter-productive because it can actually stimulate unnecessary movement of

animals in order to bypass quarantines and restriction orders. To this end, movement

controls must be tempered by the experience of local animal health teams who are

better equipped to judge the behaviour of local owners when faced with such

restrictions.

Applying serological monitoring in the field: there is an ongoing debate about the recruitment rate of newly susceptible sheep and goats into small ruminant populations following vaccination, and how this may require more frequent vaccinations than the annual ones that proved so successful during rinderpest eradication. This could be investigated through computer modelling but empirical data would be persuasive. In this context it would be useful to use serological

monitoring to investigate the levels of recruitment and to develop a proposed methodology for risk-based surveillance, which could be translated into useful actions such as targeted re-vaccination. There are wider calls for serological monitoring to be used to assess the success rates of vaccination campaigns or to invigilate the effectiveness of individual vaccination teams. However, in reality this may only be useful if immediate revaccination can be carried out, which is not often

possible. Whilst, it is important to identify and remedy technical or administrative errors or indeed administrator negligence the implementation of penalties or fines is very difficult. Ultimately, the case for detailed serological monitoring may have to be made on a case-by-case basis, depending on a cost-benefit analysis and whether the resultant data can realistically contribute to improve long-term planning.

Accurately assessing socio-economic impact: although there are many parameters available to facilitate the evaluation of the socio-economic impact of a disease, there are several drawbacks to applying these, e.g. they can be subject specific or handle only one major factor at a time, therefore lacking the ability to estimate the cumulative impact of a disease on the economy. Nevertheless, these economy-wide

considerations are crucial in implementing and funding control and eradication strategies for emerging diseases. For PPR a well-planned cost-benefit analysis of PPR comparing policies and responses that include both the direct and indirect impacts associated with PPR additional cost-benefit analysis are needed to better understand PPR impact in all settings. (Muhammad Munir et al., 2013). The annual global impacts of PPR have been estimated at between US$ 1.4 billion to US$ 2.1

billion; cost-benefit studies have also been carried in different countries; losses which clearly justify both national and global PPR eradication programmes being pursued.


15

Funding and political will: Many of the countries where PPR is now endemic

simply cannot finance an efficient, effective and sustained control/eradication

programme. Indeed, they will need significant support for operational costs, training

and meetings in order to properly implement PPR GEP. Even if livestock owners

themselves contribute more towards the costs of vaccination, there will still be a

requirement at the regional and global level for international funding to provide

technical and coordination costs, as well as member state support. In this context, it

will be necessary to explore public-private partnerships, such as those that have

proved so effective for polio, measles and malaria control.

Conclusion

PPR eradication stands within our reach and will have a positive impact on the lives of

pastoralist communities in all developing countries, directly supporting global efforts to

end poverty and hunger by 2030. The right political and financial backing coupled with a

dedicated plan of action are key to success.


16

REGIONAL ROADMAP ON PROGRESSIVE CONTROL

OF PESTE DES PETITS RUMINANTS (PC PPR) FOR

SOUTH ASIAN COUNTRIES

Nure Alam Siddiky Consultant, CAMR-ZD in BD, Bangladesh Livestock Research Institute, Savar, Dhaka-1341


PPR is a widespread, virulent and devastating transboundary animal disease of domestic

and wild small ruminants. The disease can have significant economic, food security and

livelihood impacts. SAARC region has a small ruminant’s population of about 300

million. The disease is endemic in most of the South Asian countries or reported at least

once in the recent times except Sri Lanka which is free from the disease. The immediate

response to control and contain the disease is based on clear epidemiologically defined

targeted surveillance for early detection and early warning, sound vaccination strategy,

and enhanced capacities in response. It can be further complemented by a medium to

long-term strategy to enhance the capacities of communities and small ruminant owners

so that their assets are protected through improved integrated activities targeting small

ruminant health and productivity.

Table 1: Economic impact of PPR in SAARC countries

Country

Total

Incidence

(M $)

Total

mortality

(M $)

Production

loss (M $):

Due to disease

Treatment

loss (M $):

Due to disease

Overall

loss

(M $)

Bangladesh 4.86 114.4 149.16 24.30 292.72

Bhutan 0.01 0.07 0.28 0.04 0.40

India 43 968.00 1,386.00 215.00 2,612.00

Nepal 1.95 46.14 59.62 9.76 107.47

Pakistan All together 342.00

Total 49.82 1,128.61 1,595.06 249.1 3,012.59

Regional Support Unit (RSU) for SAARC at FAO Sub-regional ECTAD Unit organized

a regional workshop to develop a regional roadmap for progressive control of PPR for

South Asian countries in 2011. The representatives from SAARC countries attending the

meeting and reviewed the status of PPR at global, regional and country level and finally

developed a roadmap for the progressive control of PPR.



17

In order to review the progress made so far and challenges to implement the agreed

roadmap by 2011-2025 in the SAARC countries, the RSU organized the second regional

workshop of PC-PPR from 19-20 December 2013 in Kathmandu, Nepal with the support

from the Government of Nepal, SAARC Secretariat and the European Union.

PPR remains endemic in most of the countries in the region except Sri Lanka. Maldives

and Bhutan had sporadic outbreaks. There is high risk of incursion of the virus through

animal movements and imports of small ruminants even in the countries, region and areas

which are free and/or have sporadic assurances. The countries in South Asian region have

varied capacities, capabilities and facilities in the fields of epidemiology, diagnosis and

vaccine production. India has for instance has well advanced capacity in diagnostic facilities

including those developed indigenously over the years and some of which are now well

recognized commercial private vaccine production entities. India also claims to be self-

sufficient in production of live attenuated homologous vaccine using safe and potent

Sungri/96 strain virus. The Regional Leading Diagnostic Laboratory (RLDL) in Dhaka,

Bangladesh developed capacities in performing cELISA, AGID and cEISA for antibody

detection, icELISA and EISA for viral antigen detection, RT-PCR, qPCR and sequencing

for viral genomic material detection, and also virus isolation on vero cells. It has now

started testing samples referred by SAARC countries.

India is implementing PPR control programme in a phased manner. The five states

were covered during first phase of 2007-2011. The entire country is likely to be

covered during the current phase of 2012-2017. Bangladesh, Nepal and Pakistan

have their localized control programmes for PPR. Bangladesh, India, Nepal, Pakistan

has developed national action plan for the eradication of PPR in accordance with global

eradication campaign. SAARC developed a regional roadmap in close consultation with

Member countries for the eradication of PPR by 2025.The salient features of SAARC

regional roadmap for the eradication of PPR has given below:

The consolidated (revised) regional roadmap for the SAARC countries for the duration of

2014-2025 spread over in three phases is as under:

Component Phase-1

(2014-2015)

Phase-2

(2016-2020)

Phase-3

(2021-2025)

Policy Small ruminant sector review

including husbandry system,

population, demographic

factors, livelihoods issue;

(BD; NP; MD*; PK*; SL*;

BH; IN)

Study report

Developing strategic

plan; (NP; MD; PK;

NP; SL***; BH;

IN*)

Developing strategic plan;

(BD; BH)

Plan document

Getting strategic plan

endorsed by

Getting strategic plan

endorsed by competent

Copy government

endorsement


18

competent forum;

(PK; SL***; IN*)

forum; (BD; NP; MD; PK;

BH)

Identification of budget sources for

Implementation of strategic plan. (BD; NP; MD; PK; SL***; IN*)

Identification of budget sources for implementation of strategic plan; (BD; NP)

List / minutes of meetings with budgetary sources

Implementing strategic plan; (NP; PK; SL***)

Implementing strategic plan;(BD; NP; MD; PK)

Yearly Work Plan

Considering zoning based on magnitude and severity of risk; (NP; MD; PK; SL***; BH; IN*)

Considering zoning based on magnitude and severity of risk; (BD ; PK)

Stop vaccination but continue surveillance for PPR virus / antibodies; (BD; MD; PK; SL***; IN)

Minute of the meeting / copy of policy decisions

Developing exit strategy; (MD)

Developing exit strategy; (PK)

Developing exit strategy; (BD; PK; SL***; IN)

Strategy document

Institutional setup and capacity building

Assessment of strengths and weakness of veterinary services at national / regional/ local level; (BD; NP; SL; MD*; PK; BH*; IN)

Assessment document

Strengthening of veterinary services;

(PK; BH; IN)

Strengthening of veterinary services; (BD; NP; MD; PK; SL; BH)

Strengthening of veterinary services; (SL; BH)

Work Plan

Conducting training need assessment in terms of area and number; (MD; PK; BH; IN)

Conducting training need assessment in terms of area and number; (BD; NP)

Needs assessment document

Identification and list of the equipment other than cold chain to be procured; (NP; MD; PK**; SL***; BH; IN)

Identification and list of the equipment other than cold chain to be procured; (BD; NP; BH)

Approved list of equipment’s

Outbreak Response

and contingency plan

Conducting training need

assessment in terms of area

and number; (MD; PK; BH;

IN)

Conducting

training need

assessment in

terms of area and

number; (BD; NP)

Needs

assessment

document


19

Developing

contingency plan.

(BD; NP; MD; PK;

SL; BH; IN)

Developing Contingency

plan. (BH)

Developing

contingency

plan. (BH)

Approved

contingency

plan document

Allocation of budget

for contingency plan;

(NP; MD; SL; BH;

IN)

Allocation of budget for

contingency

plan; (BD; PK)

Allocation of

budget for

contingency plan;

(PK)

Financial

statement /

Pink Book/

Agreement

withINGO/

NGOs etc.

Implementing

contingency plan;

(IN)

Identification and list of the

equipment

other than cold chain to be

procured; (BD;

NP; BH)

Work plan

Legislation Updating disease control Act

(NP)

Updated bill/Act

etc.

Enforcement of

import regulation

regarding

PPR; (BH)

Copy of import

regulation

Epidemiology/n

surveillance /

outbreak

investigation


assessment in terms of

area and number; (MD; PK;

BH;IN)

Conducting

training need

assessment in

terms of area and

number; (BD; NP)

Needs

assessment

document

Developing

surveillance plan and

epidemiology, sero

surveillance

/monitoring of PPR;

(NP; MD*; PK; SL;

BH; IN)

Developing surveillance plan

and

epidemiology, sero

surveillance/ monitoring of

PPR; (BD; IN)

Copy of PPR

surveillance Plan

Identification of risk

factors for PPR(BD;

NP; MD*; PK**;

SL*; BH; IN)

Study report

Mapping of key

cross-border routes

and markets and

services and facilities

available; (BD; NP;

MD*; PK*; SL*;

BH; IN)

Study report


20

Identification of hot

spots; (BD; NP;

MD*; PK**; SL***;

BH; IN*)

Identification of hot

spots;(NP)

Study Report?

Spatial

maps

Identification and list

of the equipment

other

than cold chain to be

procured; (NP; MD;

PK**; SL***; BH;

IN)

Identification and List of the

equipment


procured; (BD;

NP ; BH)

Approved list of

equipment

Disease investigation

team composition

and

SOPs for disease

investigation (NP;

BH; MD; PK; SL;

BH; IN)

Team composition and SOPs

for disease

investigation; (BD; BH)

Team

composition for

disease

investigation and

SOPs for

investigation;

(BH)

Notification by

competent

authorities.

Identification of risk

factors for area

classification /

zoning (infected,

buffer and free

zones); (MD*; NP;

SL***; BH; IN*)

Identification of risk factors

for area classification / zoning

(infected, buffer and free

zones); (BD)

Study Report

Developing sero

surveillance plan;

(NP; MD*; SL; BH;

IN)

Developing sero surveillance

Plan; (BD; NP; PK; BH)

Developing sero

surveillance plan;

(BH)

Copy of

approved sero

surveillance

plan

Implementing sero

surveillance plan;

(NP; MD;

SL; BH)

Implementing sero

surveillance plan;

(BD; NP; PK; SL; BH; IN)

Implementing

sero

surveillance plan;

(BD); PK; BH)

Work plan

Developing line of

communication;

(BD; NP; MD*; PK;

SL; BH;IN)

Developing line of

communication; (NP)

Approved copy of

organogram

Vaccine and

vaccination

Good quality vaccine, assured

cold chain and SOPs to

ensure cold chain at all level

(storage to

inoculation of vaccine); (NP;

PK**; IN)

Procurement of

Cold chain and

SOPs to ensure

cold chain at all

level (storage to

inoculation of

vaccine); (BD; NP;

MD; PK; BH)

Approved list of

equipment


21

Listing of all the

steps for the vaccine

procurement and

vaccination (SOPs);

(NP; PK; SL***;

BH*; IN)

Listing of all the steps for the

vaccine procurement and

vaccination (SOPs);

(BD; NP; MD; PK; BH)

Listing of all the

steps for the

vaccine

procurement and

vaccination

(SOPs); (BH)

Approved copy

of SOPs

Scheduling of the

field activity; (NP;

MD; PK; SL; BH;

IN)

Scheduling of the field

activity; (BD)

Work plan + Time

lines

Conducting training

need assessment in

terms of area and

number; (MD; PK;

BH; IN)


assessment in terms of area

and number; (BD; NP)

Needs assessment

document

Post vaccination

monitoring with

reference to FAO/

OIE guidelines;

(MD; PK; NP;

SL***)

Post vaccination monitoring

with reference to FAO/ OIE

guidelines; (BD; NP; BH; IN;

PK)

Post vaccination

monitoring with

reference to

FAO/OIE

guidelines; (BD;

PK; BH)

Approved plan

for post

vaccination

monitoring

Diagnosis Identification of labs for

diagnosis and diagnostic tests;

(BD; NP; MD; PK; SL;

IN*)

Identification of

labs for diagnosis

and diagnostic

tests; (NP)

Notification for

designated labs

and diagnostic

tests

Identification and list

of the equipment

other than cold chain

to be procured; (NP;

MD; PK**; SL***;

BH; IN)

Identification and list of the

equipment


procured; (BD;

NP ; BH)

Approved list of

equipment

Conducting training

need assessment in

terms of area and

number; (MD; PK;

BH; IN)


assessment in

terms of area and number;

(BD; NP)

Needs assessment

document

Impact assessment/

food security/

poverty alleviation

Listing of all of stakeholders

and their respective role; (BD;

NP; MD; PK; SL; BH)

Approved list of

stakeholders

Consultation with

stakeholders; (BD;

NP; MD; PK**; SL;

BH; IN*);

Impact Assessment; (IN) Minutes of

meeting/proceedin

gs of consultation

process/ study

report


22

Abbreviations: BD-Bangladesh, BH-Bhutan, NP-Nepal, IN-India, SL-Sri Lanka, MD- Maldives, PK-

Pakistan

Advocacy and

Communication

Seeking political

commitment; (BD; NP; MD;

PK; SL***; BH; IN)

Seeking political

commitment;

(NP; PK)

Advocacy plan

Developing Public

Awareness

Campaigns: (NP;

MD; PK**; SL; BH;

IN)

Developing Public Awareness

Campaigns; (BD; NP; PK;

BH)

Public awareness

plan

Implementing Public

Awareness

Campaigns; (NP;

MD; SL; BH; IN)

Implementing Public

Awareness Campaigns; (BD;

NP; BH)

Work plan /Tools

of awareness

campaign

Monitoring and

evaluation

Developing monitoring and

evaluation system

for respective activity /

intervention for PPR control;

(NP; MD; PK; SL***)

Developing

monitoring and

evaluation system

for respective

activity

intervention for

PPR control; (BD;

NP; BH; IN)

Developing

monitoring and

evaluation

system for

respective

activity /

intervention for

PPR control;

(BH)

Evaluation of PPR

control plan

including

surveillance and

vaccination

outcomes; (MD)

Evaluation of PPR control

plan including surveillance

and vaccination outcomes;

(BD; NP; PK; IN)

Evaluation of PPR

control plan

including

surveillance

and vaccination

outcomes; (BD;

PK)

Evaluation Plan


23

BLRI DEVELOPED PPR CONTROL MODEL

Md. Giasuddin Animal Health Research Division, Bangladesh Livestock Research Institute, Savar, Dhaka-1341


Introduction

PPR (Peste des petits Ruminants) is a highly contagious diseases which severely attacks

small ruminants (Goat and Sheep) in almost 70 countries in Africa, the Middle East and

part of Asia (OIE). The disease was first identified in this region in the year 1993. Now

the disease is endemic in this region and considered as one of the most important

transboundary animal disease. It constitutes a threat to livestock production in many

developing countries including Bangladesh. OIE categorized PPR with a group of

economically important animal diseases, which must be notified to the OIE (Diallo et al.,

2007; Sen et al., 2010).

In all regions where PPR is endemic, it constitutes a serious threat to small ruminant

production and thereby influences on the livelihood of poor farmers, the main owners of

sheep and goats (Diallo et al., 2007). Bangladesh has been blessed with an exclusive

breed of goat, the Black Bengal goat which is world famous for the quality of its meat,

skin and proliferation nature. Furthermore, the goat is considered as the poor man’s cow,

and is an important means of livelihood of rural underprivileged people of Bangladesh

(Miazi et al., 2008). In Bangladesh, PPR was first identified during a severe outbreak in

1993. Since then, the disease has become endemic in Bangladesh (Sil et al., 1995)

causing serious economic losses. In 2010, there were 84000 hospital cases, causing an

estimated economic loss of Taka 1842 million (US$ 24.6) (Islam, 2011). The actual

number of cases may be much more as cases from very rural area were not reported to the

hospital.

At present small goat farmers in Bangladesh are facing massive economic losses due to

PPR as the disease is still endemic in Bangladesh. It has been estimated that losses due to

PPR that has a direct impact to fulfillment of Sustainable Development Goals (SDGs) of

United Nations. The volume of the economic losses demands immediate interventions to

be taken for the progressive control leading to eradication of PPR by 2030.

Following issues need to be addressed for successful implementation of the model

a) Identify a laboratory for PPR diagnosis monitoring

b) Quality vaccines

c) Trained manpower

d) Farmers training

e) Resource mobilization



24

According to OIE strategic PPR control approach, BLRI researchers implemented some

specific activities to achieve the goal.

Below stage 1: We have selected an area where

There were insufficient and unstructured raw data to understand the true risks for

PPR.

No appropriate epidemiological investigations were undertaken.

No official prevention and control program was present.

Stage-1: To cross the stage-1, we have done following activities

Activities:

Selected an area or region with biological barrier which separate the region from

other areas

A base line survey was conducted for determining the number of susceptible animals.

A work plan has been developed

Farmer’s attitude, farmer’s training, demand of vaccines and trained worker for

vaccination and other health management has been assessed

A comprehensive control strategy developed in this stage

Stage-2: (Control stage): In this stage, following tools has been considered

Good quality PPR vaccine

Improvement of farm biosecurity

Animal identification or introduction of health card to identify the farm or animal

Implementation of animal movement control

Quarantine the sick or new introduced animal

Engagement of administrative and political leader

Involvement of stockholders

Activities –Following activities has been implemented to overcome the stage 2

Farmers and technicians training


25

Pre-vaccination antibody assessment

Deworming

Mass vaccination

Post-vaccination antibody assessment to know the level of antibodies

Stage-3 (Eradication stage): Following proper implementation of control stage 2 to 3

years, the area or region or country will enter into eradication stage. We have done

following activities during stage 3-

Activities

Investigation of any outbreak of PPR or PPR like diseases

Mass vaccination of all goat and sheep over the age of 2 months

Regular virological and serological surveillance

Maintain zero circulation of PPR virus in the area

Stage-4 (Post eradication stage): After maintaining 2-3 years of eradication stage, the

area will enter into post eradication stage. In this stage there will be no PPR outbreak in

the area and will be maintain for at least 24 months and apply for free status. In our BLRI

model we executed all activities in accordance with OIE prescribed guidelines.

Beyond stage-4: We have maintained zero circulation of PPR virus in post eradication

stage, now we can claim for PPR free status as with all necessary documents.

Authority may declare the area or region free status.

Requirement for successful implementation of the PPR control model

Good quality PPR vaccine

PPR diagnostic laboratory

Trained manpower

National strategic plan for PPR control

Conclusion

BLRI developed a PPR control model on the basis of the guideline of OIE Global

Strategy for the Control and Eradication of PPR. It is found very effective if the model is

properly implemented.


26

AN OVERVIEW OF GENERAL GUIDELINES FOR PPR

SAMPLE COLLECTION, PRESERVATION AND

GENOMIC ANALYSIS

Emdadul Haque Chowdhury Department of Pathology, Faculty of Veterinary Science

Bangladesh Agricultural University, Mymensingh


Peste des Petits Ruminants (PPR) also known as Goat Plague, is a disease of goats, sheep,

and other taxonomically related species. It is caused by a morbillivirus in the family

Paramixoviridae. It is clinically and pathologically similar to Rinderpest (RP), and is the

most economically important viral disease of small ruminants in the areas where it

occurs. In the field PPR virus causes disease in goats and sheep but not in cattle or pigs,

although these latter two species can be infected sub clinically by experimental

inoculation. One outbreak of PPR was also reported in India, but most of the cases

buffalo sero-converts without showing any clinical disease. Goats are usually considered

to be more susceptible than sheep, but this is not always the case. The disease is

characterized by: high fever, discharges from nose, eyes, and mouth; profuse diarrhea;

pneumonia; and, oral erosion with high morbidity and mortality rates. Strategic

vaccination along with biosecurity measures could help to control the disease.

History of outbreaks

First PPR was described in Ivory Coast in West Africa in 1942. The disease has since

been recognized as endemic in West and Central Africa and gradually spread to other part

of the Africa. The Morocco outbreak is the first case of PPR in North Africa which

followed by Tunisia in 2010. In 1987 PPR appeared in the Middle East and has since then

been confirmed in Arabia (1991), Southern India (1989), Bangladesh (1993), Pakistan

(1993), Iraq (2000), Afghanistan (2006), Turkey ( 2004), Nepal (2010), China and

Bhutan (2010) and recently outbreaks has also been reported in some part of Europe; PPR

reported in Kazakstan, 2014; On June 19, 2018, the Bulgarian National Diagnostic

Research Veterinary Medical Institute confirmed PPR in sheep; the Bulgarian PPR

received growing attention in Europe because of its continuing spreads and economic

impacts.

Distribution of virus and phylogenetic relationship

In the recent years gene sequence analysis has facilitated the classification PPRV strains

into four lineages which are prevailing on the different geographical location of the

world. Lineage I is represented mainly by Western African isolates from the 1970s and

recent isolates from Central Africa; lineage II by West African isolates from the Ivory

Coast, Guinea and Burkina Faso; lineage III by isolates from Eastern Africa, the Sudan,



27

Yemen and Oman; and lineage IV includes all viruses isolated from recent outbreaks

across the Arabian Peninsula, the Middle East, Southern Asia and recently across several

African territories. Genetic characterization of the Moroccan and Egyptian PPR virus

classified it as a lineage IV virus; this marks the first time this lineage has been detected

in Africa, where all four lineages are now present. Serological detection of antibodies to

PPRV has also been reported in samples from Vietnam. Bhutan is the latest country being

affected with PPR in South Asia and samples submitted to WRLs from Bhutan have

recently been typed as lineage IV virus.

In Bangladesh, A total 5 (five) circulating field isolates of PPR virus during a period of

2015 to 2017 were characterized in our laboratory using molecular techniques. Full

length of N, M and F genes of the five fields PPRV isolates were sequenced and was

constructed a phylogenic tree considering the full length sequence of respective PPRV

genes taken from Gene Bank (NCBI blast) by MEGA 7.0. Phylogenetically, the

Bangladeshi PPRV strains belong to the PPRV lineage IV and formed a separate

subgroup and these are closely related with China-Tibet/07 and Indian/TN/VEL/2015

PPRV isolate. A number of amino acid substitutions fluctuating from one (in 2016 PPRV

field isolate) to four (in 2017 PPRV field isolates) amino acids (AA) were found among

the N genes of five field PPRV isolates. These may indicate that PPR virus in Bangladesh

slowly evolving. The following figure 1 can give an idea about the phylogenetic

relationship of Bangladeshi PPR virus isolates along with others elsewhere.

Figure 1. Phylogenetic analysis of N gene by maximum likelihood method

(92 sequences:1574 bp nucleotides; unpublished data from our laboratory)

PPRV China isolate (2013-2015)

PPRV/Bangladesh/BD2/2008(MG581412.1)

PPRV/Tibet/Bharal/2008(JX217850.1)

PPRV/China/Tib/07(JF939201.1)

PPRV/China/33/2007(KX421388.1)

PPRV/China/Tibet/Geg/07(FJ905304.1)

PPRV/x11(GQ184299.1)

PPRV/Bangladesh/2015-1



PPRV/Bangladesh/2016


PPRV/IND/TN/VEL/2015/03(KT860064.1)

PPRV/IND/Delhi/2016/05(KX033350.1)

PPRV/IND/TN/ED/2015/04(KT860065.1)

PPRV/IND/TN/GIN/2014/01(KT270355.1)

PPRV/IND/TN/VM/2014/02(KT860063.1)

PPRV/India/TN/Gingee/2014(KR261605.1)

PPR/PRADESH/95(JN647694.1)

PPRV/Izatnagar/94(KR140086.1)

PPRV/Sungri/1996/MSD(KJ867542.1)

PPRV/Sungri/96(GQ452013.1)

PPRV/Sungri/96(AY560591.3)

PPRV/Revati/2006(GU014574.1)

PPRV/Jhansi/03(EU344738.1)

PPRV/Jhansi/2003(GU014571.1)

PPRV/Revati/2005(FJ750559.1)

PPRV/Guj/2007(JN632532.1)

PPRV/Morocco/2008(KC609745.1)

PPRV/Morocco/2008(KC594074.1)

PPRV/S15(KY885100.1)

PPRV/Smailia1/2014(KT006588.1)

PPRV/Ethiopia/2010(KJ867541.1)

PPRV/Georgia/Tbilisi/2016(MF737202.1)

PPRV/Sungri/96(KF727981.2)Vaccine

PPRV/Turkey/00(AJ563705.1)

PPRV/2005(AJ849636.2)

Lineage IV

Lineage II

Lineage I

Lineage III99

99

99

93

99

72

82

64

99

92

95

50

99

99

95

30

46

97

4

75

97

82

65

38

46

99

65

96

78

99


28

Host Range

PPR is primarily a disease of goats and sheep and goats are usually more severely

affected than sheep. But in India and the Middle East both goats and sheep are affected

with equally devastating consequences. Breed may affect the outcome of PPR virus

infection. Cattle and pigs are known to be a dead end host. Sero neutralization test for the

presence of PPR antibodies detected 4.2% in 142 camels. PPR affects wildlife animals

both under field condition and experimentally. The disease was induced experimentally

in American white deer (Odocoileus virginianus) which was found to be susceptible and

a field outbreak was reported from a zoological collection in Alain. It caused a high

mortality and severe disease in Dorcas Gazelles (Gazella dorcas), Nubian Ibex (Capra

ibex nubiana), Laristan sheep (Ovis orientalis laristani) and gemsbok (Oryx azellaa).

Subclinical involvement of Nigale (Tragelaphinae) was suspected. In another report from

Saudi Arabia, PPR was suspected on clinical and serological base in Gazaelle and deer.

Antelope and other small wild ruminant species can also be severely affected. Sero-

conversion of large ruminants occurs but no clinical disease so far, except only one

outbreak reported in buffalo. Mild clinical disease has also been reported from camel.

Etiology

PPRV is a member of the genus Morbillivirus under the family Paramyxoviridae and

order Mononegavirales. Paramyxoviruses are enveloped animal viruses which are found

almost exclusively in nucleocapsid structures. This virus shares structural, biological,

antigenic and molecular features in common with the other members of the group. It is

closely related to the rinderpest virus (RPV), the measles virus of humans, the distemper

virus of dogs and some wild carnivores, and the morbilliviruses of aquatic mammals. The

genome of PPRV is non-segmented, negative-strand unlike other member of

Morbillivirus. PPRV consist of 15948 nucleotides that encodes eight proteins: the

nucleocapsid protein (N), the phosphoprotein (P), the matrix protein (M), the fusion

protein (F), the haemagglutinin protein (H), the polymerase protein (L) and the two non-

structural proteins, C and V ( Figure 2). PPRV genome is organized into six contiguous,

non-overlapping transcription units corresponding to the gene of the six structural viral

proteins in the order of 3'-N-P-M-F-H-L-5' in the genome sense. They are separated by

short sequences of three nucleotides called intergenic regions (IG) which is CTT in most

cases. In some virus strains the H-L junction sequence is CGT. The main feature of the

genome organization that is unique to morbilliviruses is the existence of a long

untranslated region (UTR) at 3 end of M gene and at the 5 end of F gene.

Epidemiology

The discharges from eyes, nose and mouth, as well as the loose feces, contain large

amounts of the virus. Fine infective droplets are released into the air from these

secretions and excretions, particularly when affected animals cough and sneeze. For PPR

to spread, close contact between infected and susceptible animals is needed. Since the

virus is enveloped, it is extremely sensitive to inactivation by environmental factors such


29

as heat, sunlight and chemicals. The virus is very fragile and cannot survive for a long

time outside host. Its half-life has been estimated to be 22 min at 56oC and 3.3 hours at

37oC. No carrier state is known to exist. The appearance of clinical PPR may be

associated with introduction of recently purchased sick animals from markets or contact

in a closed/village flock with sheep and/or goats that had been sent to market but returned

unsold. Trade in small ruminants at markets where animals from different sources are

brought into close contact with one another increases opportunities for PPR transmission,

as does the development of intensive fattening units. Changes in weather also have been

suggested to contribute to outbreaks but the reports from different geographical regions

are not in total agreement. Young animals are more susceptible. Morbidity is an about

70%, mortality also varied from 50-100%.

Clinical signs

The disease usually appears in the acute form, with an incubation period of 4 to 5 days

followed by a sudden rise in body temperature to 104-106° F (40-41° C). Affected

animals appear ill and restless and have a dull coat, dry muzzle, and depressed appetite.

Affected animals breathe fast, sometimes so fast that they exhibit rocking movements

with both the chest and abdominal walls moving as the animal breathes. Severely

affected cases show difficult and noisy breathing marked by extension of the head and

neck, dilation of the nostrils, protrusion of the tongue and soft painful coughs. They have

obvious signs of pneumonia. A clear watery discharge starts to appear from the eyes,

nose and mouth, which progressively becomes mucopurulent. These mucopurulent

discharges tend to dry, causing matting together of the eyelids, obstruction of the nose

and difficulty in breathing. Animals that survived more days developed erosive and

necrotising stomatitis, enteritis and anorexia. At the height of development of oral

lesions, most animals manifest severe diarrhea, often profuse but not hemorrhagic. Body

temperature usually remains high for about 5-8 days, and then slowly returns to normal

prior to recovery or drops below normal before death.

Pathology

The mucosal surfaces of the esophagus, abomasums, large intestine, rectum and cecum

show congestion, severe edema, hemorrhage, necrotic plagues with erosions. Rumen,

reticulum, and omasum rarely may have lesions; erosions on pillars of rumen, congestion

of the abomasums may be seen. Small intestine lesions are usually moderate and include

extensive necrosis of Peyer's patches, resulting in severe ulceration. The large intestine is

usually more severely affected with congestion around the ileocecal valve, at the ceco-

colic junction, and in the rectum. In the posterior part of the colon and the rectum,

discontinuous streaks of congestion ("zebra stripes") form on the crests of the mucosal

folds. Liver may be moderately enlarged and pale. Indented gall bladder may be found. In

some goats rumen, reticulum, omasum and abomasum may filled with foetid watery fluid

and this watery fluid also may be found in the small intestine. Small erosions and

petechiae are visible on nasal mucosa, turbinates, larynx and trachea, while pleuritis may

be seen in lungs, resulting in hydrothorax. Respiratory tract usually contains frothy


30

exudates and the lungs are found to be severely congested with areas of consolidation,

consistently in the apical lobes and in antero-ventral lobes. Most lymph nodes throughout

the body particularly mesenteric and mediastinal lymphnodes are found enlarged,

congested, and edematous but spleen may be either enlarged and congested or slightly

atrophied. Ecchymotic and brush paint hemorrhage are seen on the epicardium. Liver and

kidney lesions were very limited earlier, recent outbreaks shows severe liver and kidney

damage (Fig.2 - 3).

Figure 2. Liver shows hemorrhages and

congestion

Figure 3. Kidney shows hemorrhages, congestion

and inflammation.

Hematological profile of PPRV infected goats

Packed cell volume, total erythrocyte count and hemoglobin concentration decrease at

later stage of the disease in the PPR infected goats. On the other hand, total leukocyte

count is increased in PPR infected goats due to significant increase in the absolute

number of lymphocytes and neutrophil. However, the other blood parameters such as

erythrocyte sedimentation rate and absolute monocytes, eosinophils and basophils counts

remain comparable between PPR infected and healthy goats.

Biochemical profile of PPRV infected goats

PPR infected goats show significantly lower level of total protein and albumin in their

sera when compared to healthy goats. No significant difference is found in the amount of

glucose, bilirubin and blood urea nitrogen between PPR infected and healthy goats.

Analysis of the serum enzymes shows significant increase in the level of creatine kinase,

aspartate transaminase and alanine transaminase in the PPR infected goats compared to

the healthy goats.

Pathogenesis

The PPRV has a particular affinity for lymphoid tissues and epithelial tissue of the

gastro-intestinal (GI) and respiratory tracts, where it produces characteristic lesions. The

At 18 dpi (Dead)


31

respiratory route is the likely portal of entry to the host. After the entry of the virus

through the respiratory system, it localizes in pharyngeal and mandibular lymph nodes as

well as tonsil and replicates. After initial replication, the virus spread to the local

lymphatic tissues and also enters into circulation (primary viremia). Primary viremia

results in the spread of virus to other lymphoid tissues and other organs including skin,

kidney and GI tract. In these various organs, the virus replicates in the endothelial cells,

epithelial cells and monocytes /macrophages and plenty of viruses enter into blood

(secondary viremia). The secondary viraemia is associated with further rise of body

temperature. Virus appeared to be released through the microvilli of the epithelial cells.

The incubation period of the disease is 4-5 days in a sero-negative herd but may range

between 3-10 days. It is suggested that death is a consequence of the combined effects of

the pathology and immuno-suppression.

Diagnosis

The routine diagnosis of PPR is based on clinical examination, gross pathology,

histopathologic findings and laboratory confirmation which include tests for the detection

of PPR antigen and PPR antibody, viral isolation, viral nucleic acid hybridization and

recently polymerase chain reaction. Either primary lamb kidney cell or vero cells are

usually used to isolate PPR virus. Isolation of PPR virus using vero cell usually takes 3-5

blind passages which may take 10-15 days. However, use of SLAM-Vero cell increases

the isolation efficiency of PPR virus. Very recently our laboratory has developed primary

goat kidney cell and is being used to isolate PPR virus from the field outbreaks.

Control

There is no specific treatment against PPR. Antibiotics may prevent secondary

pulmonary infections but this treatment is too costly in case of an outbreak. Therefore,

vaccination is the preferred method of control. However, vaccination cannot give 100%

guarantees to control the disease. Implementation of biosecurity measures along with

vaccination and early diagnosis of the disease are required for better control of the

disease. However, vaccination before any big festival in which major movements of goats

occurred across the country may limit the spread of the PPR virus in the country. PPR

infected goat that survived from infection becomes resistant against PPR virus but

becomes immuno-suppressed. Therefore may die due to other opportunistic infection and

needs special attention.

Sample collection, preservation and transportation guideline

General considerations in sample collection

The starting point for the laboratory investigation is the collection of samples. Before

taking samples, careful consideration should be given to the purpose for which they are

required. This will determine the type and number of samples needed to provide valid

results. When samples are taken from live animals, care should be taken to avoid injury

or distress to the animal or danger to the operator and attendants. Whenever handling


32

biological material, from either live or dead animals, the risk of zoonotic disease should

be kept in mind and precautions taken to avoid human infection.

Points to be considered before collection of sample

What test would be requested for? Or test to be performed.

What are the appropriate samples to collect?

What transport medium and preservative to be used?

What cool chain to be maintained?

What are the packaging and shipment requirements?

Is the laboratory ready to receive the sample?

Laboratory tests used in the investigation of PPR

The following tests usually used to detect or investigate PPR disease: isolation of virus in

laboratory host system; Detection viral antigen in tissues (Agar gel immunodiffusion test,

Antigen-capture ELISA and Immunohistochemistry); Detection of antibodies in serum

(Agar gel immunodiffusion test, Haemagglutination inhibition test, ELISA) and

Detection of viral DNA / RNA (PCR, RT-PCR etc.).

Types of samples collected for PPR investigation

The following samples need to be collected to investigate PPR: swabs (nasal, tracheal,

oropharyngeal, cloacal), blood, tissues, tissue smear, etc.

Requirement for packaging & transport

Primary container depending on sample type

Secondary container (Air tight Box/Bottle/Tube/ polythene pouch and envelope)

Cool box/ vaccine carrier

Wet ice or Ice packs (put ice packs in deep freezer before transport)

Pen or permanent marker for labeling samples

From live animal

Tears/ nasal swab

Swab from conjunctiva or posterior nasal cavity avoiding external secretion. Keeping swabs moist after collection is most important. Swab should be placed in virological transport medium or any sterile isotonic fluid, like phosphate buffered saline (PBS) or common tissue culture medium like Eagle’s MEM or 50% buffered glycerol with antibiotics (penicillin 200 IU/ml and streptomycin 200 μg/ml) can be used. Commercially available kits containing swabs and viral transport media are also acceptable. This sample can be used for isolation, PCR or any other assays related with antigen detection.

Swab/samples can be placed in 100% ethanol if other media is not available. Samples with ethanol can be only used in PCR.

Sampling also possible with filter paper. Filter (Whatman filter paper, Grade1, 5 to 6 cm in length and 0.25 cm in width) paper can be soaked with nasal


33

secretions. Air dried the filter paper avoiding direct sun light and put them in eppendorf tube or polyethane pouch. Pieces of filter paper smeared with secretions can be used in RT-PCR as template.

If none of the system available, collect swab in a tube, put the samples in

container in ice and send it to the laboratory quickly. If necessary, replace ice.

Take sample as many as possible (5-6 animals from an outbreak).

Gum debris

This material can be collected by a spatula or finger rubbed across the gum and

inside the upper and lower lips. The material collected is then scraped into a

container containing 50% buffered glycerol or described as above.

Or place the materials in 100% ethanol for PCR

Collect and shift using ice.

Blood sample

Use whole blood for isolation or RT-PCR.

Sampling also possible with filter paper. Filter (Whatman filter paper, Grade1, 5

to 6 cm in length and 0.25 cm in width) paper can be soaked with blood (pour the

blood drop wise from bottom towards the tip). Air dried the filter paper avoiding

direct sun light and put them in eppendorf tube or polyethane pouch. Pieces of

filter paper smeared with secretions can be used in RT-PCR as template.

Blood samples is good when animal is in febrile state, while nasal/other secretion

is good when animal is in febrile and non-febrile state.

Serum samples

For sero- prevalence study, blood sample should be collected considering

different age group. For example- if a herd size is 25 goat, 20 sample should be

collected in the order of, 8 from goats age < 1years old, 8 from 1-2 years old and

4 from >2 years old.

For diagnostic purpose from a PPR suspected herd, paired sample is preferred,

one is at beginning of outbreak (acute phase of the disease) and other is after 10-

15 days later (Convalescent stage).

Tissues from dead animals

The following tissues should be collected during post mortem examination:

Perform postmortem as early as possible and record all post mortem changes. Then

collect-

Bronchial Lymph nodes, pieces of lungs (mediastinal) and alimentary tract

(mesenteric) for virus isolation, RT-PCR and other immunological assays that

detect antigen.

Collect part of all tissues in 10% neutral buffered formalin for histopathology and

immunohistochemistry (when required).


34

Sample preservation

To prevent spoilage due to autolysis (refrigeration, freezing is required)

To prevent bacterial growth (antibiotics, refrigeration, freezing is required)

To prevent desiccation (fluid vehicle is required)

To prevent hypotonic shock (isotonic buffered solution need to be added)

To prevent freezing shock (glycerol, gelatin, albumin, calf serum –

cryoprotectent need to be added)

Precautions for specimen preservation and transport

Specimens for virus isolation should be refrigerated immediately after collection,

if facilities does not permit keep them chilled ( at 2 to 40C) in refrigerator or with

wet ice and shipped to the laboratory as soon as possible (within 48 hours)

Keep swab in chilled condition for short period (Maximum 4 days at 40C) and

don’t store swab sample at -200C. For longer storage keep them at -70

0C.

Filter paper sample can be kept at normal temperature and can be sent to the PPR

lab by normal post.

Serum sample can be kept at 20C or 4

0C for maximum period of two days and for

longer period store the sample at -200C

Specimens for PCR test should be kept in chilled condition in refrigerator or with

wet ice for short time (48 hours), if specimens cannot be processed immediately;

they should be kept -20°C for short period or below at -70°C for longer period.

When refrigeration facilities are unavailable preserve the specimen in Tryzol

reagent or 100% ethanol in screw cap container store at 4°C and shipped to the

laboratory as soon as possible

Use cool box with icepacks for transporting the specimen

Send the specimen preferably through messenger

Always avoid freeze thaw cycle

Labelling and packaging of specimen for transportation and storage

Specimen type/name e.g. blood, swab, PM tissue samples

Put an identification number

Place of collection

Date and time of collection

* Always submit clinical and sample collection history along with the samples. This is

mandatory. Laboratory may have prescribed format for sample collection and

submission. Before sending samples, recipient laboratory should be ready and agree

to receive the samples.


35

Table 1: Storage condition

Samples/

Time

Swab Blood/sera Tissue for

isolation or

ELISA

Sample in

filter paper

Formalin

Fixed tissue

>6

months

<-70°C or

liquid

nitrogen

<-70°C or

liquid

nitrogen

<-70°C or

liquid nitrogen

<-70°C (up

to 1 year)

Several years

in room

temperature

Up to 6

months

-20°C -20°C -20°C

1-7 days 4- 8 °C 4- 8 °C 4- 8 °C Room

temp. Shipment Maximum 12

hours in ice

Max 12 hours

in room

temp./ice

Maximum 12

hours in ice

References

Begum, S., Nooruzzaman M., Murshida, M.M., Mohanto, N., Parvin, R., Islam, M.R., and

Chowdhury, E.H. 2018. PPR virus infection showed altered hematological and serum

biochemical profile in Black Bengal goats. Onderstepoort Journal of Veterinary Research

85(1), 1595.

Rahman, M.M., Parvin, Rokshana., Bhuiyan, A.R., Giasuddin, M., Chowdhury, S.M.Z.H., Islam,

M.R., Chowdhury, E.H.2016. Genetic characterization of Peste des Petits ruminants virus

circulating in Bangladesh. British Journal of Virology, 3(4),115-122 ·

Chowdhury, E.H., Bhuiyan, A.R., Rahman, M.M., Alam., Siddique, M.S.A., and Islam, M.R..

2014. Natural Peste des Petits ruminants virus infection in Black Bengal goats: Virological,

pathological and immunohistochemically investigation. BMC Veterinary Research

11/2014; 10(1):263. doi:10.1186/s12917-014-0263-y

Siddiqui, M.S.I., Ahasan, S.A., Islam, N., Kundu., P., and Chowdhury, E.H. 2014. Peste des petits

virus antibodies in goats and cattle of the Saints Martin’s island of Bangladesh. The

Bangladesh Veterinarian, 31, 55-59.

Bhuiyan, A.R., Chowdhury, E.H., Kwaiatek, O., Parvin, R., Rahman, M.M., Islam, M.R., Albina,

E., and Libeau., G. 2014. Dried fluid spots for Peste des Petits ruminants virus load

evaluation allowing for non-invasive diagnosis and genotyping. BMC Veterinary Research,

10/2014; 10(1), 247.,doi:10.1186/s12917-014-0247-y.

Bhuiyan, A.R., Rahman, M.M., Begum, J.A., Islam, M.R., and Chowdhury, E.H. 2012.

Comparison of genes as target for molecular diagnosis of Peste des petits ruminants in

goats. The Bangladesh Veterinarian, 29(2), 56 -62. doi: http://dx.doi.org/10.3329/bvet.v

29i2.14343

Rahman, M.A., Shadmin, I., Noor, N., Parvin, R., Chowdhury, E.H., and Islam, M.R. 2011. Peste

des petits ruminants virus infection of goats in Bangladesh: Pathological investigation,

molecular detection and isolation of the virus. Bangladesh Veterinarian, 28, 1-7. doi:

http://dx.doi.org/10.3329/bvet.v28i1.8808

Rahman, M.M., Bhuiyan, A.R., Parvin,

R., Giasuddin, M., Haque, M.E., Sayem, S.M., Islam,

M.R., and Chowdhury, E.H. 2011. Immune response of goats to thermostable preparation



http://dx.doi.org/10.3329%2Fbvet.v28i1.8808


36

of conventional Peste des petits ruminants (PPR) vaccine in Bangladesh. SAARC Journal of

Agriculture, 9, 73-81. www.saarcagri.net/index.php?option=com_content

Islam, M.R., Shamsuddin, M., Rahman, M.A., Das, P.M., and Dewan, M.L. 2001. An outbreak of

Peste des petits ruminants in Black Bengal goats in Mymensingh, Bangladesh. The

Bangladesh Veterinarian, 18, 14-19.

Dialo, A., Barrett, T., Barbron, M., Meyer, G., and Lefevre, P.C. 1994. Cloning of the

nucleocapsid protein gene of PPR virus: relationship to other morbilliviruses. Journal of

Gen. Virology, 75, 233-237.

Pronab, D., Sreenivasa, B.P., Barrett, T., Corteyn, M., Singh, R.P., Bandyopadhyay, S.K., and

Dhar, P. 2002. Recent epidemiology of Peste des petits ruminants virus (PPRV). Veterinary

Microbiology, 88(2), 153-159.

Raj, G.D., Kumar, A.S., Shaila, M.S., Nachimuthu, K., and Palaniswami, K.S. 2003. Molecular

epidemiology of Peste des petits ruminants viruses from southern India. Veterinary Record,

152, 264-6.

Rowland, A.C., Scott, G.R., and Hill, D.H. 1969. The pathology of an erosive stomatitis and

enteritis in West African dwarf goats. Journal of Pathology, 98, 83-87.

Taylor, W.P. 1984. The distribution and epidemiology of Peste des petits ruminants. Preventive

Veterinary Medicine 2, 157-166.

http://www.saarcagri.net/index.php?option=com_content


37

PRINCIPLES OF PCR AND GUIDELINES FOR GOOD

LABORATORY PRACTICES

Mohammad Rafiqul Islam Senior National Technical Advisor, One Health Education

ECTAD, FAO, Bangladesh


Polymerase chain reaction (PCR)

Polymerase chain reaction is one of the most common and often indispensable tools for

molecular biology research and applications. Kary Mullis, an American biochemist,

received Nobel Prize in 1993 for PCR that he invented almost 10 years earlier. Mullis

summarized the procedure of PCR as follows: "Beginning with a single molecule of the

genetic material DNA, the PCR can generate 100 billion similar molecules in an

afternoon. The reaction is easy to execute. It requires no more than a test tube, a few

simple reagents, and a source of heat." The PCR has now become very sophisticated in

terms of instrumentation and reagents. However, the basic principle still remains the

same, which involves repeated cycles of (i) heat denaturation of double stranded DNA,

(ii) annealing of primers (short pieces of complementary DNA) to the denatured strands

of DNA, and (iii) extension or amplification of new complementary strand of DNA under

the influence of DNA polymerase enzyme (Fig. 1). Amplification of the right fragment of

DNA is confirmed by agarose gel electrophoresis of PCR products.

Figure 1. Basic principle of PCR (left) and an example of a PCR protocol (right)

The invention of PCR

Mullis developed the PCR in 1983, when he was working in Emeryville, California for

Cetus Corporation, one of the first biotechnology companies. There, he was responsible

for synthesizing short chains of DNA. He conceived of PCR while cruising along the

Pacific Coast Highway one night in his car. He was playing in his mind with a new way

of analyzing changes (mutations) in DNA when he realized that he had instead invented a



38

method of amplifying any targeted DNA region through repeated cycles of duplication

driven by DNA polymerase. The PCR method relies on thermal cycling, i.e., repeated

heating and cooling of a buffered reaction mixture of template DNA, two oligonucleotide

primers, all the four types of deoxynucleotide triphosphates (dNTPs) and DNA

polymerase enzyme. During each of the thermal cycles the two strands of DNA first

denature or separate, the primers anneal to the complimentary sequences in the denatured

template and then as primed by the annealed primers and driven by polymerase enzyme

new strands of complimentary DNA are synthesized through incorporation of dNTPs. On

each thermal cycle the number of targeted DNA increases exponentially leading to the

synthesis of thousands, millions or billions of copies of the same DNA fragment which

can be visualized on agarose gel electrophoresis and staining.

The legacy of PCR

Although Mullis is credited for the invention of PCR, he in fact had put together several

known principles of biochemistry in a noble approach of repeated cycling to obtain a

noble product, a visible quantity of targeted DNA fragment. The invention of PCR has a

long legacy. James Watson and Francis Crick received Nobel Prize in 1953 for their

discovery of DNA structure founding the field of molecular genetics. Another Nobel

laureate Arthur Kornberg identified DNA polymerase enzyme in 1957. Hor Gobind

Khorana, who received Nobel Prize for elucidation of genetic codes, also used

oligonucleotide primers and polymerase. In 1977 Frederick Sanger reported a method for

determining the sequence of DNA. The technique employed an oligonucleotide primer,

DNA polymerase, and dNTPs (native and modified). For this innovation he was awarded

the Nobel Prize in 1980. In 1971 Kjell Kleppe, a researcher in Khorana's lab, envisioned

a process very similar to PCR and he described how a two-primer system might lead to

replication of a specific segment of DNA. However, it is Kary Mullis who demonstrated

the use of two primers, template, dNTPs and polymerase in repeated cycles of heating

and cooling to synthesize copies of targeted DNA.

Refinement of PCR

The DNA polymerases initially employed for experimental PCR were unable to

withstand high temperatures needed for DNA denaturation. So the early procedures for

DNA replication were very inefficient and time consuming, and required large amounts

of DNA polymerase to be replenished at each cycle. The discovery in 1976 of Taq

polymerase - a DNA polymerase purified from the thermophilic bacterium, Thermus

aquaticus, which naturally lives in hot (50 to 80°C) environments such as hot springs -

paved the way for dramatic improvements of the PCR method. The DNA polymerase

isolated from T. aquaticus is stable at high temperatures remaining active even after DNA

denaturation, thus obviating the need to add new DNA polymerase after each cycle. This

allowed an automated thermocycler-based process for DNA amplification. Most

commonly used thermal cycling profile for PCR includes denaturation of DNA at 94°C,

primer annealing at 45-65°C and synthesis (elongation) of new strand at 68-72°C.


39

Relatively more recent advancement in PCR is the introduction of real-time PCR, which

does not require post-PCR analysis of the products by agarose gel electrophoresis.

Instead, the amplification of DNA can be observed real time on computer screen as

amplification curve generated by the signals of fluorescent dyes incorporated into the

newly amplified DNA.

Reverse transcription polymerase chain reaction (RT-PCR)

The classical PCR technique can be applied only to DNA strands. RT-PCR is a variant of

PCR, where with the help of reverse transcriptase enzyme, RNA can be transcribed into DNA

(complementary DNA, or cDNA), thus making PCR analysis of RNA molecules possible.

Reverse transcriptase was discovered in 1970 by Howard Temin at the University of

Wisconsin–Madison and David Baltimore at MIT from RNA tumour viruses

(retroviruses). For their achievements, both shared the 1975 Nobel Prize in Physiology or

Medicine (with Renato Dulbecco).

Good laboratory practices (GLPs) for PCR laboratory

PCR is an extremely sensitive procedure and hence is very prone to contamination.

Accidental contamination with DNA or RNA or the source of DNA/RNA, similar to that

expected in the test sample, could lead to a false positive reaction. The source of

contamination could be another sample, the environment, the positive control or the DNA

amplified earlier in the laboratory. Another type of contamination which is particularly

important for RT-PCR is RNase enzyme. RNase is a highly stable enzyme with

ubiquitous distribution, virtually present on every surface including human hands,

laboratory benches, glassware and plastic ware and air exposed to dusts (containing

microbes). RNase destroys RNA giving false negative results in RT-PCR, which is a

nightmare for any laboratory diagnostician. Hence to avoid unwanted false positive or

false negative reactions and to ensure laboratory reproducibility there is no alternative but

to adhere to strict GLPs. The building blocks of GLP in a PCR laboratory are (i) proper

work flow, (ii) clean working area, (iii) laboratory discipline, (iv)quality control and (v)

good housekeeping.

Work flow

A PCR involves several steps including extraction of nucleic acid, assembly of test

reaction (master-mix preparation and template addition), amplification of DNA and

finally analysis of PCR products. Activities prior to amplification can be identified as

pre-PCR steps and analysis of PCR products as the post-PCR step. It is very important to

physically separate the pre-PCR, amplification and post-PCR activities (Figure 2). This

demands designation of isolated laboratory spaces for nucleic acid extraction, reaction

assembly, amplification and PCR product analysis. However, mere separation of

activities might not bring expected benefits if a unidirectional work flow is not

established. Ideally there must not be any back flow from the post-PCR areas to the pre-

PCR areas.


40

Figure 2. Work flow in a PCR laboratory

Clean work area

The nucleic acid extraction room should meet the standard of a virology lab in terms of

biosafety and aseptic measures. A Class II biosafety cabinet should be available for

extraction of nucleic acids. The PCR reaction assembly room should meet a “clean room”

standard and should have at least a PCR hood. PCR laboratories need to be kept clean

and tidy. There should be routine cleaning and decontamination schedule. Sodium

hypochlorite solution or commercially available DNA decontaminating agents can be

used. Ethanol could clean the surface and wipe out microorganisms but would not destroy

DNA residues if remained on the surface. UV irradiation would destroy DNA but only to

a certain distance. All pipettes, tips, gloves and tubes should be protected from dust while

not in use.

Laboratory discipline

A laboratory designed for maintaining clean environment and good work flow would be

of little value unless a stringent laboratory discipline could be established. Laboratory

workers should be well informed about the laboratory rules and should follow them

rather religiously. Extraction of DNA/RNA from infectious materials must be done at a

designated place (ideally in a Class II biosafety cabinet). Dedicated and separate sets of

equipment and appliances should be used in different sections of PCR lab and they

should be clearly marked and preferably colour coded. Consumables, reagents and water

for pre- and post-PCR activities should be stored separately. Appliances, consumables,

reagents, water, marker, pen, note books, etc. must not be moved between pre- and post-

PCR sections. Separate lab coats should be used for pre- and post-PCR sections.

Disposable gloves must be used for all activities and they should be changed frequently,

particularly after touching any contaminated surface. All surfaces should be considered

potentially contaminated.

Work flow should never be reversed. If possible, it is useful to establish a daily schedule

for pre- and post PCR activities for routine diagnostic activities. One should plan and

organize work before beginning a test, such as making all calculations, filling in


41

worksheets, ensuring availability of all reagents and supplies at respective work areas.

Reagents and water should be aliquoted in convenient volumes with due precautions. It is

advisable to pulse centrifuge tubes to collect reagents at the bottom before opening the

tubes. Any reagent must not be left on the bench any longer than necessary. Pipetting

steps should be minimized by preparing master-mix, whenever applicable and any

leftover master mix must be discarded. Use of filter tips is suggested in all steps

particularly at least prior to amplification.

Quality control

PCR and RT-PCR involves the use of a number of reagents which are highly perishable

and prone to contamination. False positive and false negative results could be a matter of

great concern. It is therefore essential to check the reproducibility of PCR and RT-PCR.

This requires the use of appropriate controls such as positive control, negative control,

reagent control (no template but water), internal positive control, etc.

Apart from these, PCR laboratories should participate in quality assurance programme at

regular intervals.

Good housekeeping

Good housekeeping is an integral part of good laboratory practices. All consumables

should be stored systematically. All reagents should be properly labeled with name, date

and batch number and stored in appropriate place keeping in mind proper work flow. Log

book should be maintained where appropriate.

Proper disposal of laboratory waste is also very important. Used tips and tubes should be

discarded carefully in designated and labeled receptacles avoiding possible contamination

of work areas and then these should be disposed by incineration. Ethidium bromide used

for staining DNA is a potential carcinogen. Special precaution should be taken and

appropriate procedures should be followed in disposing agarose gel and buffer containing

ethidium bromide.


42

ELISA: AN ESSENTIAL TOOL FOR

SURVEILLANCE OF PPR

Jahangir Alam National Institute of Biotechnology, Ganakbari, Ashulia, Dhaka-1349, Bangladesh


Historical background

Enzyme-linked immunosorbent assay (ELISA) and Enzyme immunoassay (EIA) became

very familiar names for medical and veterinary laboratories, diagnostic product

manufacturers, regulatory bodies, quality assessment & proficiency-testing organizations,

etc. Three scientific research groups independently and simultaneously developed

immunolabelled technique and demonstrate its feasibility. The ELISA technique

developed by Peter Perlmann and Eva Engvall at Stockholm University, Sweden

published in September 1971 while Avrameas & Guilbert from the Pasteur Institute

published in December 1971. On the other hand the EIA technique developed by Bauke

van Weemen and Anton Schuurs at the Research Laboratories of NV Organon, Oss, The

Netherlands published article on June 1971. Radio immune assay (RIA) was first

described in July, 1960 for measurement of endogenous plasma insulin by Solomon

Berson and Rosalyn Yalow of the Veterans Administration Hospital in New York. Yalow

would later be awarded the 1977 Nobel Prize for Medicine for “the development of the

RIA for peptide hormones”, but because of his untimely death in 1972, Berson could not

share the award.

Basic principles of ELISA

ELISA is a plate-based assay

technique designed for detecting and

quantifying substances such as

peptides, proteins, antibodies,

hormones, etc. by changing

antibodies and color to identify a

substance. In an ELISA, an antigen

must be immobilized on a solid

surface and then combined with an

antibody that is linked to an enzyme.

Detection is accomplished by assessing the conjugated enzyme activity via incubation

with a substrate to produce a measureable product (Figure 1). The most crucial element

of the detection strategy is a highly specific antibody-antigen interaction. It is a common

test that detects and measure antibodies in blood.

Figure 1. Basic Procedure of ELISA (Source: https://www.cusabio.com/c-20659.html)


https://www.cusabio.com/c-20659.html


43

Type of ELISA

As its name implies, ELISA involves the use of enzymes and the specific binding of

antibody and antigen. According to how it works, ELISA can be divided into four major

types: direct, indirect, sandwich and competitive. Each type has advantages as well as

disadvantages (Table 1). On the basis of whether ELISA can quantify the level of the

target molecule, ELISA can be divided into two types, qualitative and quantitative.

Qualitative ELISA provides a simple positive or negative result for a sample, while

quantitative ELISA reflects the concentration of the target molecule in a sample via a

standard curve.

Table 1: Comparison of direct, indirect, sandwich, and competitive ELISA

Type Advantages Disadvantages

Direct ELISA Simple protocol, time-

saving, and reagents-

saving.

No cross-reactivity from

secondary antibody.

High background.

No signal amplification, since only a primary

antibody is used and a secondary antibody is

not needed.

Low flexibility, since the primary antibody

must be labeled.

Indirect ELISA Signal amplification, since

one or more secondary

antibodies can be used to

bind to the primary

antibody.

High flexibility, since the

same secondary antibody

can be used for various

primary antibodies.

Complex protocol compared with direct

ELISA.

Cross-reactivity from secondary antibody.

Sandwich

ELISA

High flexibility.

High sensitivity.

High specificity, since

different antibodies bind

to the same antigen for

detection.

The antigen of interest must be large enough so

that two different antibodies can bind to it at

different epitopes.

It's sometimes difficult to find two different

antibodies that recognize different epitopes on

the antigen of interest and cooperate well in a

sandwich format.

Competitive

ELISA

High flexibility.

High sensitivity.

Best for the detection of

small antigens, even when

they are present in low

concentrations.

Relatively complex protocol.

Needs the use of inhibitor antigen.

Source: https://www.cusabio.com/c-20659.html; https://www.thermofisher.com/bd/en/home/life-

science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-

methods/overview-elisa.html (Accessed in 11 July 2019)


https://www.thermofisher.com/bd/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/overview-elisa.html




44

ELISA and its application

Since its conception in the

early 1970’s the ELISA has

been a primary method of

analyte detection. In its four

decades it has become a

fundamental tool in a wide

range of scientific fields, its

diverse nature demonstrated by

its range of uses. It was

extensively utilized with great

success in laboratory diagnosis

and research in the medical

field, veterinary medicine,

agricultural sciences,

biotechnology and in many

fields of research where it

could be applied. In addition to

molecular technologies, there is a need to use serological confirmatory methods in a dual

approach to directly identify and characterize disease agents and to assess disease

prevalence through the measurement of specific antibodies. The use of ELISA methods

in testing the environment and animal or plant products are reported to as safe for human

and animal consumption is also a rapidly evolving area for ELISA. Although

immunoassays are both highly sensitive and specific, false positive and negative results

may occur. False-negative results may be caused by improper sample storage, reagent

deterioration, improper washing technique or prozone effect. False-positive results have

been reported for samples containing small fibrin strands that adhere to the solid phase

matrix or due to substances in the blood or urine that cross-react or bind to the antibody

used in the test. Crowther, (2009) scanned the literature involving ELISA mentioned in

all science areas from 1976 to 2004 (Figure 2) elucidates continuous rise in the number of

works using ELISA methods. He also categorized the ELISA related publication in field

wise (Table 2) and demonstrate the major areas of use in medicine and dentistry;

immunology and microbiology, molecular biology, and genetics and biotechnology. He

mentioned that the earliest exploitation of ELISA was done in the field of immunology

and microbiology and molecular biology and biotechnology.

PPR and its diagnosis

Peste des Petits Ruminants (PPR), is an acute, highly contagious, notifiable and

economically important transboundary viral disease of sheep and goats associated with

high morbidity and mortality. Clinically, the disease resembles rinderpest (RP) in cattle

and is characterized by high fever (pyrexia), conjunctivitis, oculo-nasal discharges,

necrotizing and erosive stomatitis, diarrhea, and bronchopneumonia followed by either

death of the animal or recovery from the disease. The causative agent, PPR virus (PPRV)

Figure 2. Number of literature in Science Direct

database for ELISA (1976-2004). Source:

Crowther, 2009.


45

is an enveloped RNA virus belongs to the genus Morbillivirus of the family

Paramyxoviridae (sub family Paramyxovirinae) under the order Mononegavirales. The

PPRV is genetically grouped into four lineages (I, II, III, and IV) based on the F and N

gene sequences analyses. Lineages I–III circulate in Africa, while lineage IV is generally

found in Asia. However, lineage IV is recently reported from Morocco. PPR is tentatively

diagnosed by clinical observations, characteristic symptoms, epidemiology, postmortem

lesions, and laboratory confirmation by using various serological and molecular

techniques. Serological tests include agar gel immuno-diffusion test (AGID)/AGPT,

counter- immuno-electrophoresis (CIE) and ELISA, etc.

Table 2: Subject wise distribution of published literature in science groups

Subject Year

1980-84 1985-89 1990-94 1995-99 2000-04

Agriculture and biological sciences 87 274 615 804 827

Molecular biology, genetics and

biotechnology

374 1329 1762 1845 2096

Chemistry 8 29 77 208 279

Environmental science 4 13 52 125 162

Immunology and microbiology 514 1584 2128 2450 2772

Medicine and dentistry 280 971 1639 2875 3372

Neurosciences 21 124 198 380 484

Pharmacology and toxicology 24 108 247 397 497

Veterinary sciences 71 219 522 769 853

(Source: Crowther, 2009)

Application of ELISA in PPR

For early and specific diagnosis of PPR, researchers are working towards the

development of molecular diagnostics tools. However, as a rapid, simple and sensitive

assay, ELISA has been widely used in serological profiling of PPRV in mass screening of

samples for sero-monitoring, sero-surveillance or clinical prevalence. Various workers

have used MAb produced against PPRV and RPV for detection of antibodies and

antigens in ELISA. Several researchers used neutralizing MAbs against H protein of

PPRV for specific detection of PPRV antibodies in competitive ELISA (c-ELISA) and

blocking ELISA (B-ELISA) for antibody detection. Use of the virus neutralizing MAb

was reported to more advantageous as it produced better correlation between VNT and

ELISA. The sensitivity and specificity of a B-ELISA was reported to be 90.4 and 98.8%,

respectively when compared to VNT. MAb-based c-ELISA for measurement of

antibodies to PPR and RP viruses in sheep, goat and cattle are in common use.

Researcher also used anti-‘N’ MAb to PPRV in a c-ELISA, which was reported to 94.5%


46

sensitive and 99.4% specific in comparison to VNT. Researchers also developed a MAb-

based c-ELISA for the detection of PPRV antibodies using a virus neutralizing MAb

directed against an epitope in ‘H’ protein specific MAb (4B11) to PPRV and cell culture

propagated vaccine virus antigen. This c-ELISA had high diagnostic specificity (99.8%)

and sensitivity (90.5%) for detection of PPRV antibody in convalescent sera, when

compared with VNT and also the commercially available kit, where, it had high

diagnostic sensitivity (92.2%) and specificity (98.4%). This assay is currently being

employed extensively throughout India for monitoring/sero-surveillance of PPR. Further,

a polyclonal antibody based indirect ELISA was reported for detection of antibodies to

PPRV in the serum samples of goats and sheep using Vero cell culture propagated

purified PPRV antigen with a high degree of specificity and sensitivity when compared

with c-ELISA and VNT, respectively. This may be a good alternative tool to c-ELISA for

sero-epidemiological surveys. Further, MAb-based immunocapture ELISA and sandwich

ELISA (s-ELISA) have been used extensively for detection of PPRV antigen in clinical

specimens. The immunocapture ELISA was developed in the world reference laboratory

(CIRAD–EMVT, France) and is an internationally accepted assay for PPRV antigen

detection. This assay uses a biotinylated anti-‘N’ MAb against a cross-reactive epitope of

RP/PPRV to capture or detection of PPRV antigen in clinical samples. Similarly, the s-

ELISA kit developed at Division of Virology, IVRI, Mukteswar, India used a MAb (4G6)

directed against an epitope of N protein of PPRV, which is the routinely being used for

clinical prevalence or detection of PPRV antigen in clinical specimens in India. This

assay was efficacious, with diagnostic sensitivities (89%) and specificities (93%),

comparable to the immunocapture-ELISA. Further, assay using multiple antigenic

peptides (MAPs), as isotope, for detection of PPRV antibodies in serum samples has also

been developed for serosurveillance and seromonitoring (Balamurugan et al., 2014).

Problems in ELISA

The ELISA results are generally based on the color depth of the chromogenic substrate.

The color reaction is required to be carried out at 37 °C for about 10 minutes, then the

color reaction is terminated with a stop solution, and the absorbance at a specific

wavelength is monitored with a microplate reader. Since the detection path of each

reaction well is perpendicular to the microplate reader, the bottom of each reaction well

should be kept clean when testing, and the amount of chromogenic substrate and stop

solution should be accurate. The amount of liquid will affect the final degree. There are

various problem in ELISA experiment including abnormal color reaction, standard curve,

data analysis, the repeatability of experiment, etc. Some causes and solutions related to

color development are depicted in table 3.


47

Table 3: The problems of the color reaction

Cause Solution

No signal

Assay set up incorrectly, used

incorrect reagents or incorrect

wavelength

Review protocol. Repeat assay using a positive control

and check plate reader for wavelength, filters, gain etc.

Not enough antibody used Try different concentration of the primary and/or

secondary antibody.

Incubation time too short Incubate samples overnight at 4°C or follow the

manufacturer guidelines.

Antibody stored at 4°C for several

weeks or subjected to repeated

freeze/thaw cycles

Use a fresh aliquot of antibody that has been stored at -

20°C or below.

Recognition of epitope impeded

by adsorption to plate

To enhance detection of a peptide by direct or indirect

ELISA, conjugate peptide to a large carrier protein before

coating onto the microtiter plate.

Slow color development of

enzymatic reaction

Prepare substrate solution immediately before use. Ensure

the stock solution has not expired and is not

contaminated. Allow longer incubation.

Low sensitivity or weak signal

Improper storage of ELISA kit Store all reagents as recommended. Please note that all

reagents may not have identical storage requirements.

Plate reader settings incorrect Check plate reader for wavelength, filters, gain, etc.

Inactive detection reagent or

detection reagent too dilute

Ensure reporter enzyme/flour has the expected activity, or

use a higher concentration of detection reagent.

Insufficient amount of antigen was

coated to microtiter plate

Use more antigen for coating or very coating buffer.

Not enough antibody used Increase concentration of the primary and/or secondary

antibody. Optimize antibody concentrations for your assay.

Mixing or substituting reagents

from different kits

Avoid mixing components from different kits.

Incubation temperature too low Optimize the incubation temperature for your assay.

Reagents should be at room temperature before beginning

the assay.

Assay plates were compromised or

previously used.

Be sure to refrigerate plates in sealed bags with a desiccant

to maintain stability. Prevent condensation from forming

on plates by allowing them to equilibrate to room

temperature while in the packaging. If partial plates are

used, you must be sure to label used wells to prevent reuse;

cover them with sealing tape and use the remaining wells


48

Cause Solution

as soon as possible. Do not store partially used plates with

other plates. Include a desiccant in the storage bag.

High background signal

Too much antibody used Optimize antibody concentrations for your assay with

different dilutions.

Too much detection reagent Ensure the reagent has been diluted properly or repeat

assay with a higher dilution of detection reagent.

Incubation temperature too high Try different temperature for optimizing your assay.

Reaction not stopped Use stop solution to prevent overdevelopment.

Waiting too long to read plate after

adding stop solution

Read plate immediately after adding stop solution.

Incubation with substrate carried

out in the light

Perform substrate incubation in the dark.

Non-specific binding of antibody Use a suitable blocking buffer or use an affinity-purified

antibody.

Dirty plate Clean the plate bottom carefully and reread.

Slow color development

Incubation temperature is wrong Ensure plates and reagents are kept at room temperature.

Contaminated solutions Make fresh solutions.

Detection reagent too old,

contaminated or used at the wrong

pH

Use fresh detection reagents at the correct pH.

Wrong conjugate was used,

conjugate was prepared incorrectly

or has deteriorated.

Be sure that the conjugate used is the one that came with

the kit. All conjugates are kit- and lot-specific. If

preparation of a working conjugate is needed, be sure that

the concentrate and diluent are mixed in correct volumes.

Do not prepare the working solution too far in advance

and do not save any unused portion for future use. If no

conjugate preparation is necessary, be sure to pour out

only the amount required for immediate use and do not

return any unused portion to the stock bottle.

Wash buffer contains sodium azide. Avoid sodium azide in the wash buffer.

(Source: (i) https://www.cusabio.com/c-15091.html. (ii) https://www.thermofisher.com/bd/en/home/life-

science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-

methods/overview-elisa/elisa-troubleshooting-guide.html (Accessed in 11 July 2019)


https://www.thermofisher.com/bd/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/overview-elisa/elisa-troubleshooting-guide.html




49

Factors to be considered for selection of ELISA kit

Given its convenience and effectiveness, ELISA has wide applications in disease

diagnosis, biomedical research, and various industries. Virtually any type of molecule

(protein, lipid, carbohydrate, nucleic acid, etc.) can be detected by the method of ELISA.

Choosing a commercially available right ELISA kit is very important. Several fractors

should take into consideration while choosing an ELISA kit. Some factors for choosing

the right ELISA kit are mentioned below (https://www.cusabio.com/c-20299.html)

(Accessed on 11 July 2019)

The species to be studied

If the sample is from a classical model such as human, mouse and rat, it is relatively easy

to find a validated ELISA kit. But if the sample is from a non-classical model such as

monkey, there are limited numbers of commercial ELISA kits available. In this case, you

may have to choose a kit validated on species that shows homology with the species of

your sample.

The analyte to be detected

It needs clear understanding about what kind of analyte (usually protein) to detect. A

sandwich ELISA is generally suitable for detecting large proteins with multiple epitopes

such as a cytokine. A competitive ELISA is appropriate for detecting small molecules

like hapten. Most commercial ELISA kits are validated on serum/plasma and culture

supernatants. It is important to read the product instructions in detail to ensure that the kit

is compatible with your sample. For example, the way that plasma samples are collected

(heparin or EDTA) can affect which ELISA kit should be chosen. Besides, other factors

such as hemolysis and the presence of lipids in the sample can interfere with assay

performance.

Purpose of the analysis

ELISA is a tool that can be used for both qualitative and quantitative analyses.

Qualitative ELISA provides a simple positive or negative result for a sample, while

quantitative ELISA reflects the concentration of the analyte in a sample via a standard

curve. Based on the purpose of the analysis either qualitative ELISA or quantitative kit

should be chosen.

Type of antibodies

Need to clearly know what types of antibodies are used in the kit: monoclonal or

polyclonal antibody. In sandwich ELISA, it is sometimes helpful to use a polyclonal

antibody for capture and a monoclonal antibody for detection.

Requirement of sensitivity

If there is no hint about the concentration of the analyte in the sample, ELISA kits with a

broad detection range is a better choice. If the concentration of the analyte in the sample



50

is very low, ELISA kits with high sensitivity will be better. If the concentration of the

analyte is too high, dilution of samples may be required to adapt to the detection range of

the ELISA kit.

Sample size

ELISA kits usually require from 100ul down to 10ul sample. If the amount of sample is

very small or sample is very precious, better choose ELISA kits that require less amount

of sample.

Recovery and linearity data

Recovery and linearity experiments are used to assess the performance of ELISA kits.

Recovery helps determine whether analyte detection is affected by differences in sample

matrices. High recovery is better. The linearity of dilution determines the extent to which

the dose-response of the analyte is linear in a particular diluent. Ideally, the concentration

of the samples should be similar for all dilutions. Most suppliers provide recovery and

linearity data on product specifications. Besides, other important parameters such as

sensitivity and dynamic range are also provided. ELISA kits of different manufacturers

may have different parameter data. Need careful comparison of these parameter data,

particularly recovery and linearity data, to choose a right ELISA kit.

Detection system

There are several different detection systems in ELISA, including colorimetric,

fluorescent, and luminescent methods. All ELISA involves the immobilization of the

analyte to a surface as well as the use of an enzyme label and a matching substrate.

Choosing an appropriate enzyme and a matching substrate is important. Moreover,

enzyme-substrate reaction conditions, the microplate, and the detection device should be

properly chosen.

The experimental protocol

ELISA kits with simple protocols, convenient operation and short experiment time will

make it easier to do an ELISA test.

Reference

ELISA kits that have been used by other researchers and reported in the literature are

typically more trustworthy. Additionally, manufacturers and products that have received

certification are generally more reliable.

Cost

The price is always a factor for selection, particularly when the budget is limited.

Shipping cost should also be taken into consideration.


51

Conclusion

ELISA is a rapid, simple and powerful method, offering a host of advantages over other

common proteomics techniques. However, it requires careful selection of kit for

obtaining best results from an experiment. Hopefully, this document will give some

insight about ELISA and helped to decide whether ELISA is the right method for

intended experiment.

References

Avrameas., S., and Guilbert, B. 1971. Enzymo-immunological determination of proteins with the

aid of immunoadsorbants and enzyme-labelled antigens. C R Acad Sci Hebd Seances Acad

Sci D. 1971 Dec 20; 273(25):2705-7.

Balamurugan, V., Hemadri, D., Gajendragad, M.R., Singh, R.K., and Rahman, H. 2014. Diagnosis

and control of peste des petits ruminants: a comprehensive review. Virus Diseases, 25(1),

39–56; doi. 10.1007/s13337-013-0188-2

Engvall, J.R., and Perlamann, P.1971.Enzyme-linked immunosorbent assay (ELISA). Quantitative

assay of immunoglobulin G. Immunochemistry. Sep; 8(9), 871-874.

Rosalyn, S. Yalow., and Solomon, A.B.1960. Immunoassay of endogenous plasma insulin in

man. Journal of Clinical Investestment, 1960 Jul; 39(7), 1157–1175. doi:

10.1172/JCI104130

Wild, D. 2000. Immunoassay Handbook. 2nd ed. London: Nature Publishing Group. Zweig MH,

Csako G.(1990). High-dose hook effect in a two site IRMA for measuring thyrotropin. Ann

Clin Biochem 27, 494-495


52

NATIONAL ANIMAL DISEASE REFERRAL

EXPERT SYSTEM (NADRES)

Parimal Roy ICAR-National Institute of Veterinary Epidemiology & Disease Informatics, Bengaluru, India


During 1987, The Indian Council of Agricultural Research (ICAR) established an All

India Coordinated Research Project on Animal Disease Monitoring and Surveillance,

(AICRP on ADMAS). On 1st April 2000, the AICRP on ADMAS was upgraded to Project

Directorate on Animal Disease Monitoring and Surveillance (PD_ ADMAS) (during IX

Plan). The Directorate got further impetus with the addition of five more collaborating

units in X plan and two mission mode NATP projects viz., Animal Health Information

System and Data monitoring System (AHIS_DMS) and Weather based Animal Disease

Forecasting (WB_ADF) having 17 and 20 collaborating units respectively. Combining the

input from AHIS_DMS and WB_ADF, an interactive, dynamic online animal disease

forewarning system called NADRES (National Animal Disease Referral Expert System)

was developed with overall aim to improve the early warning and response capacity to

animal disease threats in the country for the benefit of farmers. Presently NIVEDI is

having 31 AICRP centres.

Early warning of disease incidence or outbreaks and the capacity of prediction of risk of

spread to new areas is an essential pre-requisite for the effective containment and control

of epidemic animal diseases, including zoonosis. Early warning is based on the concept that

dealing with a disease epidemic in its early stages is easier and more economical than

having to deal with it once it is wide spread. From the public health prospective, early

warning of disease outbreaks with a known zoonotic potential will enable control

measures that can prevent human morbidity and mortality. National Institute of Veterinary

Epidemiology & Disease Informatics developed the software application, NADRES that

systematically collect, verify, analyse and respond to the information from designated

AICRP-ADMAS, unofficial media reports and informal networks. NADRES builds on

the added value combining the alert and response mechanisms of different organizations

like state animal husbandry departments, Departments from universities, department of

Animal husbandry, Dairying and Fisheries, AICRP on ADMAS and other agencies

including NGOS, enhancing the capacity for the benefit of the farmers in the country and

other stakeholders to assist in prediction, prevention and control of animal disease threats,

including zoonosis, through sharing information, epidemiological analysis and joint

missions to assess and control the outbreak, whenever needed. For Zoonotic disease events,

alerts of animal outbreaks or incidence can provide the direct early warning so that human

surveillance could be enhanced and preventive action can be taken. Similarly there may

be cases where human surveillance is more sensitive and alerts of human cases precede

known animal occurrence of disease. Sharing assessments of an outbreak will enable a

joint and comprehensive analysis of the disease event and its possible consequences. Joint



53

dissemination will furthermore allow harmonised communication by the Central and state

Animal departments, ICAR-NIVEDI, regarding disease control strategies.

Regarding the joint response to disease emergencies, the three organizations will be able to

respond to a larger number and cover a wider range of outbreaks or exceptional

epidemiological events with the provision of a wider range of expertise. This will improve

the national preparedness for epidemics and provide rapid, efficient and coordinated

assistance in developing disease control strategies.

Specific objectives of NADRES

Allow state and central animal husbandry departments to better prepare themselves

to prevent incursion of animal diseases/infection and enable their rapid containment.

Increase timeliness and sensitivity of alerts

Improve the detection of exceptional epidemiological events at country level

Improve the transparency among different stakeholders

Improve the national surveillance and monitoring systems and strengthen the networks of

veterinary laboratories working in the country.

Improve national preparedness for animal and zoonotic epidemics and provide rapid,

efficient and coordinated assistance to states experiencing them.

Provide the technical support to states on issues at the animal/human interface of

outbreak control.

Disease outbreak data base

Database on disease outbreaks were collected though the networks of AICRP on ADMAS

with 31 centres across the country, provide the regular outbreak information along with

date and location of outbreaks, susceptible population, deaths, attacks etc., Disease data

obtained on a format is entered in to NADRES database in a double data-entry validation

mode to achieve to zero error entry. Database contains the disease events since 1990 was

further improved by inclusion of additional 16 AICRP centres.

Risk factors database

Risk factors such as weather parameters from different sources includes the monthly

precipitation(mm), sea level pressure (millibar),minimum temperature (0C) maximum

temperature(0C) wind speed (m/s), vapour pressure (millibar), soil moisture(%) ,

perceptible water(mm), potential evaporation transpiration (mm), cloud cover(%) etc.,

extracted from National Centre for environmental prediction (NCEP), Indian Meteorological

Department(IMD),National Innovations Climate Resilient Agriculture (NICRA) and

other sources. The remote sensing variables like Normalised Difference vegetative index

(NDVI) and Land Surface temperature were extracted from MODIS/LANDSAT/LISS III

or IV satellite images. The livestock population and densities were extracted from Livestock

census 2012.


54

Statistical model

A multivariate logistic regression model was used to predict the probability disease risk in

relation to weather parameters, remote sensing variables and livestock population or

densities. The goal of logistic regression is to find the best fitting (yet biological

reasonable) model to describe the relationship between dichotomous characteristic of

interest (disease outbreak) and a set of predictors (weather parameters, RS variables and

demographics) Logistic regression generates the co-efficients (and its standard error and

significance level) of a formula to predict a logit transformation of the probability of

presence of the characteristics of interest.

where p is the probability of presence of the characteristic of interest. The logit

transformation is defined as the logged odds

ather than choosing parameters that minimize the sum of squared errors (like in

ordinary regression), estimation in logistic regression chooses parameters that maximize

the likelihood of observing the sample values.

Overall fit of model

The null model −2 Log Likelihood is given by −2 * ln (L0) where L0 is the likelihood of

obtaining the observations if the independent variables had no effect on the outcome. The

full model −2 Log Likelihood is given by −2 * ln (L) where L is the likelihood of

obtaining the observations with all independent variables incorporated in the model.

The difference of these two yields a Chi-Squared statistic which is a measure of how well

the independent variables affect the outcome or dependent variable. If the P-value for the

overall model fit statistic is less than the conventional 0.05 then there is evidence that at

least one of the independent variables contributes to the prediction of the outcome. Cox &

Snell R2 and Nagelkerke R

2 are other goodness of fit measures known as pseudo R-

squares. Note that Cox & Snell’s pseudo R-squared has a maximum value that is not 1.

Nagelkerke R2 adjusts Cox & Snell’s so that the range of possible values extends to 1.

Hosmer-Lemeshow test

The Hosmer-Lemeshow test is a statistical test for goodness of fit for the logistic

regression model. The data are divided into approximately ten groups defined by

increasing order of estimated risk. The observed and expected number of cases in each

group is calculated and a Chi-squared statistic is calculated as follows:


55

with O, Eg and ng the observed events, expected events and number of observations for the

gth risk decile group, and G the number of groups. The test statistic follows a Chi-squared

distribution with G−2 degrees of freedom.

A large value of Chi-squared (with small p-value < 0.05) indicates poor fit and small Chi-

squared values (with larger p-value closer to 1) indicate a good logistic regression model

fit.

Early warning system (EWS)

Early identification of an infectious disease outbreak is an important first step towards

implementing effective disease interventions and reducing resulting mortality and

morbidity. The geographic and seasonal distribution of many infectious diseases are

associated with climate and therefore the possibility of using seasonal climate forecasts as

predictive indicators in disease early warning system (EWS) is an interest of focus.

Geographic Information system (GIS), remote sensing (RS) and Global Positioning system

(GPS) are the three commonly used veterinary geo-informatics technologies employed in

this digital era for rapid communication of data for better management of animal diseases.

Early warning systems are combinations of tools and process embedded within

institutional structures coordinated by national or international agencies. These systems

are composed of four elements depending upon they focus on specific hazard or many,

namely, knowledge of risk, a technical monitoring and warning services, dissemination of

meaningful warnings to at-risk areas, and farmers awareness and preparedness to act.

Warning services lie at the core of these systems, and how well they operate depends on

having a sound scientific basis for predicting and forecasting. As early warning systems grow

in geographical coverage and sophistication, false alarms to in rise. High false alarms can

undermine the public confidence, breed mistrust, dilute the impact of alerts and reduce the

credibility of future warnings.

Classification table

The classification table is another method to evaluate the predictive accuracy of the

logistic regression model.

In this table the observed values for the dependent outcome and the predicted values (at

a user defined cutoff value, for example p=0.50) are cross-classified and provides the

accuracy index for assessment model performance in prediction.


56

Table 1: Accuracy of Prediction

Sl. No. Diseases Accuracy (%)

1 Anthrax 95.52

2 Babesiosis 96.14

3 Black Quarter 92.43

4 Bluetongue 97.99

5 Enterotoxemia 95.98

6 Fasciolosis 97.06

7 Foot and mouth disease 86.72

8 Haemorrhagic septicaemia 90.58

9 Peste des petits ruminants 90.12

10 Sheep & Goat pox 95.52

11 Swine fever 95.37

12 Theileriosis 96.60

13 Trypanosomosis 96.45

Internal Accuracy was performed using 10 years of data. Accuracy obtained was > 90%

except Foot and mouth disease (86.72%).

The probability of disease outbreak was categorized in 6 risk levels- No risk (NR), Very

low risk (VLR), Low risk (LR), Moderate risk (MR), High risk (HR) and Very high risk

(VHR) for enabling the stake holders to take appropriate control measures by suitably

allocating available resources.

Table 2: Probability distribution of risk

interpretations

Figure 1.Risk prediction of Anthrax for the month of June 2018

Sl.

No.

Probabilit

y of risk

Interpretation

1 0 No risk/No or inadequate

data 2 0-0.20 Very low risk

3 0.21-0.40 Low risk

4 0.41-0.60 Moderate risk

5 0.61-0.80 High risk

6 0.8-1.0 Very high risk


57

NADRES outlook

The ICAR-NIVEDI website consists of details regarding NADRES database, Monthly

prediction bulletin, Risk maps, disease maps and Disease trend analysis.

LDF-mobile application

To extend the reach of the NADRES forewarning bulletin among the various stakeholders,

a Mobile Application (app) “LDF-Mobile App” was developed. The forewarning

methodology adapted in the “mobile app” remains the same as monthly bulletin. In

addition to forewarning, the LDF-Mobile App also provides the details of clinical samples

to be collected in case of outbreaks of the listed diseases for laboratory confirmation.

Immediate preventive measures to be taken up in case of positive prediction/disease

confirmation. The LDF mobile app is available at ICAR-NIVEDI website. It will also be

made available on Google play store.


58

LABORATORY MANAGEMENT WITH BIOSAFETY

AND BIOSECURITY PRACTICES

Asadulghani Head, Biosafety and BSL-3 Laboratory, icddr,b, Mohakhali, Dhaka-1212


Safety-first” is to keep the workers and

the environment safe. In biomedical

research and diagnostic laboratories,

either in human or in animal sector,

biosafety and biosecurity (BSBS) issues

have to be prioritized as part of

laboratory management system.

Prioritizing BSBS issues, the

laboratorians’ responsibility is to ensure

Biosafety & Biosecurity

Accuracy Timeline

accuracy/ assure quality (QA) of laboratory diagnosis and research. The intended

diagnosis or research has to be completed by a defined timeline (TL). Thus, the three

priorities of biomedical and microbiological research and diagnostic laboratories, in

general, are BSBS, QA and TL. Any violation in those three may have a serious human,

animal and environmental health impact.

Biosafety is to protect not just humans from harm but Earth’s entire biosphere as well. On

this regard we follow two principles – Risk Assessment and Containment. Risk

assessment identifies biohazardous and non-biohazardous materials, infectious and non-

infectious biohazards, and among the infectious biohazards the risk group of the

infectious agents may be present in the specimens under study. While we contain the

biohazard following the risk assessment, we ensure optimized personal practices, use

safety equipment to manipulate biohazardous materials within, and we work in a

dedicated and properly designed laboratory facility.

Laboratory

Priorities



59

As a consequence, implementing biosafety policies and procedure, we mitigate

unintentional release of biohazards from the laboratory. Whereas, biosecurity is to limit

access to facilities, biological materials, and related information to protect intentional

release of biohazard. All the diagnostic and research work have to be conducted

following approved standard operating procedures. Relevant staff members have to be

trained not only to develop SOPs but also to work following SOPs strictly. To ensure

safety in quality and vice versa, SOPs have to de developed for all laboratories

procedures and operations, validated, and strictly followed.

Early diagnosis and detection of infectious diseases is the key parameter for robust

control of spread of infectious diseases. Thus, scientists are moving from culture based

techniques to culture free techniques, which are faster, more sensitive and specific. This

ultimately helps the scientists to detect and control the diseases outbreak within shortest

possible time. Research and development for disease diagnosis, control and eradication

must also have specific goals, to achieve within clearly defined and determined timeline,

to contribute in the control of spread and ultimately eradicate the infectious disease if

applicable.

This is an established fact that human errors, poor technique, and contravening the

standards contribute to unnecessary exposure and compromise the best safeguards set

into place for protection. Thus, it is necessary to implement and accordingly follow the

policies and procedures of biosafety in microbiological and biomedical research and

diagnostics to mitigate human errors, poor techniques, as part of standard practices,

optimizing safeguard at the workplace.


60

RT-PCR: THE TECHNIQUE FOR

DETECTION OF PPR

Md. Abu Yousuf*, Mohammed A Samad, Mahmudul Hasan and Md. Giasuddin Animal Health Research Division, Bangladesh Livestock Research institute, Savar, Dhaka-1341


Introduction

Peste des petits ruminants is a highly contagious and infectious viral disease of domestic wild small ruminants. PPRV is a member of the genus Morbillivirus under the family Paramyxoviridae and order Mononegavirales. It is closely related to the rinderpest virus (RPV), the measles virus (MV) of humans, the canine distemper virus (CDV) of dogs and some wild carnivores, and the morbilliviruses of aquatic mammals. PPR is primarily a disease of goats and sheep and goats are usually more severely affected than sheep. The genome of PPRV is non-segmented, negative-strand enveloped virus. PPRV consist of 15948 nucleotides that encodes eight proteins: the nucleocapsid protein (N), the phosphoprotein (P), the matrix protein (M), the fusion protein (F), the haemagglutinin protein (H), the polymerase protein (L) and the two nonstructural proteins, C and V. PPRV genome is organized into six contiguous, non-overlapping transcription units corresponding to the gene of the six structural viral proteins in the order of 3'-N-P-M-F-H-L-5' in the genome sense. It is an economically significant disease of small ruminants such as sheep and goats. In many areas of Asia, small ruminant production and therefore the livelihoods of poor farmers is threatened by transboundary animal diseases (TADs) like Peste des petits ruminants (PPR).The disease causes severe losses to small ruminant production and is presently considered as one of the major threats to about 22 million small ruminant population of Bangladesh where mortality may reach up to 100% in an outbreak. Because of its high mortality rate, PPR affects food security directly by reducing the availability of meat and milk for family consumption and of funds for purchasing other commodities and foods as these animals are the main assets for the poor. Thus its control is a major goal for the programs aimed at poverty alleviation. Rapid diagnosis is essential for effective control measures. In South Asia Global Framework for the Progressive Control of Transboundary Animal Diseases (GFTADs) has recognized three priority diseases: FMD, PPR and HPAI. Considering the importance of PPR disease, SARRC regional cooperation and other international organization like FAO came forward to establish specialized laboratory on PPR in BLRI premises to diagnosis the disease, increase coordination among the laboratory and to conduct other research related to this disease.

Bio-safety

Containment Level BSL–II should be used for overall laboratory practice. Handling of

PPR suspected samples should be carried out under a Biosafety Cabinet Class II. Though

PPR is not zoonotic disease but sample must be handled with care using Personal

Protection Equipment (PPE) like lab coat, disposable gloves.



61

Conventional PCR

One-step single tube reverse transcription PCR assay incorporating primer specific for N

of PPR virus is used to detect the virus. This assay utilizes the Nexus, eppendorf, double

house PCR machine and operations according to standard operating procedures. In house

developed internal quality control (IQC materials are used in both extraction processes

and during test run. Results inference is drawn after electrophoresis using 100kb DNA

ladder. The sequential steps of PCR has given below:

annealing 60°C

denaturation 95°C

extension 72°C

Gel electroforesis

1

2

4

8

2n

n cycles

Specificity determined by 2 primers

Traditional PCR (Semi-quantitative)

RNA isolation (Extraction)

Viral RNA will be extracted from the tissue suspension or swab samples using RNeasy

Kit (PureLink RNA mini Kit , Ambion, USA) as recommended by the manufacturer.

Add 400 μl RLT buffer (add 10 μl of β- mercaptoehthanol to 1 ml RLT buffer) and

300 μl tissue suspensions in an Eppendorf tube, vortex and incubate for 3 minutes at

room temperature.

Then add 700 μl of 70% ethanol to the supernatant, mix and transfer to an RNeasy

spin column place in a 2 ml collection tube.


62

Transfer 700 μl in spin column from Eppendorf tube, centrifuge at 12000-13000 rpm

for 30 sec, discard the flow-through at the collection tube and set again in the

collection tube

Take the rest 700 μl and repeat the procedure as above

Add 700 μl of wash buffer-1 to the column, centrifuge as above and discard the flow-

through.

Add 500 μl of wash buffer-2 (add 4 volumes ethanol to 1 volume of RPE) to the spin

column and centrifuge for 30 sec at 12000-13000 rpm.

After removal of the flow-through, add again 500 μl of wash buffer-2 in the column

and centrifuge for 2 min at 12000-13000 rpm.

Place the spin column in a 1.5 ml Eppendorf tube and add 50 μl of RNAse free water

in to the centre of the column and centrifuge for 1 min as above.

Discard the RNeasy spin column and label Eppendorf tube containing RNA and store

at -20ºC or at -70ºC for short term and long term storage, respectively.

Master mix preparation

Total Sample (including positive and negative control)

Ambion , the RNA company Quantity/ sample Sample no.

Nuclease free water (μl) 5

2x RT- PCR buffer ( μl) 13

Primer F (100 pmol/ μl) (μl) 0.5

Primer R (100 pmol/ μl) 0.5

25x RT-PCR Enzyme Mix (μl) 1.0

Total Master mix 20.0

Template RNA (μl) 5

Final Reaction Volume (μl) 25

N gene specific primer

Gene Primer Sequence Position Size References

N

NP3 5´-TCTCGGAAATCGCCTCACAGACTG-3´ 1232-

1255 351

bp

Couacy-

Hymann, et

al,2002 NP4 5´-CCTCCTCCTGGTCCTCCAGAATCT -3´ 1583-

1560


63

Picture 1. Preparation of master mix and performing conventional RT-PCR

Thermal profile

RT step Initial

denaturation

Denaturation Annealing Elongation No. of

cycles

Final

elongation

Temp 500 C 95

0 C 94

0 C 55

0 C 72

0 C

35

720 C

Time 30 min 15 min 30 sec 30 sec 30 sec 10 min

PC NC S10 S9 S8 S7 S6 S5 S4 S3 S2 S1 M

Figure 1. Agarose gel electrophoresis of PCR products (351 bp) amplified with NP3 and NP4,

PPR specific primers. Lane M:100bp DNA molecular weight marker; Lane PC: Positive

control; Lane NC: Negative control; Lane S1-S10: Field samples

351bp


64

Analyses of RT-PCR products by agarose gel electrophoresis

Prepare agarose gels 1 to1.5% (w/v) in 1x TAE or TBE buffer

Add ethidium bromide to the agarose solution at 0.5 μg/ml

Pour the agarose solution containing ethidium bromide into the gel casting tray

When the gel set completely, remove the comb and transfer the gel into the

electrophoresis tank and cover with 1x TAE buffer

Mix DNA samples with DNA loading buffer (5 vol. DNA solutions with 1 vol. of

Bromophenol blue/ xylene cyanol as loading buffer) and load them into individual slots

DNA marker mixing with loading buffer

Perform electrophoresis at 100 V for 30 min

Place on the UV transilluminator in the dark chamber of the image viewing and

documentation system

The image will be shown in the monitor and to be captured to analyses the result

Trouble shooting of PCR

Observation Possible cause Solution

No product

Incorrect annealing temperature Recalculate primer Tm values test

annealing temperature gradient starting at

50 C below the lower Tm of the primer pair.

Poor primer design Verify that primers are non-

complementary both internally and to each

other.

Poor primer specificity Verify that oligos are complementary to

proper target sequence

Missing reaction component Repeat reaction setup

Suboptimal reaction conditions Optimize annealing temperature by testing

an annealing temperature gradient, starting at

50 C below the lower Tm of the primer pair.

Poor temperature quality Check 260/280 ratio of DNA template

Presence of inhibitor in reaction Further purify starting temperature by

alcohol precipitation, drop analysis or

commercial clean up kit decrease sample

volume.

Contamination of reaction tubes

or solutions

Reaction tubes prior to use to eliminate

biological inhibitors. Prepare fresh

solutions or use new reagents and new

tubes.


65

Special practices for PCR lab

Keep pre-PCR (extraction), PCR (master mix & amplification) and post-PCR

(analysis) work as isolate as possible.

Strictly adhere to the work flow for pre-PCR, PCR and post-PCR activities. Take

utmost precaution to avoid carry-over or cross contamination of PCR reagents with

(a) previously amplified PCR products, or (b) extracted DNA/RNA.

Use separate sets of equipment, appliances and supplies for different steps of PCR.

Do not move pipette, tips, tubes, marker, pen, note books, etc. between pre-PCR,

PCR and post-PCR sections.

Store consumables, reagents and water separately for pre-PCR, PCR and post-PCR

work. Aliquot reagents and water in convenient volumes.

Sharing of reagents and supplies among a group of researchers must be based on

mutual trust and understanding under strict control of a single leader. Always use

Incorrect temperature

concentration

For low complexity temperature (plasmid,

lambda, BAC DAN), use 1 pg-10 ng of

DNA per 50 μl reaction.

For Higher temperature complexity

templates (genomic DNA), use 1 ng- μg of


Multiple or

non-specific

products

Primer annealing temperature

too low

Increase annealing temperature.

Poor primer design Verify that primers are non-complementary

both internally and to each other.

Excess primer Primer concentration can range from 005

– 1 μM in the reaction.

Incorrect temperature

concentration

For Higher temperature complexity

templates (genomic DNA), use 1 ng- μg of


Concentration with exogenous

DNA

Setup dedicated work area and pipettor for

reaction setup. Wear gloves during

reaction setup.

Incorrect

product size

Incorrect annealing temperature Recalculate primer Tm values

Mispriming Verify that primers have no additional

complementary regions within the

template DNA.

Improper Mg ++

concentration Adjust Mg++

concentration in 0.2-1 mM

increments.

Nuclease concentration Repeat reactions using fresh solutions.


66

disposable gloves and change gloves frequently based on logical judgment. Consider

all surfaces potentially contaminated and avoid touching them wearing gloves

Plan and organize work before beginning a test. Do all calculations; fill in

worksheets; ensure availability of all reagents and supplies at respective work areas

before start.

Never reverse work-flow. Ideally, nothing should come back from the post-PCR

section to the pre-PCR or PCR sections without proper decontamination. Never bring

cDNA to the extraction and master-mix room.

Clean and wipe all work surfaces with 70% alcohol before and after each session

briefly pulse-centrifuge tubes to collect reagents at the bottom before opening.

Do not leave reagents on the bench (even on ice) any longer than necessary.

Use only freshly prepared ice flakes. Do not freeze ice for re-use.

Archive all information about new clones and newly synthesized primers.

Work wrap-up

Check all your samples, buffer, media and reagents in the refrigerator, freezer and

shelves.

Discard the materials that are no longer required (consult with your supervisor).

Handover the materials to the supervisor, along with a list, if these worth keeping

(isolates, clones, selected samples for future study, developed/prepared reagents, etc.)

Don’t forget to label properly

Clean all glassware and return to respective place/person, as appropriate

Return books, dissertations, reprints, etc. that you might have borrowed


67

DETECTION OF PPRV ANTIBODY IN SERA BY

COMPETITIVE ELISA (cELISA)

Md. Abu Yousuf* Mohammed A Samad, M Rafiqul Islam1 and Md. Giasuddin

Animal Health Research Division, Bangladesh Livestock Research Institute, Savar, Dhaka-1341 1Livestock Division, Bangladesh Agricultural Research Council, Farmgate, Dhaka-1215


Introduction

Competitive ELISA (cELISA) based on monoclonal antibodies specific for N –protein

and H-protein developed for detection of antibodies in animal sera. Goats and sheep

experienced PPRV infection at younger age remained sero-positive for 1-2 years

following exposure. cELISA has been widely used to detect PPR antibodies in many

countries. In the N-protein cELISA, the serum antibodies and the MAB complete on

specific epitope on nucleoprotein generated through recombinant technology using

baculovirus expression vector system. The sera samples were tested by c-ELISA. (ID vet.

Innovative Diagnostics, France) according to the instruction.

Principle of cELISA

The wells are coated with purified recombinant PPR nucleoprotein (NP).The samples to

be tested and the controls are added to the micro wells. Anti-NP antibodies, if present

form an antigen-antibody complex which masked the NP epitopes. An anti-NP

peroxidase (PO) conjugate was added to the micro wells. It fixed to the remaining free

NP epitopes, forming an antigen conjugate peroxidase complex. After washing in order to

eliminate the excess conjugate, the substrate solution (TMB) is added. The resulting

coloration depend on the quantity of specific antibodies present in the sample to be

tested:

- In the absence of antibodies, a blue solution appeared which becomes yellow

after the addition of the stop solution

- In the presence of antibodies, no coloration appeared.

- The absorbance is read at 450 nm.


68

Requirements for sample collection

70% Alcohol/ iodine swab

5 ml sterilized disposable syringe

Eppendorf tube

Centrifuge machine (Rotor for eppendorf tube )

Pipette ( 100µl -1000 µl capacity with tips)

Wet ice or ice pack

Vaccine carrier or suitable carrier

Refrigerator

Blood sample collection and serum separation

The goat blood is collected in early morning. After controlling the goat vein was

detected. (2-3) ml of fresh blood sample was collected from each of the animal

aseptically by puncturing jugular vein after swabbing with 70% alcohol or iodine swab in

a gentle manner. The loaded syringe was remaining in inverted condition at (30-40) min

for blood clotting. In that way blood sample was taken from different aged goats. After

clotting of blood, serum was separated into eppendorf tube, numbering and packaging

was done remain in freeze condition (2-8) for further use.

Figure1. Collection of blood and performed cELISA

Sample labeling

Specimen type/name. e.g. serum, blood, swab etc.

Unique identification number

Place of collection (location)

Date of collection


69

Equipment

Samples (Serum)

cELISA kit component

96 well Micro plate

Positive control

Negative control

Tips (200 1000 , 2000 ) Single channel pipette

Wash solution

Stop solution

ELISA washer

ELISA reader

Thermomixer compact (Eppendorf, Germany)

Minimum glass-distilled or deionized water

Incubator

Micropipettes (Multichannel, Single channel )

Graduated cylinders (10-2000ml)

Graduated pipettes (1-20 ml) with suitable bulbs

Storage bottles with closures (1-100m1)

Dilution tubes (2-4 ml.)

Reagents

The competitive ELISA kit is developed by ID. Vet Innovative Diagnostics Montpellier,

France.

Kit components

Screen® PPR Competition Kit, ID vet Innovative Diagnostics contain the following

reagents and chemicals

Micro plates coated with PPR recombinant nucleoprotein

Anti-NP-HRP concentrated conjugate (10X)

Positive control

Negative control

Dilution buffer 13

Dilution buffer 4

Wash concentrate 20X)

Substrate solution

Stop solution (H2SO4 0.5 M)

Sample preparation

In order to avoid differences in incubation times between samples, prepared a 96 well

plate containing the test and control samples, before transferring them into ELISA micro


70

plate using a multi-channel pipette.

Wash solution preparation

If necessary, brought the wash concentrate (20X) to room temperature (21°C ± 5°C) and mix

thoroughly to ensure the wash concentrate is completely solubilized. Prepare the wash

solution (1X) by diluting the wash concentrate (20X) in distilled water.

Test procedure of cELISA

Allow all reagents to come at room temperature (21 0c ±5

0c) before use. Homogenize all

reagents by inversion or vortex.

1. Add:

a. 25 μl of dilution buffer 13 to each well

b. 25 μl of the positive control to well A1 and B1

c. 25 μl of the negative control to wells C1 and D1.

d. 25 μl of each sample to be tested to the remaining wells.

2. Incubate 45 min + 4 min at 370C (± 3

0C)

3. Wash each well 3 times with approximately 300 μl of the wash solution. Avoid

drying of the well between washings.

4. Prepare the conjugate 1x by diluting the conjugate 10x to 1/10 in dilution buffer.

5. Add 100 µl of the conjugate 1x to each well.

6. Incubate 30 min ± 3 min at 210 C ( ±5

0 C)

7. Wash each well 3 times with approximately 300 µl of the wash solution. Avoid

drying of the wells between washings.

8. Add 100µl of the substrate solution to each well.

9. Incubate 15 min ± 2 min 210C (±5

0 C) in the dark.

10. Add 100 µl of the stop solution to each well in order to stop the reaction.

11. Read and record the O.D. at 450 nm with the help of ELISA plate reader.

12. Finally the reading data is place into data sheet of Microsoft Excel program and

save it in the computer hard.

Validation

The test is validated if:

The mean value of the negative control O.D (ODNC) is greater than 0.7.

ODNC >0.700

The mean value of the positive control (ODPC) is less than 30% of the OD.

ODPC /ODNC <0.3


71

Interpretation

For each sample, the competition percentage is calculate using the following formula

S/N % =

x 100

Sample presenting a S/N%:

- Less than or equal to 50% are considered positive

- Greater than 50% and less than or equal to 60% are considered doubtful

- Greater than 60% are considered negative

Result Status

S/N % ≤ 50 % Positive

50 % < S/N % ≤ 60% Doubtful

S/N % > 60% Negative

Precautions

Do not pipette by mouth.

The substrate solution can be irritating to the skin.

The stop solution (H2SO4 0.5M) can cause serious burns (R35). In the event of

contact with skin or eyes, wash immediately and abundantly with water and consult a

doctor (S26).

The conjugate, the controls and the substrate solution must be stored at 5 ( 3 )

The other reagents can be stored between +2 and +26

Components bearing the same name (wash solution, dilution buffers) can be used for

the entire ID vet product range.

Do not expose the substrate solution to bright light nor to oxidizing agents.

Decontaminate all reagents before elimination.


72

CELL CULTURE FOR VIRUS ISOLATION

AND IDENTIFICATION

Md. Giasuddin*, Md. Abu Yousuf, Amal Kumar Saha Animal Health Research Division, Bangladesh Livestock Research Division, Savar, Dhaka-1341


Introduction

The scientists started try to alive animal cell in vitro before near about 140 years. Now a

days cell culture is a very vital tools of many biological research. Once it was very

difficult to test any microbes in live animal but cell culture has made it easy to testing this

type of experiment. Cell culture also has been used for vaccine development. Somewhere

it is vigorously using to heal human diseases and burn injured patient. Specially stem cell

commonly using for human patient. Cell culture is doing for investigate the normal

metabolic pathways can be investigated by applying radioactively substrates and looking

at products. It can be used for effects of compounds on specific cell types such hormones,

growth factors may be evaluated by cell culture. Virus isolation in cell culture is very

important for preservation of the isolates and its subsequent use.

Regional Leading Diagnostic Laboratory for PPR, Bangladesh is using cell culture for

PPR virus isolation, identification and vaccine development.

Vero Cell culture procedure

Equipment

1. Bio safety cabinet- class-II

2. Incubator with CO2 facilities

3. Centrifuge machine with minimum capacity 5000 RPM and 15 ml falcon tube holder.

4. Kitchen refrigerator

5. -800 deep fridge

6. Liquid Nitrogen container

7. Inverted Microscope

8. Hot air oven

9. Water bath

10. Autoclave machine

11. Distilled water plant

Prime chemical reagents of (vero/ BHK21) cell culture

1. Minimum Essential Midium (MEM) or Dulbecco Minimum Essential Midium

2. Fetal calf serum/ Bovine calf serum

3. L-Glutamin

4. Sodium Bicarbonate


73

5. Antibiotic (Penicillin Streptomycin)

6. HEPES

7. Trypsin

8. Cell storage media (DMSO)

Essential auxiliary chemicals

1) Distilled deionized water

2) PBS

3) Alcohol (As cleaning reagent)

4) Disinfectant

Plastic ware

1. Tissue culture flasks (25cm2,75cm

2)

2 . Pipettes and Pipette holder

3. Tips

4. Falcon tubes

5. Syringe

6. Syringe filter (.22 micron & .45 micron)

7. 96 well flat bottom plate

8. Tub

Glassware

1. Graduated bottle (500 ml, 250 ml, 1000 ml)

2. Measuring cylinder

3. Biker (250 ml, 500 ml, 1000 ml)

5. Cell counter slide

6. Petri dish

Other consumables

1. Hand gloves

2. Apron

3. Masks and hair cover

4. Permanent marker

5. Aluminum foil

6. Cotton

7. Tissue paper

8. Autoclable polythene bag

9. Lab. shoe

Chemical reagents storage temperature

1. MEM at + 40C

2. Fetal calf serum/ Bovine calf serum at -200

to -800C


74

3. L-Glutamin at -200C

4. Sodium Bicarbonate at +40 C

5. Antibiotic (Penicillin Streptomycin) at -200C

6. HEPES at +40 C

7. Trypsin at -200C

8. Cell storage media (DMSO) at -200C

** Growth and Maintenance media have to be kept at +40 C

N.B. a) All the stored chemical reagents are have to be kept in water bath at 370C for

melting to bring in working condition before any cell culture related tasks.

b) For cleaning all the bottle of ingredients have to be wiped by tissue paper and then

wash by 70% alcohol and keep in bio safety cabinet.

Necessary reagents for Cell growth and maintenance media preparation

1. Minimum Essential Midium (MEM) or Dulbecco Minimum Essential Midium

2. Fetal calf serum/ Bovine calf serum

3. L-Glutamin

4. Sodium Bicarbonate

5. Antibiotic (Penicillin Streptomycin)

6. HEPES

7. Double distilled sterile water

Cell culture procedure

Picture 1. Cell culture in the laboratory

Start biosafety cabinet and switch on UV light. Stop the UV light after recommended

time (3-5 minutes) and then start the airflow and normal light. At first a preserved cryo

tube of cell has to pull out from liquid nitrogen and inoculate in growth media. And keep

in 370 C incubator for 24 hours and for betterment of cell growth cell inoculated flask can

be washed by PBS and add GM again in flask at next day. It will take 3 days for 80-90%

confluent condition.


75

After 80-90% confluent of flask bottom it may ready for further tasks especially for virus

isolation, identification and vaccine development and cell multiplication (Sub-culture).

Sub culture

Confluent cell culture flask pull out from incubator and wash and clean at the same

way.

Pour out growth media from flask and wash 2-3 times by PBS.

Trypsinized and allocate in several flask and keep it for growth.

Picture 2. Cell culture in the laboratory

Add 200-300 micro liter trypsin for 252cm flask for trypsinization (trypsin quantity

will depend on flask size).

After trypsinization add some growth media and shake or pipetting for cell cluster

uniformly distribution in media. Add more GM in flask according to expected

concentration and flasks.

And keep in incubator at 370Cfor growth.

Cell counting by hemocytometer

Step 1

Take a hemocytometer which has 9 square chambers. We shall count of cell total 5

chambers among 9 chambers according to table given be low.

1 2

5

3 4

Keep 100 micro litre cell in one chamber

Add 100 micro litre typan blue in chamber


76

Count every square separately

Count viable and non-viable cell differently

Step 2

We have to count some parameters for cell number determination. Those are-

Total viable cells

Total nonviable cell

Average number of cell per square

Dilution factor

Concentration (Viable cells /ml)

Suppose we have got 54 viable cells and 3 nonviable cells. In that case calculation will

be,

1. % of viable cells = # viable cells/ Total # cells x 100 = 54/57= 0.947x100= 94.7%

2. Average # of cells/ square= Viable cells/No. Square= 54/5=10.8

3. Dilution factor= Final volume /volume of cell= 200 micro litre/100 micro litre=2

*(Cell 100 micro litre and Typan blue 100 micro litre)

4. Concentration (viable cell) = average number of cell/Dilution factor x 104

= 10.8 x 2

x 104 = 216000 cells/ ml. In scientific notation 2.16 x10

5 cell/ml

Sample collection

Many types of viral samples may come from field level. After a certain processing we

can use these collected samples for our purposes like virus isolation and identification.

Samples types and collection procedures have described below-

Swab samples

Use a cotton/dacron swabs to collect the specimen. Collect swab from posterior nasal

cavity or Conjunctiva, avoiding external secretion. Keeping swabs moist after collection

is most important. Place swab in 3-4 ml Viral Transport Media (VTM). Any sterile

isotonic fluid, like phosphate buffered saline (PBS) with antibiotics (penicillin 200

International Units/ml and streptomycin 200 μg/ml or Gentamycin 500 ug/ml), or

common tissue culture medium like Eagle’s MEM can be used. Swabs may be broken off

to fit within the capped tube containing the VTM and shipped. Alternatively,

whirl/agitate the swab in the media for several minbefore removing it and shipping the

suspension.

Commercially available kits containing swabs and viral transport media are acceptable.

Blood sample

Collect blood aseptically from Jugular vein in heparini zed tubes when the animal is in an

acute febrile stage of the disease and store at chilled condition.


77

PM tissue samples

Collect lung, mesenteric and bronchial lymph nodes aseptically from PPR suspected dead

goats during post-mortem.

Transportation of the samples

Samples should be transported in chilled condition with ice packs or with wet ice. They

should be received by the testing laboratory within 48 hours of collection. If shipment is

delayed and facilities are available, the specimens should be frozen at -70o C and shipped

on dry ice. Otherwise, store specimens in refrigerator (freezing at -20o C reduces viability

of virus).

Preparation of inoculums for virus isolation

Swab samples

Draw 0.5 ml of sample suspension into 1 cc syringe. Attach 0.2um syringe filter

(such as an Acrodisc 13) and push sample through filter directly onto cells or in an

eppendorf tube. The drawback is that some samples will not pass through the filter.

To overcome clogging the filter make sure suspension is well homogenized before

drawing into the syringe.

Blood samples

Centrifuges heparinized blood at 1000 rpm for 5 minute and collect the buffy coat.

Wash the white blood cell in Minimal Essential Medium (MEM) containing

antibiotics (penicillin 200 International Units/ml and streptomycin 200 μg/ml).

Resuspend the pellet cells in 1.0 ml of MEM plus antibiotics. Inoculate 0.5 ml of the

cell suspension into confluent Vero cells grown in 25 cm2 tissue culture flask.

Tissue samples

Use individual organ or pool the collected tissues (lung, lymph nodes) collected from

the same animal, weigh and macerate them with sterile mortar and pestle while the

tissues are still frozen.

Add PBS to make 20% (w/v) suspension and collect the suspension in a sterile tube.

Centrifuge tissue suspension at 3000 rpm for 10 minutes. Collect the supernatant in

fresh sterile Falcon tubes and add antibiotic (Gentamycin) at 500 μg/ml and store at -

700C.

Prior to inoculation, filter the suspension, using Acrodisc® Syringe filter (13 mm

diameter, 0.2 μm pore size) [Sigma-Aldrich or similar] and use the filtrate as

inoculums.

Inoculation in vero cells

Maintain the Vero cell in media M199 with bovine fetal serum (10% for growth and

5% for maintenance).

Inoculate the flask when Vero cell monolayer becomes confluent.


78

Discard the spent medium, wash the flask/s with 10 ml sterile pre-warmed PBS for 2

times.

Inoculate the cells with the prepared sample suspension at 200 μl/25 sq. cm flasks.

Run positive (with PPR Vaccine virus, 200 ul/ per flask) and negative control (200 μl

PBS)

Spreads the inoculums all over the cell sheet and incubate the flasks at 37°C for 1

hour for adsorption of the virus. Spread the inoculums by tilting the flasks every 15

minutes. After one hour, add 5ml of growth medium containing 10% fetal calf serum

to the flasks without removing the excess inoculums.

Incubate the flasks at 370 and examine twice daily for the appearance of cytopathic

effects (CPE).

Harvesting of virus

Harvest the culture medium at 5thday of each passage or when the maximum CPE

manifested.

Collect the infected tissue culture by freezing and thawing for 4 times, and centrifuge

at 2500 rpm for 5 minutes, Collected the suspension in sterile 50ml Falcon tubes.

Use the collected suspension for further passage in fresh Vero cells.

Special practice for tissue culture lab

Use laminar flow cabinet for preparation/handling of fresh and uninfected cell culture

and the biosafety cabinet for handling infectious materials.

Wipe all work surfaces liberally with a disinfectant (70% alcohol) before and after

each session of work.

Wipe hands with 70% alcohol before commencement of the sterile work.

Use only sterilized glassware/plastic ware. Decontaminate the surfaces of all

glassware with 70% alcohol before placing into the laminar flow cabinet or biosafety

cabinet.

Flame all bottles at around the neck before and after opening and before re-closing.

(Note – Use of spirit lamp or Bunsen burner is not allowed in the biosafety cabinet.

Rely on decontamination with 70% alcohol)

Store stock and working media/reagents in a separate freezer designated for tissue

culture reagents only. Divide all reagents into suitable aliquots whenthey are opened

for the first time.

Store viruses and infected cultures in clearly labeled vials and place them in separate

designated freezer.

Collect all used glassware in disinfectant (Sodium hypochlorite/Virkon) and follow

the procedure of cleaning and disinfection.

Clean and disinfect all surfaces daily at the end of the day.


79

Collect all used glassware and plastic ware (except tips and Eppendorf tubes) in a

bucket containing disinfectant (sodium hypochlorite, virkonS), leave overnight and

proceed for cleaning & sterilization or disposal. Cleaning & sterilization of used

glassware must be completed in the shortest possible time.

Waste disposal

Collect all sharp items (needle, blade, broken ampoule) in sharp disposal cans. When

full, dispose the can by incineration.

Solid waste such as flasks, micropipette tips, eppendorp tubes centrifuge tubes,

contaminated gloves, tissues, etc., should be placed inside heavy-duty Poly bag for

contaminated waste and incinerated.

Collect the biodegradable solid waste in large volume of freshly prepared disinfectant

(sodium hypochlorite, virkon S), leave overnight and dispose as garbage.

Decontaminate infectious liquid waste in large volume of freshly prepared

disinfectant (sodium hypochlorite, virkon S), leave overnight and dispose in sanitary

sewer.

Cell preservation procedure

Select confluent and healthy cell flask from incubator. Discard media and has to be

washed for 2-3 times by PBS. And then cell to be trypsinized by trypsin (300 micro litre

for 252 cm flask). After 2-3 minwe will get floating cell. Add some growth media. For

more uniform cell concentration in media has to be performed 1-4 times pipetting. Cells

allocate in 15 ml falcon tube and then it has to be centrifuged 10 minin 1500 rpm. After

centrifuge supernant has to be discarded and add supplied cell storage media and then

allocate in cryo tube for preservation. In second phase, allocated cell direct to be kept in -

800C for overnight and then it can be transported into liquid N2 for long time storage.

TCID50 Determination

Step-1

Take cell containing flask

Discard media, wash 3 times with PBS

Trypsinization of monolayer flask for cell detachment (.5ml for 252 cm flask)

Add growth media

Transfer tryposinized cell to a sterile petridish

Distribution of 100 micro litre of cell with growth media in per column for TCID50

using 96 wells flate bottom plate (NUNC plate)

Incubate at 370 C for 24-48 hrs for confluent growth of cell


80

Step-2

Thawing of virus sample

Make 10 fold dilution of virus

Take cultured cell plate from

incubator and check cell

under microscope

Discard media and wash 3

times with PBS

Add 30 micro liter virus

dilution in 96 well flat bottom

plate except last 2 columns

(Control)

Incubate for 40-45 minat 370 C

Then 200 micro litre maintenance media is added in each well. Incubate at 370C in a

incubator for 24-72 hrs.

Calculation

After completion of incubation time cell plate has to be observed for CPE under

microscope in every well to calculate TCID50 according to Reed and Munch method.

Virus dilution 10-5

10-6

10-7

10-8

Positive 10 8 4 2

Well inoculated 10 10 10 10

Percentage of infection 100 80 40 20

Calculating proportionate distance (PD)

PD = % positive at or above 50%- 50%

% positive at or above 50% - % Positive below 50%

PD= [(80-50/80-40)]

= 30/40

= .75

Calculating TCID50 /ml

50% in point titre (TCID50 per 0.1ml) =10 log total dilution above 50% - [PD*log (dilution factor)]

= 10-6-(0.75* log 10)

= 10-6-0.75*1

= 10 -6.75

TCID50= 107.75

/ml viral titre


81

List of the Participants

Dr. Md. Golam Azam Chowdhury Upzila Livestock Officer

Central Disease Investigation Laboratory

Dhaka, Bangladesh

Cell: +880-1716940769


Dr. Shukes Chandra Badhy Upzila Livestock Officer

Central Disease Investigation Laboratory

Dhaka, Bangladesh

Cell: +880-1718129848


Dr. Sonia Akter Scientific Officer

Goat and Sheep Production Research Division


Savar, Dhaka-1341

Cell: +88 01717871911


Dr. Md. Habibur Rahman Scientific Officer

Goat and Sheep Production Research Division


Savar, Dhaka-1341

Cell: +88 01716286487







82

Dr. V. Balamurugan

Principal Scientist (Veterinary Microbiologist)

ICAR-NIVEDI, Post Box No. 6450,

Yelahanka, Bengaluru-560064,

Karnataka, India

Mobile: +91- 9481807438; 9108427438


Dr. D. Muthuchelvan

Principal Scientist

In-Charge, PPR Laboratory

Indian Veterinary Research Institute

Mukteswar, Nainital Dist

Uttarakhand

Phone: +91 05942-286346


Dr. Krishna Raj Pandey Senior Veterinary Officer

Veterinary Laboratory

Surkhet, Nepal

Phone: 9849337320


Dr. (Mrs.) G. A. Gunawardana Veterinary Research Officer

Veterinary Research Institute

Sri Lanka

Mobile: 00-94-717895190

Telephone no.: 00-94-81-2388125





83

List of the Resource Persons

Dr. Nathu Ram Sarker Director General


Savar, Dhaka-1341, Bangladesh

Tel: +88027791683


Dr. Asadulghani Head, Biosafety and BSL-3 Laboratory

Biosafety Office, Icddr,b

Mohakhali, Dhaka-1212


Dr. Mohammad Rafiqul Islam

Professor

Department of Pathology, Faculty of Veterinary

Science

Bangladesh Agricultural University,

Mymensingh-2202


Bouna Diop Secretary

FAO/OIE PPR Global Secretariat

Viale delle Terme di Caracalla, 00153

Rome, Italy

Tel: +39 06570 55667


Dr. Emdadul Haque Chowdhury Professor

Department of Pathology

Faculty of Veterinary Science

Bangladesh Agricultural University

Mymensingh -2202, Bangladesh

Cell: +88 01712017381


Dr. Mohammed Abdus Samad Senior Scientific Officer &

Director, RLDL for PPR

Bangladesh Livestock Research

Institute


Call: +88 01717047877


Dr. Md. Giasuddin Head, Animal Health Research Division &

Director, National Reference Laboratory for

Avian Influenza



Call: +88 01711055597


Dr. Md. Nure Alam Siddiky National Consultant (Laboratory)

Combating the Threats of Antimicrobial

Resistance and Zoonotic Diseases to

Achieve the GHSA in Bangladesh

BLRI, Savar, Dhaka-1341

Cell: +88 01716475486

E-mail: [email protected]

Dr. Jahangir Alam Chief Scientific Officer

Animal Biotechnology Division

National Institute of Biotechnology

Ganakbari, Ashulia



Cell:+88 01712819098

Dr. Md. Abu Yousuf Scientific Officer (Bacteriology)

Animal Health Research Division


Institute

Savar, Dhaka-1341.

Cell: +88 01717449845












84

Dr. Mohammad Rafiqul Islam Principal Scientific Officer

Livestock Division

Bangladesh Agricultural Research Council

Farmgate, Dhaka-1215, Bangladesh

Cell: +88 01716350628


Dr. Mahmudul Hasan Scientific Officer

BLZAC Project


Institute

Savar, Dhaka

Cell: +88 01737152465


Dr. Parimal Roy Director

ICAR - National Institute of Veterinary

Epidemiology and Disease Informatics

(NIVEDI)

Indian Council of Agricultural Research

Post Box # 6450, Yelahanka

Bengaluru - 560064, India


[email protected]



http://[email protected]/


SAARC Regional Training - SAARC Agriculture Centre Diagnosis...SAARC Regional Training On Molecular Diagnosis and Laboratory Surveillance of PPR 21-26 July 2019 Editors Mohammed A

Documents