project title: development of a vaccine to eradicate ... · final technical report: development of...

Project Title: Development of a Vaccine to Eradicate Contagious Bovine

PleuroPneumonia in Africa

IDRC Project Number: 106929

Research Organizations Involved in the Study:

University of Saskatchewan - Vaccine and Infectious Disease Organization –

International Vaccine Centre

Kenya Agricultural Research Institute

Location of Study: Canada, Kenya

By: Dr. Andrew Potter and Dr. Volker Gerdts, VIDO-InterVac

Dr. Hezron Wesonga and Dr. Reuben Soi, KARI

Report Type: Final Technical Report

Period Covered by the Report: March 2012 – August 2014

Date: August 28, 2014

Final Technical Report: Development of a Vaccine to Eradicate Contagious Bovine PleuroPneumonia in Africa

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*Abstract: Research outputs should include an abstract of 150-200 words specifying the issue under

investigation, the methodology, major findings, and overall impact.

Contagious Bovine PleuroPneumonia (CBPP) is considered to be one of the most economically

important livestock diseases in Africa. The disease is caused by Mycoplasma mycoides subsp.

mycoides, which is transmitted between cattle by aerosol and kills up to 50% of infected animals.

The objective of our research was to develop a novel CBPP vaccine that will help to eliminate

this disease of cattle from large areas of Africa. We used a “reverse vaccinology” approach in

which all potential vaccine components were identified using computational tools and the genes

coding for potential vaccine components were cloned and expressed for the production of large

quantities of protein. These were formulated into vaccines which were tested for their ability to

protect against experimental infection with M. mycoides. Out of sixty six proteins tested, we

have identified three that provided significant protection against infection and 1-3 others that are

currently being re-tested. These positive results will set the stage for further scale-up and testing

of the vaccine. We have also carried out socio-economic studies to look at acceptance of the

vaccine and the results of those studies indicate that the current formulation would be well

received by Kenyan farmers.

*Keywords: Include up to six subject keywords separated by commas.

CBPP, vaccine, cattle, Kenya, genomics


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Table of Contents

EXECUTIVE SUMMARY ............................................................................................................ 4

1.0 THE RESEARCH PROBLEM ................................................................................................ 6

2.0 PROGRESS TOWARDS MILESTONES ............................................................................... 7

2.1 Twelve Month Milestones: ............................................................................................... 7

2.2 Twenty Four Month Milestones ....................................................................................... 8

2.3 Thirty Month Milestones .................................................................................................. 9

3.0 SYNTHESIS OF RESEARCH ACTIVITIES AND RESULTS ........................................... 11

3.1 Objectives of the Research Project ................................................................................. 12

3.1.1 Objective 1: Identify all potential surface-localized and secreted proteins of Mmm

using bioinformatics tools. .................................................................................... 12

3.1.2 Objective 2: Clone and express the genes coding for potential vaccine targets in

Escherichia coli for production of antigens. ......................................................... 17

3.1.3 Objective 3: Test pools of proteins for their vaccine potential in a bovine model

for CBPP and identify formulations containing protective antigens. .................... 18

3.1.4 Objective 4: Test the immunogenicity and protective capacity of 2-4 antigens

formulated with different adjuvants to enhance the immune response. ................ 25

3.1.5 Objective 5: Analyze a priori, the social and economic factors, including

willingness to pay and gender constraints that will influence the acceptability and

adoption of the vaccine by livestock keepers. ....................................................... 25

CONCLUSIONS ................................................................................................................... 30

4.0 SYNTHESIS OF RESULTS TOWARDS AFS OUTCOMES ............................................. 31

5.0 PROBLEMS AND CHALLENGES ..................................................................................... 33

6.0 RECOMMENDATIONS ....................................................................................................... 34

APPENDICES .............................................................................................................................. 35

Appendix 1: Clinical and Pathology Data from Efficacy Trial 1. ........................................ 36



Appendix 4: Mean group temperatures 4 weeks after challenge. ........................................ 53

Appendix 5: Group average proliferation stimulation indices in vaccinated and control

animals. .......................................................................................................... 54

Appendix 6: Serological response to immunization in Trial 1. ............................................. 57



Appendix 9: Participation in various conferences/symposia. ................................................ 60


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EXECUTIVE SUMMARY

The objective of this research project is to deliver a novel vaccine against Contagious Bovine

Pleuro-Pneumonia (CBPP) that will help to eliminate this disease of cattle from large areas of

Africa. The major outcomes of the research will be reduced mortality and morbidity of cattle

associated with CBPP, facilitation of trade and cross-border movement of animals, and a reduced

allocation of national veterinary services towards management of this disease. An effective

vaccine will protect assets and increase food security among pastoralist communities vulnerable

to the impact of livestock disease, ultimately leading to increased profitability.

Traditional Mycoplasma mycoides vaccines have been successfully used in a number of countries

to eradicate the disease. However, they present several problems in the African context,

including a relatively short shelf life and a strict requirement for an effective cold chain to be

maintained. We have therefore used a “reverse vaccinology” approach to subunit vaccine

development in which the genomic sequence of the organism is analyzed using computational

tools for those components which interact with the immune system of the host. They are then

tested for efficacy in animal models. During this project a total of 69 genes of Mycoplasma

mycoides, the causative agent of CBPP, were identified as coding for potential vaccine

candidates and they were cloned into bacterial expression vectors to produce the large quantities

of protein necessary for vaccine trials. We were able to complete this for 66 M. mycoides target

proteins which were produced in sufficient quantities for prototype vaccine formulations as well

as for assays of samples obtained from field and experimental animals. Infection models were

established in Kenya to evaluate the vaccine in the target species and breeds, including both

Boran and Zebu cattle. The logistics of working with large numbers of cattle at the experimental

station in Muguga were optimized and personnel were trained to provide care and handling of

the animals. Prototype vaccines were formulated in Canada using pools of five proteins plus

adjuvants and then tested in both Kenya and Canada. Three trials using total of 170 head of

Boran cattle were performed in Kenya. Concurrently eighty Canadian cattle were immunized at

VIDO and their immune responses to each of the vaccine proteins characterized. The results of

the vaccine trials indicate that ~8 proteins were capable of protecting cattle against experimental

infection, thus setting the stage for further development work.

In parallel to the laboratory work, the socio-economic components of the project were assessed.

This analysis was conducted in close interaction with Canadian-South African collaboration

(CIFSRF project 106930 developing vaccines for 6 viral livestock diseases). Large surveys were

conducted to address future uptake of the vaccine by small holder farmers, women in particular,

to ensure that the vaccine will be used effectively by this target group. In addition, the

preferences for the type of vaccine and its properties (method of administration, stability,

duration of immunity, etc) were determined. Data collection for impact evaluation is complete

and has been analyzed and it indicates that the preferred CBPP vaccine and its delivery by

livestock keepers in Ijara sub-County is one that includes an indicator (vial vaccine monitor), has

maximum efficacy and safety, administered once a year by the Government and is compulsory.

Farmers were willing to pay more for the vaccine and vaccination with preferred attributes than

the market price (KSh. 34.6) calculated in a previous study in Narok County of Kenya i.e they

valued the vaccine and vaccination higher than the market price.


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Also coordinated with CIFSRF 106930 was the preparation for vaccine launch. The possibilities

for vaccine production in Kenya and South Africa were surveyed and discussions were held with

Kenya Veterinary Vaccines Production Institute regarding their capability and desire to produce

the CBPP vaccine. Initial evaluation of KEVAVAPI production facilities and equipment was

made, resulting in a list of necessary equipment for pre-production and initial production. Plans

were made for the next stages of development, field trials, and safety tests. In response to a call

from IDRC a Phase 2 project was developed and submitted.

The data obtained by this project has potential commercial value and therefore, publication of the

results has been delayed until all details related to protection of intellectual property have been

finalized. The process of filing patent applications has been initiated and we expect that they will

have been submitted in early October 2014.

The milestones of the project were all achieved on or before their due dates, with the exception

of finishing the final 2-4 protein vaccine prototype test which was delayed because one trial had

to be repeated. Interestingly, the most protective vaccine formulation is one that had a relatively

low ranking of its vaccine potential by computational analyses and demonstrates the power of

this approach relative to conventional technologies used for the selection of vaccine antigens.


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1.0 THE RESEARCH PROBLEM

Contagious Bovine PleuroPneumonia (CBPP) is considered by the African Union’s Inter-African

Bureau for Animal Resources as one of the most economically important livestock diseases in

Africa, an opinion echoed by the OIE. The disease is caused by a small bacterium, Mycoplasma

mycoides subsp. mycoides (Mmm), in an earlier nomenclature specified as Small Colony type,

which is transmitted by aerosol and kills up to 50% of infected animals when newly introduced

into a population. About 26 countries are affected by the disease (see Figure 1) and the extent of

endemically infected regions is increasing.

Over 24 million people are at risk from its effects. The

impact on the economies is very difficult to calculate, but has

been estimated to be 2 billion dollars per annum at a

minimum. CBPP was once endemic in Europe, especially

during the 19th

century when widespread trade in live animals

became commonplace. However, the disease was eradicated

in most parts of Europe during the 20th

century through

traditional methods for disease control ranging from changes

in management practices and antibiotic treatment to selective

killing of infected animals. In recent years, both live and

killed vaccines for CBPP have been tested but have shown

either poor efficacy or, in the case of live vaccines, issues

with stability and safety.

Epidemiologists have predicted that with the current efficacy of the live vaccine, control of

CBPP is not possible in African conditions, where policies such as movement restriction or

slaughter are difficult or impossible to implement. In addition, this vaccine has a strict

requirement for a cold chain to be in place, something that is not always possible in rural regions.

Thus, we proposed to develop a novel vaccine with greater efficacy and stability that is easier to

handle under field conditions. Such a vaccine has the potential to dramatically enhance our

ability to control the disease and lead to greater economic viability of the cattle sector.

As described in our research proposal, we have taken a “reverse vaccinology” approach to CBPP

vaccine development in which all potential surface components of the organism were to be tested

for their ability to protect against disease. Those showing protection will ultimately be combined

in a formulation with an adjuvant and its use optimized for conditions in Africa. The overall

project achievements are within the planned timelines and the milestones have been achieved.

Figure 1. Distribution of CBPP in Africa


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2.0 PROGRESS TOWARDS MILESTONES

2.1 Twelve Month Milestones:

2.1.1 The Scientific Advisory Board will be established and their first meeting held.

An internationally-renowned Scientific Advisory Board was assembled, including Dr. Joseph

Musaa (Ministry of Livestock and Fisheries Development, Department of Veterinary Services,

Kabete, Kenya; retired), Dr. Willie Donachie (Moredun Research Institute, Penicuik, Edinburgh

EH530QA), Dr. Adrian Hill (The Jenner Institute, Oxford 0X3 7DQ, UK), Dr. Robin Nicholas

(Animal Health and Veterinary Laboratories Agency, Addlestone, Surrey KT15 3NB UK) and

Dr. Dieter Schillinger (Animal Health Consultancy, D-81247 Munich, Germany). The Board was

present at the project launch and annual meetings providing recommendations and advice at each

stage of the project. This Board was shared with the second vaccine-related CIFSRF project on

viral vaccines in order to help identify areas of potential overlap and synergies that could result

from conceptually similar activities.

2.1.2 Signing of agreements between VIDO-InterVac and KARI and between KARI and

ILRI.

The agreement between KARI and ILRI was signed in 2012 and a collaborative research

agreement between KARI and VIDO-InterVac was signed shortly afterwards.

2.1.3 Report on the Project Inception Workshop in Nairobi outlining comprehensive

work plan and monitoring and evaluation plan.

The inception workshop took place in the Jacaranda Hotel in Nairobi, Kenya on July 1-2, 2012

and a comprehensive report was submitted to IDRC, the SAB and all project participants

following the meeting. The workshop spanned three days and included joint sessions between

both vaccine projects as well as project-specific meetings.

2.1.4 Project Managers (Kenya, Canada) will be hired and on site. Recruiting of students

and postdoctoral fellows will be completed.

A Project manager (Dr. Emil Berberov) was hired in Canada and has traveled to Nairobi to

discuss further activities with the KARI and ILRI teams and with the IDRC office in Kenya. In

consultation with members of KARI and the SAB, it was decided not to hire an additional

Project Manager for Kenya but rather to have a single individual take care of both sets of

activities. Students were hired in Nairobi (Martin Mwirigi, Isabel Nkando, Teresia Maina) with

one of them (Teresia Maina) transferred to VIDO-InterVac at the University of Saskatchewan for

training and later technology transfer to KARI. Technical personnel were hired at VIDO-

InterVac to complete the computational work as well as laboratory research activities (Yejun

Wang, Tracy Prysliak).


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2.1.5 Orders will have been placed for all equipment required for the research in both

Nairobi and Saskatoon.

A Luminex 200 multiplex analyzer was purchased at VIDO-InterVac for the measurement of

immune responses. Equipment in Nairobi was purchased as described in the proposal.

2.1.6 Bioinformatic analysis complete, open reading frames selected for cloning and

expression (Objective ( i).

The bioinformatics analysis on the genome of M. mycoides was completed on time and

identified a total of 69 gene products as targets for gene expression and vaccine testing.

2.1.7 Cloning and expression of surface and secreted antigens initiated with 40% of

eligible open reading frames completed (Objective ii).

Of the 69 gene products identified as potential vaccine antigens, a total of 66 have been

expressed and tested for immunogenicity and protective efficacy.

2.1.8 Areas in Kenya and farmer households will have been selected for socio-economic

analysis (Objective v).

Areas and farmer households in Kenya were selected in accordance with statistical and socio-

economical criteria. These areas include locations in Ijara sub-county from Garissa County

which have been selected from the CBPP infected zone of Kenya. These counties represent areas

where pastoralists know about the impact of CBPP and are aware of its control through

vaccination. Data collection tools for both qualitative and quantitative studies were prepared.

2.1.9 Procurement of cattle for testing to be commenced in Kenya.

Boran and Zebu cattle for preliminary studies were procured and used in experiments in KARI

facilities.

2.2 Twenty Four Month Milestones:

2.2.1 Cloning and expression of genes coding for M. mycoides surface-localized and

secreted antigens completed for all but re-synthesized genes (Objective ii).

Cloning and expression was completed for all genes identified by bioinformatics analysis as

potential vaccine candidates. A total of 66 proteins were expressed in E. coli and their production

was optimised in order to obtain the necessary amounts for prototype vaccine testing and for

analytical assays. Purification was carried out using nickel-chelate affinity chromatography. The

proteins were ranked for testing using a variety of parameters including type of molecule,

cellular localization, size, charge, and recognition by the immune response found in naturally-

infected cattle as described in the original proposal. Based on the ranking, pools of five proteins

each were selected and co-formulated with adjuvants to increase the magnitude and quality of the

immune response. The prototype vaccines were tested in Canadian cattle for immunogenicity

according to CFIA guidelines and shipped to KARI for efficacy trials.


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2.2.2 Initial testing of pools of antigens in a bovine disease model complete (Objective iii).

Initial testing was completed for all proteins. Vaccination and challenge trials were conducted in

Kenya in the KARI facility at Muguga. A total of 3 trials by 60 animals (50 for trial 3) were

conducted, each trial having vaccinated groups of 10 animals each and a control (placebo) group.

After Trial 3 was completed, we chose to repeat Trial 1 where the results for the control group

showed low efficiency of the challenge with Mmm.

In parallel, immunogenicity testing was conducted at VIDO-InterVac. These trials were

organized in similar manner to the vaccination and challenge trials in Kenya, with the main

difference being a smaller group size (5 animals per group).

2.2.3 Interviews with farmers complete, socio-economic data ready for analysis

(Objective v)

Focus group discussions and key informant interviews for collection of qualitative data using

participatory methodologies is complete. The results of this qualitative study have been used to

refine data collection tools for the cross sectional, preferences and willingness to pay (WTP)

study. In addition a CBPP outbreak investigation was carried out between January and February

2014 to collect empirical data that will demonstrate the socio-economic impact of the disease at

community and herd level in Laikipia County.

2.3 Thirty Month Milestones:

2.3.1 Cloning and expression completed for all antigens (Objective ii).

Cloning and expression of the genes coding for all antigens selected by bioinformatic analyses

was completed. Antigens were isolated in sufficient quantities for vaccine testing and analysis of

immune responses.

2.3.2 A final vaccine formulation containing 2-4 antigens combined with an appropriate

adjuvant will have been tested in an M. mycoides efficacy model (Objective iv).

Testing of the final vaccine formulation was delayed because its time slot was used for a repeat

of Trial 1 due to the low levels of disease observed in the control group cattle in the trial. We

will be completing this during October/November 2014 in Canada at no cost to IDRC. However,

we have identified a total of 3-4 protective antigens in trial 3 and we believe an equal number

will be identified from the repeat of trial one which is currently underway.

2.3.3 Data on the uptake of a CBPP vaccine by farmers will have been analyzed, providing

a pathway forward for field testing of a vaccine formulation (Objective v).

Training was carried out on questionnaire administration and GPS reading. A total of 312

questionnaires were administered to collect data on demographics, socio-economic

characteristics as well as preferences and WTP for vaccine and vaccination attributes. Data were


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managed in Microsoft Access and analysed with SPSS. The results were also a basis for the

development strategy when writing the proposal for phase 2 of this project. A cross-sectional

study was carried out in which 518 cattle were sampled in 32 households to establish the

prevalence of CBPP and the associated risk factors in Ijara sub-County. The samples were

analysed at the Central Veterinary Laboratory, Kabete, Kenya and data entered in Microsoft

Excel. Team members participated in 3 conferences with three oral presentations and two

posters.


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3.0 SYNTHESIS OF RESEARCH ACTIVITIES AND RESULTS

Infectious diseases remain the leading cause of death in humans, responsible for >30% of all

deaths on the planet, and are the single most important cause of economic loss in the agricultural

livestock sector worldwide. These losses to animal producers are due not only to direct effects on

animal health, but also to their effect on international trade, especially for those diseases which

have been either eradicated or controlled in western countries. Livestock production is a critical

industry in sub-Saharan Africa through the provision of food and animals for export, with 25%

of the GDP of some countries coming from this sector. At the present time, CBPP remains an

issue affecting the health and movement of animals in sub-Saharan Africa, with estimated

economic losses of $2 billion per year.

Vaccines have historically been the most cost effective and sustainable method of disease

control. There are numerous types of live and killed vaccines, most of which require

refrigeration. We have chosen to pursue a subunit vaccine to circumvent many of the issues

faced with mass vaccination in Kenya. The selection of microbial components, or antigens, for

inclusion in a subunit vaccine has traditionally been carried out empirically following the

identification of specific molecules which are recognized by antibodies from infected animals.

This approach has proven remarkably successful given its limitations. First and foremost, the use

of this approach generally examines only those antigens which are produced by bacteria grown

under laboratory conditions rather than those components produced during an infection. We now

know that many protective proteins, including those involved in the acquisition of nutrients (e.g.

iron, carbohydrates) are not produced by bacteria grown outside of the host yet have been shown

to be protective vaccine components for other pathogens. One way of circumventing this

problem is to systematically screen all antigens in an unbiased fashion, an approach generally

referred to as reverse vaccinology. All that one needs to carry out this type of vaccine

development approach is the sequence of the pathogen‘s genome and a method for identifying

which portions of the sequence code for proteins which are located on the cell surface or are

secreted, since they are usually targets of the immune response.

Figure 2. Bioinformatics and biological methods for identification of vaccine components using

a reverse vaccinology approach.


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The objectives of our research project were established as shown in Figure 2: identification of

proteins which are potential targets for immune response using computational methods;

laboratory production of the selected proteins in sufficient quantities for analytical assays and for

injectable formulations used to determine their immunogenicity and protective efficacy;

experimental evaluation of the immunogenicity and protective efficacy of the formulations in

cattle, and analysis of the social and economic factors that will influence the acceptability and

adoption of the vaccine by livestock keepers.

3.1 Objectives of the Research Project

3.1.1 Objective 1: Identify all potential surface-localized and secreted proteins of Mmm

using bioinformatics tools.

From the genome sequence of M. mycoides strain MMC-95010, 922 genes were analyzed using

bioinformatics software as is shown in Figure 3. Analysis was not limited to those strains alone,

but also included other strains and isolates where data was available: PG1, Glaysdale, IS22,

138/5, 9809, and 8676/93. The PSORTb algorithm developed at Simon Fraser University and the

University of British Columbia was used as the primary tool to identify non-cytoplasmic

proteins. After the analysis, 410 non-cytoplasmic proteins were selected and 271 potential

extracellular sequences were identified. Further analysis revealed adhesion probability and

vaccine potential, based on multiple factors including homologues in other strains, secretion,

localization on the microbial surface, etc. ,

Figure 3. Bioinformatic process for analyzing M. mycoides genome.


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These genes were further analyzed using the following criteria: codon usage bias, GC content,

CpG dinucleotides content, mRNA secondary structure, cryptic splicing sites, premature PolyA

sites, internal chi sites and ribosomal binding sites, negative CpG islands, RNA instability motif

(ARE), repeat sequences (direct repeat, reverse repeat, and Dyad repeat) and restriction sites that

may interfere with cloning were also excluded. The resulting corrected sequences were translated

in silico to protein and BLAST-compared to the original Mmm-95010-derived protein sequence.

After this verification, the sequences, now optimized for E. coli expression, were ordered for

synthesis and sub-cloning in maintenance and expression plasmids.

In the first year of our work we identified 69 potential candidates, which satisfied a stringent set

of requirements as potential targets for vaccine development as described above. At the end of

the first year the production of these proteins was well under way with sixty six proteins having

undergone all the optimization steps necessary for gene expression and protein production. The

next step was to test the antibody immune responses to these proteins in sera from naturally

exposed (CBPP positive) and non-exposed (CBPP negative) animals (Table 1). Those showing a

higher response were ranked higher for vaccine testing.

Table 1. Mean titres of the selected proteins with CBPP-positive sera.

Protein Mean Titre Protein Mean Titre Protein Mean Titre

MSC 0500 136865 MSC 0397 31153 4400581 803

MSC 0957 272527 MSC 0266 3025 4400580 4403

MSC 1058 4085 MSC 0265 10619 4400559 19852

MSC 1005 1022 MSC 0240 5757 4400534 5278

MSC 0927 701 MSC 0184 4016 4400446 389

MSC 0816 43581 MSC 0163 791 4400371 896

MSC 0813 4551 MSC 0160 20934 4400368 2587

MSC 0804 3897 MSC 0139 836 4400300 3157

MSC 0798 4051 MSC 0136 335391 4400296 753

MSC 0790 5578 MSC 0052 6022 4400291 4391

MSC 0782 3445 MSC 0014 9306 4400226 942

MSC 0776 29419 MSC 0013 79343 4400204 1181

MSC 0775 25091 MSC 0011 6691 4400171 501

MSC 0653 42194 MMS A0415 1312 4400127 1610

MSC 0610 13805 MMS A0381 6164 4400021 1148

MSC 0575 33308 MMS A0108 7485 4399939 155

MSC 0519 120112 4400622 3639 4399927 1381

MSC 0499 224663 4400620 3238 4399914 1173

MSC 0456 3208 4400616 9091 4399851 222

MSC 0453 4399 4400615 2491 4399807 15318

MSC 0431 82662 4400610 1180 4399790 1030

MSC 0401 3000 4400602 2583 4000004 1215


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The 66 proteins were individually tested against 35 CBPP-positive and 15 CBPP-negative sera

from Kenya by multiplex ELISA assays. The proteins were ranked according to the IgG1 titres

of the 35-positive animals, i.e. the higher the titres, the higher the rank. To ensure that the

ranking approach was relevant, for each protein the sum of its ranks (ranksum) was plotted

against the median (the most frequent value) of its rank. In a relevant ranking such a plot should

produce a linear dependency as shown in Figure 4 below. The correlation (R2=0.9672) between

the Ranksum and the median of the ranks indicated that our ranking approach was appropriate.

Figure 4. Test for effectiveness of protein ranking method. The comparison between the

ranksum and median of ranks for the serum IgG1 titres against each antigen in 35 CBPP-positive

animals is shown. The correlation coefficient between median of ranks and ranksum of titres

indicates that the approach taken to rank the proteins was effective. See text for details.

Sets of five proteins were assembled into pools for vaccine formulations according to their

ranking order, i.e. the first five proteins were included in pool A, the second five in pool B and

so on. The proteins were combined with CpG2007 ODN and 30% EmulsigenTM

and used as

prototype vaccines (Tables 2-4).

R² = 0.9672

0

10

20

30

40

50

60

70

0 500 1000 1500 2000 2500

Me

dia

n o

f ra

nks

Ranksum

Sorted by ranksum


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Table 2. Proteins used in efficacy Trial 1.

Name Function Size Yield/100ml #MHC-I Median

rank

Group A

MSC_0136 lipoprotein 66kDa 563.100 20 58





Group B






Group C

YP_004400559.1 Hypothetical protein 18kDa 24.200 4 48



MSC_0160 Elongation factor Tu 75kDa 528.000 14 46


Group D



MSC_0265 Pyruvate dehydrogenase E1 74kDa 581.450 12 42



Group E



YP_004400534.1 Transmembrane protein 229kDa 16.200 95 37




16



rank

Group G



MMS_A0381 lipoprotein 100kDa 1318.450 28 34



Group H

MSC_0453 FKBP-type peptidyl-prolyl

isomerase 81kDa 517.725 46 32

MMS_A0108 lipoprotein 71kDa 258.750 34 32




Group I



YP_004400300.1 Lipoprotein 98kDa 10.800 49 27



Group J



MM_A0415 lipoprotein 45kDa 812.400 0 22



Group K


YP_004400371.1 Permease 84kDa 5.500 45 20


YP_004400021.1 PTS transporter 36kDa 29.000 2 20

MSC_0163 Leucyl aminopeptidase 82kDa 468.725 26 19


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rank

Group M


YP_004400296.1 Lipoprotein 101kDa 10.600 42 19

YP_004399851.1 PTS transporter 19kDa 222.000 4 18


Group N





Group O

YP_004400171.1 ABC transporter 41kDa 123.400 19 13

MSC_0139 Fructose-bisphosphate aldolase

class II 65kDa 380.800 9 12



Group P YP_004400446.1 Hypothetical protein 20kDa 7.500 6 8



YO-00440368 Hypothetical protein 105kDa 0.060 306

At the same time as the antigens were being assembled in Canada, the team in Kenya finalized

and optimized the challenge model using Boran and Zebu breeds, which allowed us to reproduce

the disease in experimental conditions and thus compare the susceptibility to CBPP of vaccinated

and non-vaccinated animals. The logistics of purchase, transportation handling and care of

relatively large groups of animals (up to 60 per trial, often with two trials taking place

simultaneously) also required some adjustments and training of personnel.

3.1.2 Objective 2: Clone and express the genes coding for potential vaccine targets in

Escherichia coli for production of antigens.

Of 69 genes selected, 66 produced yields appropriate for further evaluation as vaccine

components when the genes were cloned into Escherichia coli gene expression vectors. The

expression vectors contained a histidine-tag which was used for purification of the proteins by

metal-chelate affinity chromatography. Following production and purification of the proteins,

we then tested sera from naturally-infected animals for immune responses to each individual

protein in order to assist in the ranking of proteins for vaccine testing; i.e. those with greater

reactivity would be ranked higher than those with little or no response. These results are

described above. This was used in assigning priority in formulating pools of proteins to be tested

as prototype vaccines. In parallel to the testing for efficacy against experimental infection in

Kenya, the proteins were also tested for their immunogenicity in vaccine formulations at VIDO-

InterVac.


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3.1.3 Objective 3: Test pools of proteins for their vaccine potential in a bovine model for

CBPP and identify formulations containing protective antigens.

3.1.3.1 Immunogenicity testing.

Testing of the pools of proteins was first conducted at VIDO-InterVac using north

American cattle breeds in order to ensure that the formulations used in the efficacy trials

below were capable of inducting a robust immune response. The number of trials, group

designation and vaccine prototype formulations were the same for both the

immunogenicity trial in VIDO-InterVac and the protective efficacy trials in KARI

(groups described in Tables 2-4). The main difference between the trials was the smaller

group size (5 animals per group in Canada, 10 in Kenya) and the breed (Angus in Canada

and Boran in Kenya).

In the 3 trials for immunogenicity there were no signs of adverse reactions to the

prototype vaccines. Blood samples were collected on days 0, 21 and 35 and serum titers

of IgG1, IgG2 and IgA were measured using multiplex assay with a Luminex 2000

Instrument.

The data collected was used to evaluate immunogenicity of the individual proteins in the

test vaccines and determine the potential of these proteins as candidates for the final

prototype vaccine. The results of the immunogenicity experiments conducted in Canada

are shown below in Tables 5-7.


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Table 5. Trial 1 - Mean serum antibody titers against vaccine antigens 35 days post vaccination.

Group Protein Vaccinated Animals Control Animals

IgG1 IgG2 IgA IgG1 IgG2 IgA

A MSC 0136 209244 22841 222880 9779 1283 3425

A MSC 0957 187746 23119 147652 8240 2063 2866

A MSC 0499 848654 76440 474260 9643 553 3087

A MSC 0776 910569 148983 423281 4288 537 2669

A MSC 0431 683175 34774 450795 4624 344 4151

B MSC 0519 475566 97938 275650 7894 2432 7081

B MSC 0500 883723 21620 371942 8377 1135 8943

B MSC 0575 1234431 67090 691488 26672 2686 21139

B MSC 0653 342181 46650 128803 8078 156 2179

B MSC 0397 934167 67829 431276 13904 646 9547

C 4400559 2323 2065 5682 237 206 2370

C 4399807 1029448 14340 249957 33927 81 37739

C MSC 0816 1139304 215202 754599 10629 868 9952

C MSC 0160 647865 55103 480554 11371 814 8405

C MSC 0775 877537 32244 221877 10347 641 4714

D MSC 0013 1009194 149502 1180301 15864 684 7498

D MSC 0610 927039 89165 626014 14444 501 3181

D MSC 0265 850300 33294 350395 7064 740 8535

D MSC 0052 1084081 25891 371153 12490 1244 7957

D MSC 0240 997706 62687 271678 6135 88 1288

E MSC 0014 1340970 156850 1405303 8031 689 1983

E MSC 0011 662442 21750 1006065 9840 158 3056

E 4400534 839283 127773 529196 7296 436 1443

E MSC 0813 799341 10720 269131 13596 814 7371

E MSC 0184 896520 35498 194194 13247 2025 6882


20




G MSC 0782 1205076 45743 1075028 3205 379 669

G MSC 0401 1667336 538017 2318981 6501 445 25708

G MMS A0381 1211971 287588 962156 5511 1169 11774

G MSC 1058 1663564 291450 699285 18795 1740 5384

G MSC 0790 1181428 120510 709172 12728 619 5490

H MSC 0453 2170593 345141 1246249 15249 5126 6544

H MSC 0798 509370 11832 126591 31755 143 5809

H MMS A0108 891905 163713 1104256 23818 644 17401

H MSC 0266 1064645 13327 322127 5987 1 5926

H MSC 0456 3112581 55555 889204 15928 1344 5795

I 4400602 577971 13081 147323 9044 287 6400

I 4400291 1495617 15135 192158 20872 321 1474

I 4400300 1376206 339215 778702 6267 215 1369

I 4400620 1732109 57594 1354588 7754 443 9995

I MSC 1005 1579098 1150713 1562593 7647 837 4705

J 4400616 2210311 91601 1363513 19285 965 16084

J 4400615 787007 16147 281760 6305 785 4343

J MMS A0415 2051456 129809 1122610 7578 965 1297

J MSC 0927 887504 27602 309879 7295 3000 1959

J MSC 0804 2495929 380054 1505288 4463 190 1313

K 4400622 781063 91891 1391039 18258 2007 51798

K 4400371 447147 8174 149103 11801 1124 4623

K 4400226 319168 437 206673 3468 104 3693

K 4400021 1961288 512500 1421188 9837 309 7857

K MSC 0163 1240798 94500 920148 19403 798 7969


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M 4400581 128219 7148 68485 2840 233 34

M 4400296 481132 379898 478994 1985 134 0

M 4399851 740596 25392 284215 1198 169 0

M 4399914 937540 104906 547962 6240 728 4

N 4400127 291728 1894 69421 5052 66 2132

N 4399790 1202229 16977 1227230 12283 662 1843

N 4400580 1270394 31897 981954 5854 975 3272

N 4400610 1420027 63574 835755 6313 215 4614

O 4400171 1069238 35759 258817 1715 97 500

O MSC 0139 190571 15609 107875 1299 6 532

O 4399939 1129185 34322 131662 5641 16 1103

O 4000004 1196044 51347 146649 4754 459 1816

P 4400446 641318 93835 900495 6158 25 4771

P 4399927 38338 787 15766 2465 529 1319

P 4400204 872682 39952 719530 12056 600 14584

P 4400368 443935 8678 409220 5726 62 1087

From the data in Tables 5-7, all proteins were capable of inducing immune responses to

varying levels, although there were differences in the antibody isotypes produced in

response to immunization with the various antigens.

3.1.3.2 Evaluation of the Protective Capacity of Recombinant Antigens

A total of three vaccine trials were conducted to test the protective capacity of the

recombinant proteins in cattle. Pools of five antigens (four for the third trial) were

formulated as described above (Tables 2-4) using Emulsigen plus CpG as an adjuvant. A

control group which received Emulsigen plus CpG with no antigen was also included in

each trial. Groups of ten Boran cattle were immunized intramuscularly, boosted with the

same vaccine three weeks later and two weeks after that, all groups were challenged with

M. mycoides by intrapulmonary installation.

All animals were examined daily for clinical signs of disease, rectal temperatures and

general health. Mortality was also recorded, although it was low (approximately 10% in

controls) as expected due to the timeframe of trial. The team in Kenya was most

interested in the post mortem pathology results as their feeling is that this would be an

accurate predictor of long term clinical outcomes associated with infection.


22

The antigens were tested in order of their in silico ranking using approximately 25

antigens per trial. The first trial resulted in few clinical signs of disease and very limited

pathology and is currently being repeated. We expect the results from this repeat trial to

be available sometime in October 2014 at which time the data will be reanalyzed. In all

cases, the groups which showed a reduction in pathology were further examined by

analyzing immune responses against the individual components in order to eliminate

those which did not appear to induce a robust response. The ideal outcome would be to

have no more than two groups showing a protective response, as further development

could incorporate up to eight antigens through the use of chimeric technologies.

Pathology Scores: The pathology score was calculated using a) the lesion score, b) a

score of 2 was added to the lesion score whenever Mycoplasma mycoides was isolated

from the lung lesion and c) The total is multiplied by a factor determined by the average

diameter of the lesion.

Lesion Scores: The lesion score scores were assigned as follows:

The presence of a resolving lesion with only fibrous tags or pleural fibrous adhesions

only was rated 1;

If other types of lesions were present, namely consolidation, acute, necrotic or

sequestrated tissue, these lesions were rated 2;

In addition, if MmmSC was isolated a score of 2 was added.

A factor between of 1 and 3 was used to multiply the lesion score (above) and was

determined as follows: i) 1 was used if the lesion size was under 5cm; ii) 2 was used if

the lesion size was over 5 and under 20cm) and iii) 3 was used if the lesion size is

20cm and above.

From this, the maximum pathology score is (2+2)3=12 for each animal. This formula was

used to obtain the mean pathology score of each group of cattle. The mean pathology

score of vaccinated cattle was divided by the mean pathology score of the control group.

The result was subtracted from 1 and multiplied by a hundred to obtain efficacy (or

percentage protection) of the vaccine under test.

This is a minor variation of that published by Hudson and Turner (J. R. Hudson and A.

W. Turner in Australian Veterinary Journal, Vol. 39, October 1963, pp373-385), who

used multiple organs for sampling while we used only lung samples.

Blood samples were taken from all animals before vaccination, after vaccination, after

boost, and after challenge. Sera derived from the samples were examined by complement

fixation test, ELISA for IgG immune responses and finally for their capacity to stimulate

cellular immunity (WBC proliferation).

Summaries of the clinical trials are given below with more details, clinical data and

immunological information included in the appendices.


23

Protective efficacy Trial 1:

The trial was conducted using 60 Boran cattle of average age of 2.5 years. Vaccination groups

were designated A to E, while group F received a control vaccine containing only the adjuvant.

The study was conducted at the KARI experimental station in Muguga where the animals were

housed. All animals were examined for adverse reactions to the prototype vaccines and none

were observed. The dose of bacteria delivered to each animal was 1010

CFU and the onset of

CBPP began two weeks after experimental infection, with respiratory symptoms being more

pronounced than fever. The group average temperature did not exceed the normal maximum

value of 39.5oC, although some individual animals were above this value. The control group

showed fewer symptoms and after necropsy had fewer lesions than most of the vaccinated

groups (see Figure 5. and Appendix 1), making it difficult to evaluate the protective capacity of

any of the vaccine formulations. The one group which tended to have lower scores was Group

C, which contained the proteins 4400559, 4399807, MSC 0816, MSC 0160, and MSC 0775.

The pathology scores for this group were very low, albeit not statistically significant due to the

low levels of disease in the control group and we are keenly interested in the response of animals

in the trial which is currently underway in Kenya. Interestingly, one of the antigens in the group,

Tuf (Elongation Factor Tu) has been shown to activate inflammatory cytokines in Francisella

tularensis in macrophages and may also be involved in bacterial invasion. While the protein is

involved in protein translation, an intracellular process, it is also found on the cell surface in

some Mycoplasma species, making it a potential vaccine target. Tuf has also been shown to be a

potential vaccine target for Burkholderia cepacia where animals immunized with it had a lower

bacterial load in the lung following infection. Finally, we also identified this protein as a major

target for the immune system in a bovine respiratory disease model using Mannheimia

haemolytica during the 1980s. The mean lesion scores and pathology index for the groups in this

trial are shown in Figure 5 and the detailed data is appended in Appendix 1.The serological

response to immunization was also measured and all of the animals seroconverted as illustrated

in Table 2 and the appendices.Also of interest was the response of animals in group E which

were worse than the controls and all other groups in the trial in terms of lesion scores and

pathology. This is not unexpected, as increases in susceptibility to disease following vaccination

have been reported previously for selected Mmm antigens.

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Figure 5. Mean lesion scores and pathology indices of groups in Trial 1.


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Trial was conducted on 60 Boran cattle of average age 2.5 years. Vaccination groups were

designated G to L with Group L serving as a control as described above. Clinical examinations

were carried out as described above, and like Trial 1, no adverse responses to immunization were

observed by clinical staff. The challenge dose and route were as described above.

The disease onset was similar to Trial 1, with respiratory symptoms being more pronounced than

fever. The overall efficiency of the challenge with Mmm was higher in this trial than the first,

with the control group having more animals with stronger symptoms and pathology indices than

the controls in Trial 1. However in this trial there was no vaccine groups which showed any

protective effect relative to the control animals, and therefore we conclude that these antigens do

not induce protective immunity.

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The third vaccine trial was conducted using 50 Boran cattle of average age 2.5 years and vaccine

groups were designated M to Q, with the latter serving as a negative control. Once again, there

were no adverse reactions to vaccination observed. The disease onset was also similar to the

previous trials. However the control group of animals responded to challenge more efficiently

with only 2 of the 10 animals having no lung lesions. The animals in most of the groups

vaccinated with prototype vaccines did develop lung lesions, although fewer animals in those

groups had lesions in comparison to the control group. One particular prototype vaccine (group

N) had only fibrous adhesions in 4 animals, while all other animals in the group had no lesions.

The differences between Group N and the Control group were statistically significant.


25

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Calculating the protective efficacy for the vaccine candidates in Group N by the Hudson and

Turner method described above suggested that the vaccine demonstrated 73.334% efficacy in

this trial. We conclude that this pool of antigens composed of protein ID’s 4400127, 4399790,

4400580 and 4400610 provided significant protection against M. mycoides infection. From the

immune responses shown in Table 4 above and the appendices, the response against antigen ID

4400127 was very poor relative to the others in the pool and therefore we believe that the

protection observed was due to the responses against antigens 4399790, 4400580 and 4400610

and that these antigens would be useful for further development of a vaccine for CBPP. Each of

these three antigens also induced a modest cellular immune response as is shown in Appendix 5.

3.1.4 Objective 4: Test the immunogenicity and protective capacity of 2-4 antigens

formulated with different adjuvants to enhance the immune response.

This test has not been conducted yet because we needed to repeat Trial 1 in order to screen all

recombinant antigens. This data will be available in September 2014 at which stage we will

carry out an immunogenicity trial at VIDO-InterVac at no cost to IDRC.

3.1.5 Objective 5: Analyze a priori, the social and economic factors, including willingness

to pay and gender constraints that will influence the acceptability and adoption of

the vaccine by livestock keepers.

While vaccines are powerful tools for disease control, they have little value if the needs of the

end users are not met. Thus, studies were carried out to determine socio-economic factors which

would influence vaccine uptake.

The objective was to provide data and recommendations on the best possible policy, including

distribution, cost and packaging; as well as the equitable access to information and knowledge on

CBPP control and management by men and women livestock keepers. Finally, the data generated

through this research project will help advice on CBPP control policy in the country.


26

The initial qualitative study was conducted in Ijara sub-county of Garissa county, Kenya (Figure

8.). A sub-location was used as the sampling unit where twelve sub-locations from a total of 31

sub-locations in Masalani and Ijara divisions were selected due to population of cattle and recent

cases of CBPP reported by the veterinary office.

Figure 8. Areas in Kenya, selected for the socio-economic survey

This participatory study consisted of focus group discussions (FGD) with pastoralist livestock

keepers and key informant interviews. The FGDs took place in 12 sub-locations, including 32

informant groups (20 men and 12 women). In each sub-location, focus groups were convened

which consisted of between six and twelve people, all of whom were ethnically Somali. This has

generated qualitative data on Knowledge, Attitudes, Perceptions and Practices (KAPP) on CBPP

and its control in the study community as well as information on constraints in cattle keeping in

Ijara.


27

Figure 9: Map of Ijara Sub-County showing its divisions and locations.

These results have been used to develop data collection tools (Questionnaires are in appended

files) for household surveys to determine the preferences and WTP for new vaccines, and the

gender and socio-economic factors that will influence them. Empirical data to demonstrate the

socio-economic impact of CBPP at community and herd level has been collected in Laikipia

County where an active outbreak occurred. The data was also used to carry out a qualitative risk

assessment for CBPP across the various production systems in Laikipia County (pastoralism and

agro-pastoralism, mixed farming, zerograzing and paddock system and ranching). A three-day

training on questionnaire administration and GPS reading was carried out in April 2014. After

field pretesting, questionnaires were administered to 312 households (261 male-headed and 51

female-headed) selected randomly in the study area, Ijara sub-County; in 14 sub-locations

(Figure 9). The respondents were both men (52%) and women (48%) across both male-headed

and female-headed households. The questionnaires included data on household head

demographics, Household socio-economic characteristics (sources of livelihood, income,

expenditure, leadership and group membership), diseases of cattle, livestock herd and

productivity data, general opinions, attitudes and beliefs about CBPP, valuation of CBPP vaccine

and vaccination attributes. The vaccine and vaccination attributes were presented to the farmers

at two levels each for vaccine vial monitor (VVM); inclusion or non inclusion, efficacy (100%

and 60%), vaccine administration/delivery (private and government), frequency (annual and

biannual), stability (>2hrs and 2hrs) and nature of vaccine administration/delivery (elective and

compulsory) Price was presented at four levels (KSh. 10, 30, 50 and 120). In this study, VVM

and efficacy of the vaccine are unique attributes included among the vaccine and vaccination

profiles not included in previous studies.


28

The data were entered and stored in Microsoft Access and analysed using Statistical package for

Social Scientists (SPSS) to give information on:

The preferences of women and men farmers for a new CBPP vaccine and the relative

importance of the attributes

The willingness to pay (WTP) of women and men farmers for the current and new CBPP

vaccine and vaccination and demand for vaccines.

The emerging results are that:

The preferred CBPP vaccine and its delivery by livestock keepers in Ijara sub-County is one

that includes an indicator (vial vaccine monitor), with 100% efficacy, 100% safety,

administered once a year by the Government and is compulsory. The attributes of inclusion

of an indicator and efficacy were given the highest priority by both types of households.

Farmers were willing to pay more for the vaccine and vaccination with preferred attributes

than the market price (KSh. 34.6) calculated in a previous study in Narok County of Kenya

i.e they valued the vaccine and vaccination higher than the market price. In addition, female-

headed households would pay more (KSh. 367) for the preferred vaccine than male-headed

households (KSh. 297).

The valuation for the current vaccine is negative indicating that livestock keepers in Ijara

may require compensation or persuasion to allow their animals to be vaccinated.

Analysis on individual valuation shows that 81.1% of the livestock keepers in Ijara had a

positive willingness to pay for the preferred vaccine at the market price of KSh. 34.6

whereas only 31.8% had positive WTP for the current vaccine.

The study points out that both MHH and FHH had preference for particular vaccine and

vaccination attributes. The high WTP compared to the market price indicates a high potential

demand for a vaccine and delivery system with the preferred attributes. It is hence urgent that

these preferences are incorporated in vaccine development and delivery so that uptake of the new

vaccine and CBPP control is enhanced. The WTPs are high for the preferred vaccine. This may

only represent the value placed on the vaccine and the affordable price needs to be worked out

given their incomes and expenditure. Both income and expenditure data has been collected.

Other activities carried out included:

1. Participation in the planning and writing of a proposal for further vaccine development,

including socio-economic factors, which formed the basis for a Phase 2 funding submission.

2. A cross sectional study in Ijara district to establish the prevalence of CBPP, identify some of

the risk factors associated with CBPP, establish any association between trypanosomiasis in

cattle and CBPP and compare the performance and degree of agreement of Complement

Fixation Test (CFT) and competitive ELISA. Sera were collected from 518 animals in 32

households. Animal biodata were collected in an animal sampling form. Blood smears were

also made from all the animals sampled to determine the prevalence of haemoparasites,


29

particularly trypanosomes which are thought to enhance prevalence of CBPP due to their

immunosuppressive activity. A questionnaire was administered to assess the impact of risk

factors in the cattle sampled. Amongst the risk factors to be investigated were age, sex,

breed, the grazing patterns, disease outbreaks, animal acquisition, animal movements, herd

size and composition, and vaccination status. The serum samples have been analysed using

cELISA and CFT in parallel at the Central Veterinary Laboratory, Kabete, Nairobi to

determine the total prevalence of CBPP in the various sub-locations. Data from the resulting

analysis have been entered in Microsoft Excel along with the risk factor data from 32

questionnaires administered to the owners of the sampled animals to allow multivariate

logistic regression analysis, Chi-square and ANOVA to determine the association between

prevalence and risk factors, the strength of association and the difference in mean prevalence

of CBPP and haemo-parasites between the various sub-locations. All samples have been

analysed for CBPP and the slides stained for observation of haemoparasites. The results

indicate that the cELISA and CFT used in parallel resulted in 14.9% sero-positivity and light

microscopy showed that 33.3% of animals were positive for haemoparasites, 66.3% of

which were Anaplasma species and 33.7% Theileria species.

The conclusion is that CBPP is still present in Ijara sub-County of Kenya and animals are

also infected with haemoparasites which may compromise the immunity of the animals,

contributing to CBPP susceptibility. Further studies are required to examine the significance

of this association.

3. Outbreak investigation in Laikipia County of Kenya

CBPP was reported in a ranch in Laikipia County. Farm managers reported a pneumonia

not responding to treatment early August 2013 with deaths. Laikipia is in the CBPP clean

area of Kenya and is a flagship county earmarked for creation of Disease Free

Zone/Livestock export Zone. The outbreak was investigated to obtain data for use in

providing information on the impact of the disease. In addition, a CBPP qualitative risk

assessment was carried out in the rest of the county. Animals in the affected ranch and in-

contact ranches were screened for the disease using the mobile laboratory and a field

screening CFT. Samples were also collected from animals in the rest of the county and

tested at the Central Veterinary Laboratory using CFT and cELISA in series. Farmers were

interviewed using semi-structured questionnaires. The overall seropositivity of the animals

throughout the County was 19.6% and 67.1% in the affected ranch. The total cost of the

outbreak was KSh, 5.1 million (1.6 million in production losses, and 3.5 million in

intervention costs). Laikipia County has various cattle production systems. The risk of future

outbreaks of CBPP was high in the pastoral and mixed production systems, low in ranches

and negligible in paddock/zero-grazing systems. Thus CBPP is a disease that can cause high

negative impacts and its control is important. Entry into a clean area should be prevented.


30

CONCLUSIONS

The principal objective of this project was to develop a prototype subunit vaccine formulation for

the prevention of CBPP using a reverse vaccinology approach. We screened a total of 66

antigens for their protective capacity and have identified 3 which will be useful for further

development as well as 4 potentially useful ones from the first vaccine trial which is currently

being repeated. A total of seven individual proteins would not be cost effective to produce, but

through the construction of chimeric antigens, this number would be feasible. The overall

efficacy of the three antigens identified in the third efficacy trial was 73.33%, an acceptable level

of protection given that the choice of adjuvant, dose and immunization schedule has not been

determined. We would expect this would increase to >85% with optimization of the vaccine

formulation, a level which is comparable or better than most vaccines currently on the market.

We have also carried out a number of surveys and focus group discussions to determine if this

type of vaccine would be acceptable for use and the conditions under which it would be used.

We conclude that from the data generated that the subunit vaccine would fit all of the

requirements for use, and would also have significant other benefits relative to the live vaccine

currently in use in some areas, including its stability, ease of use, efficacy level and a less strict

requirement for a cold chain.

The next steps in this project will be to refine the production of the antigens so that the levels are

increased, determine the optimal formulation for use including antigen dose, adjuvant selection

and immunization schedule. Following these experiments, a field efficacy trial will be carried

out using material manufactured in Kenya.


31

4.0 SYNTHESIS OF RESULTS TOWARDS AFS OUTCOMES

1. New technologies and/or farming systems and practices. At the current stage the

project is not offering new technologies for general use. We do expect within the next

phase of the work to have new vaccine technologies which will offer reduced price,

increased stability and the flexibility to combine with other products.

2. Dietary diversity & nutrition. The project is aimed at improving animal health, thus

improving food safety and quality. There will be no direct impact on dietary diversity and

nutrition.

3. Engagement of Canadian researchers with Southern researcher organizations (for

CIFSRF-funded projects only). Canadian expertise is increased by first-hand knowledge

and experience in prevention of foreign animal diseases. Drs. Andrew Potter, Volker

Gerdts, Jose Perez-Casal and Emil Berberov have travelled to Kenya a number of times for

planning meetings as well as research activities and gained understanding on the potential

and capabilities of Kenyan research institutions. Drs Hezron Wesonga, Reuben Soi and

Ephraim Mukisira have also travelled to Canada and appreciated the capabilities of the

Canadian institution. The established professional relationships will have a positive impact

on any future work aimed at improving productivity and solving animal health-related

problems in African countries. Interestingly, while M. mycoides is not a problem in

Canada, the closely related pathogen Mycoplasma bovis is a significant cause of economic

losses in the cattle and bison production sectors and the engagement of researchers from

VIDO-InterVac with those in Kenya has led to new insights into this domestic disease and

potential methods of control.

4. Research groups. The project is intensifying connections and collaboration between

Canadian and African research centres, thus improving the capacity of both sides to

prevent dangerous animal infectious diseases. Six Kenyan students (two in social sciences:

1 PhD and MSc; and four: 3 PhD and 1 MSc; in vaccine development technical processes)

have been trained in livestock vaccine research, an expertise lacking in many countries in

Africa. The active partnership with the viral vaccines project (CIFSRF 106930 which

involves Canadian and South African research institutions) has been one that has added

considerable value to this project and vice versa. Indeed, resulting in a more efficient use

of research beyond the limits of a single project or country.

5. Food distribution. No direct impact.

6. Food processing and storage. No direct impact.

7. Risk-mitigation. The contribution to risk mitigation is mainly through increased potential

for prevention of animal disease, a leading cause of economic losses to livestock producers

in Africa. The project will also serve as a platform technology which can rapidly be

applied to other existing and emerging infectious disease threats, the latter of which is an

issue in many parts of the world due to climate change and other factors.


32

8. Access to resources. This project has no direct impact on economic factors, other than the

potential for increase of animal products supply.

9. Income generation. The improvement of animal health will lead to improved stability and

profitability of small holder farmers, thus directly benefitting both income and nutrition of

women and children. The current estimate of the economic losses due to CBPP is about 2

billion dollars annually and an effective vaccine would ensure that much of this loss is

prevented. In addition, the manufacture of vaccines such as that for CBPP will have an

impact on the manufacturing and biotechnology sectors, leading to new jobs as well as

opportunities for trade. A reduction in antibiotic use alone would be expected to save

individual farmers a significant amount and would also have beneficial environmental

effects (see below).

10. Policy options. The project will provide the opportunity for vaccination campaigns of

animal diseases with significant impact on food security. Ultimately the successful

development of a CBPP vaccine will permit options for continent-wide eradication of the

disease, although that is far in the future.

11. Information and Communication Technologies (ICTs). The project has no direct impact

on communication technologies.

12. Gender. Many of the small farmers in Sub-Saharan Africa are women, and thus improved

animal health has direct beneficial effect on multiple aspects of their status. The study on

“willingness to pay” and new vaccine preferences was gendered and the results will

influence the approach to gender related issues when implementing vaccinations in future.

Composition of the project team has remained gender sensitive and members have also

participated in the Gender Learning Workshop organized by CIFSRF.

13. Environment. Vaccine usage for prevention and control of CBPP will have positive

environmental impact by reducing antibiotic use, which will result in lowering the

contamination of the environment (soil, groundwater etc.) with antibiotics and all the

negative downstream consequences. This is a significant issue worldwide, in both small-

and large-scale agricultural settings. The CBPP vaccine being developed is composed of

protein, carbohydrate and nucleic acid, all of which are completely biodegradable. The use

of killed products rather than live vaccines will also reduce the load of infectious agents in

the environment. Finally, the participating institutions have environmental policies,

focused on sustainability. This project does not have any aspects, which are not covered by

the policies and practices of the participating institutions. Results of the socio-economic

study will be analysed to establish social determinants of environmental disaster

vulnerabilities of the studied community. The community often requires outside assistance

to cope with environmental extreme events, in particular droughts which are cyclical in

nature. The project intends to make recommendations with mitigation measures.


33

5.0 PROBLEMS AND CHALLENGES

The challenges faced by this collaborative project include both those that are typical of research

projects of this kind as well as those that are unique. The typical problems include things such as

the timely recruitment of personnel, purchase of equipment, and administrative issues related to

coordinating research teams on two continents. None of these issues are out of the ordinary;

however, in this case a 30 month timeframe to implement and complete the research was

challenging. For example, the ordering of highly specialized equipment, installation and training

can take 6-12 months, or 40% of the project lifetime.

The project was a truly collaborative one which involved not only the exchange of personnel, but

also reagents and biological samples. The complexity of dealing with regulatory agencies in

both Canada and Kenya did prove challenging at times and was the source of delays in

experiments as well as data analysis. Unfortunately, in a 30 month timeframe, it was not

possible to circumvent these issues, although we did identify alternative procedures which would

mitigate some of the risks.

The project involved a significant amount of work with animals in both Kenya and Canada and

this did prove to be challenging as well. The market availability of cattle of the appropriate

breed, age and health status was problematic in both countries and did lead to some delays in the

vaccine testing. However, the sheer number of vaccine candidates tested within the 30 month

timeframe was impressive in our view and the view of our Scientific Advisory Board and this

was due to the dedication of the team in Kenya.

Finally, although there were numerous face-to-face meetings, routine communication by

telephone or Skype was problematic at times due to connection issues in Nairobi. There is no

question in our minds that some of the data analysis would have been facilitated with more

effective infrastructure.


34

6.0 RECOMMENDATIONS

An international multi-lateral project of such scale takes time, specifically for synchronization of

the administrative practices of institutions in different countries. An additional lead period of 6

months may help to distribute the overall effort more evenly throughout the project term.

Often agencies funding international projects are able to place orders on behalf of beneficiary

organizations, thus avoiding the administrative delays. One such example was the ISTC. Similar

practice could be considered as an opportunity to accelerate the ordering process where reagent

and equipment orders are known to take long and unpredictable periods to complete.


35

APPENDICES


36

Appendix 1: Clinical and Pathology Data from Efficacy Trial 1.

Animal

ID Group

Fever

≥39.5⁰C

Clinical:

cough, nasal

discharge,

dyspnea

Lung Lesions Lesion

Score

Pathology

Index

Mmm

culture

1006 A No Yes

oedema; distended

interlobular septa

with fibrinous

exudate

1 1 No

1010 A No Yes No 0 0 No

1016 A No Yes No 0 2 Positive


1027 A Yes Yes

Fibrous tags;

distended

interlobular septa

with fibrinous

exudate

1 3 Positive

1034 A No Yes No 0 2 Positive

1035 A No No No 0 2 Positive

1046 A No No Fibrous adhesions;

fibrous tags 1 1 No


1058 A Yes Yes No 0 0 No


37

Appendix 1 (cont`d)

Animal

ID Group

Fever

≥39.5⁰C

Clinical:

cough, nasal

discharge,

dyspnea

Lung Lesions Lesion

Score

Pathology

Index

Mmm

culture

1014 B No Yes No 0 0 No

1018 B No Yes No 0 2 Positive



1030 B No No No 0 0 No


1053 B Yes Yes No 0 0 No

1057 B Yes No Consolidation;

fibrous adhesions 2 4 Positive




38

Appendix 1 (cont`d)

Animal

ID Group

Fever

≥39.5⁰C

Clinical:

cough, nasal

discharge,

dyspnea

Lung Lesions Lesion

Score

Pathology

Index

Mmm

culture

999 C No Yes No 0 0 No

1001 C No Yes No 0 2 Positive

1017 C No Yes Fibrous adhesions 1 1 No

1026 C No No No 0 0 No



1047 C No No No 0 0 No

1052 C No Yes No 0 2 Positive




39

Appendix 1 (cont`d)

Animal

ID Group

Fever

≥39.5⁰C

Clinical:

cough, nasal

discharge,

dyspnea

Lung Lesions Lesion

Score

Pathology

Index

Mmm

culture

998 D No Yes No 0 2 Positive

1005 D No No No 0 0 No

1007 D No Yes

Gray and red

hepatization; fibrous

tags

2 2 No

1023 D No Yes No 0 0 No


1033 D No Yes Sequestra (8x5);

fibrous adhesions 2 4 No



1044 D No No No 0 0 No



40

Appendix 1 (cont`d)

Animal

ID Group

Fever

≥39.5⁰C

Clinical:

cough, nasal

discharge,

dyspnea

Lung Lesions Lesion

Score

Pathology

Index

Mmm

culture

1000 E No Yes Fibrous adhesions 1 2 Positive

1003 E No No No 0 0 No

1011 E Yes Yes

oedema; distended

interlobular septa

with fibrinous

exudate

1 1 No

1022 E No No No 0 0 No

1031 E No Yes

Sequestra (6x4); PF;

distended interlobular

septa with fibrinous

exudate

2 8 Positive

1039 E No Yes No 0 0 No

1040 E No Yes Sequestra(40x28);

fibrous adhesions 2 6 No

1042 E No Yes Sequestra (24x14) 2 12 Positive

1049 E No Yes No 0 0 No

1061 E No Yes

Distended

interlobular septa

with fibrinous

exudate

1 3 Positive


41

Appendix 1 (cont`d)

Animal

ID Group

Fever

≥39.5⁰C

Clinical:

cough, nasal

discharge,

dyspnea

Lung Lesions Lesion

Score

Pathology

Index

Mmm

culture

997 F No Yes

PF; distended

interlobular septa

with fibrinous

exudate

1 3 Positive

1002 F No Yes No 0 0 No




1020 F No Yes Sequestra (3x2; 6x4);

consolidation 2 4 No

1037 F No No No 0 0 No





42


Animal

ID Group

Fever

≥39.5⁰C

Clinical:

cough, nasal

discharge,

dyspnea

Lung Lesions Lesion

Score

Pathology

Index

Mmm

culture

1080 G No Yes PF 1 1 No

1090 G No No

Sequestra 2x2cm;

Fibrous

adhesions&tags

2 4 No

1093 G No Yes No 0 0 No

1096 G No Yes No 0 2 Positive

1098 G No Yes

Multiple sequestra

(13x6;

6x6;7x6;3x2cm;

3x2); Fibrous

adhesions; PF

2 12 Positive

1102 G No Yes No 0 2 Positive

1105 G Yes Yes No 0 2 Positive

1110 G No No No 0 0 No

1129 G No Yes No 0 0 No

1133 G

Yes

Multiple sequestra

(5x5; 2x2; 1.5x1.5);

PF

2 8 Positive


43

Appendix 2 (cont`d)

Animal

ID Group

Fever

≥39.5⁰C

Clinical:

cough, nasal

discharge,

dyspnea

Lung Lesions Lesion

Score

Pathology

Index

Mmm

culture

1077 H No No No 0 2 Positive

1084 H

Yes PF 1 2 Positive

1087 H No Yes No 0 0 No

1089 H No No

Oedematous lobes;



exudate

1 1 No

1097 H No No No 0 2 Positive

1103 H No Yes No 0 2 Positive

1122 H No Yes Fibrous tags 1 3 Positive

1126 H Yes Yes Sequestra (3x2; 3x2);

PF 2 8 Positive

1128 H No Yes Fibrous adhesions 1 1 No

1136 H No Yes Sequestra (5x4; 3x2);

PF 2 8 Positive


44

Appendix 2 (cont`d)

Animal

ID Group

Fever

≥39.5⁰C

Clinical:

cough, nasal

discharge,

dyspnea

Lung Lesions Lesion

Score

Pathology

Index

Mmm

culture

1078 I No Yes Fibrous tags 1 1 No

1081 I No Yes No 0 2 Positive

1088 I No No

Oedematous lobes;



exudate

1 3 Positive

1095 I No Yes No 0 0 No

1100 I No Yes No 0 0 Positive

1104 I No No Sequestra (4x3; 4x3) 2 8 Positive

1116 I No Yes No 0 0 No

1121 I Yes Yes

Sequestra(30x30;

22x19);Fibrous

adhesions; PF

2 12 Positive

1123 I

Yes Fibrous adhesion 1 3 Positive

1131 I No No

Oedematous lobes;



exudate

1 3 Positive


45

Appendix 2 (cont`d)

Animal

ID Group

Fever

≥39.5⁰C

Clinical:

cough, nasal

discharge,

dyspnea

Lung Lesions Lesion

Score

Pathology

Index

Mmm

culture

1083 J No No No 0 0 No

1086 J No Yes Fibrous adhesion &

tags; PF 1 1 No

1101 J

Yes

Multiple

sequestra(13x17;10x6

; 10x6); Fibrous

adhesions

2 6 No

1107 J Yes Yes No 0 2 Positive

1108 J No Yes No 0 2 Positive

1111 J No Yes No 0 0 No

1120 J No Yes Sequestra(2x2cm);

PF 2 8 Positive

1125 J No Yes Fibrous tags; multiple

nodules/necrosis; PF 2 2 No

1130 J Yes Yes

Sequestra(15x13;

13x12); hepatization;

fibrous adhesions: PF

2 12 Positive

1135 J No Yes No 0 0 No


46

Appendix 2 (cont`d)

Animal

ID Group

Fever

≥39.5⁰C

Clinical:

cough, nasal

discharge,

dyspnea

Lung Lesions Lesion

Score

Pathology

Index

Mmm

culture

1085 K No 0 Sequestra(12x11);

hepatization 2 12 Positive

1091 K No Yes No 0 0 No


1106 K No 0 No 0 2 Positive

1109 K No 0

Oedematous lobes;



exudate

1 3 Positive


1115 K Yes Yes

Fibrous tags;

oedematous lobes;



exudate

1 1 No

1124 K No Yes No 0 2 Positive

1132 K Yes 0 Sequestra(17x11);

hepatization; PF 2 12 Positive

1134 K No Yes No 0 2 Positive


47

Appendix 2 (cont`d)

Animal

ID Group

Fever

≥39.5⁰C

Clinical:

cough, nasal

discharge,

dyspnea

Lung Lesions Lesion

Score

Pathology

Index

Mmm

culture

1079 L No Yes Fibrous tags 1 1 No

1082 L No 0 No 0 0 No

1092 L No 0 PF 1 0 No

1099 L No Yes No 0 0 No

1112 L No Yes PF 1 3 Positive

1113 L No 0 No 0 2 Positive

1117 L No Yes No 0 2 Positive

1118 L

Yes

Multiple

sequestra(15x17;

7x4; 6x6);

hepatization; fibrous

adhesions

2 12 Positive




48


Animal

ID Group

Fever

≥39.5⁰C

Clinical:

cough, nasal

discharge,

dyspnea

Lung Lesions Lesion

Score

Pathology

Index

Mmm

culture

1142 M Present Present No 0 0 No

1148 M

Present

Multiple sequestra

(18x18; 3x3; 3x2;

2x2cm); Fibrous

adhesions

2 6 No


1154 M Present Present No 0 2 Positive

1158 M Present

Sequestra (14x14cm);

Fibrous adhesions 2 8 Positive


1171 M

Present No 0 0 No

1182 M

No 0 2 Positive

1186 M Present Present Sequestra (30x25cm);

Fibrous adhesions 2 12 Positive

1189 M Present Present

Multiple sequestra

(35x25; 7x7cm);

fibrous adhesions

2 12 Positive


49

Appendix 3 (cont`d)

Animal

ID Group

Fever

≥39.5⁰C

Clinical:

cough, nasal

discharge,

dyspnea

Lung Lesions Lesion

Score

Pathology

Index

Mmm

culture

1144 N Present Present No 0 0 No

1147 N Present Present Fibrous adhesions 1 1 No




1165 N Present

No 0 0 No






50

Appendix 3 (cont`d)

Animal

ID Group

Fever

≥39.5⁰C

Clinical:

cough, nasal

discharge,

dyspnea

Lung Lesions Lesion

Score

Pathology

Index

Mmm

culture

1143 O Present

No 0 2 Positive

1150 O Present Present No 0 0 No



1161 O

Present

Sequestra (30x14cm);

fibrous

adhesions;distended

interlobular septi filled

with fibrinous exudates

2 12 Positive

1168 O Present Present Sequestra (35x33cm);

Fibrous adhesions 2 6 No

1173 O Present

Fibrous adhesions;


septi

1 2 No

1175 O Present Present No 0 2 Positive

1177 O Present


1187 O



51

Appendix 3 (con`t)

Animal

ID Group

Fever

≥39.5⁰C

Clinical:

cough, nasal

discharge,

dyspnea

Lung Lesions Lesion

Score

Pathology

Index

Mmm

culture

1141 P Present Present Fibrous adhesions 1 1 No


1146 P

No 0 0 No

1152 P Present Present No 0 0 No

1160 P

Sequestra (14x6cm);




1176 P Present Present No 0 0 No

1181 P Present

No 0 0 No

1184 P Present

Sequestra (6x6cm);

fibrous adhesions

(15x11cm)

2 8 Positive


52

Appendix 3 (cont`d)

Animal

ID Group

Fever

≥39.5⁰C

Clinical:

cough, nasal

discharge,

dyspnea

Lung Lesions Lesion

Score

Pathology

Index

Mmm

culture

1151 Q Present Present

Distended interlobular

septi filled with

fibrinous exudate;

Fibrous adhesions

1 1 No

1159 Q Present Present Fibrous adhesions 1 1 No

1164 Q

Fibrous adhesions;


septi filled with

fibrinous exudates

1 1 No

1167 Q

Present Fibrous adhesions 1 1 No

1169 Q

Present

Distended interlobular

septi filled with

fibrinous exudate;

fibrous adhesions

1 1 No


1179 Q

Present - 0 0 No

1180 Q Present Present

Multiple sequestra

(7x6; 2x2; 2x2;

1x1cm); fibrous

adhesions

2 8 Positive

1188 Q Present Present - 0 0 No



53

Appendix 4: Mean group temperatures 4 weeks after challenge.

Gro

up

A

Gro

up

B

Gro

up

C

Gro

up

D

Gro

up

E

Gro

up

F

3 7 .0

3 7 .5

3 8 .0

3 8 .5

M e a n g r o u p te m p e r a tu r e

T r ia l 1 g ro u p s

W e e k 1

W e e k 2

W e e k 3

W e e k 4

Gro

up

G

Gro

up

H

Gro

up

I

Gro

up

J

Gro

up

K

Gro

up

L

3 7 .0

3 7 .5

3 8 .0

3 8 .5



W e e k 1

W e e k 2

W e e k 3

W e e k 4

Gro

up

M

Gro

up

N

Gro

up

O

Gro

up

P

Gro

up

Q

3 7 .0

3 7 .5

3 8 .0

3 8 .5



W e e k 1

W e e k 2

W e e k 3

W e e k 4


54

Appendix 5: Group average proliferation stimulation indices in vaccinated and control animals.

Protein

Vaccination

Group

Control

Group

Day 0 Day 35 Day 0 Day 35

MSC0136

MSC 0957

MSC0499

MSC0431

MSC 0776

A

15.7250 6.5063 9.4836 3.6515

6.6529 5.3360

3.8993 2.7745

12.1449 5.0489

6.5644 3.9771

11.6603 6.3795

7.5121 5.0269

20.5772 2.8758

10.4453 2.1005

MSC0519

MSC 0500

MSC 0575

MSC 0653

MSC 0397

B

5.6094 7.9083 6.7499 5.1498

7.6779 13.5029

13.1552 7.2227

8.0181 19.3064

14.7636 8.2918

7.8402 4.9128

12.2672 2.8836

11.1702 1.6111

10.3976 0.3907

4400559

4399087

MSC 0816

MSC 0160

MSC 0775

C

14.9155 8.2816 11.0556 4.5328

16.6559 6.7822

10.3518 3.6426

10.8709 6.6859

7.7864 2.4080

14.7463 7.6397

8.8523 3.4534

18.6418 12.5553

11.4032 8.3979

MSC 0013

MSC 0610

MSC 0265

MSC 0052

MSC 0240

D

6.9805 18.0844 9.2763 4.7680

7.5359 13.3600

10.7231 5.7113

6.1716 19.1601

9.8531 5.1086

5.5634 13.0433

9.3665 2.8457

8.6932 8.7028

6.6647 3.7371

MSC 0014

MSC 0011

4400534

MSC 0813

MSC 0184

E

4.8483 11.7658 10.4058 2.9181

4.3181 15.6995

11.2094 7.7941

3.3524 9.4443

4.9889 3.8043

3.5662 12.9675

6.7954 3.7912

4.1671 13.9985

7.4398 3.1621


55

Appendix 5 (cont`d)

Protein

Vaccination

Group Control

Group


MSC0782

G

1.8671 26.6260 0.9690 2.6280

MSC0401 2.0033 21.8100

0.8186 2.1100

MMSA0381 2.2649 32.1280

1.0711 2.8160

MSC1058 1.4283 20.2680

0.7984 1.3680

MSC0790 1.8990 35.9820

1.2004 3.1220

MSC0453

H

1.1348 4.9320 1.2097 3.7311

MSC0798 0.8873 4.2500

4.1489 4.8742

MMSA0108 0.7910 4.1440

1.7557 3.8606

MSC0266 1.1886 3.2940

1.2521 3.1637

MSC0456 1.1907 7.1580

1.0820 4.8399

4400602

I

0.9984 3.5003 1.5059 3.1258

4400291 1.1257 4.6621

1.8339 2.8100

4400300 0.9161 6.0198

2.1356 4.8194

4400620 1.3034 4.4032

2.3960 3.1250

MSC1005 1.2750 7.9380

2.0159 3.4059

44000616

J

0.8017 8.3786 0.9588 1.7983

4400615 0.9812 7.7199

0.9813 1.0766

MMSA0415 0.7500 10.5692

0.9698 2.6789

MSC0927 0.8872 15.8012

1.2304 1.2984

MSC0804 0.7955 15.4425

1.0075 3.5338

4400622

K

0.7366 5.3142 1.0572 1.9133

4400371 0.9193 9.1955

1.3729 2.9845

4400226 0.7020 2.8001

1.4032 5.0231

4400021 0.8490 6.8100

1.2210 3.4437

MSC0163 0.6691 8.2623

1.3060 3.5459


56

Appendix 5 (cont`d)

Protein

Vaccination

Group

Control

Group


4400581

M

3.3505 6.6412 2.9307 3.3682

4400296 2.7375 6.4076

4.7834 5.0640

4399851 5.0129 8.1043

3.9284 4.5708

4399914 2.4063 3.4866

4.0050 2.6188

MSC0453

N

1.4599 2.4719 6.3984 4.3824

4399790 1.1797 2.8555

4.3148 5.3872

4400580 1.0066 3.9188

4.4586 6.2667

4400610 1.2198 2.5670

2.5755 3.3413

4400171

O

1.0450 2.6026 0.9507 0.8436

MSC0139 0.8708 4.3440

7.4379 1.2901

4399939 0.7906 2.6299

5.9656 4.6327

44000004 0.7067 3.2141

2.3092 2.2037

4400446

P

2.4848 1.8830 4.8467 4.2718

4399927 1.7190 1.7388

4.5505 3.1769

4400204 2.9223 1.4345

3.0266 2.0874

4400368 3.9400 1.8975

5.4858 1.4994


57

Appendix 6: Serological response to immunization in Trial 1.

Vaccination

Group Protein

OD 1/400 day 35 after

primary vaccination1

A

MSC0136

MSC 0957 2.061

MSC0499 2.005

MSC0431 2.294

MSC 0776 1.347

B

MSC0519 2.244

MSC 0500 ND2

MSC 0575 ND

MSC 0653 3.165

MSC 0397 ND

C

4400559 1.393

4399087 1.088

MSC 0816 1.623

MSC 0160 3.442

MSC 0775 2.679

D

MSC 0013 3.132

MSC 0610 2.903

MSC 0265 2.217

MSC 0052 2.124

MSC 0240 2.882

E

MSC 0014 ND

MSC 0011 3.106

4400534 1.393

MSC 0813 1.295

MSC 0184 ND

1 Since the clinical scores and pathology results for Trial 1 were very low, the ELISA’s were carried out using a

single 1/400 dilution of each serum sample rather than full endpoint titrations as done for the other trials. The mean

value for the control animals was 1.35.

2 ND: Assays not completed.


58


Vaccination

Group Protein

3Titre day 35 after

primary vaccination

G

MSC0782 1840

MSC0401 10240

MMSA0381 5440

MSC1058 7840

MSC0790 6400

H

MSC0453 46720

MSC0798 ND4

MMSA0108 ND

MSC0266 ND

MSC0456 ND

I

4400602 ND

4400291 ND

4400300 ND

4400620 ND

MSC1005 ND

J

44000616 39040

4400615 4000

MMSA0415 56320

MSC0927 94720

MSC0804 35200

K

4400622 110080

4400371 25600

4400226 27520

4400021 ND

MSC0163 5440

3 Data is expressed as the mean of the reciprocal of the last dilution which gave a positive response (2-fold dilutions

used).

4 ND: Assays not completed


59


Vaccination

Group Protein

Titre day 35 after


Control

Group

Titre day 35 after


M

4400581 820 4400581 1000

4400296 3040 4400296 1240

4399851 23680 4399851 610

4399914 4480 4399914 100

N

MSC0453 760 MSC0453 300

4399790 14080 4399790 80

4400580 1000 4400580 180

4400610 10240 4400610 330

O

4400171 1480 4400171 1470

MSC0139 1000 MSC0139 ND

4399939 8320 4399939 8320

44000004 1720 44000004 ND

P

4400446 8800 4400446 ND

4399927 5170 4399927 ND

4400204 7840 4400204 ND

4400368 13000 4400368 ND

5 Data is expressed as the mean of the reciprocal of the last dilution which gave a positive response (2-fold dilutions

used). 6 ND: Assays not completed.


60

Appendix 9: Participation in various conferences/symposia.

Dialogue on International Food Security 2014, 30 April-2 May, University of Alberta,

Edmonton, Canada Risk assessment and economic impact of an outbreak of Contagious Bovine

Pleuropneumonia in Laikipia County of Kenya under the economics, value chains and

policy theme.

Research to Feed Africa Symposium 23-27 June 2014 Great Rift Valley Lodge,

Naivasha, Kenya

Gender and socio-economic factors influencing preferences and willingness to pay for

new livestock vaccines with reference to CBPP-oral presentation

A gendered analysis of constraints to cattle production in Ijara sub-county, Kenya-

Poster

CIFSRF end of project symposium 7-8 July 2014 Sankara Hotel, Nairobi

Development of a Subunit Vaccine for Contagious Bovine Pleuropneumonia in Africa

Project: Socio-economics component overview

A cross sectional estimation of prevalence and associated risk factors of CBPP in ijara

sub-county of Kenya

A gendered analysis of socio-economic factors influencing adoption of new CBPP in

Ijara sub-county

Pending work and analysis which will be completed by the gender PhD student within the next 6

months (by March 2015) includes:

An analysis of socio-economic factors that could influence preferences and willingness to

pay for CBPP vaccine and vaccination

Interviews with policy makers, regulatory bodies and key informants along the CBPP

vaccine delivery chain and survey of animal health service providers in Ijara sub-county

CBPP outbreak investigations in Ijara sub-county

Two presentations at the University of Nairobi 9th

Biennial Scientific Conference 3-5th

September 2014 as follows

Prevalence, risk and impact of Contagious Bovine Pleuropneumonia in Laikipia County

of Kenya

Gendered preferences and WTP for CBPP vaccine and vaccination among pastoralists in

Ijara sub-county, Kenya

Pending work and analysis which will be completed by the epidemiology MSc student includes:

Regression of prevalence against postulated risk factors which include age, sex, breed,

intercurrent diseases, management and production system and CBPP vaccination to

determine the risk factors for CBPP in Ijara sub-county


61

Pearson’s Chi square to determine the strength of association between CBPP prevalence

and the risk factors

Pending publications:

The publications likely to come out of the socio-economic and gender studies are:

The prevalence and risk factors of CBPP in Ijara, Kenya

Knowledge, attitudes and perceptions of CBPP in Ijara, Kenya

A gendered analysis of the constraints of cattle production in Ijara, Kenya

Gendered preferences for contagious bovine pleuropneumonia vaccine and vaccination

among pastoralists in Ijara Sub-County, Kenya

Gendered preferences and willingness to pay for contagious bovine pleuropneumonia

vaccine and vaccination among pastoralists in Ijara Sub-County, Kenya

Prevalence, risk and impact of Contagious Bovine Pleuropneumonia in LaikipiaCounty

of Kenya

project title: development of a vaccine to eradicate ... · final technical report: development of...

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