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 2: Clinical and Pathology Data from Efficacy Trial 2. ........................................ 42
Appendix 3: Clinical and Pathology Data from Efficacy Trial 3. ........................................ 48
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 7: Serological response to immunization in Trial 2. ............................................. 58
Appendix 8: Serological response to immunization in Trial 3. ............................................. 59
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
MSC_0957 lipoprotein 79kDa 29.100 79 55
MSC_0499 lipoprotein 111kDa 1202.450 29 54
MSC_0431 lipoprotein 70kDa 442.275 8 54
MSC_0776 lipoprotein 120kDa 28.000 31 52
Group B
MSC_0519 lipoprotein 99kDa 61.600 10 51
MSC_0500 lipoprotein 138kDa 30.825 8 50
MSC_0575 lipoprotein 69kDa 29.600 14 50
MSC_0653 lipoprotein 75kDa 2100.000 24 49
MSC_0397 lipoprotein 45kDa 316.000 10 49
Group C
YP_004400559.1 Hypothetical protein 18kDa 24.200 4 48
YP_004399807.1 Hypothetical protein 41kDa 27.000 10 48
MSC_0816 lipoprotein 76kDa 164.000 19 47
MSC_0160 Elongation factor Tu 75kDa 528.000 14 46
MSC_0775 lipoprotein 81kDa 343.000 0 44
Group D
MSC_0013 lipoprotein 92kDa 687.800 25 44
MSC_0610 lipoprotein 64kDa 250.000 24 42
MSC_0265 Pyruvate dehydrogenase E1 74kDa 581.450 12 42
MSC_0052 lipoprotein 111kDa 647.900 15 41
MSC_0240 lipoprotein 94kDa 10.000 11 38
Group E
MSC_0014 lipoprotein 91kDa 408.550 36 38
MSC_0011 lipoprotein 91kDa 221.100 17 37
YP_004400534.1 Transmembrane protein 229kDa 16.200 95 37
MSC_0813 lipoprotein 88kDa 660.400 5 36
MSC_0184 lipoprotein 149kDa 534.200 24 36
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Table 3. Proteins used in efficacy Trial 2.
Name Function Size Yield/100ml #MHC-I Median
rank
Group G
MSC_0782 lipoprotein 101kDa 25.200 23 35
MSC_0401 lipoprotein 34kDa 398.250 0 35
MMS_A0381 lipoprotein 100kDa 1318.450 28 34
MSC_1058 lipoprotein 45kDa 286.000 0 33
MSC_0790 lipoprotein 85kDa 42.400 16 33
Group H
MSC_0453 FKBP-type peptidyl-prolyl
isomerase 81kDa 517.725 46 32
MMS_A0108 lipoprotein 71kDa 258.750 34 32
MSC_0798 lipoprotein 107kDa 690.000 10 30
MSC_0266 lipoprotein 68kDa 382.400 6 30
MSC_0456 lipoprotein 125kDa 114.950 32 29
Group I
YP_004400602.1 Transmembrane protein 15kDa 12.800 10 29
YP_004400291.1 Transmembrane protein 82kDa 10.600 10 29
YP_004400300.1 Lipoprotein 98kDa 10.800 49 27
YP_004400620.1 Hypothetical protein 24kDa 21.600 9 25
MSC_1005 lipoprotein 77kDa 1641.000 5 24
Group J
YP_004400616.1 Hypothetical protein 18kDa 9.000 5 24
YP_004400615.1 Hypothetical protein 23kDa 284.000 3 24
MM_A0415 lipoprotein 45kDa 812.400 0 22
MSC_0927 lipoprotein 45kDa 440.000 9 21
MSC_0804 lipoprotein 83kDa 521.400 17 21
Group K
YP_004400622.1 Hypothetical protein 24kDa 21.600 0 21
YP_004400371.1 Permease 84kDa 5.500 45 20
YP_004400226.1 Hypothetical protein 15kDa 30.300 16 20
YP_004400021.1 PTS transporter 36kDa 29.000 2 20
MSC_0163 Leucyl aminopeptidase 82kDa 468.725 26 19
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Table 4. Proteins used in efficacy Trial 3.
Name Function Size Yield/100ml #MHC-I Median
rank
Group M
YP_004400581.1 Transmembrane protein 52kDa 237.000 44 19
YP_004400296.1 Lipoprotein 101kDa 10.600 42 19
YP_004399851.1 PTS transporter 19kDa 222.000 4 18
YP_004399914.1 Hypothetical protein 16kDa 101.000 1 17
Group N
YP_004400127.1 Hypothetical protein 27kDa 318.000 3 16
YP_004399790.1 Hypothetical protein 38kDa 145.000 1 16
YP_004400580.1 Hypothetical protein 15kDa 68.000 1 15
YP_004400610.1 Hypothetical protein 23kDa 15.800 2 14
Group O
YP_004400171.1 ABC transporter 41kDa 123.400 19 13
MSC_0139 Fructose-bisphosphate aldolase
class II 65kDa 380.800 9 12
YP_004399939.1 Transmembrane protein 55kDa 38.000 29 12
YP_004400004.1 Transmembrane protein 41kDa 49.000 0 9
Group P YP_004400446.1 Hypothetical protein 20kDa 7.500 6 8
YP_004399927.1 Hypothetical protein 41kDa 74.900 3 3
YP_004400204.1 Hypothetical protein 149kDa 21.200 39 1
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
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Table 6. Trial 2 - Mean serum antibody titers against vaccine antigens 35 days post vaccination.
Group Protein Vaccinated Animals Control Animals
IgG1 IgG2 IgA IgG1 IgG2 IgA
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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Table 7. Trial 3 - Mean serum antibody titers against vaccine antigens 35 days post vaccination.
Group Protein Vaccinated Animals Control Animals
IgG1 IgG2 IgA IgG1 IgG2 IgA
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.
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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.
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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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Protective efficacy Trial 2:
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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Figure 6. Mean lesion scores and pathology indices of groups in Trial 2.
Protective efficacy Trial 3:
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.
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Figure 7. Mean lesion scores and pathology indices of groups in Trial 3.
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.
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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.
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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.
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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,
Final Technical Report: Development of a Vaccine to Eradicate Contagious Bovine PleuroPneumonia in Africa
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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.
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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.
Final Technical Report: Development of a Vaccine to Eradicate Contagious Bovine PleuroPneumonia in Africa
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.
Final Technical Report: Development of a Vaccine to Eradicate Contagious Bovine PleuroPneumonia in Africa
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.
Final Technical Report: Development of a Vaccine to Eradicate Contagious Bovine PleuroPneumonia in Africa
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.
Final Technical Report: Development of a Vaccine to Eradicate Contagious Bovine PleuroPneumonia in Africa
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.
Final Technical Report: Development of a Vaccine to Eradicate Contagious Bovine PleuroPneumonia in Africa
35
APPENDICES
Final Technical Report: Development of a Vaccine to Eradicate Contagious Bovine PleuroPneumonia in Africa
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
1024 A No Yes No 0 0 No
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
1048 A No Yes No 0 0 No
1058 A Yes Yes No 0 0 No
Final Technical Report: Development of a Vaccine to Eradicate Contagious Bovine PleuroPneumonia in Africa
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
1021 B No Yes No 0 2 Positive
1025 B No Yes No 0 2 Positive
1030 B No No No 0 0 No
1041 B No Yes No 0 0 No
1053 B Yes Yes No 0 0 No
1057 B Yes No Consolidation;
fibrous adhesions 2 4 Positive
1059 B No Yes No 0 2 Positive
1060 B No Yes No 0 0 No
Final Technical Report: Development of a Vaccine to Eradicate Contagious Bovine PleuroPneumonia in Africa
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
1028 C No Yes No 0 0 No
1038 C No Yes No 0 0 No
1047 C No No No 0 0 No
1052 C No Yes No 0 2 Positive
1055 C No Yes No 0 0 No
1056 C No Yes No 0 0 No
Final Technical Report: Development of a Vaccine to Eradicate Contagious Bovine PleuroPneumonia in Africa
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
1032 D No Yes No 0 0 No
1033 D No Yes Sequestra (8x5);
fibrous adhesions 2 4 No
1036 D No Yes No 0 0 No
1043 D No Yes No 0 0 No
1044 D No No No 0 0 No
1054 D No Yes No 0 0 No
Final Technical Report: Development of a Vaccine to Eradicate Contagious Bovine PleuroPneumonia in Africa
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
Final Technical Report: Development of a Vaccine to Eradicate Contagious Bovine PleuroPneumonia in Africa
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
1008 F No Yes No 0 0 No
1012 F No Yes No 0 0 No
1019 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
1045 F No Yes No 0 0 No
1050 F No Yes No 0 0 No
1051 F No Yes No 0 0 No
Final Technical Report: Development of a Vaccine to Eradicate Contagious Bovine PleuroPneumonia in Africa
42
Appendix 2: Clinical and Pathology Data from Efficacy Trial 2.
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
Final Technical Report: Development of a Vaccine to Eradicate Contagious Bovine PleuroPneumonia in Africa
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;
distended interlobular
septa with fibrinous
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
Final Technical Report: Development of a Vaccine to Eradicate Contagious Bovine PleuroPneumonia in Africa
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;
distended interlobular
septa with fibrinous
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;
distended interlobular
septa with fibrinous
exudate
1 3 Positive
Final Technical Report: Development of a Vaccine to Eradicate Contagious Bovine PleuroPneumonia in Africa
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
Final Technical Report: Development of a Vaccine to Eradicate Contagious Bovine PleuroPneumonia in Africa
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
1094 K No Yes No 0 0 No
1106 K No 0 No 0 2 Positive
1109 K No 0
Oedematous lobes;
distended interlobular
septa with fibrinous
exudate
1 3 Positive
1114 K No Yes No 0 0 No
1115 K Yes Yes
Fibrous tags;
oedematous lobes;
distended interlobular
septa with fibrinous
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
Final Technical Report: Development of a Vaccine to Eradicate Contagious Bovine PleuroPneumonia in Africa
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
1119 L No Yes No 0 0 No
1127 L No Yes No 0 0 No
Final Technical Report: Development of a Vaccine to Eradicate Contagious Bovine PleuroPneumonia in Africa
48
Appendix 3: Clinical and Pathology Data from Efficacy Trial 3.
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
1149 M Present Present No 0 0 No
1154 M Present Present No 0 2 Positive
1158 M Present
Sequestra (14x14cm);
Fibrous adhesions 2 8 Positive
1170 M Present Present No 0 0 No
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
Final Technical Report: Development of a Vaccine to Eradicate Contagious Bovine PleuroPneumonia in Africa
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
1155 N Present Present No 0 0 No
1157 N Present Present No 0 0 No
1163 N Present Present No 0 0 No
1165 N Present
No 0 0 No
1166 N Present Present No 0 0 No
1172 N Present Present Fibrous adhesions 1 1 No
1183 N Present Present Fibrous adhesions 1 1 No
1185 N Present Present Fibrous adhesions 1 1 No
Final Technical Report: Development of a Vaccine to Eradicate Contagious Bovine PleuroPneumonia in Africa
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
1153 O Present Present No 0 0 No
1156 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;
distended interlobular
septi
1 2 No
1175 O Present Present No 0 2 Positive
1177 O Present
Fibrous adhesions 1 1 No
1187 O
Fibrous adhesions 1 1 No
Final Technical Report: Development of a Vaccine to Eradicate Contagious Bovine PleuroPneumonia in Africa
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
1145 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);
Fibrous adhesions 2 4 No
1162 P Present Present Fibrous adhesions 1 1 No
1174 P Present Present Fibrous adhesions 1 1 No
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
Final Technical Report: Development of a Vaccine to Eradicate Contagious Bovine PleuroPneumonia in Africa
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;
distended interlobular
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
1178 Q Present Present 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
1190 Q Present Present Fibrous adhesions 1 1 No
Final Technical Report: Development of a Vaccine to Eradicate Contagious Bovine PleuroPneumonia in Africa
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
M e a n g r o u p te m p e r a tu r e
T r ia l 2 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
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
M e a n g r o u p te m p e r a tu r e
T r ia l 3 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
Final Technical Report: Development of a Vaccine to Eradicate Contagious Bovine PleuroPneumonia in Africa
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
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Appendix 5 (cont`d)
Protein
Vaccination
Group Control
Group
Day 0 Day 35 Day 0 Day 35
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
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Appendix 5 (cont`d)
Protein
Vaccination
Group
Control
Group
Day 0 Day 35 Day 0 Day 35
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
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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.
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Appendix 7: Serological response to immunization in Trial 2.
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
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Appendix 8: Serological response to immunization in Trial 3.
Vaccination
Group Protein
Titre day 35 after
primary vaccination5
Control
Group
Titre day 35 after
primary vaccination6
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.
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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
Final Technical Report: Development of a Vaccine to Eradicate Contagious Bovine PleuroPneumonia in Africa
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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