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					Towards the Foot-and-mouth disease control in West
Africa: investigating spatio-temporal epidemiological
  patterns in Mali – a community based approach




                    A project proposal submitted to the

EUROPEAN COMMISSION FOR THE CONTROL OF FOOT-AND-MOUTH DISEASE (EUFMD)
    FOOD AND AGRICULTURE ORGANIZATION OF THE UNITED NATIONS (FAO)




                             PREPARED BY
                            Traore, A. (LCV)
                           Di Nardo, A. (IAH)




      LABORATOIRE CENTRAL VÉTÉRINAIRE DU MALI (LCV), BAMAKO – MALI

      INSTITUTE FOR ANIMAL HEALTH (IAH), PIRBRIGHT LABORATORY – UK
SECTION 1 – BACKGROUND

1.1 Introduction

        Livestock rearing plays a key role in the economies of West African countries
providing, at times, 44% of agricultural GDP. With 60 million heads of cattle and 160 of small
ruminants, the Sahel and West Africa is an exceptional region for livestock rearing. In
numbers, and in comparison with the entire sub-Saharan Africa region, the Sahel and West
Africa contain 25% of the cattle, 33% of the sheep, and 40% of the goats. Livestock rearing is
one of the main economic activities on which the poorest population depend for the food
and income. It is useful to refer to livestock systems in Sahel and West Africa according to
their agro-ecological zones: arid, semi-arid, sub-humid and humid (Fig. 1). The classification
of livestock system recognises these agro-ecological zones in distinguish among the various
mixed agriculture-livestock system that are fairly widespread in West Africa: pastoral
system, off-land system, agro-pastoral system.




Figure 1 – Agro-ecological zones in West Africa.

The pastoral system depends essentially on the use of natural resources (grasslands and
shrub-land) grazed by livestock on uncultivated land mainly in the arid and semi-arid zones
of Burkina Faso, Mali, Niger and Nigeria, stretching as far as north in Senegal and
Mauritania. Livestock are reared in mixed or unmixed herds of cattle, sheep, goats,
camelids, donkeys and horses. In traditional extensive system the availability of grazing
dictates the herd movements that distinguish nomadic or transhumant production system:
transhumance is defined as an oscillating, seasonal movement of livestock under the care of
herders, following precise routes in order to exploit pastoral resources; it is distinguished
from nomadism, which is characterised by more random movements and is followed by the
herder’s whole family. Dairy production occupies a central place in the management of this
system and, although the sale of live animals is not the main aim, it is the primary source of
cash income.

Cross-border transhumance is thus the
dominant feature of pastoralism in the
region and, although, there are no reliable
statistics to quantify the numbers of
animals involved, estimates suggests that
more than two million of cattle are driven
yearly in transhumance to Benin, Burkina
Faso, Chad, Mali, Mauritania and Nigeria. In
Mauritania, the flows of transhumant
animals towards Mali and Senegal are
estimated at more than one million head.
Pastoralism has always played an important
role in the regional trade in livestock, and a Figure 2 – Cross-border transhumance routes in West Africa.
general observation is that most West and Central African countries are affected by cross-
border transhumance, either as departure countries, or as reception or transit countries. In
this view, it is possible to define two types of cross-border transhumance routes within the
region (Fig. 2): north-south routes are the more numerous and indicate dry-season
movements in the departure zones, while south-north routes are less numerous and
indicate wet-season movements.

The evolution of an intra- and extra-regional
trade is mainly linked to the pastoral
system. It is possible to define three major
traditional trading basins for livestock, with
flows going from the Sahel to coastal
countries, showing certain interdependence
between Sahel countries and those of the
coast (Fig. 3). The first trading basin
comprises the countries on the Atlantic
seaboard – Cape Verde, Gambia, Guinea-
Bissau, Guinea, Liberia, Mauritania, Senegal
and Sierra Leone. The zone has little trade Figure 3 – Main trade-flow routes in West Africa, according
with other zones (some between Mali and to bovine density data.
Senegal, Mauritania and Western Sahara, or between Guinea and Mali) and a little trade
among the countries themselves (except for sheep between Mauritania and Senegal, or
Gambia and Senegal). The second basin is the central area, comprising Burkina Faso, Ivory
Coast, Ghana, Mali and Togo. Some trade flows going from Mali to Western Sahara through
Mauritania. The third basin comprises Benin, Cameroon, Chad, Niger and Nigeria. Nigeria
and to lesser extent Cameroon are poles of attraction for livestock. Some rare trade flows
come from Burkina Faso and Mali, while other even less frequent flows exist from Chad and
Niger toward the countries of North Africa. Cameroon and the Central African Republic have
been included in this zone, since the majority of their trade goes to Nigeria, although there
are smaller flows from Cameroon to Gabon and from Central African Republic to the Congo.
The value of trade in live cattle has estimated to USD 150 million in 2000. With regard to
specific trade among countries, Burkina Faso and Mali are the main exporter of live cattle to
Ivory Coast; the main suppliers of the Nigerian live cattle market are Niger, Chad and
Cameroon; Burkina Faso and to lesser extent Niger are the two main source of live cattle to
Ghana; Benin and Togo represent very minor markets for export from the Sahel.

1.2 Foot-and-mouth disease in West Africa

        Based on the prevalence, serotype and topotype data, and according to expert
evaluation of factors such as animal movement
patterns, impact of wildlife and farming system,
FMD distribution in Africa seems to follow
regional pattern of virus pools, circulating within
specific epidemiological clusters (Fig. 4).
Accordingly, West Africa can be divided into two
clusters, the Sudan/Sahel and the Coastal West
Africa, which may overlap in some way.

The Sudan/Sahel cluster comprises Western
Sudan, Niger, Chad, Burkina Faso, Mali, Northern
Nigeria, Senegal and Mauritania. This cluster
probably contains important FMD primary Figure 4 – Foot-and-mouth distribution in West
                                                   Africa, showing epidemiological cluster of virus
endemic foci. As previously stated, the farming pools.
system in this ecosystem is predominantly
pastoral, characterised by long distance movement due to either transhumance or trade:
this poses a risk in the spread of FMD serotypes whitin the region and between East and
West Africa, as confirmed for rinderpest in the past. Therefore, it may be an important
disease corridor cluster, linking the Northern East Africa cluster with West Africa and
probably West Africa with North Africa: for example, the 1999 FMD strain in Algeria was
found to be related to the West Africa type O topotype.

The Coastal West Africa cluster comprises Cameroon, Southern Nigeria, Benin, Togo, Ghana,
Ivory Coast, Liberia, Guina, Guinea-Bissau, Gambia and Senegal. Although the epidemiology
of FMD in the coastal region of West Africa has not been deeply studied, it seems that this
cluster probably gets infected from the Sudan/Sahel cluster and/or from the North East
Africa cluster by the livestock movement routes. Four of the seven FMD serotypes (A, O, SAT
1, SAT 2) have been found circulating in West Africa, with different ecological patterns and
topotypes distribution within the region (Fig. 5). Accordingly, phylogenetic analysis has been
showed that severeal isolates from West Africa are strictly related with those isolated from
the East and Central Africa, meaning that some overlapping in the viral circulation exists




Figure 5 – Foot-and-mouth A, O, SAT 1 and SAT 2 topotypes distribution in West Africa.
between those regions.

The high mobility of the pastoral herds and trade-related animal movements within the sub-
Saharian region may provide a reasonable source of virus spread. However, no data are
available which may support this hypothesis, as provided for the Egyptian serotype A
outbreak in 2006. Therefore, field studies are required to gather reasonable information
regarding the current FMD distribution and the circulating serotypes/strains in West Africa,
in order to provide detailed epidemiological information, to assess the transmission and
spreading routes within the sub-Saharan regions and to support the development of
appropriate vaccines to control the disease.

1.3 Foot-and-mouth disease in Mali

          Three of the seven serotypes have been found circulating in Mali: these include
serotype A, O and SAT 2. FMD was first reported in Mali in 1991 in outbreaks attributed to
type SAT 2 virus and, subsequently, other serotypes were identified (A, 1999; O, 2005).
Although no extensive studies have been conducted in Mali, the onsets of FMD seem to
follow a seasonal occurrence: during June-July-August (rain season) in the entire country,
and during January-February (dry season) in target areas of Gao and Timbouctou regions.
The livestock husbandry in Mali is divided between three different systems: the sedentary
system in the Soudanian area, where focal points of transhumance occur during the rain
season; the semi-nomadic system in the Sahelian area; and the nomadic system in the
                                                          Saharian area. It should be
                                                          noticed that, however, the
                                                          pastoral     system     is    the
                                                          predominant husbandry system
                                                          in sub-Saharian Africa and many
                                                          of these flocks traverse national
                                                          borders in search of water and
                                                          better pastures without any
                                                          recourse to effective quarantine
                                                          or movement control measures.
                                                          Most of the cattle and small
                                                          ruminat population enter into
                                                          Mali from Mauritania, Guinea,
                                                          Ivory Coast, Burkina Faso and
                                                          Niger; on the other hand
                                                          transboundary movement also
                                                          exists from Mali to the above
                                                          countries and also to Algeria.
                                                          The Fig. 6 shows the main
                                                          transboundary movement in
Figure 6 – Cross-border transhumant movement in Mali.      Mali by regions. Therefore, the
regular congregation of herds from different sources without essential health check
predisposes all herds to FMD and to the introduction and spreading of new strains.
Furthermore, presence of forest buffalo (Syncerus caffer nanus) in some regions of Mali
(southern part of Sikasso; north-eastern part of Sikasso and Ségou) and potential contact
with livestock during transhumance movements pose a serious risk in spreading and
transmission of FMD between wildlife and livestock species.

SECTION 2 – PURPOSE AND OBJECTIVES
        FMD remains a major Transboundary Animal Disease (TAD) for Mali and the whole
West Africa region, and no studies have been undertaken to establish the currently
circulating serotypes/strains as a prelude to the application of appropriate vaccines to
control the disease. Since vaccination is an important consideration in the control of FMD in
West Africa, and the best vaccination program should involve vaccine that target the strains
and serotypes circulating within the region, effective surveillance that will provide
information in order that correct vaccines are developed and selected will be needed to
identify all of the strains and serotypes circulating within the sub-regions. Similarly, the
adverse climatic conditions, nomadic lifestyles of pastoralists mean that transhumance
cattle movement will continue for the unforeseeable future. Therefore, for effective control
and eradication of FMD in Mali, and by extension, West Africa, vaccines that incorporate the
antigenic strains from the sub-region must be developed. The overall objective of the study
is to identify areas of endemic maintenance of FMD virus, if they exist and improve the
understanding of the epidemiology of FMD in the region.

2.1 Main purposes

    To determine the prevalence of FMD and to identify the circulating serotypes and
     strains in Mali by the means of a cross-sectional survey and monitoring of clinical
     cases
    To investigate the epidemiological patterns of FMD in Mali by the means of a
     community based approach using Participatory Epidemiology tools
    To investigate the risk associated between livestock movement and FMD distribution
     within the countries
    To investigate the FMD prevalence and potential virus serotypes/strains circulating
     in forest buffalo (Syncerus caffer nanus) resident populations of Mali. Detection of
     FMDV carrier wild buffaloes by newly validated methods (PCR, VI and IgA antibody
     test) and sequence analysis of viruses obtained from oro-pharyngeal fluids of wild
     buffaloes may facilitate to enlighten the role of transmission of FMDV to domestic
     cattle
    To provide valuable information on FMD epidemiological situation in Mali, in order
     to appropriately assist the government agencies in the surveillance and control of
     the disease
    To evaluate the economical cost-benefit impact of different vaccine control
     strategies in Mali by the means of a participatory vaccine trial

2.2 Specific objectives

    To generate scientific and updated information on FMD in Mali and to build
     appropriate tissue, sera and data banks, providing an important backup for the
     disease situation in Mali
     To develop comprehensive epidemiological map, including the identification of
      potential FMD hotspots in Mali, and to create cattle trek route maps which will assist
      in the development of appropriate FMD control strategies
     To train laboratory staff and field teams on the effective sampling procedure and
      diagnosis of FMD and to promote diagnostic-based technology transfer (including
      NSP-ELISA, LPB-ELISA, and molecular analysis techniques)
     To estabilish laboratory network and to enanche collaboration between LCV and
      WRLFMD and with FAO for promoting regional assistance in the surveillance and
      control of FMD
     To identify and select suitable vaccines based on the information on strains and
      topotypes present in Mali gathered from the project with the final aim of controlling
      the FMD through extensive and structured vaccination campaigns

SECTION 3 – PLAN OF INVESTIGATION
        The study will assess the prevalence of FMD in Mali using serological and clinical
disease survey based on a random sampling approach by the means of applied Geographic
Information System (GIS) method. Furthermore, Participatory Epidemiology (PE) component
will be integrated within the survey activities in order to gather relevant epidemiological
information on the past events of FMD, the seasonal variation in the disease occurrence,
and to investigate the risk associated with livestock movement and FMD prevalence. The
data collected by combining quantitative epidemiological tools would provide useful
information in exploring the FMD serotypes circulating in the region, the spatio-temporal
pattern of the disease occurrence, the livestock movement, the contact rate and the
dynamics of aggregation and dispersion between herds, with the ultimate aim of identifying
potential presence of hotspots and endemic foci of FMD within the region.

3.1 Sampling design

3.1.1 Livestock component
         The study will be based on a two-stage cluster sampling design, covering 9 identified
target areas corresponding to each region of Mali. The sampling strategy has been
developed on population size and structure basis, where sampling sites will be
proportionally allocated according to the livestock density. Using the formula provided by
Thrusfield (2007), the sample size has been
calculated for: (i) 50% expected prevalence
(Pexp), (ii) 95% confidence interval and (iii) 0.05
desired absolute precision (Fig. 7). The
between cluster variance (Vc = 0.00679435) has
been estimated from available data from FMD
endemic countries and utilized for the sample
size calculation for the entire study area. The
1485 serum, saliva and nasal samples will be
collected from 99 sampling sites (primary
sampling units). In each sampling site at least
15 serum samples will be collected from
                                                    Figure 7 – Sample size calculation (n=1485) for Mali
eligible animals (cattle 6-12 months, 1 to 2 based on the Two-stage cluster sampling method.
years old, 2 to 3 years old, >4 years old). The randomisation of the primary sampling units
will be obtained by generating the needed-number of random map coordinates, allowing
the application of weight factors to different sub-units of the survey area. This method
consists in generating sampling sites at random and then sampling the required number of
animals from the nearest flock to each point. In reality, a generated site is materialized only
as a point on the map and therefore a fixed radius (10 km) will be defined around each point
in order to determine a geographical area within which the sampling will be carried out.
When using this sampling procedure,
it is likely that quite a high
proportion of selected sites will have
no herds or animals within the
specified radius. In order to cope
with this scenario, “spare” sampling
sites (above the needed number of
primary sampling units) will be
generated. The total number of
“spare”      sampling     sites    will
represents 30% of the total number
of target sampling sites assigned.
These “spare” sampling sites will be
used by the survey teams to replace
sampling locations where no animals
will be found within the given radius.
In addition, 27 primary target towns Figure 8 – Random allocation of sampling sites (n=99) and primary
will be sampled and a total of 405 target towns (n=27).
sera will be collected (15 samples from each). The location and distribution of sampling site
are showed in Fig. 8, where a detailed list is enclosed in Annex IV.

3.1.2 Wildlife component

        The study will be based on a
simple random sampling design, covering
2 identified target area (Fig. 9). Using the
formula provided by Thrusfield (2007), the
sample size has been calculated for: (i)
50% expected prevalence (Pexp), (ii) 95%
confidence interval and (iii) 0.05 desired
absolute precision. The 377 serum, saliva,
nasal and probang samples will be
collected from the 2 identified target area,
in specific the southern part of Sikasso
bordering with Ivory Coast, and the Figure 9 – Forest buffalo (Syncerus caffer nanus) distribution
eastern part of Ségou bordering with in Mali: target sampling areas.
Burkina Faso. In each area at least 188 samples will be collected from forest buffaloes,
allocating the total number of samples required according to age groups (<12 months, 1-2
years old, 2-3 years old, >4 years old).
3.2 Participatory Epidemiology investigation

       During the survey implementation, PE activities will be carried out in order to collect
information on epidemiological patterns of FMD, herds’ structure and animal movement for
each target survey area. A toolkit will be used for this purpose, comprising interviewing,
ranking, mapping, and scoring methods.

Semi-structured interviews will be administered by the means of a questionnaire to every
herdsman, where groups of key informants for each survey areas will be identified for other
activities (informants will be recruited according to who are respected by their communities
for possessing specialist knowledge). In every case, prior informed consent will be obtained
verbally before interview will be conducted and an explanation of the methodology, aims,
and expected outcomes of the study will be given to participants. Questionnaires will be
used to gather information about herd structure, past events of FMD, control strategies
applied and animal movement patterns, along with pictorial form [Annex II].

Matrix scoring will be used to gain the understanding of the pastoralist and local perception
of animal diseases, ranking for priorities. Pair-wise of FMD and other four control diseases
(CBPP, PPR, LSD, and Mastitis) will be conducted to identify locally perceived indicators
(signs). The five diseases indicated above will be presented using everyday objects and
placed along the tope X-axis of the matrix. Each of the five diseases in the matrix will be
scored against a list of 17 clinical signs or cause of the diseases. The indicators will be
illustrated along the Y-axis [Annex II]. For each indicator, informants will be asked to score
each disease by dividing 25 stones against the five diseases.

Proportional pilling will be used to estimate the relative incidence and mortality caused by
the five diseases indicated above in the past [Annex II]. Before performing the assay,
informants will be asked to classify the animals into different age groups, that fits into the
real cattle husbandry system (i.e. calves aged 0-2 years, weaner 2-3 years, young group 3-4
years, >4 years old); then, informants will be allowed to maintain a pile of 100 stones for
each group of age, before splitting the pile of stones into two relative numbers of sick and
healthy stocks during the past one year. The pile of stones representing the sick animals will
be then subdivided into the five diseases to show the relative number of animals affected
during the year. Subsequently, informants will be asked to remove some of the already
allocated stones representing the sick animals to indicate the number of dead/survival
animals during the year.

Seasonal calendar will be used to describe the seasonality and variation of the diseases’
incidence previously identified by the matrix scoring. The methodology for constructing
seasonal calendar is similar to that of the matrix scoring. The seasons’ local names of long
rain, short rain, cold dry and long dry will be identified by the informants before. Seasons
will be represented on the X-axis, where diseases and causes will be placed along the Y-axis
[Annex II].

3.3 Survey implementation
        The survey activities will be carried out by contracted survey teams. Each
investigating team will comprise of a Team Leader and a Monitor. All activities will be
carried out under the close supervision of the Laboratoires Central Vétérinaires (LCV),
Bamako – Mali, and the World Reference Laboratory for FMD (WRLFMD), Institute for
Animal Health (IAH), Pirbright – UK. A total of two field campaigns for collection of serum
samples will be carried out at six months interval (the first during the dry season and the
second one during the rainy season). Field investigation and monitoring of clinical cases will
be carried out every month over the entire duration of the study (12 months) [Annex I].

Initial training will be carried out for team leaders and monitors and it will include: (i)
purpose and structure of the survey; (ii) use of Global Positioning System (GPS) device for
navigation and geo-referencing; (iii) sampling procedures; (iv) sample processing and
dispatch; (v) interviewing and methods used for the PE component; (vi) clinical inspection
and collection of relevant samples from FMD suspected cases; (vii) filling of relevant forms,
and (viii) preparation of plan of action.

3.3.1 Sample collection

3.3.1.1 Sera and saliva/nasal samples
         Each Team Leader will have the task to implement the survey activities. He will be
assisted by a veterinary assistant and a monitor who will have the mandate of collecting and
dispatching all collected samples and relevant survey forms to the LCV every three days. The
monitor will also ensure the maintenance of the cold chain by replenishing ice packs for the
survey teams. The survey teams will be provided with a GPS where all coordinates
identifying the sampling sites for each team will be entered during the initial training. The
teams will use the GPS to navigate their way to the target sampling sites. Each sampling
sites, identified by number, will be plotted on a map that will assist the survey teams to
reach the neighbourhood of the target site. At each specific target site, the investigating
teams will identify the closest herd. The distance around the target site in which animals can
be bled is set at 10 km radius. Once the herd is identified the coordinates of the actual
sampling site will be recorded and a total of 15 eligible animals (cattle <12 months, 1 to 2
years old, 2 to 3 years old, >4 years old) will be bled. In order to cater for poor clotting or
spoilage of samples, another 2 animals will be bled at each site. Thus each team will be
expected to collect a total of 17 samples at each site.

Blood samples will be collected using plain vacutainer tubes (Venoject® 10 ml) and sterile
needles (20 G; one for each animal) and will be allowed to clot for a maximum of 24 hours in
the shade or a cool place. Two aliquots of sera will be transferred into pre-labelled cryovials
identified by progressive number and team identification code (ID). Sera will be decanted
using sterile disposable Pasteur pipettes (3.5 ml). Serum samples collected at each site will
be packed and labelled according to standards defined at the initial training course and
stored in vaccine carriers at +4°C until they will be dispatched to the LCV by the monitors.
Saliva/nasal samples will be collected (swab dipped into 0.5 ml PBS) along with blood
samples. Serum and saliva/nasal samples will be accompanied by the original survey forms
(one for each site). In each sampling site, animals will be clinically inspected and relevant
samples for antigen detection will be collected if FMD suspected cases will be identified.
If no herds or animals will be found within the specified radius (10 km) the target sampling
site will be replaced by a “spare” location.

Questionnaire will be administered by the team leader to every herdsman after sample
collection, while drugs (i.e. anthelminithics, trypanocides, vitamin supplement, etc.) will be
provided to respondents as motivation. At least two different groups of informants will be
recruited for each survey areas and participatory investigations (matrix scoring, proportional
pilling and seasonal calendar) will be carried out as previously stated. In addition, the same
methodology will be administered to DVOs of each survey areas for cross-checking and
triangulation of the results. All the participatory appraisal activities will be implemented
within the first field campaign; however, the animal movement data will be recorded in the
second campaign as well.

Prior to sampling forest buffaloes, measures will be taken to perform events safely for the
animals and the staff involved. Immobilization drugs will be administered by remote
intramuscular injection using Dan-inject dart gun. Ethorphine Hydrocloride (5 mg) combined
with Xylazine Hydrocloride (30 mg) will be administered in a single dart. All the animals will
be revived using Diprenorphine Hydrocloride at a dosage rate of three times the dose of
Ethorphine and tenth the initial Xylazine dose. The immobilized animal will be monitored to
ensure the stable condition of physiological parameters (respiration, pulse and body
temperature). The dart will be removed and the dart wound will be treated using Cloxacillin
and sprayed using Oxytetracycline spray. The animals will be examined for presence of any
lesions and any other signs of FMD. Doxapram Hydrochloride will be administered in cases
of respiratory depression.

Each serum sample collected will be checked at the LCV and the WRLFMD for quality and
quantity. Only samples collected according to agreed standards and accompanied by
properly filled survey forms will be considered eligible.

All data recorded in the survey forms will be entered in specifically designed database and
the distance between actual and target sampling site will be measured in order to monitor
the accuracy of field operations.

3.3.1.2 Probang samples
        Prior to sampling, a 2 ml amount of 0.08 M phosphate buffer containing 0.01%
bovine serum albumin, phenol red (0.002%) and antibiotics adjusted to pH 7.2 will be added
to one bijou bottle for each of the animals to be sampled. After collection, the sample will
be poured from the probang cup into a wide-necked bottle (20 ml Universal bottle) and
examined visually for quality. 2 ml, containing some visible cellular material, will then be
added to the previously prepared bijou bottle containing an equal volume of buffer and
thoroughly mixed by gentle shaking. Between collections from each animal, probangs will be
disinfected in a bucket containing 4% Na2CO3 or 0.2% citric acid. After disinfection, the
probang will be thoroughly rinsed in clean water.

3.3.1.3 Clinical samples
        During the two survey campaigns, animals will be clinically inspected and relevant
samples for virus isolation (i.e. epithelium, skin, and vesicular fluid) will be collected if FMD
suspected and/or confirmed cases will be identified. In addition, surveillance campaign will
be put in place every month for the entire period of the study, aiming at monitor the clinical
situation in each target areas in order to early detect new FMD cases as they occur.

All epithelia tissues will be collected in transport media (glycerine and 0.04 M phosphate
buffer pH 7.2-7.6) with added antibiotics (Penicillin, Mycostatin, Neomycin, Polymyxin). The
samples will be stored at -20°C prior to submission and dispatch to the WRLFMD.

3.3.2 Testing of samples

        Sera samples will be tested in parallel at the LCV and at the IAH. NSP-ELISA test
(Ceditest®) will be used to assess presence of antibodies to FMD virus, where titration and
serotyping of positive samples will be carried out using the LPB-ELISA. Saliva/nasal samples
will be tested using the IgA indirect-ELISA, currently under development by IAH. Samples for
virus detection will be tested for the presence of FMD virus RNA at the WRLFMD using RT-
PCR, and positive samples will be sequenced.

3.3.3 Data analysis

        Results from the laboratory testing and participatory appraisal will be entered in two
different databases, the first linked to the serology and clinical samples data and the second
to the participatory investigations.

The survey data will be analysed using the svymean function in STATA® 10 MP. The
observed between cluster variance will be taken into account in the analysis of the data for
the calculation of the standard error used for the construction of the confidence intervals
for the observed animal prevalence. Sensitivity and specificity of the test will be used to
calculate the true prevalence from the observed test prevalence. Herd prevalence and
within-herd prevalence will also be calculated. Moreover, to account for differential
probability of selection due to the design and to ensure proper survey estimate, the
sampling base weight will be calculated for each sampling site as the inverse of the sampling
fraction. A herd will be considered positive if at least one animal within the herd is found
positive.

The seroprevalence distribution map will be generated using the Kriging method. Briefly, the
observed prevalence at each sampling site (z) will be used to generate the krigged surface
utilizing as location the coordinates (x, y) of the sampling locations. The krigged surface will
be created by first fitting an n polynomial transurface to the data. Then, the covariance
structure of the data will be modelled via a covariance function. The Akaiake’s Information
Criteria (AIC) will be used to compare the fit of the different transurface (e.g 1st to 4th order
polynomial) and covariance functions (eg. Gauss, Exponential, Spherical and Matern).
Mapping and spatial analysis will be carried out using ArcGIS® 9.3 and R 2.10. Spatial and
temporal clusters of FMD prevalence will be analysed by computing the purely spatial
discrete Poisson and the space-time permutation models of the scan statistic test, using
SaTScan™ 8.0.2. Results obtained will be mapped using ArcGIS® 9.3.
Participatory data will be analysed using SPSS® 17. Agreement among the scores of
informant groups will be assessed using the Kendall’s Coefficient of Concordance (W). The
level of agreement between FMD diagnosis by pastoralists and conventional diagnosis by
way of NSP ELISA will be analysed using kappa statistic and positive predictive value. Finally,
logistic regression will be used to determine the risk factors associated with seropositivity of
the disease in the study area.

Animal movement data will be collated and analysed by the means of Social Network
Analysis methods. The contact network between nomadic herds will be constructed using
NetDraw 2.090 with the point locations of town, villages, watering points, grazing areas and
primary/secondary markets extracted from the participatory database, and including the
sampling points from the survey campaigns. The home-range (hr) for each sampling points
will be estimated, along with attributes generated through spatial queries and analysis in
ArcGIS® 9.3 (as type of point, proximity to roads, proximity to trade routes, home range
radius, herd density, herd size). Accordingly, the following general parameters of the
symmetric binary network will be estimated: number of nodes, number of links, density,
giant weak component and average distance among reachable pairs. For individual node,
the following centrality measures will be extracted: degree, betweenness, closeness and
clustering coefficient. All the network analysis will be carried out using Ucinet 6.232.

SECTION 4 – PARTICIPATORY VACCINE TRIAL
        In order to assess the economic impact of vaccination on FMD control in Mali, a pilot
interventional study will be established after relevant data capture and analysis of
circulating strains and FMD prevalence following the implementation of the first survey. The
study will be a randomised double-blind controlled trial, designed to determine the efficacy
of different vaccine treatments administered in different doses and protocols to cattle
groups. After receiving informed consent for conducting the study in the community, cattle
selected from Touba village (Koulikoro region), located in FMD endemic area, will be
enrolled into the study according to the inclusion criteria below:
    1. No clinical signs of FMD
    2. Not previously vaccinated against FMD
    3. FMD seronegative
    4. Heterogeneous group
    5. Sedentary husbandry system

Cattle will be randomly allocated into three groups (2 case groups; 1 control group) using
permuted block randomization process. The three groups will differ according to the
treatment intervention and protocol (allocated group and treatment intervention details are
provided in Fig. 10). The vaccines used in the trial will be administered as:
    - High potency FMD vaccine (≥6 PD50)
    - Normal commercially supplied FMD vaccine (≥3 PD50)
Then, each of the above case group will be split into two further groups treated with
different protocols, as:
    - Protocol 1 (single full dose, 2ml)
    - Protocol 2 (first shot with half dose, 1ml, plus booster at 44 days with half dose, 1ml)
Each of the five treatment groups will consist of 20 animals, homogenously distributed
according to four age/groups: 6-12 months; 1-2 years old; 2-3 years old; >4 years old (5
animals each). Animals will be monitored until the next FMD cases are recorded. Blood
samples will be collected at day 0 (prior to immunisation), and then weekly. Immune
response and antibody rate will be analysed. Participatory investigation will be used to
assess if the observed benefits will be sufficient that owners would be prepared to pay. For
this purpose, a scoring system will be developed using results obtained from the
Participatory Epidemiological survey. Agreement between immune response rate,
protection gained and the participative scoring system will be analysed using the Kendall’s
Coefficient of Concordance (W). Furthermore, Willingness to Pay (WTP) study will be
conducted at the end of the trial.




                     Figure 10 – Groups allocation according to treatment intervention.


SECTION 5 – PROJECT PARTNER INSTITUTIONS
Laboratoires Central Vétérinaire du Mali (LCV), Bamako – Mali
         The LVC is the result of an intensive and lasting collaboration between Mali and the
United States of America. Since 1979, the LVC has excelled in research, surveillance
activities, vaccine production and diagnoses of livestock diseases within the countries with
the ultimate aim of control and eradication of infectious diseases. The LVC has an ongoing
collaboration with international organisations including CIRAD, IAEA, WRLFMD and FAO and
had successfully carried out surveillances, researches and diagnostic activities to the above
organisations in the past on CBPP, PPR, RVF, AI and Newcastle disease. The lead researchers
have an established record of delivery on key scientific tasks. The LVC has also facilities for
ELISA and RT-PCR and is currently building capacity for FMD diagnosis.
Dr. Saidou Tembley (Director)
Dr. Mamadou Niang (Scientific coordinator)
Dr. Abdallah Traore (Vétérinaire et Ingénieur d’Élevage/Attaché de recherche)
Direction Nationale des Services Vétérinaires
Direction Nationale des Industrie et des Productions Animales
Direction Nationale des Eaux et Forets
ECTAD-FAO
Representation OIE pour l’Afrique de l’Ouest et du Centre
       All the above governmental agencies and organisations will help in the
implementation of the survey, concerning the logistic and field support.

World Reference Laboratory for FMD (WRLFMD), Institute for Animal Health (IAH),
Pirbright laboratory – UK
        The IAH is a world-leading centre of excellence for research into exotic and endemic
infectious diseases of farm animals sponsored by the Biotechnology and Biological Science
Research Council (BBSRC). The IAH research extends from fundamental through strategic to
applied sciences, from genes all the way through to animal populations. The FMD research
programme at IAH, probably the biggest on FMD worldwide, is concerned with both
improving disease preparedness in developed countries and working towards international
FMD control, including eventual eradication from developing countries. In addition to
fundamental and strategic research, the IAH Pirbright Laboratory provides a vital service in
real time at times of outbreaks of disease, both nationally and internationally. Specifically,
the Institute provides Reference Laboratories in respect of 10 exotic viral diseases of
livestock. These Reference Laboratories work on behalf of national (DEFRA) and
international organisations (FAO, OIE and EU). The WRLFMD at IAH provides a diagnostic
service to the world and maintains a global surveillance for FMD to warn of its presence and
help prevent its spread to neighbouring countries and trading partners. Research at IAH
using samples of FMDV from Africa, Asia and South America has provided a unique insight
into the evolution and natural history of the virus in its natural environment.
Dr. David Paton (Head of FMD Programme)
Dr. Don King (Head of Molecular and Characterisation Group)
Dr. Nick Knowles (Senior Molecular Biologist)
Dr. Satya Parida (Head of FMD Vaccine Differentiation Group)
SECTION 6 – BUDGET†
                                                                Unit       Cost/Unit ($)      Total Cost ($)
Consumable
                 Description   1. Sampling kits:                  2             4315.86            8631.73
                               - Venoject® tubes
                               - Venoject® needles
                               - Venoject® holders
                               - Salivette
                               2. FMD vaccines                   80                 1.65                132
Equipment
                 Description   1. NSP ELISA kit (PrioCHECK®)     21             1160.91           24379.11
                               2. LPB ELISA kit (IAH)             3             4828.84           14486.52
                               3. GPS devices                     9               142.2             1279.8
Travel Expenses
                                                         *
             Description       1. IAH staff visiting Mali         2                2200               4400
                                                            *
                               2. LCV staff training in IAH       1                2200               2200
Other
                 Description   1. Fuel for survey                 9                4620              41580
                               - 20l/210 days (0.7£/l)
                               2. Shipment to WRLFMD              2                 500               1000
                               3. Miscellaneous                                    1000               1000

Total Cost (£)                                                                                    99087.14
†
    Calculated at exchange rate of £/$ 1.6548
*
    Total cost include air fare (Air France London Heathrow/Bamako ~1200$) plus DSA (1000$)


SECTION 7 – ANNEXES
                                                           ANNEX I – Survey Time-Frame

                                                 Month 1             Month 2             Month 3             Month 4                Month 5             Month 6
Location                Activity
                                            W1   W2  W3    W4   W1   W2  W3    W4   W1   W2   W3   W4   W1   W2  W3       W4   W1   W2  W3    W4   W1   W2   W3   W4

Bamako      Initial Training

            1     Implem.          Team A
Bamako
            QC & Data Entry

            2     Implem.          Team B
Gao
            QC & Data Entry

            3     Implem.          Team C
Kayes
            QC & Data Entry

            4     Implem.          Team D
Kidal
            QC & Data Entry

            5     Implem.          Team E
Koulikoro
            QC & Data Entry

            6     Implem.          Team F
Mopti
            QC & Data Entry

            7     Implem.          Team G
Ségou
            QC & Data Entry

            8     Implem.          Team H
Sikasso
            QC & Data Entry

            9     Implem.          Team I
Timbouct.
            QC & Data Entry



CVL         Preparation & Dispatching
CVL         Sample Testing                                                                                   Until Completed
IAH         Sample Testing                                                                                   Until Completed
                                                            ANNEX I – Survey Time-Frame

                                                Month 7               Month 8             Month 9             Month 10             Month 11
Location              Activity
                                          W1    W2  W3      W4   W1   W2  W3    W4   W1   W2   W3   W4   W1   W2   W3    W4   W1   W2   W3    W4

            1     Implem.        Team A
Bamako
            QC & Data Entry

            2     Implem.        Team B
Gao
            QC & Data Entry

            3     Implem.        Team C
Kayes
            QC & Data Entry

            4     Implem.        Team D
Kidal
            QC & Data Entry

            5     Implem.        Team E
Koulikoro
            QC & Data Entry

            6     Implem.        Team F
Mopti
            QC & Data Entry

            7     Implem.        Team G
Ségou
            QC & Data Entry

            8     Implem.        Team H
Sikasso
            QC & Data Entry

            9     Implem.        Team I
Timbouct.
            QC & Data Entry

            Case group 1
Vaccine
            Case group 2
trial
            Control group

CVL         Preparation & Dispatching
CVL         Sample Testing                Until completed
IAH         Sample Testing                Until completed
                                                                NOTE

Training Activities – team role; survey implementation; participatory investigation; data entry and management; diagnostic analysis
Field Implementation – sera, saliva/nasal, probang, tissue sampling and participatory investigation
Field Implementation – monitoring and animal movement data recording
Database – data entry and storage of survey data
Dispatching – samples and forms preparation for dispatching to LCV and WRLFMD
Testing – sera, saliva/nasal, probang and clinical samples testing; serology and phylogentic characterisation; vaccine matching
Vaccine Trial – day 0, blood collection and inoculation
Vaccine Trial – booster inoculation (Protocol 2 groups)
Vaccine Trial – daily monitoring and weekly blood sampling
    ANNEX II – Participatory Epidemiology form – Questionnaire

SECTION 1 – GENERAL INFORMATION


 Date                                         Team ID
 Region                                       Site ID
 District                                     Latitude (N)
 Location                                     Longitude (E)


1.1 OWNER

 Name                                          Village
 Sex                                           Tribe


1.2 HERD

                Type                    Grazing                     Production
 Settled                Open                             Dairy
 Semi-nomadic           Enclosed                         Beef
 Nomadic                Open/Enclosed                    Breeding

                                        Structure
 Species
 Breed                  Male                             Young
 Size                   Female                           Adult

 Species
 Breed                  Male                             Young
 Size                   Female                           Adult

 Species
 Breed                  Male                             Young
 Size                   Female                           Adult

 Species
 Breed                  Male                             Young
 Size                   Female                           Adult

 Species
 Breed                  Male                             Young
 Size                   Female                           Adult
       ANNEX II – Participatory Epidemiology form – Questionnaire

SECTION 2 – EPIDEMIOLOGICAL INFORMATION
2.1 DISEASE CHARACTERISTICS AND EVENTS

      Has he/she ever had FMD in his/her herd?                   Can the owner report signs consistent with FMD?


Q1.                                              YES   NO   Q12. What are the clinical signs of the
                                                            disease?

Q2. What the name do you call FMD in
your dialect?
                                                            Q13. Fall in milk production                     YES     NO
Q3. Does the owner consider FMD to be            YES   NO
an important problem?
                                                            Q14. Nasal discharge                             YES     NO


Q4. Year of the last outbreak                               Q15. Vesicles (mouth, feet, teats)               YES     NO
Q5. No of animal affected
Q6. Year of the last FMD vaccination                        Q16. Lameness                                    YES     NO


Q7. Have there been any FMD outbreaks            YES   NO   Q17. Heat intolerance                            YES     NO
in the area in the last two years?

                                                            Q18. What species/breeds are affected
                                                            most during outbreak?
Q8. (If YES) How many months ago?
                                      Region                                                           %
                                      District
                                 Location                   Q19. What age groups are affected most          Young   Adult
                                                            during outbreak?

Q9. How many infected animals?                                                                         %


Q10. How frequent does the outbreak occur?                  Q20. Do you encounter mortalities during         YES     NO
                                                            outbreak?
                                Every year
                             Occasionally                                                              %
                                Seasonally
                                                            Q21. Does your animal have contact with          YES     NO
                                                            wildlife during grazing?
Q11. If seasonally, please specify
                                Short rain                                                   Infrequent
                                 Long rain                                                  Occasionally
                                     Cold dry                                                    Frequent
                                 Long dry                                                         Species
       ANNEX II – Participatory Epidemiology form – Questionnaire
2.2 DISEASE CONTROL

  Has he/she ever used vaccines for FMD control?                 What FMD control policies are in place?


Q22. Are your animals vaccinated?           YES   NO   Q29. How do you confirm FMD outbreaks?
                                                                                       Clinical signs
Q23. Date of the last vaccination                                              Laboratory diagnosis


Q24. Vaccine producer                                  Q30. Who do you report to during FMD outbreak?
                                                                                 Community leader
Q25. Type of vaccine                                                                          Police
                                        A                                                      NGO
                                       O                                                       DVO
                                    SAT 1
                                    SAT 2              Q31. How urgent does the authority respond?
                                                                                        Very urgent
Q26. Where do you obtain the vaccine?                                                        Urgent
                                                                                         Not urgent
Q27. Is the vaccine only used for           YES   NO
outbreak control?
                                                       Q32. Do you receive incentives for animal        YES   NO
                                                       health from Government or NGO?

Q28. Number of inoculation per year
       ANNEX II – Participatory Epidemiology form – Questionnaire

SECTION 3 – ANIMAL MOVEMENT INFORMATION
3.1 MOVEMENT PATTERNS

                 Pattern of movement                                                Point location

Q33. Type of movement                                      Q42. Do you cross country borders?                YES   NO
                             Long distance      YES   NO   Q43. (If YES) Please, specify where
                            Short distance      YES   NO                                          Region
                                 Seasonal       YES   NO                                          District
                                                                                                 Location
Q34. If seasonal, please specify
                                    Long rain   YES   NO   Q44. Do you have specific grazing areas?          YES   NO
                                   Short rain   YES   NO   Q45. (If YES) Please, specify where
                                     Cold dry   YES   NO                                         Region
                                     Long dry   YES   NO                                         District
                                                                                                Location
Q35. How far you travel in one day?
                                 1-2 km                    Q46. Do you have specific watering points?        YES   NO
                                 3-5 km                    Q47. (If YES) Please, specify where
                                6-10 km                                                          Region
                                 >10 km                                                         District
                                   More                                                        Location

Q36. How many animals do you move?                         Q48. Do you have specific stop-points?            YES   NO
                                                           Q49. (If YES) Please, specify where
Q37. Herd/flock composition                                                                      Region
                       Male           Young                                                      District
                                       Adult                                                   Location
                        Female        Young
                                       Adult               Q50. Do you have specific market?                 YES   NO
                                                           Q51. (If YES) Please, specify where
Q38. Reason for movement                                                                          Region
                     Grazing/watering                                                             District
                                Trade                                                            Location
                            Slaughter
                                                           Q52. Are there areas where herds/flocks           YES   NO
Q39. Do you have contact with other             YES   NO   congregate?
herds/flocks?                                              Q53. (If YES) Please, specify where
                                                                                                  Region
Q40. Do you have contact with                   YES   NO                                          District
herds/flocks coming from abroad?                                                                 Location
Q41. (If YES) Please, specify
                                From where                 Q54. Do you have contact with wildlife            YES   NO
                                    Region                 during movement?
                                    District               Q55. (If YES) Please, specify
                                   Location                                                        Region
                                                                                                  District
                                                                                                 Location
                                                                                                  Species
                            ANNEX II – Participatory Epidemiology form – Mapping
3.2 GRAZING/WATERING RELATED MOVEMENT
                             ANNEX II – Participatory Epidemiology form – Mapping
3.3 TRADE REALTED MOVEMENT
                             ANNEX II – Participatory Epidemiology form – Matrix Scoring

 INDICATORS                   FMD              CBPP              PPR               LSD     Mastitis
                Coughing

               Salivation

                Abortion

               Lameness

                Mortality

Reduced milk production

     Loss of body weight

             Skin lesions

             Teat lesions

        Hair over growth

                  Panting

              Seek shade

        Decrease fertility

  Decrease market value

   Disease affect wildlife

     Transmitted by tick

Transmitted with contact
                           ANNEX II – Participatory Epidemiology form – Proportional Pilling

              AGE GROUP                   FMD            CBPP            PPR            LSD    Mastitis
                                Dead
                 Sick
  Calves                      Survival
(0-2 years)
                 Healthy
                                Dead
                 Sick
 Weaner                       Survival
(2-3 years)
                 Healthy
                                Dead
                 Sick
  Young                       Survival
(3-4 years)
                 Healthy
                                Dead
                 Sick
  Adult                       Survival
(>4 years)
                 Healthy
                            ANNEX II – Participatory Epidemiology form – Seasonal Calendar

                                                                     SEASONS

                                    Long rain           Short rain                 Cold dry           Long dry
                               M       A        M   J       J        A         S      O       N   D      J       F

FMD


CBBP


PPR


LSD


Mastitis


Rainfall


Increased animal movement


Wildlife contact
                               ANNEX III – Sample Database – Serum

No.   Owner’s Name   Species       Sex   Age   Vaccinated   Blood No.   Serum No.   Team_ID   Remarks/Comments


1                                              Yes   No
2                                              Yes   No
3                                              Yes   No
4                                              Yes   No
5                                              Yes   No
6                                              Yes   No
7                                              Yes   No
8                                              Yes   No

9                                              Yes   No
10                                             Yes   No
11                                             Yes   No
12                                             Yes   No
13                                             Yes   No
14                                             Yes   No

15                                             Yes   No
16                                             Yes   No
17                                             Yes   No
                          ANNEX III – Sample Database – Saliva/Nasal

No.   Owner’s Name   Species     Sex   Age   Vaccinated   Saliva No.   Nasal No.   Team_ID   Remarks/Comments


1                                            Yes   No
2                                            Yes   No
3                                            Yes   No
4                                            Yes   No
5                                            Yes   No
6                                            Yes   No
7                                            Yes   No
8                                            Yes   No

9                                            Yes   No
10                                           Yes   No
11                                           Yes   No
12                                           Yes   No
13                                           Yes   No
14                                           Yes   No

15                                           Yes   No
16                                           Yes   No
17                                           Yes   No
                          ANNEX III – Sample Database – Probang

No.   Region   Location       Latitude      Longitude     Species   Sex   Age   Probang No.   Team_ID


1
2
3
4
5
6
7
8

9
10
11
12
13
14

15
16
17
                                                  ANNEX III – Sample Database – Clinical

                                                                                                                                               Sample’s
No.       Owner’s name             Species        Sex    Age   Vaccinated     Sample No.     Team_ID       Clinical Signs*    Age of lesions
                                                                                                                                                type*


1                                                               Yes     No                               FM ND VS LS                           ES    VF
2                                                               Yes     No                               FM ND VS LS                           ES    VF
3                                                               Yes     No                               FM ND VS LS                           ES    VF

4                                                               Yes     No                               FM ND VS LS                           ES    VF
5                                                               Yes     No                               FM ND VS LS                           ES    VF
6                                                               Yes     No                               FM ND VS LS                           ES    VF

7                                                               Yes     No                               FM ND VS LS                           ES    VF
8                                                               Yes     No                               FM ND VS LS                           ES    VF
9                                                               Yes     No                               FM ND VS LS                           ES    VF
10                                                              Yes     No                               FM ND VS LS                           ES    VF
11                                                              Yes     No                               FM ND VS LS                           ES    VF
12                                                              Yes     No                               FM ND VS LS                           ES    VF
13                                                              Yes     No                               FM ND VS LS                           ES    VF
14                                                              Yes     No                               FM ND VS LS                           ES    VF
15                                                              Yes     No                               FM ND VS LS                           ES    VF
16                                                              Yes     No                               FM ND VS LS                           ES    VF
17                                                              Yes     No                               FM ND VS LS                           ES    VF
*Note (FM = Fall in milk production; ND = Nasal discharge; VS = Vesicles; LS = Lameness; ES = Epithelium/Skin; VF = Vesicular fluid)
                   ANNEX IV – Sampling Site Code-List

SITE    REGION       ID_NAME    LATITUDE LONGITUDE       ID_0   ID_1 TEAM_ID
  1     Bamako         BAM01    12.616079    -8.056296   146    1844      TA
  2     Bamako         BAM02    12.612811    -7.949557   146    1844      TA
  3     Bamako         BAM03    12.519505    -7.933945   146    1844      TA
  4     Bamako         BAM04    12.557990    -7.994939   146    1844      TA
  5     Bamako         BAM05    12.661824    -7.945200   146    1844      TA
  6     Bamako         BAM06    12.700083    -7.951315   146    1844      TA
  7     Bamako         BAM07    12.577950    -8.053754   146    1844      TA
  8     Bamako         BAM08    12.643671    -7.912888   146    1844      TA
  9     Bamako         BAM09    12.578004    -7.932709   146    1844      TA
 10     Bamako         BAM10    12.628060    -7.993850   146    1844      TA
 11     Bamako         BAM11    12.658193    -8.043589   146    1844      TA
 12         Gao        GAO01    17.680524     4.150852   146    1845      TB
 13         Gao        GAO02    15.967455     2.992012   146    1845      TB
 14         Gao        GAO03    15.564380    -0.056243   146    1845      TB
 15         Gao        GAO04    14.959768     0.901060   146    1845      TB
 16         Gao        GAO05    17.932446     3.571432   146    1845      TB
 17         Gao        GAO06    15.690341     2.362207   146    1845      TB
 18         Gao        GAO07    15.942263     0.145294   146    1845      TB
 19         Gao        GAO08    16.986719     0.109757   146    1845      TB
 20         Gao        GAO09    17.075912     1.379712   146    1845      TB
 21         Gao        GAO10    16.118609     3.596624   146    1845      TB
 22         Gao        GAO11    17.856870     2.563744   146    1845      TB
 23       Kayes         KAY01   15.085729   -10.359848   146    1846      TC
 24       Kayes         KAY02   15.136113    -8.873509   146    1846      TC
 25       Kayes         KAY03   14.556693    -9.881196   146    1846      TC
 26       Kayes         KAY04   12.868816   -10.662154   146    1846      TC
 27       Kayes         KAY05   13.649774   -10.284271   146    1846      TC
 28       Kayes         KAY06   12.984707    -8.680142   146    1846      TC
 29       Kayes         KAY07   15.236882   -11.493496   146    1846      TC
 30       Kayes         KAY08   12.339780   -10.410232   146    1846      TC
 31       Kayes         KAY09   13.598876   -11.926900   146    1846      TC
 32       Kayes         KAY10   13.649774    -9.755235   146    1846      TC
 33       Kayes         KAY11   12.415357   -11.065229   146    1846      TC
 34        Kidal        KID01   18.486674     4.050083   146    1847      TD
 35        Kidal        KID02   18.159176     3.495855   146    1847      TD
 36        Kidal        KID03   18.184368     1.732402   146    1847      TD
 37        Kidal        KID04   18.537059     1.001829   146    1847      TD
 38        Kidal        KID05   18.612635     2.009516   146    1847      TD
 39        Kidal        KID06   18.889749     3.017204   146    1847      TD
 40        Kidal        KID07   18.353743     0.400332   146    1847      TD
 41        Kidal        KID08   18.940134     2.437783   146    1847      TD
 42        Kidal        KID09   18.941497     0.499392   146    1847      TD
 43        Kidal        KID10   16.017840     1.631633   146    1847      TD
 44        Kidal        KID11   18.347139     2.718330   146    1847      TD
 45    Koulikoro       KOU01    14.959768    -8.470434   146    1848      TE
                 ANNEX IV – Sampling Site Code List

46   Koulikoro       KOU02     15.262074   -6.530635   146   1848   TE
47   Koulikoro       KOU03     13.977273   -8.142935   146   1848   TE
48   Koulikoro       KOU04     13.423045   -8.193320   146   1848   TE
49   Koulikoro       KOU05     13.649774   -7.790245   146   1848   TE
50   Koulikoro       KOU06     12.440549   -7.387170   146   1848   TE
51   Koulikoro       KOU07     12.390165   -8.294088   146   1848   TE
52   Koulikoro       KOU08     15.463612   -7.487938   146   1848   TE
53   Koulikoro       KOU09     13.397852   -7.160440   146   1848   TE
54   Koulikoro       KOU10     13.952080   -7.160440   146   1848   TE
55   Koulikoro       KOU11     15.388035   -8.470434   146   1848   TE
56      Mopti        MOP01     14.531501   -5.145065   146   1849   TF
57      Mopti        MOP02     14.002465   -3.860263   146   1849   TF
58      Mopti        MOP03     14.688534   -3.645508   146   1849   TF
59      Mopti        MOP04     15.441388   -3.440784   146   1849   TF
60      Mopti        MOP05     13.826120   -4.540453   146   1849   TF
61      Mopti        MOP06     15.071565   -2.232256   146   1849   TF
62      Mopti        MOP07     14.959768   -4.842759   146   1849   TF
63      Mopti        MOP08     15.211690   -1.240276   146   1849   TF
64      Mopti        MOP09     14.682654   -2.298348   146   1849   TF
65      Mopti        MOP10     15.513996   -4.490068   146   1849   TF
66      Mopti        MOP11     13.523813   -3.482381   146   1849   TF
67      Ségou         SEG01    14.682654   -5.674101   146   1850   TG
68      Ségou         SEG02    15.110921   -5.220642   146   1850   TG
69      Ségou         SEG03    13.196315   -4.263338   146   1850   TG
70      Ségou         SEG04    13.574198   -4.464876   146   1850   TG
71      Ségou         SEG05    13.775735   -5.976407   146   1850   TG
72      Ségou         SEG06    13.019970   -6.883326   146   1850   TG
73      Ségou         SEG07    15.362139   -5.923881   146   1850   TG
74      Ségou         SEG08    13.800927   -6.606212   146   1850   TG
75      Ségou         SEG09    13.498621   -5.497756   146   1850   TG
76      Ségou         SEG10    14.061154   -5.197443   146   1850   TG
77      Ségou         SEG11    12.753566   -4.979512   146   1850   TG
78     Sikasso         SIK01   11.609207   -7.689476   146   1851   TH
79     Sikasso         SIK02   11.709976   -6.908518   146   1851   TH
80     Sikasso         SIK03   11.886321   -7.160440   146   1851   TH
81     Sikasso         SIK04   11.684784   -5.346603   146   1851   TH
82     Sikasso         SIK05   11.987090   -5.976407   146   1851   TH
83     Sikasso         SIK06   12.113051   -5.195449   146   1851   TH
84     Sikasso         SIK07   12.667279   -5.699293   146   1851   TH
85     Sikasso         SIK08   11.472395   -6.518239   146   1851   TH
86     Sikasso         SIK09   10.248829   -6.152753   146   1851   TH
87     Sikasso         SIK10   10.651904   -7.815437   146   1851   TH
88     Sikasso         SIK11   10.752673   -7.059671   146   1851   TH
89 Tombouctou        TOM01     16.496491   -4.187762   146   1852    TI
90 Tombouctou        TOM02     16.017840   -2.902960   146   1852    TI
91 Tombouctou        TOM03     17.554563   -5.497759   146   1852    TI
                     ANNEX IV – Sampling Site Code List

 92   Tombouctou         TOM04    16.118609    -2.122003   146   1852    TI
 93   Tombouctou         TOM05    15.110921    -0.686048   146   1852    TI
 94   Tombouctou         TOM06    16.068224    -4.464876   146   1852    TI
 95   Tombouctou         TOM07    16.546876    -5.195449   146   1852    TI
 96   Tombouctou         TOM08    15.943290    -0.852024   146   1852    TI
 97   Tombouctou         TOM09    15.586675    -2.313904   146   1852    TI
 98   Tombouctou         TOM10    16.546876    -0.761624   146   1852    TI
 99   Tombouctou         TOM11    16.412173    -3.456393   146   1852    TI
100     Soudanian        SOU01    12.650000    -8.000000   146   1844   TA
101     Soudanian        SOU02    13.034900    -9.489500   146   1848   TE
102     Soudanian        SOU03    12.862734    -7.559849   146   1848   TE
103     Soudanian        SOU04    11.933333    -8.416667   146   1848   TE
104     Soudanian        SOU05    10.966667    -5.700000   146   1851   TH
105     Soudanian        SOU06    12.482200    -6.791100   146   1848   TE
106     Soudanian        SOU07    11.313500    -5.669700   146   1851   TH
107     Soudanian        SOU08    10.550000    -5.766667   146   1851   TH
108     Soudanian        SOU09    12.391726    -5.464209   146   1851   TH
109     Soudanian        SOU10    12.353600    -4.776100   146   1851   TH
110     Soudanian        SOU11     11.18333    -8.150000   146   1851   TH
111       Sahelian        SAE01   15.229317    -9.592771   146   1846   TC
112       Sahelian        SAE02   14.540000    -9.192100   146   1846   TC
113       Sahelian        SAE03   14.441700   -11.434100   146   1846   TC
114       Sahelian        SAE04   15.168751    -7.284660   146   1848   TE
115       Sahelian        SAE05   14.489094    -4.192827   146   1849   TF
116       Sahelian        SAE06   14.063998    -3.075387   146   1849   TF
117       Sahelian        SAE07   13.904500    -4.559900   146   1849   TF
118       Sahelian        SAE08   14.995100    -2.951700   146   1849   TF
119       Sahelian        SAE09   13.437100    -6.215700   146   1850   TG
120       Sahelian        SAE10   14.252600    -5.993000   146   1850   TG
121       Sahelian        SAE11   13.303346    -4.895615   146   1850   TG
122       Saharian        SAH01   16.271667    -0.044722   146   1845   TB
123       Saharian        SAH02   15.659700     0.502200   146   1852    TI
124       Saharian        SAH03   16.770456    -3.005588   146   1845   TB
125       Saharian        SAH04   15.711800    -4.911800   146   1852    TI
126       Saharian        SAH05   15.932200    -3.990600   146   1852    TI
                    ANNEX IV – Sampling Site Map Location

TEAM C – Kayes Region




                                                            Target sites
                                                            Target towns
ANNEX IV – Sampling Site Map Location

                        TEAM E – Koulikoro Region




                                    Target sites
                                    Target towns
                          ANNEX IV – Sampling Site Map Location

TEAM H – Sikasso Region




                                                                  Target sites
                                                                  Target towns
        ANNEX IV – Sampling Site Map Location

TEAM A – Bamako District




                       Target sites
                       Target towns
             ANNEX IV – Sampling Site Map Location

TEAM G –Ségou Region




                                                     Target sites
                                                     Target towns
                       ANNEX IV – Sampling Site Map Location

TEAM F –Mopti Region




                                                               Target sites
                                                               Target towns
               ANNEX IV – Sampling Site Map Location



                                                       TEAM I –Tombouctou Region




Target sites
Target towns
                     ANNEX IV – Sampling Site Map Location

TEAM B –Gao Region




                                                             Target sites
                                                             Target towns
                       ANNEX IV – Sampling Site Map Location




TEAM D –Kidal Region




                                                               Target sites
                                                               Target towns

				
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