Data Collection

2.4.1 Ethics statement

Ethical approval for the study was obtained from the University of Liverpool research ethics committee (reference RETH000410). A later amendment was made after the study design was adjusted to collect follow-up data on the birds, to allow participants’ names to be recorded. Due to low literacy levels among participants, a statement explaining the study

objectives and outlining sampling methods was read to the farmers before they elected to participate. They were also informed that they could withdraw from the study at any time, and asked to contact their local development agent (DA) in the event of any subsequent problems. DAs are employees of the agricultural extension service provided by the Ethiopian Government whose role is to provide farmers with access to training, research and technologies (http://www.moa.gov.et/policies-and-strategies). Oral permission was obtained for data collection and bird sampling. All data were anonymised, and only very limited personal data was collected from the study participants. Local ethical approval for animal sampling was not required.

Addis Ababa Harari Somalia Eritrea Djibouti Sudan Kenya Oromiya 0 50 100 150 200 kilometers Debre Zeit Jarso Lafin Fedo Aman Bedhasa Afgug Haro Agar Bonnee Abuna Didibe & Kirstana Doyoo Barisoo Horro Ethiopia

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2.4.2 Rapid rural appraisal (RRA)

In January 2011, a rapid rural appraisal (RRA) was conducted in four of the study kebeles; one from each of four market sheds. These were carried out by some of the Ethiopian members of the study team, accompanied by district veterinarians or officials from the local agriculture office. Focus group discussions (FGD) comprised of up to 50 farmers, of which between 20 and 50% were female. Group discussions typically took two hours. Following the FGD, field visits were conducted in each of the kebeles to directly observe village poultry production. Discussions with key informants, such as district administrators and animal extension workers, were used to collect additional information and triangulate the FGD. The results of these were fed back to the rest of the study team and informed questionnaire development and fieldwork planning.

2.4.3 Sample selection

A pilot study was performed in one kebele in the Horro region in April 2011, where questionnaires and sampling techniques were trialled in the field. Sera collected during the pilot study were used in the development of laboratory tests. Questionnaires and data collection techniques were further refined before the study commenced.

Each kebele was visited on four occasions; in May/June and October/November in each year of the study (2011-2012). These visits were timed for before and after the main rainy season, which occurs between June and September. Each visit to the kebele took place over 2-4 days. Lists of farmers in each kebele, grouped by village or area, were compiled by the local DAs. Within each kebele, a number of contiguous sub-areas were selected in order to produce a list of approximately 400-700 households.Systematic random sampling was used to select 104 potential participants in each kebele by selecting every nth name from each list with a starting place chosen at random. The selected names were split into four groups, one for each sampling season, so that different households were selected to

sample on each visit to the kebele. Approximately 30% more names than were required were selected to allow for exclusions or non-participation.

Ethiopian staff were recruited to collect the field samples, and conduct the questionnaires in the local language. Training was provided beforehand, and where necessary in the field if problems were identified. Selected households were visited by the study team, and farmers were interviewed to confirm that they had no exotic birds or vaccinations used in their flock. They also needed to own two indigenous-type birds of at least six months of age in order to be included in the study.

Two birds were selected from each household to be sampled, and where possible one cock and one hen were chosen; otherwise two hens were sampled. If the household owned more than one cock over six months of age, each bird was allocated a number then, using random number tables, one was selected for sampling. Hens were sampled using the same method, but if possible, birds related in the first degree to the cock were excluded, based on the farmer’s knowledge of their birds. This was to minimise the relatedness of birds where possible for the genetics study. Once the team had made the selection from all available birds, they were caught, usually by members of the household, and placed securely in baskets.

2.4.4 Questionnaires

A questionnaire interview was carried out with a member of the household, in order to collect data on the management practices and the disease history of the flock. Interviews were conducted in the local language either with the person with main responsibility for caring for the birds or with the head of the household, and answers recorded in English. A copy of the questionnaires used is in General Appendix A. Basic questions for this study remained the same throughout, but some additional questions were asked for one or more seasons, as complimentary projects by other students were carried out simultaneously.

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Household latitude and longitude were recorded using handheld Global Positioning System (GPS) units. Altitude was not recorded, due to poor agreement between individual units, but was subsequently found from the location data using Google Earth software.

2.4.5 Clinical examination

A form was provided with a checklist of the information which needed to be collected for each bird (General Appendix A) for fieldworkers to complete. This form was labelled with a unique code to identify the household and bird, and all samples collected from that bird were labelled with the same code, in the form of pre-printed stickers. Bird codes were also linked to the household questionnaire.

Information about the clinical history of the two sampled birds was obtained by interviewing a member of the household. A series of photographs were taken of each bird, comprising a lateral, dorsal and frontal view, and close-up shots of the head, feet and skin. These were identified by taking a photograph of the label with the bird’s unique identifying code before commencing the photo series. Photos were also imprinted with the date and time, to help with identification. Photographs were primarily to help with characterisation of the bird phenotypes, but also served a useful role in identifying individuals.

The birds were each then subjected to a clinical examination, and scoring for ectoparasites. The checklist provided comprised a series of tick boxes for fieldworkers to complete, to ensure that birds were examined thoroughly and consistently. Information was collected on various clinical signs exhibited, and also on certain morphological traits, such as feathering, for the genetics study. Birds were weighed in the baskets, and ascribed a body condition score on a 0 – 3 grading system (Gregory and Robins 1998).

A scoring system was also devised for selected ectoparasites. Ticks, fleas or mites were scored by counting the number found on an examination of the most likely places to find them – the naked areas on the face, under the wings or around the vent. Lice were scored

by performing a timed count of 3 areas of the body – one side of the keel, the back and the rump - and a total count of lice under the tail feathers and at the base of the flight feathers of one wing, as described by Clayton and Drown (2001). Birds were also scored for scaly leg mite, by grading the amount of hyperkeratosis; none, mild or severe. If ectoparasites were observed or suspected, a sample of the parasite or a skin scraping from the leg was collected and stored in 70% ethanol.

A 1.5ml blood sample was taken from the wing vein of each bird. This was collected into a syringe which had been flushed with sodium citrate. The sample was gently mixed by inverting the syringe prior to being transferred to eppendorf tubes for storage. In addition, some blood was placed on FTA cards and, for birds sampled in the first two sampling periods, two blood smears were made at the time of sampling and fixed in methanol for 30 seconds. Before releasing sampled birds, they were fitted with a leg ring, the colour and number of which was recorded on the checklist.

Wherever possible, a faecal sample was collected from the basket where the bird had been confined, or where the bird was observed to defecate after release. If it could not be collected from the bird, a sample was taken from the household environment. Fieldworkers were required to record in the checklist whether or not the faecal sample was known to come directly from the sampled bird. Blood and faecal samples were stored and transported in a refrigerated container to the laboratory in Debre Zeit, where plasma was separated from the cell pellets and stored at -20°C. The faecal samples were kept at 4°C until processed. Ectoparasite samples were stored in ethanol at room temperature.

2.4.6 Follow-up data collection

A follow-up visit was carried out for households interviewed during the first three rounds. This visit occurred around 6 months later, at the time of the next visit to the kebele. Farmers took part in a short interview questionnaire (General Appendix A) to ascertain

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what further disease events they had experienced in the intervening period, and the fate of the two previously sampled birds was determined. Surviving birds were re-examined, including body condition scoring and re-weighing, and a second photograph series was taken. If a bird had not survived, farmers were asked to report what had happened to it, and to provide some brief description of signs if it had died from disease.