The aim, design and setting of the study
Abuja is the capital of Nigeria, located in the central region of the country, north of the confluence of rivers Niger and Benue. It has a projected population of 3,564,126 in 2016 (23). Abuja consists of six local councils, comprising the city of Abuja; Abuja Municipal Area Council (AMAC), Abaji, Gwagwalada, Kuje, Bwari and Kwali. The projected population of 2016 for Gwagwalada was 402,000 while that of Kuje was 246, 400(24).
We conducted a cross-sectional study using qualitative (PDS) and quantitative study methods. The entire study period was between from 1st of December 2020 to 30th January, 2022.
The general objective of the study was to determine the presence of ATB, the knowledge, attitude, and practices (biosecurity measures) observed by poultry farmers that could enable transmission of ATB within Gwagwalada and Kuje LGAs. Specific objectives were to detect the presence of ATB in layers within Gwagwalada and Kuje LGAs using participatory disease surveillance (PDS), detect the presence of M. avium infection using avian purified protein derivative (PPD) in layers within Gwagwalada and Kuje LGAs, to determine the presence of gross pathological lesions in layers that tested positive to avian PPD, confirm the presence of M. avium in avian PPD positive layers using polymerase chain reaction within Gwagwalada and Kuje LGAs.
Participatory disease surveillance
Participatory disease surveillance was conducted in Gwagwalada live bird market (LBM) and twelve farms that met the inclusion criteria for sampling and gave their consent to be sampled within Gwagwalada and Kuje LGAs. A pre-advocacy visit was conducted at Gwagwalada LBM and all the poultry farms in various settlements that were sampled. Meeting was arranged with the poultry butchers at the LBM and poultry management staff and workers at the various farms according to the suitability of time, place, and convenience. Cardboards, counters (beans), permanent markers and android phone were used for the study. The geographical coordinates of each meeting point were obtained using an android mobile phone. Each team member was assigned a role (note taker, observer, tool applicator and the facilitator) before moving out. To avoid bias, the PDS Team did not mention ATB during the discussion process. Pictures on ATB lesions on the android phone was presented to the farmers by the facilitator to aid in the identification of the disease. We adapted a check list based on previous PDS studies(25–27) with some modifications to suite the objectives of our study. The following tools were used during the PDS(28): (a) check list consisting of mutual introduction, identification of respondents, sources of chickens and feed, poultry diseases, source of water, biosecurity on farm (foot dip, disinfection of vehicles, presence of other animals on farm and movement on farm), questions and advice. (b) Methods used in data collection were, scoring and ranking, simple ranking, and proportional pilling(28). (c) Visualization includes transect walk/ walk survey around the farms(28).
Our inclusion criteria were all backyard and commercial farms that had layers above 24 weeks old within Gwagwalada and Kuje LGAs while the exclusion criteria were layers 24 weeks old or less during the period of the study.
As part of the biosecurity measures, we ensured that not more than two farms were visited in a day and observed all biosafety measures. We calculated our sample size using the method of Thrusfield for cross sectional surveys (29), a total of 395 layers were sampled. Administration of avian PPD was done using systematic sampling method to select the bird to be tested from twelve farms (10 commercial and 2 backyard poultry) within Gwagwalada and Kuje LGAs. Administration of avian PPD, was conducted by 2 trained research assistants; one research assistant was responsible for restraining the layers properly for the PPD administration while the other assistant was responsible for marking the birds with permanent markers to indicate those layers that were administered avian PPD for easy identification between 48 − 72 hours after administration.
Onderstepoort Biological Products; Avian PPD 2500 with Reg. N0. G4085 (Act 36/1947) from Onderstepoort Veterinary Institute (OVI), South Africa was used for the test. Exactly 0.1ml (containing 2500 International Units (IU) of standard avian purified protein derivative (PPD) was administered using needle of approximately 10 x 0.5mm2 (insulin syringe). The avian PPD was administered intradermally into one of the wattles of the layers, the other wattle was used as control. The test was read between 48 hours to 72 hours. A positive reaction was detected by swelling or induration at the site of inoculation ranging from a small firm nodule approximately 5mm in diameter to gross edema extending into the other wattle and down the neck(30).
Ante-mortem and Post-mortem Inspection
Layers that tested positive after avian PPD administration were bought from the owners and transported to the laboratory for post-mortem inspection. For the post-mortem inspection, visual examination, palpation of tissues and organs and incisions were conducted for nodular and granulomatous lesions. The entire procedures was carried out based on the standard methods of postmortem examination in poultry(31, 32). Factors such as sources of birds, breed, body conformation, predilection site/sites of lesions (specified organ) and body condition score to determine the impact of these factors on the disease picture were obtained.
Tissue Sample collection and Transportation
Infected tissues and organs with gross pathological lesions were collected, packaged using standard procedures and stored (refrigerated at-4OC) in the laboratory at the University of Abuja Veterinary Teaching Hospital throughout the 3 months’ period of sample collection (December 2020 to February 2021). Thereafter, in June 2021, stored samples were packaged using ice packs and cold boxes and were transported to the National Veterinary Research Institute (NVRI), Vom, Plateau State for PCR analysis. Processing of Tissue Samples for PCR
Stored tissue samples were removed from the freezer and thawed before the commencement of the DNA extraction process. ZYMO RESEARCH Quick-DNA™ Tissue/Insect Miniprep Kit; was used for extraction of DNA from the lesions that were collected. The DNA extraction was done according to the manufacturers protocol.
Polymerase chain reaction (PCR)
Two primers (P) were used in the PCR; M. avium F and R; P1 (5´-GCCGCCGAAACGATCTAC) and P2 (5´--AGGTGGCGTCGAGGAAGAC)(33) to define the host range of IS1245 and to generate a 427 bp fragment. PCR amplifications were performed in a Gene Amp PCR system 9700 (PE Applied Biosystems, Foster City, Calif.), according to the method described by Telenti et al.(34): 30 cycles of 1 minute at 94OC, 65 OC and 72OC, with an extension cycle of 10 minutes at72OC.
An advocacy visit was made to each farm and LBM (poultry butchers) for sensitization and fixing of suitable time to ensure full participation of the key informants/focus group discussion where applicable on the subject matter. Team members were trained on application of PDS tools on the field and the various functions of the PDS. Direct observation such as transect walks or walking surveys were used in assessing available infrastructure, farm environment, activities on farm such as slaughter and dressing of chickens, working conditions of farm workers and biosecurity measures; potential drivers of disease (dumping of poultry manure on farm premises, presence of water bodies, animal movements and interactions). Distance examination of animals for signs of disease was conducted(15).
All procedures (administration of avian PPD, antemortem and postmortem inspection) were conducted based on standard procedures. Research assistants were trained before the commencement of sampling. Systematic random sampling was used. Mycobacterium avium positive control obtained from OVI was optimized and used for the PCR analysis.
Data from PDS was analyzed based on the analytical procedure of PDS data(28); simple ranking and proportional pilling were used in obtaining the poultry diseases occurring on farms based on their importance (predominance of occurrence on farm) followed by ranking and scoring. A table was created where all diseases scored on each of the farms were imputed and computed(28). Computation indicated in S1 Table. Microsoft Excel was used in the descriptive analysis of the results.