Operational area
The exercise took place in 12 Ghana Armed Forces barracks in the country from 7 to 28 April 2011. These barracks are located in the coastal, tropical rain forest and northern savannah belts (Figure 1).
Education
Seminars on pandemic avian influenza were conducted to sensitize all ranks of GAF personnel from the Army, Navy and Air Force, civilian employees of the Ministry of Defense, spouses and dependants of the uniformed personnel and civilians living within or near the catchment areas of the military barracks. The educational seminars were held in all garrisons. Attendance sheets were provided at the entrance of seminar rooms for the attendees to register their names, units, ranks and signatures. Within the Western region, the Army, Navy and Air Force were brought together at the second battalion (2BN) facilities for the seminar, while in the far north, troops from detachments in Bazua and Bawku came together for the seminar in Bawku. In the Northern region, separate seminars were held for the Air Force and the Airborne Force at the Barwah Barracks.
Education was done in four parts; showing a dramatization of a hypothetical pandemic, conducting didactic lectures, showing an AI biosecurity educational program, and question and answer sessions. The dramatization “Fatal Contact,” was developed in the US and depicts an avian influenza pandemic starting in China and spreading to the US and Africa, leading to many deaths. The didactic lectures were conducted by military and civilian veterinarians, public health officials and scientists on the different types of avian influenza viruses, factors contributing to emerging and re-emerging zoonotic diseases, signs and symptoms of bird influenza, surveillance techniques, outbreaks and outbreak zoning. An AI biosecurity video, developed by the Canadian Poultry Feed Agency, was screened. The final part was an interactive section where the troops were allowed to ask questions and were answered by the resource persons.
Sampling
Poultry production in Ghana can be classified into three categories by installed capacity (bird population), marketing system and level of integration of its operations. These categories include commercial farms, semi-commercial farms and backyard/village poultry producers. On animal health services delivery, the GAF has one veterinary clinic that provides regulatory, field and clinical services in the capital city Accra.
A cross sectional study using an active avian influenza surveillance approach was conducted within 12 military barracks. A convenience sampling method based on the availability and consent of a household member was applied. Where a member or owner of the household was available and consented to the study, a maximum of 30-60 minutes was allotted for animal sampling and the individual interviewed. Households were then classified according to the installed capacity. Using criteria for eligibility based on birds whether apparently healthy or with respiratory signs or gastroenteritis or nervous illness, subjects were conveniently selected for either tracheal or cloacal swabbing. Verbal consent was obtained from all poultry owners to take swabs from their birds. Backyard poultry owners and household members were interviewed. A semi-structured questionnaire was administered and information on demographics, basic hygiene practices, quantity of poultry owned, poultry death reporting, practices when deaths occurred, knowledge of the cause of death, and knowledge of avian influenza was collected.
A total number of 680 birds from 102 households were sampled. At Teshie and Burma Camp both tracheal and cloacal samples were taken from each bird, while at the other sites cloacal samples were taken from ducks and tracheal samples from any other birds. All samples collected were appropriately labelled and stored in dry shippers containing liquid nitrogen and transported to the National Influenza Centre (NIC) at the Noguchi Memorial Institute for Medical Research (NMIMR) for processing. At the laboratory, all samples were transferred to -70°C for storage until ready for processing.
Sample processing
Processing of samples took place in the Biosafety level-3 laboratory. Samples from 14 fowls found with pox-like lesions and respiratory abnormalities were separated and processed. The remaining samples were pooled according to swab site (tracheal or cloacal), bird type and household. The pools ranged from a single sample to 11. In all 94 pools obtained, there were 58 tracheal, 35 cloacal and 1 feather thallus (domesticated wild bird) samples.
RNA extraction
Viral RNA was extracted from 140 μl of pooled or individual avian tracheal or cloacal swabs using the QIAmp viral RNA mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. RNA was eluted in 60 μl of elution buffer and 8 μl used as template for real time Reverse Transcription-Polymerase Chain Reaction (rRT-PCR).
Real time RT-PCR
Two rRT-PCR protocols described by the Centers for Disease Control and Prevention (CDC), Atlanta, Georgia, USA and Spackman et al., 2002 for influenza viruses, were used to screen all the samples [16, 17]. RNA was amplified by rRT-PCR using the AgPath-ID One-Step RT-PCR Kit (Ambion, Austin, Texas, USA) in a 25 μl reaction mixture or Qiagen One Step RT-PCR Kit (Hilden, Germany). The RT step conditions for all primer sets were 30 min at 50°C and 15 min at 94°C. A two-step PCR cycling protocol was used for the matrix gene primer set as follows: 45 cycles of 94°C for 0 sec and 60°C for 30 sec and for the AgPath-ID as 45 cycles of 95°C for 2 min and 55°C for 30 sec. All temperature transition rates were set at the maximum transition rate of 20. Fluorescence data were acquired at the end of each annealing step.
The rRT-PCR assays were performed on real time PCR instruments (Applied Biosystems, Singapore) with SDS software version 1.4 (Applied Biosystems, Singapore). Results of the rRT-PCR assays were determined by the analyses of cycle threshold values generated by SDS auto analysis on samples against reference positive and negative controls.
Data analysis
Demographic data was entered in an electronic database file (Microsoft Excel, 2003). Basic analyses were performed using Microsoft Excel to generate frequencies, graphs and tables.
Ethical approval
Military to Military influenza surveillance is considered a public health activity and is conducted with the Ghana Armed Forces Health Directorate, Veterinary Services Directorate and Ghana Health Service in a manner consistent with Ghanaian standards. It has served to facilitate identification of potential sources of avian influenza around military bases, through educational seminars, and active and passive processing of bird samples. This activity is under the Surveillance for influenza virus in acute respiratory illness in Ghana project which was initiated in 2007 under the integrated disease surveillance and response system of the Ghana Health Service with ethical clearance 015/06-07 from the Noguchi Memorial Institute of Medical Research Institutional Review Board.