Samples were collected exclusively in live-bird markets and backyard farms. The latter were preferred to commercial farms because the outbreaks of HPAI (H5N1) reported during 2006–2008 occurred most often in backyard flocks (for 11/12, 4/5, and 2/3 outbreaks in Côte d’Ivoire, Benin, and Togo, respectively) or on small farms (308–7,771 birds per farm) (13,14). Sampling sites were selected in the 3 countries for 1) their density of poultry farms (backyard and commercial, even though we focused on the backyard sector, as in the district of Abidjan and in the Middle-Comoé region in Côte d’Ivoire; Lokossa in Benin; Lomé and the Maritime Province in Togo), 2) the presence of water bodies and the possible contact of domestic birds with wild waterfowl (South-Comoé in Côte d’Ivoire; Malanville in Benin); and 3) their past outbreaks of HPAI (H5N1) (district of Abidjan in Côte d’Ivoire; Lomé and the Maritime Province in Togo).

In Côte d’Ivoire, samples were collected during January 2009–December 2010. Three regions were selected (Figure 1). Specimens were collected in the district of Abidjan (i.e., Bingerville, Marcory, Treichville, Port-Bouet, Koumassi, Yopougon), the Middle-Comoé region (i.e., Agnibilékro, Takikro, Abengourou, Niablé), and the South-Comoé region (i.e., Aboisso, Adiaké). In Benin, samples were collected during November 2008–September 2010 in live-poultry markets in Malanville, Gogounou, and Dérasi in the provinces of Borgou and Alibori in the north of the country (Figure 1). A total of 200 swab samples were collected from birds in Lokossa (Mono Province) in 2009. The specimens from pigs were collected from animals in slaughterhouses in Parakou (Borgou Province).

In Togo, swab specimens from birds were collected in January and March 2009 and during February–December 2010. Locations were live-poultry markets in Adidogomé, Aklakou, Tabligbo, Vogan, Agoé, Akodesséwa, Aného, Tsévié, Adawlato, and Gbossimé, in Lomé and in the Maritime Province (Figure 1).

In each region in Côte d’Ivoire, a minimum of 5 villages were randomly selected among those willing to participate. Birds from live-bird markets were randomly selected before sampling (5 randomly selected birds per vendor, number of vendors randomly selected depending on the total number of specimens to collect in a given market).

At each sampling site, >25 birds were clinically examined, and tracheal and cloacal swab samples were collected at least monthly. In Côte d’Ivoire, nasal swab samples from pigs were collected monthly in 2009 and every 3 months in 2010. In backyard flocks in Côte d’Ivoire, serum was collected every 3 months. Each selected market was visited 1×/month in Togo and 2×/month in Benin.

The samples were collected in viral transport media as described (15) and then stored in liquid nitrogen or on ice during sampling and transportation to the laboratory, which never exceeded 1 day. Swabs were then immediately stored either at –80°C in Côte d’Ivoire and Benin or in liquid nitrogen in Togo before further processing. Serum was stored at –20°C before further processing.

Serum was screened for influenza antibodies by performing ELISAs and/or hemagglutination inhibition (HI) assays. ELISAs were performed by using the FlockChek AI MultiS-Screen Ab Test Kit (Idexx, Westbrook, ME, USA) according to the manufacturer’s instructions. HI assays to detect influenza virus were performed as described (15,16) by using inactivated H5 (A/whooper swan/Mongolia/244/05), H6 (A/turkey/Massachussetts/65), H7 (A/ruddy turnstone/New Jersey/65/85), and H9 (A/duck/Hong Kong Special Administrative Region, People’s Republic of China/Y280/97) antigens and positive- and negative-control serum. Serum samples were screened for Newcastle disease virus (NDV)–specific antibodies by performing HI assays with inactivated reference antigen and positive- and negative-control serum.

Tracheal and cloacal swabs were processed as described (17,18). The samples were screened either individually or in pools of 2 or 5 swabs. RNA was isolated by using the RNeasy mini kit (QIAGEN, Valencia, CA, USA), the QIAmp viral RNA minikit (QIAGEN), or the MagMAXTM-96 AI/ND viral RNA isolation kit (Applied Biosystems/Ambion, Austin, TX, USA) with a Kingfisher Flex magnetic particle processor (Thermo Scientific, Rockford, IL, USA). RNA was eluted in 50 μL of nuclease-free water.

The swab samples from Côte d’Ivoire were tested by using 2-step reverse transcription PCRs (RT-PCRs). The RT step was performed by using random hexamers (Invitrogen, Carlsbad, CA, USA) with 10 μL of extracted RNA and the First-Strand cDNA Synthesis kit (GE Healthcare Europe GmBH, Orsay, France,) according to the manufacturer’s protocol. Next, 5 µL of the cDNA obtained was used as the template for the PCR step. The PCR was performed by using the Gene Amp PCR System 2400 (Perkin-Elmer, Applied Biosystem, Paris, France) as described (14).

The swabs from Benin and Togo were tested by using 1-step RT-PCRs performed with the Qiagen 1-step RT-PCR kit (QIAGEN) with either an ABI 9700, ABI 2720 (Applied Biosystems, Vienna, Austria) or ABI 7500 (Stratagene; Applied Biosystems, Carlsbad, CA, USA) thermocycler. For conventional RT-PCR screenings, we used primers that target the influenza A matrix gene (19) and the following cycling conditions: 1 cycle of 50°C for 30 min; 1 cycle of 95°C for 15 min; 40 cycles of 95°C for 30 sec, 60°C for 30 sec, and 72°C for 1 min; and 1 cycle of 72°C for 10 min. For real-time RT-PCR screenings, we used influenza A primers (18) with the following cycling conditions: 1 cycle of 50°C for 30 min; 1 cycle of 95°C for 15 min; and 40 cycles of 95°C for 10 sec and 60°C for 30 sec.

Swab samples from birds were screened for other avian pathogens. We used PCR to screen for DNA viruses (infectious laryngotracheitis [ILTV], Marek’s disease virus [MDV], and chicken anemia virus [CAV]) and 2-step RT-PCR to screen for RNA viruses (NDV, infectious bronchitis virus [IBV], avian metapneumovirus [aMPV], and infectious bursal disease virus [IBDV]) as described (primers available on request).

We collected 25,136 swab and 1,819 serum samples from birds and 1,610 swab and 457 serum samples from pigs during the 2-year survey in the 3 countries. Of the bird samples, 70% were from live-poultry markets and 30% from backyard flocks (Table 1 and Table 2). Specimens were collected year-round, and monthly samples ranged from 20 to 160 and from 218 to 1,778 per month, from swine and poultry respectively.

The 26,746 total swab samples collected from birds and pigs in Côte d’Ivoire, Benin, and Togo all tested negative for influenza A genome by RT-PCR, irrespective of collection month or host, and the annual prevalence per country was null (95% CI 0.04–4.79%) (Table 1, Table 2, Table 3). To verify that cold-chain or storage problems had not simply degraded our samples’ nucleotides, we screened a subset of 2,427 swab samples collected from birds during early 2009 and 2010 from Benin and Togo for other RNA avian viruses (NDV, IBV, IBDV, or aMPV) and DNA viruses (CAV, ILTV, or MDV). Of the 2,427 samples collected in Benin and Togo, the prevalence of the other viral pathogens ranged from 0 for MDV and IBDV to 4.9% for NDV (119 positive samples), 2.8% for ILTV (68), 2.1% for CAV (51), 1.4% for IBV (34), and 0.3% for aMPV (7) (Figure 2). In addition, 3,330 swab samples collected from birds in Côte d’Ivoire in 2010 were screened for NDV; NDV prevalence ranged from 0.3% to 1.4% depending on time (data not shown). Taken together, these results show the fair quality of our specimens. Cold-chain and sample quality were unlikely to account for the absence of detected influenza virus RNA.

Because influenza virus infection might last only a few days in birds and pigs, we could have missed the virus in the animals sampled. Therefore, we conducted serologic screening, which provides insight into the infection history of an animal’s entire life. None of the serum samples collected in birds in Côte d’Ivoire, Benin, and Togo were positive for influenza antibodies by ELISA or HI assay. Although 16 of 457 pig serum samples from Côte d'Ivoire were weakly positive by ELISA, none were confirmed positive by HI; they most likely were all negative for influenza antibodies. NDV antibodies were detected in 20% of serum samples from birds in Côte d’Ivoire and in 32% of bird serum samples from Togo (data not shown).

Determining the factors contributing to the seasonality of influenza has been difficult because some countries report having influenza activity year-round and others report having 2 peaks of activity or a combination of these patterns. To follow up on recent results (20) seemingly confirming the year-round activity hypothesis that states that influenza spread in the tropics is due to contact rather than aerosol transmission, we compared the average livestock production, temperature, and relative humidity (RH) levels in Côte d’Ivoire, Benin, and Togo with those of Nigeria, Egypt, Vietnam, and Indonesia. Côte d’Ivoire, Benin, and Togo produce significantly less bird and pig meat and fewer bird eggs than do Nigeria, Egypt, Vietnam, or Indonesia (Table 4). West Africa is hot and humid all year, with temperatures ranging from 22°C to 32°C and RH ranging from 63% to 82% (Table 4).