Prevalence of influenza A viruses in livestock and free-living waterfowl in Uganda

Avian influenza (AI) viruses may cause natural infection in a variety of domestic and wild bird species throughout the world and have particularly been reported to occur in poultry either in the highly pathogenic or low pathogenic forms [1]. In addition to the sixteen previously known haemagglutinin (H1–H16) and nine neuraminidase (N1–N9) subtypes identified from avian species, H17N10 and H18N11 subtypes have recently been detected in bats [2] representing the entire pool of influenza A viruses known today. AI viruses have been isolated from at least 105 wild bird species of 26 different families [3]. Among all species where influenza viruses have been isolated, waterfowl and shorebirds are regarded the main reservoirs of these viruses [3]. The prevalence of these viruses has been reported in waterfowl [3], domestic birds and pigs in several parts of Africa [4, 5].

Uganda is a seasonal shelter of about 240,000 of the estimated 50 billion birds that make predictable seasonal movements between the temperate zone and the tropics [6] including about 5.4 million ducks [7]. Due to the favourable climate and numerous fresh waterbodies, Uganda serves as a migratory destination for both Palaearctic and intra-Africa migratory species of birds with many wintering in the area for several months [8]. Among the regularly observed birds are eight bird species which are regarded high risk species in the spread of influenza viruses around the world [9, 10]. These include Tufted duck (Aythya fuligula), long-tailed Cormorant (Phalacrocorax africanus) Great Cormorant (Phalacrocorax carbo), Northern Shoveler (Anas clypeata), Garganey (Anas querquedula), Black-headed Gull (Chroicocephalus ridibundus) and Eurasian Wigeon (Anas penelope). Waterfowl can be a source of low pathogenic avian influenza (LPAI) viruses for domestic avian populations [11], in which they can evolve into highly pathogenic avian influenza (HPAI) strains [12]. Spread of these viruses to domestic species can be favoured by the large number of non-wetland dependant groups (“bridge species” such as cattle and little egrets) that move between free-living waterfowl and human settlements and may interact with domestic birds [11]. The other factors that may facilitate spread of these viruses are the numerous live bird markets (LBMs) scattered across the country. Outbreaks due to HPAI subtypes cause death in poultry and sometimes also in humans. This study was undertaken to establish evidence of exposure of poultry, pigs and wild birds in Uganda to influenza A viruses.

Influenza A viruses have been detected in waterfowl [11, 21], domestic birds and pigs in several parts of Africa [4, 5]. Different tropical countries in Africa and Asia have also reported prevalence of antibodies against avian influenza viruses in poultry [22, 23] and swine [24, 25]. Although isolation of influenza A virus from RT-PCR positive samples in this study was not successful, RT-PCR results provide the first evidence of occurrence of these viruses in waterfowl, poultry and swine in Uganda. Previous studies have reported that isolation in embryonated chicken eggs is less sensitive than RT-PCR using primers designed based on the most highly conserved regions of the matrix gene, which is up to 100-fold times more sensitive [17]. Detection of influenza A viruses has in several studies been achieved without concurrent isolation in embryonated eggs and mammalian cell cultures [17], which has mainly been attributed to low titres. In cases of failure of isolation or detection of influenza A viruses by RT-PCR assay, serologic results have been used to provide evidence of presence of the same viruses in animal and bird populations [26]. The PCR and ELISA tests used in the study are highly sensitive and specific tests [17, 27] that produce reliable results.

A prevalence of 1.3% of avian influenza reported of free-living waterfowl in our study was expected. It is comparable with prevalence of 0.3% - 3.0% among wild birds in North America and is close to 3.0% - 4.0% in other African countries [11, 28]. Since some of the species observed at the different sites of sample collection were those that have been reported to harbour LPAI virus in different parts of Africa [9, 11], detecting influenza viruses in a country where such birds reside may not be of surprise. Anseriformes (ducks, geese, and swans) and Charadriiformes (shorebirds, gulls and terns) have previously been described to have a high prevalence of influenza A viruses [11] and a number of species belonging to the two orders of birds have regularly been sighted in Uganda [29]. During a study by Gaidet and colleagues [14], 6.6% and 2.8% of samples taken from Eurasian and African ducks, respectively, were positive to influenza A viruses. Similarly, 3.8% of the samples from Gulls and 1.3% from Terns were RT-PCR positive for the same viruses [14]. These risky bird species were observed in large numbers at study sites during the time samples that turned positive by RT-PCR were collected. Palaearctic migratory birds were associated with the introduction of the HPAI viruses in the wetlands in Nigeria during November and December 2005 before outbreaks of the HPAI in the country later in 2006. Ducks were particularly hypothesized to have acted as bridge species [30].

Variation in prevalence of influenza A viruses in different regions observed in this study is not uncommon and could probably be associated with migration of wild birds and poultry movement. This observation is comparable to previous studies, which have indicated that the spread of HPAI H5N1 virus from Russia and Kazakhstan to the Black Sea basin was consistent in space; and with the hypothesis that birds in the Anatidae family seeded the virus along their autumn migration routes [31]. In China spatial distribution of H5N1 has been observed and epidemiologically linked to poultry trade and wild bird migrations [32].

Seasonal variation in positive samples observed in our study could be expected. Regardless of the host source of samples, no single positive sample was obtained during the months of May – August. In Uganda, these months match with the period when most of the migrant species are either absent or are in significantly small numbers in the country [10, 29]. September –April, during which positive samples were collected, is when resident populations are boosted by the many Palaearctic migrants that spend the northern hemisphere winter in Africa [8]. This also broadly coincides with East Africa’s rainy season. Increase in precipitation, high humidity and low temperature could together influence the seasonal variation in prevalence of these viruses. In Uganda the two rainy seasons are March - May and October - December [33] with amount of rainfall averaging 500 mm - 2200 mm/year. In April and November, during which most positive samples were obtained, are months particularly associated with high amount of rainfall, lower temperatures (19°C - 26°C) and higher relative humidity (65% - 96%). Presence of positive samples during September - April period could easily be associated with seasonal presence of migratory waterbird communities [14] during a weather conditions favourable for survival of influenza A viruses. While low relative humidity of 20%–35% has been reported to be most favorable in transmission of influenza viruses [34], no RT-PCR positive sample was obtained during the dry, hot and low humidity season. This discrepancy may on the contrary re-strengthen the notion that high temperatures of Afro-tropical region decrease the potential survival of influenza viruses in the environment [35]. Although seasonal prevalence cycles of influenza A viruses, especially in wild birds, has previously been observed to be consistent with season [31], it can easily be hypothesized that when migratory waterfowl from Europe are present, the influenza A virus circulation considerably increases irrespective of humidity.

The seasonal prevalence of these viruses observed in waterfowl in the dry (0.7%) and wet season (2.2%) was not very different from that obtained in other studies. In a study by Gaidet and others [14], 3.5% prevalence was observed in a large-scale surveillance of water birds in 12 countries in Africa. Proportions of 15% for ducks and geese and 2% for other species have also been reported in an earlier study [1]. In our study, the seasonal prevalence for these viruses in poultry in the dry (0.3%) and wet season (1.3%) and seroprevalence of 0.5% and 0.1% in the same group of species for each of dry and wet season, respectively, were expected in a country that has never had outbreak of HPAI. Similar and varying levels of seroprevalence due to influenza viruses have been reported in poultry and swine [5].

While one of the earlier studies [36] reported higher prevalence of influenza A viruses among juveniles, our results show more positive samples in adult domestic ducks. Similarly, all our positive samples were solely from adult chickens despite the fact that most species of birds, regardless of ages and breed, are considered equally susceptible to influenza A viruses [37]. In a study by Takemae et al. [38] in which influenza A viruses were solely isolated from young pigs (4 to 12 weeks); in this study positive serological results were only in adult pigs. Despite having collected most samples from pigs kept under semi-intensive and free-range type of management system, all positive samples were restricted to pigs on intensive system. Intensive system is where larger numbers of pigs are kept.

Detecting influenza A viruses H5 subtype in wild and domestic birds of Uganda was consistent with results of other studies. Work, in which different influenza A virus subtypes have been detected, including that of Gaidet and others in Mali, Munster and colleagues in North America and Caron with others in Southern Africa have been reported in a review by Fouchier and Munster [11]. H5 influenza A virus subtypes (including the H5N1) have particularly been reported to occur in domestic poultry in Africa between 2006 and 2008 [39]. Our haemagglutination inhibition test results showed that all the 19 ELISA-positive swine samples were positive for H1 sub-type, which was not unexpected [40]. While H5N2 HPAI virus has been known to cause disease outbreaks in poultry [41], the LPAI virus H5N2 has been isolated from apparently healthy chickens during routine surveillance in Taiwan [42]. Occurrence of H5 could however be worse for Africa, if the circulating serotype is one of the HPAI form.

The result that the antibodies against influenza A viruses in poultry sera were not sub-typed was an expected observation. While the IDEXX AI Ab Test™ kit used in the study had a panel for detecting antibody reactivity to subtypes of H1-H14, antigens used for HI test were limited to only H1, H3, H5, H6, H7 and H9. Hemagglutinins are subtype-specific and it is possible that hemagglutination inhibition (HAI) test may miss some particular sub-types not tested for. It is very likely that antibodies in the sera were due to influenza subtypes different from those tested by HAI test.

This study has demonstrated occurrence of influenza viruses in animals in Uganda. Results have specifically suggested that increase in volumes of migratory waterfowl in the country could be associated with increased prevalence of influenza A viruses in free-living waterfowl and poultry, although this may require further study. Swine and domestic birds, especially domestic ducks, would still be ideal for more intensive and longer (longitudinal) studies in order to clearly understand the epidemiology of this group of viruses in Uganda. This could be combined with wild bird capture for cloacal samples to increase chances of virus isolation for complete characterization of the occurring influenza viruses.