Saliha Hammoumi

Duck Migration and Past Influenza A (H5N1) Outbreak Areas

To the Editor: In 2005 and 2006, the highly pathogenic avian influenza (HPAI) virus subtype H5N1 rapidly spread from Asia through Europe, the Middle East, and Africa. Waterbirds are considered the natural reservoir of low pathogenic avian influenza viruses (1), but their potential role in the spread of HPAI (H5N1), along with legal and illegal poultry and wildlife trade (2), is yet to be clarified. The garganey (Anas querquedula) is the most numerous duck migrating between Eurasia and Africa: ≈2 million gather in the wetlands of Western Africa every northern winter (3). We report on a spatial correlation between the 2007 migration path of a garganey monitored through satellite telemetry and areas that had major HPAI (H5N1) outbreaks from 2005 through 2007. Seven garganeys were captured, sampled, and fitted with a 12-g satellite transmitter in northern Nigeria (Hadejia-Nguru Wetlands; 12°48′N; 10°44′E) in the period February 7–15, 2007. All cloacal and tracheal swabs tested negative for avian influenza virus by real-time reverse transcription–PCR analysis of the matrix gene. One second-year (>9-month-old) female garganey migrated from northern Nigeria to Russia in April–May 2007 (Appendix Figure), where she remained until the end of July. During this 6-week spring migration over the Sahara Desert, Mediterranean Sea, and Eastern Europe, this duck stopped at 3 main stopover sites in Crete, Turkey (Bosphorus region), and Romania (Danube River delta). The duck migrated back to the Danube delta in August, where it remained until November, when the signal was lost. Other garganeys we monitored stopped transmitting before initiating spring migration (n = 3) or remained in West Africa during spring and summer (n = 3), which suggests a stress linked to capture or constraint from the transmitter attachment. This transcontinental migration path connects several areas of past major HPAI (H5N1) outbreaks (Appendix Figure). The wintering area in Nigeria where this duck was caught and remained for 8 weeks before spring migration is located where a large number of outbreaks have occurred repeatedly since February 2006 (the closest being 30 km away). This bird reached its breeding ground in Russia near Moscow and stayed for 2 months in an area that had several outbreaks in backyard poultry in February 2007 (the closest being 30 km away). Finally, the Danube delta, used as a resting ground for 3 months in late summer and autumn, is also an area with recurring outbreaks since October 2005 in wild and domestic birds, with the most recent case reported in November 2007 (the closest being 10 km away). The initial spread of HPAI virus (H5N1) from Eurasia to Africa occurred in autumn and winter 2005–06. The migratory movements we observed during spring and summer in this study were not temporally correlated with any reported HPAI (H5N1) outbreak, either in sequence or period; hence, they should not be interpreted as evidence of the role of wild bird in expansions of the virus. During spring migration from Nigeria to Russia, the garganey stopped several days in wetlands situated close to areas of past outbreaks in the Danube delta (4 days at a distance of 1–4 km from October 2005 outbreaks) and Lake Kus, Turkey (8 days at a distance of 10–30 km from October 2005 outbreaks). The occurrence of past outbreaks indicates that the duck used wetlands favorable to HPAI virus (H5N1) transmission as stopover sites. The relatively long stopover periods enabled prolonged contact of migratory ducks with local domestic and wild bird populations or through shared water, thus prolonging the potential for virus transmission. Considering the persistence of infectivity of HPAI virus (H5N1) in aquatic habitats (4), the number of migratory ducks congregating at stopover sites from various geographic origins and destinations, and the asynchronous timing of the arrival and departure of migratory ducks (5), we believe that these sites may provide locations for disease transmission and possible spread upon movement of wild birds. The satellite-fitted female garganey covered distances between stopover sites of >2,000 km in <2 days, traveling at an estimated speed of 60 km/h. This large-scale movement in a short period, coupled with experimental exposure trials demonstrating viral shedding of up to 4 days in ducks with no clinical signs of infection (6), is consistent with potential viral transmission over great distances. These facts illustrate how a pathogen such as HPAI virus (H5N1) can potentially be transported rapidly by migratory birds across continents. However, the physiologic impact of an HPAI (H5N1) infection on the ability of birds to migrate long distances is still unknown (7) and to date, most empirical evidence suggests that wild birds have only moved short distances (a few hundred kilometers) likely carrying HPAI virus (H5N1) (8). Despite extensive global wildlife surveillance efforts and with the exception of a few reported cases of HPAI (H5N1) infection in apparently healthy wild ducks (9,10), evidence of wild bird involvement in the spread of HPAI virus (H5N1) over long distances is still lacking.

Investigating Avian Influenza Infection Hotspots in Old-World Shorebirds

Heterogeneity in the transmission rates of pathogens across hosts or environments may produce disease hotspots, which are defined as specific sites, times or species associations in which the infection rate is consistently elevated. Hotspots for avian influenza virus (AIV) in wild birds are largely unstudied and poorly understood. A striking feature is the existence of a unique but consistent AIV hotspot in shorebirds (Charadriiformes) associated with a single species at a specific location and time (ruddy turnstone Arenaria interpres at Delaware Bay, USA, in May). This unique case, though a valuable reference, limits our capacity to explore and understand the general properties of AIV hotspots in shorebirds. Unfortunately, relatively few shorebirds have been sampled outside Delaware Bay and they belong to only a few shorebird families; there also has been a lack of consistent oropharyngeal sampling as a complement to cloacal sampling. In this study we looked for AIV hotspots associated with other shorebird species and/or with some of the larger congregation sites of shorebirds in the old world. We assembled and analysed a regionally extensive dataset of AIV prevalence from 69 shorebird species sampled in 25 countries across Africa and Western Eurasia. Despite this diverse and extensive coverage we did not detect any new shorebird AIV hotspots. Neither large shorebird congregation sites nor the ruddy turnstone were consistently associated with AIV hotspots. We did, however, find a low but widespread circulation of AIV in shorebirds that contrast with the absence of AIV previously reported in shorebirds in Europe. A very high AIV antibody prevalence coupled to a low infection rate was found in both first-year and adult birds of two migratory sandpiper species, suggesting the potential existence of an AIV hotspot along their migratory flyway that is yet to be discovered.

New avian paramyxoviruses Type I strains identified in Africa provide new outcomes for phylogeny reconstruction and genotype classification

Newcastle disease (ND) is one of the most lethal diseases of poultry worldwide. It is caused by an avian paramyxovirus 1 that has high genomic diversity. In the framework of an international surveillance program launched in 2007, several thousand samples from domestic and wild birds in Africa were collected and analyzed. ND viruses (NDV) were detected and isolated in apparently healthy fowls and wild birds. However, two thirds of the isolates collected in this study were classified as virulent strains of NDV based on the molecular analysis of the fusion protein and experimental in vivo challenges with two representative isolates. Phylogenetic analysis based on the F and HN genes showed that isolates recovered from poultry in Mali and Ethiopia form new groups, herein proposed as genotypes XIV and sub-genotype VIf with reference to the new nomenclature described by Diel’s group. In Madagascar, the circulation of NDV strains of genotype XI, originally reported elsewhere, is also confirmed. Full genome sequencing of five African isolates was generated and an extensive phylogeny reconstruction was carried out based on the nucleotide sequences. The evolutionary distances between groups and the specific amino acid signatures of each cluster allowed us to refine the genotype nomenclature.

Saving resources: Avian influenza surveillance using pooled swab samples and reduced reaction volumes in real-time RT-PCR

The occurrence of highly pathogenic (HP) avian influenza (AI) H5N1 in Asia and its spread to Africa and Europe prompted costly monitoring programs of wild birds and domestic poultry. AI virus excretion is tested by examining avian swab samples by real-time reverse transcription PCR (RT-qPCR). In this study, pools of swab samples and a reagents volume reduction per RT-qPCR were evaluated as measures of economization. Viral transport medium and faecal matrices were spiked with different low pathogenic AI virus strains and tested for loss of target RNA during all processing steps as individual rayon swabs or in sample pools of 5, 10 and 15 swabs. Fresh faeces from Mallard ducks and other aquatic bird species as sample matrix resulted in loss of AIV RNA of about 90% compared to transport medium. Due to sample RNA dilution in pools the likelihood of detection of single positive samples is decreasing with increasing size of sample pools. However, pools of five samples containing only one positive sample consistently gave positive results. Similarly, no differences in detection rates were obtained when analyzing 1030 wild bird swab samples either individually or in pools of five. Reducing the reaction volume of influenza A virus generic as well as of subtype-specific RT-qPCRs to 12.5 μl (2.5 μl template) instead of 25 μl did not adversely affect the limit of detection of these RT-qPCRs. A significant economic benefit without impeding detection efficacy can be achieved when sample pools of five samples are analyzed by RT-qPCR using a reduction of the reaction mix to the half of the original volume.

Avian influenza in backyard poultry of the Mopti region, Mali.

This study reports the first evidence of circulation of avian influenza viruses (AIV) in domestic poultry in Mali. In the Mopti region, where AIV have already been isolated in migratory water birds, we sampled 223 backyard domestic birds potentially in contact with wild birds and found that 3.6% had tracheal or cloacal swabs positive by real-time reverse transcription PCR (rRT-PCR) for type A influenza viruses (IVA) and that 13.7% had sera positive by commercial ELISA test detecting antibodies against IVA. None of the birds positive by rRT-PCR for IVA was positive by rRT-PCR for H5 and H7 subtypes, and none showed any clinical signs therefore indicating the circulation of low pathogenic avian influenza. Unfortunately, no virus isolation was possible. Further studies are needed to assess the temporal evolution of AIV circulation in the Mopti region and its possible correlation with the presence of wild birds.