The authors have declared that no competing interests exist.
Conceived and designed the experiments: UH NM MB EPR PA A. Allepuz A. Alba. Performed the experiments: EPR SDS UH NB A. Allepuz A. Alba FA VA MB XG. Analyzed the data: PA EPR A. Allepuz A. Alba. Wrote the paper: EPR PA A. Allepuz XG A. Alba NB SDS VA FA MB NM UH.
Studies exploring the ecological interactions between avian influenza viruses (AIV), natural hosts and the environment are scarce. Most work has focused on viral survival and transmission under laboratory conditions and through mathematical modelling. However, more integrated studies performed under field conditions are required to validate these results. In this study, we combined information on bird community, environmental factors and viral epidemiology to assess the contribution of biotic and abiotic factors in the occurrence of low pathogenic AIV in Spanish wetlands. For that purpose, seven locations in five different wetlands were studied during two years (2007–2009), including seven sampling visits by location. In each survey, fresh faeces (n = 4578) of wild birds and water samples were collected for viral detection. Also, the vegetation structure, water physical properties of wetlands, climatic conditions and wild bird community composition were determined. An overall AIV prevalence of 1.7%±0.4 was detected in faecal samples with important fluctuations among seasons and locations. Twenty-six AIV were isolated from the 78 RRT-PCR positive samples and eight different haemagglutinines and five neuraminidases were identified, being the combination H3N8 the most frequent. Variation partitioning procedures identified the combination of space and time variables as the most important pure factor – independently to other factors – explaining the variation in AIV prevalence (36.8%), followed by meteorological factor (21.5%) and wild bird community composition/vegetation structure (21.1%). These results contribute to the understanding of AIV ecological drivers in Spanish ecosystems and provide useful guidelines for AIV risk assessment identifying potential hotspots of AIV activity.
Avian influenza viruses (AIV) belonging to Influenzavirus A genus infect a broad variety of vertebrates, mainly avian species, but also diverse mammals including humans
Currently, there is limited scientific information about the interface between the ecology and the epidemiology of AIV in wild birds and their persistence in natural ecosystems
The Iberian Peninsula is strategically located in the Mediterranean area in relation to migratory flyways and many of its wetlands are important reserves and major stop-over points for breeding and migratory birds between Eurasia and Africa. For this reason, Spanish wetlands are important locations for disease surveillance and of great interest for the study of LPAIV epidemiology under Mediterranean conditions
The aim of this study was to determine the influence of a wide range of ecological factors (climatic conditions, density and diversity of wild birds, water physico-chemical properties and shelter and food availability) on AIV dynamics in Spanish wetlands under field conditions. The analysis of the field data in this study represents an approach that adds novel information to the last generated mathematical models
The study was carried out in seven sampling locations from five Spanish wetlands (see
Faecal sampling and collection of environmental and bird population data were carried out from winter 2007 to autumn 2009. According to host ecology of the most important AIV reservoirs (Anseriformes), three sampling periods corresponding to autumn migration and wintering (AM/W; from August to January), spring migration (SM; from February to April) and breeding/moult (BM; from May to July) were established. In total, 7 sampling visits were carried out to each wetland (AM/W 2007, SM 2008, BM 2008, AM/W 2008, SM 2009, BM 2009 and AM/W 2009).
In every wetland, suitable sampling sites (where wild birds aggregate to feed or rest) were identified prior to sample collection with the help of ornithologists. Only faeces that appeared freshly passed as judged by appearance of surface, colour and moisture were sampled. We obtained fresh droppings in every visit, excluding two visits to the Castrejón dam wetland where no fresh samples could be obtained.
Factor | Code of predictor | Definition |
Water characteristics | Mean temperature | °C |
Mean pH | ||
Mean conductivity | Ms/cm | |
Mean turbidity | FTU | |
Wild bird communities | Census of wild birds* | Total number of wild birds |
Species richness | Number of wild bird species | |
White storks ( |
Density of White storks (ind/km |
|
Flamingos ( |
Density of Flamingos (ind/km |
|
Others* | Density of wild birds other than Anseriformes, White storks and Flamingos | |
Anseriformes | Density of Anseriformes (ind/km |
|
Dabbling ducks | Percentage of Anseriformes that are dabbling ducks | |
Dabbling ducks + Flamingos |
Percentage of Anseriformes and Flamingos | |
Meteorological data | Mean monthly humidity at 00 h | % RH |
Mean monthly humidity | Average of mean highest and lowest daily humidity (% RH) | |
Mean monthly temperature | °C | |
Monthly mean highest daily temperature MMHDT | °C | |
Monthly mean lowest daily temperature MMLDT | °C | |
Average of MMHDT and MMLDT | °C | |
Total monthly rainfall | mm ×10 | |
Vegetation structure | Vegetation thickness (%) | Percentage of the transect length offering shelter to birds |
Vegetation thickness in the lake shoreline (%) | Percentage of transects with shelter on 2m from the lake shoreline | |
Feeding grounds (%) | Percentage of feeding grounds in 1km radius around the wetland |
Variables marked with * were excluded as highly correlated (Spearman's coefficient ≥ |0.6|) with other variables within their factor.
Flamingos and White storks were considered separately due to previous epidemiological data obtained in the area that identified these species as important AIV carriers
In this index we grouped dabbling ducks and flamingos due to similar feeding habits, as both avian groups feed on surface water, which has been identified as a risk factor in AIV epidemiology
In total, 4578 samples were collected. Approximately 0.1 g of faecal matter were placed in 1 ml of transport medium (Hanks or PBS buffered saline solution with 10% glycerol plus antibiotics and antifungal agents [1000 U/ml penicillin, 1000 U/ml streptomycin, 100 μg/ml gentamicin and 50 μg/ml nystatin]) and transferred in a refrigerated container (4 to 10°C) to the corresponding diagnostic laboratories in less than 24 h. Upon arrival, samples were stored at −80°C until analysis. In the case of Basque Country wetlands, complete faeces were collected and maintained in refrigeration without transport medium due to proximity of the sampled wetlands to the laboratory (less than 2 h).
Two litres of wetland water were collected in every visit at two different points of the water body (4 litres for every location and visit). Temperature, pH, conductivity and turbidity of water were registered directly in the field (
Molecular analyses were performed in three different laboratories located in the three sampled regions, assuring minimal transport time of samples from the field. In laboratories located in Catalonia and Basque Country samples were screened following a TaqMan real time RT-PCR (RRT-PCR) specific for the matrix gen (gene M) in the segment 7 of AIV using primers previously described
In the corresponding laboratory in Castilla-La Mancha, RNA was extracted using commercial kits (High Pure RNA isolation kit, Roche Diagnostics, Germany) according to the manufacturer's instructions. AIV was detected using a RRT-PCR assay targeting the matrix gene as described by Ward et al.
Although two different protocols were used for RNA extraction and RRT-PCR, equal sensitivity and specificity in AIV detection was assured by means of an interlaboratorial assay controlled by the Spanish National Reference Laboratory.
In all cases, pools of five individual samples were processed and upon identification of any AIV positive pool, RNA extraction and RRT-PCR procedures were repeated for the individual samples within each positive pool. Individual RRT-PCR positive samples were subsequently used for virus isolation.
For AIV isolation from RRT-PCR positive samples, 100–200 μl of the original material were inoculated into the allantoic cavity of 9–11 day-old embryonated specific pathogen free chicken eggs following OIE recommendations
The haemagglutinin (HA) and neuraminidase (NA) were identified, when possible, by sequencing or by direct PCR techniques following the protocols described by Hoffmann et al.
Although AIV transmission and perpetuation in an ecosystem is dependent on many different factors, abundance and density of susceptible hosts is a key factor in the epidemiology of the virus
To estimate wild bird species richness and abundance, focal counts of wild birds were undertaken during morning hours in each visit. A point counting approach with several experienced observers was used, in accordance with the waterbird monitoring protocols proposed by the Agreement on the Conservation of African-Eurasian Migratory Waterbirds
These data were used to estimate density of Anseriformes, White storks, Flamingos and other wild birds as well as to quantify the percentage of dabbling ducks and the number of wild bird species (see
The availability and spatial disposition of food and shelter determines the abundance and aggregation of wild birds in and around wetlands
Aggregation of birds in foraging areas greatly increases the contact rates among individuals, creating ideal conditions for disease transmission
Weather conditions have been recognized as an important parameter with regard to migratory bird movements and AIV environmental survival
The 95% confidence intervals for the proportion of positive samples detected in each location and period was estimated by the exact binomial method with Epicalc 2000 (Brixton Health).
The effect of the ecological factors on AIV positivity in every wetland and visit was assessed using logistic regression
The final model was partitioned in order to enhance its explanatory capacity and improve the reliability and interpretation of multiple regressions in the presence of multicollinearity between predictors
During the 2-year period, 78 out of 4578 analysed faecal samples were positive by RRT-PCR (global prevalence of 1.7% [95% CI: 1.3% – 2.1%]). Detection rates by period and location are exposed in
Dates | Period | Positive samples | N | Prevalence (%) ±95%CI |
Sept 2007-Feb 2008 | AM/W | 5 | 592 | 0.8±0.7 |
Mar 2008-May 2008 | SM | 4 | 651 | 0.6±0.6 |
Jun-Aug 2008 | BM | 20 | 503 | 4.0±1.7 |
Sept 2008-Feb 2009 | AM/W | 11 | 1315 | 0.8±0.5 |
Mar-May 2009 | SM | 1 | 585 | 0.2±0.3 |
Jun-Aug 2009 | BM | 5 | 515 | 1.0±0.8 |
Sept-Dec 2009 | AM/W | 32 | 417 | 7.7±2.5 |
TOTAL | 78 | 4578 | 1.7±0.4 |
BM: Breeding/moult; AM/W: Autumn migration/wintering; SM: Spring migration.
Sampling Location | BM | AM/W | SM | Total | ||||||||
Pos | N | Prev (%) ±95%CI | Pos | N | Prev (%) ±95%CI | Pos | N | Prev (%) ±95%CI | Pos | N | Prev (%) ±95%CI | |
1 | 2 | 67 | 3.0±4.0 | 2 | 553 | 0.3±0.5 | 0 | 292 | 0 | 4 | 912 | 0.4±0.3 |
2 | 13 | 46 | 28.2±13 | 30 | 369 | 8.1±2.8 | 1 | 252 | 0.4±0.8 | 44 | 667 | 6.6±0.4 |
3 | 0 | 140 | 0 | 0 | 239 | 0 | 4 | 125 | 3.2±3.0 | 4 | 504 | 0.8±1.9 |
4 | 1 | 139 | 0.7±1.4 | 2 | 249 | 0.8±1.1 | 0 | 121 | 0 | 3 | 509 | 0.6±0.8 |
5 | 1 | 140 | 0.7±1.4 | 5 | 235 | 2.1±1.8 | 0 | 104 | 0 | 6 | 479 | 1.2±0.6 |
6 | 1 | 115 | 0.9±1.7 | 3 | 288 | 1.0±1.2 | 0 | 47 | 0 | 4 | 450 | 0.9±1.0 |
7 | 7 | 371 | 1.9±1.4 | 6 | 391 | 1.5±1.2 | 0 | 295 | 0 | 13 | 1057 | 1.2±0.6 |
Total |
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BM: Breeding/moult; AM/W: Autumn migration/wintering; SM: Spring migration.
Sampling location | Period | Virus subtypes |
1 | BM; AM/W | H13N8 (x2), H3N8 (x1) |
2 | BM; AM/W | H5N2 (x1), H7N? (x8), H7N8 (x1), H3N8 (x11), H11N? (x4), H11N2 (x1) |
3 | n/d | n/d |
4 | n/d | n/d |
5 | AM/W | H6N1 (x2) H8N4 (x1) |
6 | n/d | n/d |
7 | BM | H4N6 (x2) |
n/d: not detected; BM: Breeding/moult; AM/W: Autumn migration/wintering; SM: Spring migration.
In the exploratory stage and with regards to location and season, differences in prevalence were found among study sites (with highest detection rates in wetlands from the Basque Country) and periods (being postbreeding/moult and autumn migration/wintering those with higher probability of AIV detection) (
The final model, in which location, season and year were included as fixed variables, retained three additional predictors related with meteorological conditions, one related to vegetation structure and two related to wild bird community (see
Values shown in diagrams are the percentages of explained variation.
ΔAICc | AICc | Model |
64.92 | 185.42 | Null model (including location, season and year) |
51.87 | 172.37 | Total monthly rainfall (V1) |
42.75 | 163.25 | V1+ Mean monthly temperature (V2) |
32.82 | 153.32 | V1 + V2 + Monthly mean of lowest daily temperature (V3) |
26.92 | 147.42 | V1 + V2 + V3 + Vegetation thickness (V4) |
9.58 | 130.08 | V1 + V2 + V3 + V4 + Anseriformes density (V5) |
0 | 120.50 | V1 + V2 + V3 + V4 + V5 + Species richness |
Variables | Coefficient | Wald | P-value |
Intersect | −2.76 | 5.969 | 0.015 |
Location | - | 48.761 | <0.001 |
Year | - | 0.147 | 0.701 |
Period | - | 19.465 | <0.001 |
Total monthly rainfall | −0.10 | 9.509 | 0.002 |
Mean monthly temperature | 0.70 | 14.237 | <0.001 |
Monthly mean lowest daily temperature | −0.31 | 25.243 | <0.001 |
Vegetation thickness | −0.23 | 21.359 | <0.001 |
Anseriformes density | −0.002 | 19.308 | <0.001 |
Wild bird species richness | −0.39 | 17.069 | <0.001 |
AIV transmission and persistence among wild birds are difficult to assess because both depend on a broad variety of factors, including host community (abundance and diversity of wild birds), environmental parameters (viral tenacity in natural environments) and multiple interactions between them
Faecal sampling for AIV monitoring in wild bird populations has been suggested as a valid alternative to the more-invasive and capture dependent methods based on cloacal sampling
The highest AIV prevalence was found in wetlands from the Basque Country, especially in late summer, autumn and winter (BM and AM/W). The wetlands sampled in this area are the smallest of the study as from total surface and water volume. Small waterbodies in areas with little availability of wetlands could favour AIV transmission due to elevated host densities and lower viral dilution that increase opportunity for AIV exposure
With regards to virus subtypes identified during the study, a noteworthy finding is the relatively high H7 prevalence detected (26.5%) among the identified AIV strains, as compared with previous results in wild birds in Spain
Variation partitioning procedure has been widely used in conservation biology (e.g.,
Regarding the effect of the temporal factor, numerous studies have previously evidenced seasonal and interannual fluctuations in AIV dynamics
The pure effect of the meteorological factor was the next in importance in explaining AIV prevalence, being retained in the final model both precipitation and temperature indexes. These results are in accordance with previous studies, since temperature and humidity levels have been recognized as critical parameters on the environmental tenacity of AIV
Finally, the factor related with wild bird communities and vegetation structure had a smaller contribution to the explanation of total variation. Within this factor, main explanatory power was attributed to wild hosts (i.e. density and richness of wild birds). The presence of suitable hosts (wild birds), and especially those from the orders Anseriformes that are considered the main reservoir of AIV, is essential for viral transmission and environmental perpetuation in the ecosystem
The vegetation structure barely explained the model's deviance, showing a limited pure effect. Vegetation thickness, that was the only variable of this factor retained in the final model, was included in the analysis as a measure of shelter availability for wild birds. Presence of dense vegetation around wetlands, offering shelter and food for birds, would potentially lead to higher density and aggregation of hosts
The essential role of water-borne transmission in AIV epidemiology has been widely proven by experimental and field studies
None of the variables included in the “water” factor were retained in the final model, even though the abiotic parameters registered in every visit (pH, temperature, salinity and turbidity of water) are thought to be the main determinants of virus survival in aquatic ecosystems
In conclusion, the results of this work are useful to better understand the ecological drivers that may modulate the occurrence of AIV in wetlands. The integrated approach presented in this study can be applied to different epidemiological scenarios and provide useful guidelines for AIV risk assessment, identifying potential hotspots of AIV activity and contributing to optimize surveillance systems in wild birds.
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The authors would like to thank A. Valeria Gutierrez, Virginia Gamino and CReSA personnel for their invaluable help in the field work. We also thank P. Bustamante, T. Velasco, Alcázar Ornithologist group, Centro de Estudios Ambientales (CEA) and Plaiaundiko Parke Ekologikoa for collaboration in wild bird censuses. We are grateful to AEMET and EUSKALMET for providing meteorological data. We would also like to thank Toni Curcó and the personnel of the Delta de l'Ebre Park for their invaluable support in the project development.