Guangdong Key Laboratory of Animal Conservation and Resource Utilization, Guangdong Public Laboratory of Wild Animal Conservation and Utilization, Guangdong Institute of Applied Biological Resources, Guangzhou 510260, China
2.
Guangdong Nanling National Nature Reserve, Ruyuan 512727, China
Funds:
Guangdong Science and Technology Plan Project2013B02031005
Guangdong Academy of Science (GDAS) Special Project of Science and Technology Development2017GDASCX-0107
Guangdong Academy of Science (GDAS) Special Project of Science and Technology Development2018GDASCX-0107
Guangdong Forestry Special Project0877-16GZTP01D060
Guangdong Forestry Special Project1210-1741YDZB0401
Special Fund of Guangdong Nature ReserveRYCG12-14
Special Fund of Guangdong Nature ReserveGDHS15SGFX07060
Special Fund of Guangdong Nature ReserveCabotos Tragopan monitoring
The reliability of long-term population estimates is crucial for conservation and management purposes. Most previous studies assume that count indices are proportionally related to abundance; however, this assumption may not hold when detection varies spatially and temporally. We examined seasonal variations in abundance of three bird species (Cabot's Tragopan Tragopan caboti, Silver Pheasant Lophura nycthemera, and White-necklaced Partridge Arborophila gingica) along an elevational gradient, using N-mixture models that take into account imperfect detection in our bird data.
Methods
Camera-trapping was used to monitor temporal activity patterns of these species at Guangdong Nanling National Nature Reserve from December 2013 to November 2017 (4 seasons per year). For abundance analysis (N-mixture modeling), we divided a year into 4 seasons, i.e. 3 months per season, and performed the analysis by season. Elevation was incorporated into the N-mixture model as a covariate that may affect abundance. We compared the N-mixture model with a null model (no covariate model) and selected the better model based on AIC values to make an inference.
Results
From 24 sampling sites, we obtained 6786 photographs of 8482 individuals of 44 bird species and 26 mammal species. Silver Pheasant was photographed much more frequently and showed higher temporal activity frequency than White-necklaced Partridge or Cabot's Tragopan. Silver Pheasant was camera-captured most frequently in summer, and other two species in winters. All three species had two daytime activity peaks: between 6:00 a.m. and 10:00 a.m., and between 5:00 p.m. and 7:00 p.m., respectively. Our estimated abundance and detection probability from the N-mixture model were variable by season. In particular, all three species showed greater abundance in summer than in winter, and estimated abundance patterns of all three species were more similar with observed camera-trapping counts in summers. Moreover, in winter, elevation had a positive impact on abundance of Silver Pheasant and Cabot's Tragopan, but not on White-necklaced Partridge.
Conclusions
Our results demonstrate that the N-mixture model performed well in the estimation of temporal population abundance at local fixed permanent plots in mountain habitat in southern China, based on the modeling of repeated camera-trapping counts. The seasonal differences in abundance of the three endemic bird species and the strong effect of elevation on abundance of two species in winter were only indicative of variations in spatio-temporal distribution within species and between species. In identifying suitable habitat for endemic pheasants, the positive elevational effect also suggests that more attention should be paid to conservation of areas with higher elevation in the Nanling Mountains.
Influenza is an animal and human disease caused by the following genera of Orthomyxoviridae family: influenza virus A, influenza virus B, influenza virus C. Among them, only influenza A viruses have a wide range of hosts with waterfowl being the main hosts. Influenza A viruses are also isolated from pigs, horses, dogs, rodents, some marine mammals, cats, minks, and humans (Webster et al. 1992).
The diversity of avian influenza viruses (AIV) is maintained by wild birds, which are the natural reservoir of influenza (Stallknecht and Shane 1988; Neumann and Kawaoka 2006). Anseriformes and Charadriiformes play a leading role in the natural circulation of influenza viruses. Anseriformes and Charadriiformes are long-distance migrants, and some species can cover up to several thousand kilometers and carry the virus over long distances, which is very important in terms of influenza epidemiology (Erohov 1988).
The life cycle of Anseriformes and Charadriiformes includes stopover on water bodies where a great number of birds from various regions can gather during migration. This can result in the active exchange of influenza viruses, reassortment, the emergence of new variants, and their further spread. Influenza infections follow a seasonal pattern and usually peak in late autumn (Dowell 2001). This may be caused by the presence of many naive young birds in bird populations in autumn. Direct or indirect contacts between wild waterfowl and poultry are also very important as they usually result in influenza transmission and poultry outbreaks (Downie et al. 1977). Seasonal migrations of wild birds promote the spread of multiple influenza A viruses to distant areas and provide their long-term persistence in various ecosystems. It is therefore important to conduct the AIV surveillance in natural conditions.
Water appears to be the optimal medium to maintain and transmit influenza viruses in nature, which explains the wide spread of this pathogen in waterfowl. In fresh water, the virus can retain infectivity for up to 4 days at 22 ℃, for more than 30 days at 0 ℃, and for a much longer period in ice and frozen soil (Rogers et al. 2004). It is likely that infected birds shed the virus in their feces. The virus gets into cold water and soil and persists for a whole winter. In spring, when birds come back, they are exposed to melted water and soil and reinfected (Marchenko et al. 2010a, b).
The main breeding grounds of many migratory Anseriformes and Charadriiformes are located in the north of Russia. The West Siberian Plain is a territory of particular importance (Gilbert et al. 2006; Sharshov et al. 2007, 2010; Donchenko et al. 2010) as there are a lot of rivers and lakes in the south of Western Siberia that are situated on migratory routes and serve as breeding grounds for many species that are ecologically connected with water bodies. Lake Chany is the biggest lake of the region and is known to be the site of the largest migratory stopover of birds (Veen et al. 2005). The south of Western Siberia is crossed by several significant flyways, namely, Central Asian Flyway, Asian-East African Flyway, and East Asian‒Australasian Flyway. These flyways unite the populations of birds wintering in different regions such as Europe, Africa, Middle East, Central Asia, India, South-East Asia, Australia, and Oceania. These conditions favor the wide spread of different influenza viruses and the study of influenza A ecology in the south of Western Siberia. Such study may help to understand the reasons behind wide genetic diversity of influenza A viruses and the spread of this pathogen in Eurasia (Sharshov et al. 2011; Zaykovskaya et al. 2011).
Methods
We conducted regular AIV surveillance in the south of Western Siberia. Cloacal swabs were collected from hunt-harvested wild birds or from wild birds captured using nets during spring and autumn migration. The samples were taken using dry cotton swabs and put into numbered vials with sterile transport medium. One-mL portions of prepared medium were packed in sterile 2-mL plastic vials. All procedures (component mixing and packing) with medium were performed under sterile conditions. Vials with medium were stored at +4 ℃ or room temperature for not more than 2 days. If a longer storage was necessary, vials were stored at ‒20 ℃. Collected samples were stored in liquid nitrogen or freezer at ≤‒70 ℃ before analysis.
Virus isolation
AIV isolation from cloacal swabs was performed in embryonated chicken eggs using three serial passages according to the WHO procedure and the national training course on animal influenza diagnosis and surveillance of China. After each passage, allantoic fluid was tested for hemagglutination activity in hemagglutination assay.
Hemagglutination assay
Two-fold serial dilutions of tested allantoic fluid were prepared in 96-well plate using phosphate-buffered saline. Final volume of diluted allantoic fluid in each plate was 50 µL. A volume of 50 µL of erythrocyte suspension was added to each well and mixed by pipetting. Plates were incubated for 30 min at 4 ℃ before reading. The presence of hemagglutinating agents in samples of allantoic fluid was determined by suspended distribution of erythrocytes, and HA was considered positive. The well with last two-fold dilution, where HA was still positive, was considered as titration end point. The haemagglutination titre of the allantoic fluid was the reciprocal of this dilution. One hemagglutinating unit (HAU) was defined as the amount of AIV in titration end point. For hemagglutination inhibition assay, virus-containing allantoic fluid was diluted to 4 HU in 25 µL of fluid.
PCR amplification and sequencing
Viral RNA was isolated from allantoic fluid using SV Total RNA Isolation System (Promega Corporation, USA). Uni12 universal primers and AMV-reverse Transcriptase (Fermentas, Lithuania) were used for reverse transcription. Gene-specific primers were used for PCR. Amplicons were extracted using QIA quick gel extraction kit (Qiagen, USA). RNA extraction, reverse transcription, amplification, and amplification product purification were performed in accordance with manufacturer's protocols. Previously described primers were used for subtyping (Hoffmann et al. 2001).
Results
Biological diversity of AIV in wild birds in the south of Western Siberia was studied in 2007‒2014. Annual field works were carried out during spring and autumn migration of wild birds to collect biological material. A total of 2300 samples from wild birds of the following 8 orders were collected: Anseriformes, Charadriiformes, Passeriformes, Ciconiiformes, Gruiformes, Podicipediformes, Pelecaniformes, and birds of prey. Majority of samples were collected from birds of 3 orders: Anseriformes, Charadriiformes, and Passeriformes. Samples from Anseriformes accounted for more than 70% of the total number, which is due to preferred sampling of birds from water and wetland ecological complexes. Sampling preferences were determined by literature data suggesting that Anseriformes play a pivotal role in AIV circulation. Most sampled birds belonged to Anseriformes and were represented primarily by wild ducks such as Garganey Teal (Anas querquedula), European Teal (Anas crecca), Pochard (Aythya ferina), Mallard Duck (Anas platyrhynchos), Gadwall (Anas strepera), and Shoveler (Anas clypeata). The number of studied Anseriformes samples is shown in Table 1.
Table
1.
Sampled wild birds (Anseriformes) in the south of Western Siberia by species
These bird species are the most widespread in the south of Siberia and migrate there for nesting. They are considered the main natural reservoir of AIV. A total of 185 AIV isolates were obtained from biological material collected from Anseriformes. The following species dominate among Anseriformes from which AIV was isolated: Garganey Teal, European Teal, and Shoveler. The percent of viral carriers among Anseriformes ranged from 5.6 to 20% in this period (Table 2).
Table
2.
AIV isolation from Anseriformes in 2007‒2014
There were also a lot of Charadriiformes among the sampled birds. A total of 556 Charadriiformes were sampled. The most frequent sampled species, which belong to family Laridae, were Common Gull (Larus canus), Black-headed Gull (L. ridibundus), and European Herring Gull (L. argentatus). These species accounted for more than 65.55% of sampled Charadriiformes. A significant number of sampled birds belong to family Scolopacidae (waders, peewit, Ruff Calidris pugnax, Black-tailed Godwit Limosa limosa, and curlew) and accounted for 21.04% of sampled Charadriiformes. Although there were few birds of other Charadriiformes species, it is important to study these species as carriers of the virus to determine the host range of AIV.
Discussion
Our findings suggest that wetland birds, namely Anseriformes, play a key role in the maintenance of natural AIV circulation. We compared the numbers of AIV carriers within wild bird populations in the south of Western Siberia in 2007‒2014. European Teal is one of the most widespread species of Anseriformes in the south of Western Siberia. European Teal winters in western Europe, the Mediterranean, and the Caspian Sea. Various subtypes (H1N1, H2N1, H3N8, H4N6, H5N1, H8N4, H8N4, and H15N4) were isolated from European Teals during influenza surveillance in Russian Federation (Sivay et al. 2011, 2013, 2014; Marchenko et al. 2010a, 2012a, b; Donchenko et al. 2012; Sharshov et al. 2012; Sayfutdinova et al. 2012). Lower virus isolation rates are typical for Charadriiformes (Webster et al. 1992; Stallknecht and Shane 1988; Neumann and Kawaoka 2006). In our study, the percent of viral carriers among Charadriiformes was 1.4%. Literature data suggest that AIV isolation rate in Charadriiformes ranges from 0.51 to 10.07% (Hurt et al. 2006; Germundsson et al. 2010). The AIV isolation rate for Coot (Fulica atra), which was the only sampled member of the order Gruiformes, was 2.6%.
Our findings demonstrate that the highest percent of viral carriers among Anseriformes was reported in 2012 and 2014, which may be related to climatic conditions. In particular, aquatic conditions (hydrological regime of water bodies, seasonal dryness, etc.) are most important for Anseriformes. Migration period in each year is also very important for AIV transmission and influences the density of migratory birds in gathering sites. Mass gatherings of birds are occasionally observed in some habitats in spring and autumn. A number of species form monospecies and multispecies colonies consisting of several thousand birds during nesting period. Local gatherings including up to 50 species and more than 20, 000 birds at a time occur in post-nesting periods and during flight. Mass gatherings involve contacts between different species or populations and create conditions that favour the spread of viral and infectious diseases (Downie et al. 1977; Webster et al. 1992; Veen et al. 2005; Neumann and Kawaoka 2006; Marchenko et al. 2010a, b).
Phylogenetic analysis of M gene of all AIV strains isolated in the study has shown that the viruses are avian-like. The following two main groups of viruses can be distinguished: avian influenza viruses of Eurasian lineage (Eurasian clade) and gull influenza viruses. In terms of isolation dates, all viruses on M gene phylogenetic tree are chaotically positioned, which is suggestive of persistence of different M gene lineages in populations of wild birds in the south of Western Siberia. Phylogenetic analysis shows that avian-like viruses circulate in the south of Western Siberia in wild birds of orders Anseriformes, Ciconiiformes, Gruiformes, and Charadriiformes. Gulls are one of the main reservoirs of low pathogenic avian influenza A viruses. However, mechanisms of transmission and maintenance of certain influenza subtypes in gull populations remain poorly understood. Despite the fact that most existing influenza subtypes were isolated from gulls, only two hemagglutinin subtypes (H13 and H16) mainly circulate in gulls, and available evidence of multiple observations suggest that in nature these two subtypes are primarily maintained in gull populations (Fouchier et al. 2005). It has been shown recently that gull influenza viruses have specific gene pool and undergo frequent gene reassortment (Sharshov et al. 2014a, b).
The south of Western Siberia is located in the center of Eurasia, crossed by three main flyways of migratory birds and unites the migratory flyways of European, African, Asian, and Oceanic birds (Veen et al. 2005). The climate of the region favors prolonged viral persistence in soil and water (Sivay et al. 2012a, b; De Marco et al. 2014).
Conclusions
Western Siberia plays an important role in persistence, evolution, and geographical spread of avian influenza viruses. Our findings warrant further influenza surveillance program in wild birds of Western Siberia belonging to different taxonomic and ecological groups. This study may bring valuable data on influenza diversity, evolution, and geographical spread.
Authors' contributions
KAS and AMS conceived and designed the experiments. KAS, XL, WW, and AMS performed the experiments. KAS, WW, LL, YB, WL, TS, and HO analyzed the data. KAS, XL, and AMS contributed reagents/materials/analysis tools. KAS, WW, LL, YB, WL, TS, and HO wrote the paper. KAS, AKY, XL, and AMS collected data and biological samples. KAS, WW, LL, YB, WL, TS, and HO contributed to fruitful discussion and critical revision of the manuscript. All authors read and approved the final manuscript.
Acknowledgements
The study was supported by RFBR Grant 16-34-00306\16 mol. We thank Vladimir Petrov from Research Institute of Experimental and Clinical Medicine for valuable suggestions regarding the language of the manuscript.
Competing interests
The authors declare that they have no competing interests.
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