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African swine fever (ASF) was diagnosed for the first time in Romania in a backyard holding in Satu‐Mare County in July 2017. Since then, more than 3800 outbreaks occurred in the entire country. Disease control strategies in the backyard sector rely almost exclusively on reactive measures implemented upon appearance of clinical signs and laboratory confirmation of ASF. In our descriptive study, infection course and outbreak investigation data of 56 affected backyard holdings in Satu‐Mare County has been investigated. Early disease detection based on clinical signs appeared to be efficient. In the majority of outbreaks, ASF was detected within the first 2 weeks after the estimated virus introduction. A clinical phase of 2–8 days was observed before pigs either succumbed to the disease or control measures were implemented on affected farms. A moderate on‐farm transmissibility of ASF virus between pigs was observed. Four clusters of outbreaks were identified indicating virus perpetuation and transmission from farm to farm. To suspend infection chains, rapid intervention by isolating affected farms combined with effective biosecurity measures is required. However, due to the backyard peculiarities, quick and effective implementation of control measures has shown to be rather difficult., In our case report, infection course and outbreak investigation data of 56 affected backyard holdings in Satu‐Mare County has been investigated. Early disease detection based on clinical signs appeared to be efficient. In the majority of outbreaks, ASF was detected within the first two weeks after the estimated virus introduction.

The present study described the cytopathic effect of PPR virus presently being used in serial passages at the level of 60th in Vero cells and infected tissue culture fluid was used in this study as viral inoculum. Vero cells were grown on cover slipwere infected with tissue culture fluid at a fixed multiplicity of infection (MOI) 0.01. The infected cover slip along with control were stained with HE stain at periodic intervals and cytopathic effect was studied with microscope. The cytopathic effect (CPE) was visible at first from 24 hpi and the Vero cells showed initial cell rounding, aggregation, and syncytial development. Development of inclusion bodies and cell degradation was noticed by 72 hpi. Complete detachment of the cell monolayer was observed by 84 hpi. It is concluded that, development of numerous inclusion bodies are the indication of well adaptationextensive multiplication of PPRV in Vero cells.

The present study described the cytopathic effect of PPR virus presently being used in serial passages at level of 60th in Vero cells and infected tissue culture fluid was used in this study as viral inoculum. Vero cells were grown on cover slip & were infected with tissue culture fluid at a fixed multiplicity of infection (MOI) 0.01. The infected cover slip along with control were stained with H&E stain at periodic intervals and cytopathic effect was studied with microscope. The cytopathic effect (CPE) was visible at first from 24 hpi and the Vero cells showed initial cell rounding, aggregation and syncytial development. Development of inclusion bodies and cell degradation was noticed by 72 hpi. Complete detachment of the cell monolayer was observed by 84 hpi. It is concluded that, development of numerous inclusion bodies is the indication of well adaptation & extensive multiplication of PPRV in Vero cells.

Understanding the transmission patterns of African swine fever (ASF) among wild boar (Sus scrofa) is an issue of major interest, especially in the wake of the current ASF epidemic. Given the high stability of ASF-virus, there is concern about scavengers spreading infectious carcass material in the environment. Here, we describe scavenging activities on 32 wild boar carcasses in their natural habitat in Germany. Using digital cameras, we detected 22 vertebrates at the study sites, thereof two mammal and three bird species scavenging. The most frequently detected species was the raccoon dog Nyctereutes procyonoides (44% of all visits). Raccoon dogs, red foxes (Vulpes vulpes), and buzzards (Buteo buteo) scavenged in the warm and the cold season, while ravens (Corvus corax) and white-tailed eagles (Haliaeetus albicilla) scavenged only in the cold season. In summer, however, insects removed most of the carcass biomass. Although most of the material was consumed on the spot, foxes, raccoon dogs and ravens left the study sites in rare cases with a small piece of meat in their mouths or beaks. We conclude that scavengers represent a minor risk factor for spreading ASF, but may contribute to reducing local virus persistence by metabolizing infected carcasses.

African swine fever (ASF) is one of the most threatening diseases for the pig farming sector worldwide. Prevention, control and eradication remain a challenge, especially in the absence of an effective vaccine or cure and despite the relatively low contagiousness of this pathogen in contrast to Classical Swine Fever or Foot and Mouth disease, for example. Usually lethal in pigs and wild boar, this viral transboundary animal disease has the potential to significantly disrupt global trade and threaten food security. This paper outlines the importance of a disease-specific legal framework, based on the latest scientific evidence in order to improve ASF control. It compares the legal basis for ASF control in a number of pig-producing regions globally, considering diverse production systems, taking into account current scientific evidence in relation to ASF spread and control. We argue that blanket policies that do not take into account disease-relevant characteristics of a biological agent, nor the specifics under which the host species are kept, can hamper disease control efforts and may prove disproportionate.

Understanding the transmission patterns of African swine fever (ASF) among wild boar (Sus scrofa) is an issue of major interest, especially in the wake of the current ASF epidemic. Given the high stability of ASF-virus, there is concern about scavengers spreading infectious carcass material in the environment. Here, we describe scavenging activities on 32 wild boar carcasses in their natural habitat in Germany. Using digital cameras, we detected 22 vertebrates at the study sites, thereof two mammal and three bird species scavenging. The most frequently detected species was the raccoon dog Nyctereutes procyonoides (44% of all visits). Raccoon dogs, red foxes (Vulpes vulpes), and buzzards (Buteo buteo) scavenged in the warm and the cold season, while ravens (Corvus corax) and white-tailed eagles (Haliaeetus albicilla) scavenged only in the cold season. In summer, however, insects removed most of the carcass biomass. Although most of the material was consumed on the spot, foxes, raccoon dogs and ravens left the study sites in rare cases with a small piece of meat in their mouths or beaks. We conclude that scavengers represent a minor risk factor for spreading ASF, but may contribute to reducing local virus persistence by metabolizing infected carcasses.

Since the first introduction of African swine fever (ASF) into the European wild boar population in 1957, the question of virus survival in carcasses of animals that succumbed to the disease has been discussed. The causative African swine fever virus (ASFV) is known to be very stable in the environment. Thus, carcasses of infected wild boar could play a major role as ASFV reservoir and thereby help to locally maintain and spread the disease in wild boar populations. To minimize this risk, removal of wild boar carcasses in ASF affected areas is regarded to be crucial for effective disease control. If removal is not feasible, carcasses are usually disposed by burial on the spot to avoid direct contact of wild boar to the infection source. In this study, carcasses of ASFV infected wild boar buried in Lithuania at different time points and locations have been excavated and retested for the presence of infectious ASFV by in vitro assays and for viral genome by qPCR. Soil samples potentially contaminated by body fluids have been additionally tested for viral genome. In seventeen out of twenty burial sites, samples of excavated carcasses were positive for ASFV genome. However, in none of the carcass samples ASFV could be isolated. On seven sites soil samples contained ASF viral DNA. These results unexpectedly negate the long-term persistence of infectious ASFV in wild boar carcasses independent from the burial time. In this context, sensitivity of ASFV isolation from carcass samples versus susceptibility of animals and doses needed for oral inoculation has to be further investigated. Furthermore, research is required to consider alternative ASF infection sources and drivers in the infection cycle among wild boar.

The outbreak of highly pathogenic avian influenza H5Nx viruses in winter 2016/2017 was the most severe HPAI epizootic ever reported in Germany. The H5N8 and H5N5 viruses detected in birds in Germany in 2016/2017 represent a reassortant swarm of at least five distinct genotypes, which carried closely related HA segments derived from clade 2.3.4.4b. The genotypes of these viruses and their spatio-temporal distribution indicated a unique situation with multiple independent entries of HPAIV into Germany.

In November 2016, an influenza A(H5N8) outbreak caused deaths of wild birds and domestic poultry in Germany. Clade 2.3.4.4 virus was closely related to viruses detected at the Russia–Mongolia border in 2016 but had new polymerase acidic and nucleoprotein segments. These new strains may be more efficiently transmitted to and shed by birds.

Outbreaks of highly pathogenic avian influenza A virus (HPAIV) subtype H5N8, clade 2.3.4.4, were first reported in January 2014 from South Korea. These viruses spread rapidly to Europe and the North American continent during autumn 2014 and caused, in Germany, five outbreaks in poultry holdings until February 2015. In addition, birds kept in a zoo in north-eastern Germany were affected. Only a few individual white storks (Ciconia ciconia) showed clinical symptoms and eventually died in the course of the infection, although subsequent in-depth diagnostic investigations showed that other birds kept in the same compound of the white storks were acutely positive for or had undergone asymptomatic infection with HPAIV H5N8. An exception from culling all of the 500 remaining zoo birds was granted by the competent authority. Restriction measures included grouping the zoo birds into eight epidemiological units in which 60 birds of each unit tested repeatedly negative for H5N8. Epidemiological and phylogenetical investigations revealed that the most likely source of introduction was direct or indirect contact with infected wild birds as the white storks had access to a small pond frequented by wild mallards and other aquatic wild birds during a period of 10 days in December 2014. Median network analysis showed that the zoo bird viruses segregated into a distinct cluster of clade 2.3.4.4 with closest ties to H5N8 isolates obtained from mute swans (Cygnus olor) in Sweden in April 2015. This case demonstrates that alternatives to culling exist to rescue valuable avifaunistic collections after incursions of HPAIV.

The amylase gene (AMY), which codes for a starch-digesting enzyme in animals, underwent several gene copy number gains in humans (Perry et al., 2007), dogs (Axelsson et al., 2013), and mice (Schibler et al., 1982), possibly along with increased starch consumption during the evolution of these species. Here, we present comprehensive evidence for AMY copy number expansions that independently occurred in several mammalian species which consume diets rich in starch. We also provide correlative evidence that AMY gene duplications may be an essential first step for amylase to be expressed in saliva. Our findings underscore the overall importance of gene copy number amplification as a flexible and fast evolutionary mechanism that can independently occur in different branches of the phylogeny., eLife digest Many mammals can digest starch by using an enzyme called amylase, but different species eat different amounts of starchy foods. Amylase is released by the pancreas, and in certain species such as humans, it is also created by the glands that produce saliva, allowing the enzyme to be present in the mouth. There, amylase can start to break down starch, releasing a sweet taste that helps the animal to detect starchy foods. Curiously, humans have multiple copies of the gene that codes for the enzyme, but the exact number varies between people. Previous research has found that populations with more copies also eat more starch; if this correlation also existed in other species, it could help to understand how diets influence and shape genetic information. In addition, it is unclear how amylase came to be present in saliva, as the ancestors of mammals only produced the protein in the pancreas. Pajic et al. analyzed the genomes of a range of mammals and found that the more starch a species had in its diet, the more amylase gene copies it harbored in its genome. In fact, unrelated mammals living in different habitats and eating different types of food have similar numbers of amylase gene copies if they have the same level of starch in their diet. In addition, Pajic et al. discovered that animals such as mice, rats, pigs and dogs, which have lived in close contact with people for thousands of years, quickly adapted to the large amount of starch present in human food. In each of these species, a mechanism called gene duplication independently created new copies of the amylase gene. This could represent the first step towards some of these copies becoming active in the glands that release saliva. In people, having fewer copies of the amylase gene could mean they have a higher risk for diabetes; this number is also tied to the composition of the collection of bacteria that live in the mouth and the gut. Understanding how the copy number of the amylase gene affects biology will help to grasp how it also affects health and wellbeing, in humans and in our four-legged companions.

In contrast to previous incursions of highly pathogenic avian influenza (HPAIV) H5 viruses, H5N8 clade 2.3.4.4b viruses caused numerous cases of lethal infections in white-tailed sea eagles (Haliaeetus albicilla) affecting mainly young eagles (younger than five years of age) in Germany during winter 2016/2017. Until April 2017, 17 HPAIV H5N8-positive white-tailed sea eagles had been detected (three found alive and 14 carcasses) by real-time RT-PCR and partial nucleotide sequence analyses. Severe neurological clinical signs were noticed which were corroborated by immunohistopathology revealing mild to moderate, oligo- to multifocal necrotizing virus-induced polioencephalitis. Lethal lead (Pb) concentrations, a main factor of mortality in sea eagles in previous years, could be ruled out by atomic absorption spectrometry. HPAIV H5 clade 2.3.4.4b reportedly is the first highly pathogenic influenza virus known to induce fatal disease in European white-tailed see eagles. This virus strain may become a new health threat to a highly protected species across its distribution range in Eurasia. Positive cloacal swabs suggest that eagles can spread the virus with their faeces.

In contrast to previous incursions of highly pathogenic H5 viruses, H5N8 clade 2.3.4.4b caused numerous lethal infections in white-tailed sea eagles (Haliaeetus albicilla) in Germany during the winter 2016/2017. Until April 2017, 17 HPAIV H5N8-positive white-tailed sea eagles had been detected (three alive and 14 dead). Mainly young eagles died (before reaching the adult plumage at 5 years), often with severe neurological symptoms, where histopathology revealed mild to moderate, oligo- to multifocal necrotizing polioencephalitis. Lethal lead (Pb) concentrations, proven as main mortality factor of the sea eagles could be ruled out since values measured in liver or kidney tissue were all within background levels (< 1 ppm). Since the fall of 2016, the epizootic of HPAIV H5 clade 2.3.4.4b reportedly induced, for the first time, fatal disease in European white-tailed see eagles. The virus strain may become a new threat to a highly protected species across its distribution range in Eurasia. Positive cloacal swaps have proven that the eagles can spread the virus with their faeces.

The amylase gene (AMY), which codes for a starch-digesting enzyme in animals, underwent several gene copy number gains in humans1, dogs2, and mice3, presumably along with increased starch consumption during the evolution of these species. Here we present evidence for additionalAMYcopy number expansions in several mammalian species, most of which also consume starch-rich diets. We also show that these independentAMYcopy number gains are often accompanied by a gain in enzymatic activity of amylase in saliva. We used multi-species coalescent modeling to provide further evidence that these recurrentAMYgene copy number expansions were adaptive. Our findings underscore the overall importance of gene copy number amplification as a flexible and fast adaptive mechanism in evolution that can independently occur in different branches of the phylogeny.

Here, we report on the occurrence of highly pathogenic avian influenza (HPAI) H5Nx clade 2.3.4.4b in Germany. Between November 8, 2016, and September 30, 2017, more than 1,150 cases of HPAI H5Nx clade 2.3.4.4b in wild birds and 107 outbreaks in birds kept in captivity (92 poultry holdings and 15 zoos/animal parks) were reported in Germany. This HPAI epidemic is the most severe recorded in Germany so far. The viruses were apparently introduced by migratory birds, sparking an epidemic among wild birds across Germany with occasional incursions into poultry holdings, zoos and animal parks, which were usually rapidly detected and controlled by stamping out. HPAI viruses (mainly subtype H5N8, in a few cases also H5N5) were found in dead wild birds of at least 53 species. The affected wild birds were water birds (including gulls, storks, herons, and cormorants) and scavenging birds (birds of prey, owls, and crows). In a number of cases, substantial gaps in farm biosecurity may have eased virus entry into the holdings. In a second wave of the epidemic starting from February 2017, there was epidemiological and molecular evidence for virus transmission of the infections between commercial turkey holdings in an area of high poultry density, which caused approximately 25% of the total number of outbreaks in poultry. Biosecurity measures in poultry holdings should be adapted. This includes, inter alia, wearing of stable-specific protective clothing and footwear, cleaning, and disinfection of equipment that has been in contact with birds and prevention of contacts between poultry and wild water birds.

RNA extraction and purification is a fundamental step that allows for highly sensitive amplification of specific RNA targets in PCR applications. However, commercial extraction kits that are broadly used because of their robustness and high yield of purified RNA are expensive and labor-intensive. In this study, boiling in distilled water or a commercial lysis buffer of different sample matrices containing avian or porcine influenza viruses was tested as an alternative. Real-time PCR (RTqPCR) for nucleoprotein gene fragment was used as read out. Results were compared with freshly extracted RNA by use of a commercial extraction kit. Different batches of virus containing materials, including diluted virus positive allantoic fluid or cell culture supernatant, and avian faecal, cloacal or oropharyngeal swab samples were used in this study. Simple boiling of samples without any additional purification steps can be used as an alternative RNA preparation method to detect influenza A virus nucleoprotein RNA in oropharyngeal swab samples, allantoic fluid or cell-culture supernatant. The boiling method is not applicable for sample matrices containing faecal material.

Avian influenza viruses (AIV) potentially transmit to swine as shown by experiments, where further reassortment may contribute to the generation of pandemic strains. Associated risks of AIV inter-species transmission are greater in countries like Nigeria with recurrent epidemics of highly pathogenic AI (HPAI) in poultry and significant pig population. Analysis of 129 tracheal swab specimens collected from apparently healthy pigs at slaughterhouse during presence of HPAI virus H5N1 in poultry in Nigeria for influenza A by RT-qPCR yielded 43 positive samples. Twenty-two could be determined by clade specific RT-qPCR as belonging to the H5N1 clade 2.3.2.1c and confirmed by partial hemagglutinin (HA) sequence analysis. In addition, 500 swine sera were screened for antibodies against influenza A virus nucleoprotein and H5 HA using competition ELISAs and hemagglutination inhibition (HI) tests. Serologically, 222 (44.4%) and 42 (8.4%) sera were positive for influenza A virus NP and H5 antibodies, respectively. Sera reacted to H5N1 and A/H1N1pdm09 strains by HI suggesting exposure of the Nigerian domestic pig population to these viruses. We report for the first time in Nigeria, exposure of domestic pigs to H5N1 virus. This poses potential public health and pandemic risk due to interspecies transmission of avian and human influenza viruses.

The behaviour of free ranging wild boar ( Sus scrofa ) towards carcasses of their conspecifics potentially infected with African swine fever (ASF) may significantly influence the course of an ASF epidemic. This study aims to better understand the behaviour of wild boar towards their dead fellows. Thirty-two wild boar carcasses on nine study sites in northeast Germany were monitored under field conditions by photo-trapping from October 2015 until October 2016. During this period, a total of 122 160 pictures were taken, thereof 16 111 pictures of wild boar. In both winter and summer, wild boar seemed to be particularly interested in the soil next to and underneath the carcasses. About one third of the visits of wild boar led to direct contact with dead conspecifics. The contacts consisted mostly in sniffing and poking on the carcass. Under the given ecological and climatic conditions, there was no evidence for intra-species scavenging. However, piglets were observed several times chewing bare bones once skeletonization of the carcasses was complete. It must be assumed that all these types of contact may represent a risk of transmission. Both the high tenacity of ASF virus and the long time wild boar carcasses can remain in the environment, allow the persistence of the virus for several months or even years. We therefore consider the rapid detection and removal (or destruction on the spot) of contaminated carcasses as an important control measure against ASF in wild boar.

Background Mallard ducks and other waterfowl represent the most important reservoirs of low pathogenic avian influenza viruses (LPAIV). In addition, mallards are the most abundant duck species in Eurasia that migrate over long distances. Despite extended wild bird monitoring studies over the past decade in many Eurasian countries and investigating hundreds of thousands of wild bird samples, no mallard duck was found to be positive for avian influenza virus of subtype H16 in faecal, cloacal or oropharyngeal samples. Just three cases of H16 infections in Anseriformes species were described worldwide. In contrast, H16 viruses have been repeatedly isolated from birds of the Laridae family. Objective Here, we tested the hypothesis that mallards are less permissive to infection with H16 viruses. Methods Groups of mallard ducks of different age were inoculated via the oculo-nasal-oral route with different infectious doses of an H16N3 AIV. Results The ducks did not show any clinical symptoms, and no virus shedding was evident from cloacal and respiratory routes after experimental infection as shown by negative RT-qPCR results. In addition, all serum samples taken on days 8, 21 and 24 post-inoculation were negative by competitive NP-ELISA. Conclusions This study provided evidence that mallards are resistant to infection with H16N3 LPAIV.

In this study we describe the use of a rope-in-a-bait sampling method (“pSWAB”: pathogen sampling wild animals with baits) for non-invasive saliva sampling aimed at the detection of foot-and-mouth disease (FMD) viral genome in wild boar. The pSWABs are produced in the form of a standardized product by embedding a 10 cm long cotton rope in a cereal-based bait matrix. To assess the general suitability of this novel sampling technique an animal experiment was conducted to detect FMD viral genome in saliva of infected wild boar. Two juvenile animals were inoculated in the bulb of the heel with a recent wild boar FMD virus isolate and kept together with three noninoculated wild boar of the same age. Over a period of 29 days, the animals were sampled by using five pSWABs per day in addition to the collection of blood and conventional saliva swabs taken every three to four days. Viral RNA in pSWABs was identified already 24 h after infection during the incubation period and until 23 dpi. Comparison of the results of pSWAB sampling with those of conventional saliva swabs or serum samples showed satisfactory sensitivity. These experimental data demonstrate the suitability of non-invasive sampling of wild boar by using pSWABs as a sensitive, cheap and feasible sample collection technique independent of hunting activities. In addition, the use of non-invasive sampling in an appropriate surveillance strategy is discussed.

Several classical swine fever (CSF) epidemics in wild boar and domestic pigs in Europe during the last decades have been caused by CSF virus (CSFV) strains of genotype 2.3. This genotype is known to be virulent leading to high morbidity and mortality. We experimentally infected two eight months old wild boar with 10(5,5) TCID50 of CSFV genotype 2.3 and kept the animals together with five noninoculated wild boar of the same age. Our original purpose was to evaluate a non-invasive sampling method based on saliva collection using "rope-in-a-bait" sampling baits. While expecting high morbidity, high level of virus shedding and some mortality, we actually observed a subclinical course of infection with an unexpected low contagiosity. The two inoculated animals infected only three contact animals while two contact animals remained uninfected. These findings substantially add to our epidemiological understanding of CSFV circulation in wild boar populations. CSFV infected animals older than six months and in good condition may not shed sufficient virus to transmit infection to all seronegative in-contact animals. The contagiosity in relation to the animal's age is discussed. This supports the hypothesis of silent perpetuation of CSFV in wild boar populations for several months if the wild boar density is sufficiently high. The feasibility of the "rope-in-a-bait" sampling method could be proven during the short viraemic phase of infected animals during the second week of infection.

A serological survey for Avian Influenza Virus AIV subtype H H and H antibodies in domestic ducks was carried out in at Central Veterinary Laboratory CVL Kathmandu Nepal A total of serum samples were tested from various localities of districts of Nepal categorized as to be at high risk for the introduction of Highly Pathogenic Avian Influenza Virus HPAIV of H N subtype Sero positive result against AIV H was found in three districts of eastern region with the highest prevalence in Jhapa p lt Likewise sero positive samples against AIV H were detected in Chitwan Kapilbastu Nawalparasi and Kaski districts However none of the samples was found to be sero positive against AIV H All the samples positive against H were sampled in December exclusively p lt In contrast two samples collected before September and three samples after this time until January were sero positive against AIV H Our findings indicate higher prevalence of AIV subtype H compared to H and H in duck species of Nepal

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.

SUMMARYPrevalence monitoring of avian influenza in wild bird populations is important to estimate risks for the occurrence of potentially zoonotic and economically disastrous outbreaks of highly pathogenic avian influenza virus (AIV) in poultry worldwide. A targeted, cost-effective monitoring method for AIV in wild birds was developed, which is based on monitoring results for AIV in Germany and information on the distribution and abundance of wild bird species in selected habitat types. Spatial data were combined with virological and outbreak data for the period of 1 January 2006 to 31 December 2010. Using Germany as an example, we identified 11 indicator species. By concentrating monitoring efforts on these species in spatially confined locations, we propose a targeted and more cost-effective risk-based AIV monitoring approach that can be adapted universally for the identification of wild bird indicator species worldwide with the perspective of reducing sample sizes (and costs) without impairing the validity of the results.