Effect of 2020-21 and 2021-22 Highly Pathogenic Avian Influenza H5 Epidemics on Wild Birds, the Netherlands.

The number of highly pathogenic avian influenza (HPAI) H5-related infections and deaths of wild birds in Europe was high during October 1, 2020-September 30, 2022. To quantify deaths among wild species groups with known susceptibility for HPAI H5 during those epidemics, we collected and recorded mortality data of wild birds in the Netherlands. HPAI virus infection was reported in 51 bird species. The species with the highest numbers of reported dead and infected birds varied per epidemic year; in 2020-21, they were within the Anatidae family, in particular barnacle geese (Branta leucopsis) and in 2021-22, they were within the sea bird group, particularly Sandwich terns (Thalasseus sandvicensis) and northern gannet (Morus bassanus). Because of the difficulty of anticipating and modeling the future trends of HPAI among wild birds, we recommend monitoring live and dead wild birds as a tool for surveillance of the changing dynamics of HPAI.

Risk for Low Pathogenicity Avian Influenza Virus on Poultry Farms, the Netherlands, 2007-2013.

Using annual serologic surveillance data from all poultry farms in the Netherlands during 2007-2013, we quantified the risk for the introduction of low pathogenicity avian influenza virus (LPAIV) in different types of poultry production farms and putative spatial-environmental risk factors: distance from poultry farms to clay soil, waterways, and wild waterfowl areas. Outdoor-layer, turkey (meat and breeder), and duck (meat and breeder) farms had a significantly higher risk for LPAIV introduction than did indoor-layer farms. Except for outdoor-layer, all poultry types (i.e., broilers, chicken breeders, ducks, and turkeys) are kept indoors. For all production types, LPAIV risk decreased significantly with increasing distance to medium-sized waterways and with increasing distance to areas with defined wild waterfowl, but only for outdoor-layer and turkey farms. Future research should focus not only on production types but also on distance to waterways and wild bird areas. In addition, settlement of new poultry farms in high-risk areas should be discouraged.

Confirmation of spatial patterns and temperature effects in Bluetongue virus serotype-8 transmission in NW-Europe from the 2007 reported case data.

Two separate analyses were carried out to understand the epidemiology of Bluetongue virus serotype 8 (BTV-8) in 2007 in North West Europe: First, the temporal change in transmission rates was compared to the evolution of temperature during that season. Second, we evaluated the spatio-temporal dynamics of newly reported outbreaks, to estimate a spatial transmission kernel. For both analyses, the approach as used before in analysing the 2006 BTV-8 epidemic had to be adapted in order to take into account the fact that the 2007 epidemic was not a newly arising epidemic, but one advancing from whereto it had already spread in 2006. We found that within the area already affected by the 2006 outbreak, the pattern of newly infected farms in 2007 cannot be explained by between-farm transmission, but rather by local re-emergence of the virus throughout that region. This indicates that persistence through winter was ubiquitous for BTV-8. Just like in 2006, we also found that the temperature at which the infection starts to spread lies close to 15 °C. Finally, we found that the shape of the transmission kernel is in line with the one from the 2006 epidemic. In conclusion, despite the substantial differences between 2006 and 2007 in temperature patterns (2006 featured a heat wave in July, whereas 2007 was more regular) and spatial epidemic extent, both the minimum temperature required for transmission and the transmission kernel were similar to those estimated for the 2006 outbreak, indicating that they are robust properties, suitable for extrapolation to other years and similar regions.

Schmallenberg virus antibody persistence in adult cattle after natural infection and decay of maternal antibodies in calves.

BACKGROUND: Schmallenberg virus (SBV) has swept through the major part of Europe in the period 2011-2013. A vaccine against SBV has been developed and may be a possible preventive instrument against infection. Presently, there is no data available to refute the assumption that natural SBV infection results in long-term immunity. In that respect, it is of interest to know how long (protecting) virus-neutralizing antibodies are present in naturally infected animals. New-born calves acquire passive immunity from their dams by ingestion and absorption of antibodies present in colostrum, which can block the production of serum antibodies when vaccine is administered to calves with maternally derived antibodies. In that respect, it is useful to know how long it takes for maternal antibodies against SBV to disappear in young animals born from infected dams. RESULTS: Longitudinal whole-herd serological monitoring using virus neutralization test (VNT) indicated that 80% of adult dairy cows still had measurable antibodies against SBV at least 24 months after the estimated introduction of the virus into the herd. Median 2Log VNT titer of the adult dairy cows (≥1 year) dropped from 8.6 to 5.6 in a period of 17 months. Median 2Log VNT maternal antibodies titers of calves sampled within 30 days after birth was 8. Calves lost their maternally-derived antibodies after 5-6 months. There was a definite positive relationship between the VNT titer of the dam and the VNT titer of the corresponding calf (age ≤ 30 days) of dam-calf combinations sampled on the same day: the higher the VNT titer of the dam, the higher the VNT titer (maternal antibodies) of the calf. CONCLUSIONS: Our field data support the assumption that natural SBV infection in adult cows results in persistence of specific antibodies for at least two years. Based on the observed decay of maternally-derived antibodies in calves, it is presumed safe to vaccinate calves against SBV at an age of approximately 6 months.

Spatial Transmission Characteristics of the Bluetongue Virus Serotype 3 Epidemic in The Netherlands, 2023.

A devastating bluetongue (BT) epidemic caused by bluetongue virus serotype 3 (BTV-3) has spread throughout most of the Netherlands within two months since the first infection was officially confirmed in the beginning of September 2023. The epidemic comes with unusually strong suffering of infected cattle through severe lameness, often resulting in mortality or euthanisation for welfare reasons. In total, tens of thousands of sheep have died or had to be euthanised. By October 2023, more than 2200 locations with ruminant livestock were officially identified to be infected with BTV-3, and additionally, ruminants from 1300 locations were showing BTV-associated clinical symptoms (but not laboratory-confirmed BT). Here, we report on the spatial spread and dynamics of this BT epidemic. More specifically, we characterized the distance-dependent intensity of the between-holding transmission by estimating the spatial transmission kernel and by comparing it to transmission kernels estimated earlier for BTV-8 transmission in Northwestern Europe in 2006 and 2007. The 2023 BTV-3 kernel parameters are in line with those of the transmission kernel estimated previously for the between-holding spread of BTV-8 in Europe in 2007. The 2023 BTV-3 transmission kernel has a long-distance spatial range (across tens of kilometres), evidencing that in addition to short-distance dispersal of infected midges, other transmission routes such as livestock transports probably played an important role.

Comparison of the Clinical Manifestation of HPAI H5Nx in Different Poultry Types in the Netherlands, 2014-2022.

This study describes clinical manifestations of highly pathogenic avian influenza (HPAI) H5N1, H5N8 and H5N6 outbreaks between 2014 and 2018 and 2020 and 2022 in the Netherlands for different poultry types and age groups. Adult duck (breeder) farms and juvenile chicken (broiler and laying pullet) farms were not diagnosed before 2020. Outbreaks in ducks decreased in 2020-2022 vs. 2014-2018, but increased for meat-type poultry. Neurological, locomotor and reproductive tract signs were often observed in ducks, whereas laying- and meat-type poultry more often showed mucosal membrane and skin signs, including cyanosis and hemorrhagic conjunctiva. Juveniles (chickens and ducks) showed neurological and locomotor signs more often than adults. Diarrhea occurred more often in adult chickens and juvenile ducks. Mortality increased exponentially within four days before notification in chickens and ducks, with a more fluctuating trend in ducks and meat-type poultry than in layers. For ducks, a mortality ratio (MR) > 3, compared to the average mortality of the previous week, was reached less often than in chickens. A lower percentage of laying flocks with MR > 3 was found for 2020-2022 vs. 2014-2018, but without significant differences in clinical signs. This study provides a basis for improvements in mortality- and clinical-sign-based early warning criteria, especially for juvenile chickens and ducks.

Schmallenberg virus in Culicoides spp. biting midges, the Netherlands, 2011.

To determine which species of Culicoides biting midges carry Schmallenberg virus (SBV), we assayed midges collected in the Netherlands during autumn 2011. SBV RNA was found in C. scoticus, C. obsoletus sensu stricto, and C. chiopterus. The high proportion of infected midges might explain the rapid spread of SBV throughout Europe.

Culicoides (Diptera: Ceratopogonidae) Abundance Is Influenced by Livestock Host Species and Distance to Hosts at the Micro Landscape Scale.

The vector/host ratio and host preference are important parameters for the modelling of vector-borne livestock diseases. It can be anticipated that Culicoides abundance is not homogeneously distributed in the landscape. We investigated the influence of host species (dairy cow, sheep, and a light-trap (LT) as a surrogate host) and distance of measurement to hosts on Culicoides abundance using a randomized block-design with 12 measuring days and seven 3-min aerial sweep-netting sessions per whole hour at three distances to the host (0, 10, and 25 m), from five hours before to and including one hour after sunset. Dairy cows were found to be a far stronger attractor of Culicoides midges than sheep, while both hosts were far stronger attractors of midges than the LT. Culicoides abundance declined significantly with increasing distance from the livestock hosts; this phenomenon was much stronger for dairy cows than for ewes. In contrast, Culicoides abundance increased with increasing distance from the LT, pin-pointing the apparent shortcomings of the LT as a surrogate host to lure midges. Our data indicate that livestock host species and the distance from these hosts have a profound effect on Culicoides abundance in the landscape.

Seroprevalence of Schmallenberg virus antibodies among dairy cattle, the Netherlands, winter 2011-2012.

Infections with Schmallenberg virus (SBV) are associated with congenital malformations in ruminants. Because reporting of suspected cases only could underestimate the true rate of infection, we conducted a seroprevalence study in the Netherlands to detect past exposure to SBV among dairy cattle. A total of 1,123 serum samples collected from cattle during November 2011-January 2012 were tested for antibodies against SBV by using a virus neutralization test; seroprevalence was 72.5%. Seroprevalence was significantly higher in the central-eastern part of the Netherlands than in the northern and southern regions (p<0.001). In addition, high (70%-100%) within-herd seroprevalence was observed in 2 SBV-infected dairy herds and 2 SBV-infected sheep herds. No significant differences were found in age-specific prevalence of antibodies against SBV, which is an indication that SBV is newly arrived in the country.

Wild Bird Densities and Landscape Variables Predict Spatial Patterns in HPAI Outbreak Risk across The Netherlands.

Highly pathogenic avian influenza viruses' (HPAIVs) transmission from wild birds to poultry occurs globally, threatening animal and public health. To predict the HPAI outbreak risk in relation to wild bird densities and land cover variables, we performed a case-control study of 26 HPAI outbreaks (cases) on Dutch poultry farms, each matched with four comparable controls. We trained machine learning classifiers to predict outbreak risk with predictors analyzed at different spatial scales. Of the 20 best explaining predictors, 17 consisted of densities of water-associated bird species, 2 of birds of prey, and 1 represented the surrounding landscape, i.e., agricultural cover. The spatial distribution of mallard (Anas platyrhynchos) contributed most to risk prediction, followed by mute swan (Cygnus olor), common kestrel (Falco tinnunculus) and brant goose (Branta bernicla). The model successfully distinguished cases from controls, with an area under the receiver operating characteristic curve of 0.92, indicating accurate prediction of HPAI outbreak risk despite the limited numbers of cases. Different classification algorithms led to similar predictions, demonstrating robustness of the risk maps. These analyses and risk maps facilitate insights into the role of wild bird species and support prioritization of areas for surveillance, biosecurity measures and establishments of new poultry farms to reduce HPAI outbreak risks.

Monitoring Wind-Borne Particle Matter Entering Poultry Farms via the Air-Inlet: Highly Pathogenic Avian Influenza Virus and Other Pathogens Risk.

Wind-supported transport of particle matter (PM) contaminated with excreta from highly pathogenic avian influenza virus (HPAIv)-infected wild birds may be a HPAIv-introduction pathway, which may explain infections in indoor-housed poultry. The primary objective of our study was therefore to measure the nature and quantity of PM entering poultry houses via air-inlets. The air-inlets of two recently HPAIv-infected poultry farms (a broiler farm and a layer farm) were equipped with mosquito-net collection bags. PM was harvested every 5 days for 25 days. Video-camera monitoring registered wild bird visits. PM was tested for avian influenza viruses (AIV), Campylobacter and Salmonella with PCR. Insects, predominantly mosquitoes, were tested for AIV, West Nile, Usutu and Schmallenberg virus. A considerable number of mosquitoes and small PM amounts entered the air-inlets, mostly cobweb and plant material, but no wild bird feathers. Substantial variation in PM entering between air-inlets existed. In stormy periods, significantly larger PM amounts may enter wind-directed air-inlets. PM samples were AIV and Salmonella negative and insect samples were negative for all viruses and bacteria, but several broiler and layer farm PM samples tested Campylobacter positive. Regular wild (water) bird visits were observed near to the poultry houses. Air-borne PM and insects-potentially contaminated with HPAIv or other pathogens-can enter poultry air-inlets. Implementation of measures limiting this potential introduction route are recommended.

Quantitative analysis of transmission parameters for bluetongue virus serotype 8 in Western Europe in 2006.

The recent bluetongue virus serotype 8 (BTV-8) epidemic in Western Europe struck hard. Controlling the infection was difficult and a good and safe vaccine was not available until the spring of 2008. Little was known regarding BTV transmission in Western Europe or the efficacy of control measures. Quantitative details on transmission are essential to assess the potential and efficacy of such measures.To quantify virus transmission between herds, a temporal and a spatio-temporal analysis were applied to data on reported infected herds in 2006. We calculated the basic reproduction number between herds (Rh: expected number of new infections, generated by one initial infected herd in a susceptible environment). It was found to be of the same order of magnitude as that of an infection with Foot and Mouth Disease (FMD) in The Netherlands, e.g. around 4. We concluded that an average day temperature of at least 15 °C is required for BTV-8 transmission between herds in Western Europe. A few degrees increase in temperature is found to lead to a major increase in BTV-8 transmission.We also found that the applied disease control (spatial zones based on 20 km radius restricting animal transport to outside regions) led to a spatial transmission pattern of BTV-8, with 85% of transmission restricted to a 20 km range. This 20 km equals the scale of the protection zones. We concluded that free animal movement led to substantial faster spread of the BTV-8 epidemic over space as compared to a situation with animal movement restrictions.

Mortality Levels and Production Indicators for Suspicion of Highly Pathogenic Avian Influenza Virus Infection in Commercially Farmed Ducks.

(1) Background: Highly pathogenic avian influenza (HPAI) is a viral infection characterized by inducing severe disease and high levels of mortality in gallinaceous poultry. Increased mortality, drop in egg production or decreased feed or water intake are used as indicators for notification of suspicions of HPAI outbreaks. However, infections in commercial duck flocks may result in mild disease with low mortality levels, thereby compromising notifications. (2) Methods: Data on daily mortality, egg production, feed intake and water intake from broiler and breeder duck flocks not infected (n = 56 and n = 11, respectively) and infected with HPAIV (n = 13, n = 4) were used for analyses. Data from negative flocks were used to assess the baseline (daily) levels of mortality and production parameters and to identify potential threshold values for triggering suspicions of HPAI infections and assess the specificity (Sp) of these thresholds. Data from infected flocks were used to assess the effect of infection on daily mortality and production and to evaluate the sensitivity (Se) of the thresholds for early detection of outbreaks. (3) Results: For broiler flocks, daily mortality > 0.3% (after the first week of production) or using a regression model for aberration detection would indicate infection with Se and Sp higher than 80%. Drops in mean daily feed or water intake larger than 7 g or 14 mL (after the first week of production), respectively, are sensitive indicators of infection but have poor Sp. For breeders, mortality thresholds are poor indicators of infection (low Se and Sp). However, a consecutive drop in egg production larger than 9% is an effective indicator of a HPAI outbreak. For both broiler and breeder duck flocks, cumulative average methods were also assessed, which had high Se but generated many false alarms (poor Sp). (4) Conclusions: The identified reporting thresholds can be used to update legislation and provide guidelines to farmers and veterinarians to notify suspicions of HPAI outbreaks in commercial duck flocks.

Efficacy of an automated laser for reducing wild bird visits to the free range area of a poultry farm.

In the Netherlands, free-range layer farms as opposed to indoor layer farms, are at greater risk with regard to the introduction of avian influenza viruses (AIVs). Wild waterfowl are the natural reservoir hosts of AIVs, and play a major role in their transmission to poultry by contaminating free-range layer areas. The laser as a wild bird repellent has been in use since the 1970s, in particular around airfields to reduce bird-strike. The efficacy of laser for reducing wild bird numbers in and around free-range poultry areas has however not been investigated. During the autumn-winter, wild bird visits to the free-range area of a layer farm was surveilled by video-camera for a month without laser, followed by a month with laser. The automated laser (Class-III B qualification) was operated in two separate areas (i) within the poultry free-range area that directly bordered the poultry barn between 5:00 p.m. and 10:00 a.m. when poultry were absent (free-range study area, size 1.5 ha), and (ii) in surrounding grass pastures between 10:00 a.m. and 5:00 p.m. The overall (all bird species combined) efficacy of the laser for reducing the rate of wild birds visiting the free-range study area was 98.2%, and for the Orders Anseriformes and Passeriformes, respectively, was 99.7% and 96.1%. With the laser in operation, the overall exposure time of the free-range area to wild bird visits, but specifically to the Order Anseriformes, was massively reduced. It can be concluded that the Class-III B laser is highly proficient at keeping wild birds, in particular waterfowl, away from the free-range area of layer farms situated along a winter migration flyway.

Seasonal risk of low pathogenic avian influenza virus introductions into free-range layer farms in the Netherlands.

Poultry can become infected with avian influenza viruses (AIV) via (in) direct contact with infected wild birds. Free-range chicken farms in the Netherlands were shown to have a higher risk for introduction of low pathogenic avian influenza (LPAI) virus than indoor chicken farms. Therefore, during outbreaks of highly pathogenic avian influenza (HPAI), free-range layers are confined indoors as a risk mitigation measure. In this study, we characterized the seasonal patterns of AIV introductions into free-range layer farms, to determine the high-risk period. Data from the LPAI serological surveillance programme for the period 2013-2016 were used to first estimate the time of virus introduction into affected farms and then assess seasonal patterns in the risk of introduction. Time of introduction was estimated by fitting a mathematical model to seroprevalence data collected longitudinally from infected farms. For the period 2015-2016, longitudinal follow-up included monthly collections of eggs for serological testing from a cohort of 261 farms. Information on the time of introduction was then used to estimate the monthly incidence and seasonality by fitting harmonic and Poisson regression models. A significant yearly seasonal risk of introduction that lasted around 4 months (November to February) was identified with the highest risk observed in January. The risk for introduction of LPAI viruses in this period was on average four times significantly higher than the period of low risk around the summer months. Although the data for HPAI infections were limited in the period 2014-2018, a similar risk period for introduction of HPAI viruses was observed. The results of this study can be used to optimize risk-based surveillance and inform decisions on timing and duration of indoor confinement when HPAI viruses are known to circulate in the wild bird population.

Within-farm transmission characteristics of bluetongue virus serotype 8 in cattle and sheep in the Netherlands, 2007-2008.

In 2006 and 2007, sheep and cattle farms in the Netherlands were affected by an epidemic of bluetongue virus serotype 8 (BTV-8). In order to obtain insight into the within-farm spread of the virus, five affected cattle and five affected sheep farms were longitudinally monitored between early 2007 and mid or late 2008. The farms were visited between four and seven times to collect blood samples. During each visit, all animals present in the flock or herd were sampled. The samples were analysed for the presence of BTV-8 antibodies (ELISA) and BTV-8 antigen (rRT-PCR). The observed patterns of RT-PCR positives indicate a rapid within-farm virus spread during the vector season. During vector-free periods we observed a complete rRT-PCR positivity decline within a few months. During the vector season a lower bound estimate of the basic reproduction number (R0) ranges from 2.9-6.9 in the cattle herds (one herd not analysed), and from 1.3-3.2 in the sheep flocks in this study.

Highly pathogenic avian influenza subtype H5Nx clade 2.3.4.4 outbreaks in Dutch poultry farms, 2014-2018: Clinical signs and mortality.

In recent years, different subtypes of highly pathogenic avian influenza (HPAI) viruses caused outbreaks in several poultry types worldwide. Early detection of HPAI virus infection is crucial to reduce virus spread. Previously, the use of a mortality ratio threshold to expedite notification of suspicion in layer farms was proposed. The purpose of this study was to describe the clinical signs reported in the early stages of HPAI H5N8 and H5N6 outbreaks on chicken and Pekin duck farms between 2014 and 2018 in the Netherlands and compare them with the onset of an increased mortality ratio (MR). Data on daily mortality and clinical signs from nine egg-producing chicken farms and seven Pekin duck farms infected with HPAI H5N8 (2014 and 2016) and H5N6 (2017-2018) in the Netherlands were analysed. In 12 out of 15 outbreaks for which a MR was available, MR increase preceded or coincided with the first observation of clinical signs by the farmer. In one chicken and two Pekin duck outbreaks, clinical signs were observed prior to MR increase. On all farms, veterinarians observed clinical signs of general disease. Nervous or locomotor signs were reported in all Pekin duck outbreaks, but only in two chicken outbreaks. Other clinical signs were observed less frequently in both chickens and Pekin ducks. Compared to veterinarians, farmers observed and reported clinical signs, especially respiratory and gastrointestinal signs, less frequently. This case series suggests that a MR with a set threshold could be an objective parameter to detect HPAI infection on chicken and Pekin duck farms at an early stage. Observation of clinical signs may provide additional indication for farmers and veterinarians for notifying a clinical suspicion of HPAI infection. Further assessment and validation of a MR threshold in Pekin ducks are important as it could serve as an important tool in HPAI surveillance programs.

Probability of exporting infected carcasses from vaccinated pigs following a foot-and-mouth disease epidemic.

Emergency vaccination is an effective control strategy for foot-and-mouth disease (FMD) epidemics in densely populated livestock areas, but results in a six-month waiting period before exports can be resumed, incurring severe economic consequences for pig exporting countries. In the European Union, a one-month waiting period has been discussed based on negative test results in a final screening. The objective of this study was to analyze the risk of exporting FMD-infected pig carcasses from a vaccinated area: (1) directly after final screening and (2) after a six-month waiting period. A risk model has been developed to estimate the probability that a processed carcass was derived from an FMD-infected pig (P(carc)). Key variables were herd prevalence (P(H)), within-herd prevalence (P(A)), and the probability of detection at slaughter (P(SL)). P(H) and P(A) were estimated using Bayesian inference under the assumption that, despite all negative test results, > or =1 infected pigs were present. Model calculations indicated that P(carc) was on average 2.0 x 10(-5) directly after final screening, and 1.7 x 10(-5) after a six-month waiting period. Therefore, the additional waiting time did not substantially reduce P(carc). The estimated values were worst-case scenarios because only viraemic pigs pose a risk for disease transmission, while seropositive pigs do not. The risk of exporting FMD via pig carcasses from a vaccinated area can further be reduced by heat treatment of pork and/or by excluding high-risk pork products from export.

Quantification of visits of wild fauna to a commercial free-range layer farm in the Netherlands located in an avian influenza hot-spot area assessed by video-camera monitoring.

Free-range poultry farms have a high risk of introduction of avian influenza viruses (AIV), and it is presumed that wild (water) birds are the source of introduction. There is very scarce quantitative data on wild fauna visiting free-range poultry farms. We quantified visits of wild fauna to a free-range area of a layer farm, situated in an AIV hot-spot area, assessed by video-camera monitoring. A total of 5,016 hr (209 days) of video recordings, covering all 12 months of a year, were analysed. A total of 16 families of wild birds and five families of mammals visited the free-range area of the layer farm. Wild birds, except for the dabbling ducks, visited the free-range area almost exclusively in the period between sunrise and the moment the chickens entered the free-range area. Known carriers of AIV visited the outdoor facility regularly: species of gulls almost daily in the period January-August; dabbling ducks only in the night in the period November-May, with a distinct peak in the period December-February. Only a small fraction of visits of wild fauna had overlap with the presence of chickens at the same time in the free-range area. No direct contact between chickens and wild birds was observed. It is hypothesized that AIV transmission to poultry on free-range poultry farms will predominantly take place via indirect contact: taking up AIV by chickens via wild-bird-faeces-contaminated water or soil in the free-range area. The free-range poultry farmer has several possibilities to potentially lower the attractiveness of the free-range area for wild (bird) fauna: daily inspection of the free-range area and removal of carcasses and eggs; prevention of forming of water pools in the free-range facility. Furthermore, there are ways to scare-off wild birds, for example use of laser equipment or trained dogs.

Estimating the introduction time of highly pathogenic avian influenza into poultry flocks.

The estimation of farm-specific time windows for the introduction of highly-pathogenic avian influenza (HPAI) virus can be used to increase the efficiency of disease control measures such as contact tracing and may help to identify risk factors for virus introduction. The aims of this research are to (1) develop and test an accurate approach for estimating farm-specific virus introduction windows and (2) evaluate this approach by applying it to 11 outbreaks of HPAI (H5N8) on Dutch commercial poultry farms during the years 2014 and 2016. We used a stochastic simulation model with susceptible, infectious and recovered/removed disease stages to generate distributions for the period from virus introduction to detection. The model was parameterized using data from the literature, except for the within-flock transmission rate, which was estimated from disease-induced mortality data using two newly developed methods that describe HPAI outbreaks using either a deterministic model (A) or a stochastic approach (B). Model testing using simulated outbreaks showed that both method A and B performed well. Application to field data showed that method A could be successfully applied to 8 out of 11 HPAI H5N8 outbreaks and is the most generally applicable one, when data on disease-induced mortality is scarce.