To investigate the association of primary acute cerebral venous thrombosis (CVT) with COVID-19 vaccination through complete ascertainment of all diagnosed CVT in the population of Scotland.Case-crossover study comparing cases of CVT recently exposed to vaccination (1–14 days after vaccination) with cases less recently exposed. Cases in Scotland from 1 December 2020 were ascertained through neuroimaging studies up to 17 May 2021 and diagnostic coding of hospital discharges up to 28 April 2021, linked to national vaccination records. The main outcome measure was primary acute CVT.Of 50 primary acute CVT cases, 29 were ascertained only from neuroimaging studies, 2 were ascertained only from hospital discharges, and 19 were ascertained from both sources. Of these 50 cases, 14 had received the Astra-Zeneca ChAdOx1 vaccine and 3 the Pfizer BNT162b2 vaccine. The incidence of CVT per million doses in the first 14 days after vaccination was 2.2  for ChAdOx1 and 1  for BNT162b2. The rate ratio for CVT associated with exposure to ChAdOx1 in the first 14 days compared with exposure 15-84 days after vaccination was 3.2 .These findings support a causal association between CVT and the AstraZeneca vaccine. The absolute risk of post-vaccination CVT in this population-wide study in Scotland was lower than has been reported for populations in Scandinavia and Germany; the explanation for this is not clear.The online version contains supplementary material available at 10.1186/s12879-021-06960-5. The first reports in international media of an association of COVID-19 vaccines with thrombotic events appeared on 7 March 2021, when the Austrian Federal Office for Safety in Health Care announced that it had suspended use of a batch of AstraZeneca ChAdOx1 vaccine after cases of thromboembolic events. On 7 April 2021 the UK Medicines and Healthcare products Regulatory Agency (MHRA) and the Joint Committee on Vaccines and Immunisation concluded that there was “a possible link” between the vaccine and cerebral venous thrombotic events and issued new guidance . An undeto estimate incidence of diagnosed primary acute CVT in Scotland during the vaccination programmeto ascertain whether diagnostic coding of hospital discharges or deaths adequately captures diagnosed CVT and allows differentiation of secondary and chronic cases from primary acute cases.to investigate the association of diagnosed CVT with vaccination.Risk/benefit assessments of the use of the AstraZeneca ChAdOx1 vaccine depend on estimating the incidence of CVT and TTS syndrome post-vaccination and evaluating evidence for causality. However, most such estimates have come from voluntary reporting schemes such as the MHRA Yellow Card scheme. These reporting schemes have limitations: vaccine-associated cases may be under-ascertained if the connection with vaccine exposure is not made by the patient or clinician and, once there has been publicity about any possible association with a particular medicine, reporting can be biased thereafter. Although studies based on linking vaccination records to hospital discharge records ascertained either directly  or via pIn brief, we retrieved all potentially relevant scan reports from the nationwide Picture Archiving and Communication System (PACS) to identify diagnoses of CVT from the study start date of 1 December 2020 up to 17 May 2021. The scan reports contain a summary of the clinical findings, the radiologist’s initial report, and a review by the consultant if the first radiologist was not a consultant. To allow the sensitivity and specificity of discharge coding to be assessed against scan reports as ground truth, we also retrieved all hospital discharge and death records with diagnostic codes for CVT in this time period. As all health care records in Scotland use the Community Health Index (CHI) as a unique identifier, we were able to link PACS reports and hospital discharge and death records to the daily updated national vaccination programme database held by Public Health Scotland/National Services Scotland. We pre-specified a case-crossover design that would compare exposure to vaccine in a recent time window (1 to 14 days since vaccination) with exposure in a less recent time window. The methods are described in detail below.The COVID-19 vaccination programme in Scotland began on 8 December 2021. Initial priority groups included care home residents and staff, front line health and social care workers and clinically extremely vulnerable individuals. Initially the Pfizer BNT162b2 product (hereafter Pfizer vaccine) was used. From 8 January 2021 the AstraZeneca ChAdOx1 product (hereafter AstraZeneca vaccine) was introduced and from 7 April 2021 the Moderna mRNA-1273 product (hereafter Moderna vaccine) was introduced. CHI number, date of administration, age at vaccination, vaccine product name, whether first or second dose were extracted for all 6894008 vaccination records from 4 December 2020 to 15 July 2021 from the national vaccination database.The key first-line investigation for patients presenting with any acute neurological syndrome is a non-contrast computed tomography (CT) scan of the head. However, this is positive in only about 30% of patients with CVT . For thiAdditional file For three of the 14 health boards in Scotland, the PACS database contained the radiology report only as an image that could not be queried. For these boards extractions were carried out directly from the local radiology information system that feeds the national PACS instead. For Lanarkshire (12.1% of the Scottish population) the same query was run as for the national PACS . From the two other boards that do not encode reports as text in PACS—Forth Valley and Dumfries and Galloway, covering 8.3% of the Scottish population—only the initial RIS code query for venograms up to 13 April 2021 was completed; a subsequent broader query could not be obtained at the time of this study.The extracted scan reports were filtered to retain any scan likely to be informative for the presence of CVT i.e. all those with a study type or study description including a venogram or where any of the following strings appeared in the the radiologist’s report: “sinus thrombosis”, “sinus thrombus”, “venous thrombosis”, “venous thrombus”, “venogr”. For any individual with at least one scan meeting these criteria all scan reports for that person since 1 Dec 2020 were retained for review.After arraying scans for each individual chronologically, each clinical event was scored by a doctor as primary acute, possible, follow-up, chronic, no valid result, secondary or negative  from 1 December 2020 to 2021-04-28 and National Register of Scotland death registrations from 1 December 2020 to 2021-04-16 were queried for any mention of a CVT code as discharge diagnosis or cause of death. The ICD-10 codes used were: I63.6 ; I67.6 ; and G08 . Records with mention of a local secondary cause such as infection, intracranial abscess or brain tumour were excluded by matching of the regular expressionThe case-crossover design compares event rates in time windows of recent and less recent exposure; this is equivalent to a matched case-control study in which the case and the control are the same person in different time windows . We pre-To allow comparison with other studies that have used a 28-day time window, we also tabulated events and rates for the period 15–28 days. All post-vaccination cases were within 84 days of vaccination, so for the case-crossover analysis the 1–14 day time window is compared with the 15–84 day time window. For each vaccine product and each time window, the number of person-fortnights at risk was calculated to the end of follow up. To allow for the variation between health boards in latest date of case ascertainment via scans, the person-days in each exposure category from 14 April to 17 May 2021 were multiplied by 0.917  and the person-days from 17 May 2021 to 1 June 2021 were multiplied by 0.121 (the proportion covered by Lanarkshire where records up to 1 June 2021 were extracted).As the numbers of observed events were too small for confidence intervals to be equivalent to credible intervals, Bayesian credible intervals were calculated directly from the quantiles of the posterior distribution. With a flat prior on the logarithm of the event rate and As shown in Additional file The yield of cases through SMR01 discharge reports up to the latest discharge in the SMR01 extract is shown in Table Table Combining the 49 cases ascertained through scans and scored as primary acute with the two additional cases with CVT coded as main diagnosis that had been ascertained only through SMR01, 50 cases classified as primary acute were retained for analysis, together with 18 scan-ascertained cases coded as possible.Table Table Seventeen definite CVT events and 25 definite or possible CVT events occurred post vaccination: the others were in individuals who had not been vaccinated at the time of onset. Of the 7 cases with onset within 14 days of exposure to AstraZeneca vaccine, 6 were in people aged less than 60 years and 4 were in women.The observed rate of definite (primary acute) CVT diagnoses in the 14 days after exposure to AstraZeneca vaccine is estimated as 2.2  per million doses. For the Pfizer vaccine, the corresponding rate was 1  per million doses. There were no cases in the small numbers exposed to Moderna vaccine. For comparison, the observed rate of primary acute CVT in the 15–84 day time window after exposure was 12 per million per year . The rate ratio associated with exposure in the last 14 days to the AstraZeneca vaccine was estimated as 3.2  for primary acute CVT, and 2.9  for primary acute or possible cases.For comparison with other studies that used a 28-day time window, the rate of primary acute CVT within 28 days of vaccination with the AstraZeneca product, based on 9 events in 2926114 28-day time intervals, was 3.1  per million doses. In those aged under 60 years the rate, based on 8 events in 1319795 28-day time intervals was 7  per million doses.Figure In this population-wide study we have captured all diagnosed CVTs nationally since before the start of the vaccination programme. We have shown that ascertainment of CVTs from diagnostic codes on hospital discharge records has low sensitivity for CVT; systematic review of scan reports is required for complete ascertainment of diagnosed cases and to distinguish primary acute from secondary cases. The number of events in those recently exposed to vaccine is small but we can still calculate a credible interval for the incidence rate and thus an upper bound. For the first 14 days after AZ vaccination, we calculate an upper bound of 4.9 per million doses on the absolute rate, and 9.1 for the rate ratio. Although the rate of venogram studies increased markedly in early April 2021 after the association of CVT with vaccination had received wide publicity, most of the vaccine-associated cases in this study were diagnosed before this, and thus it is unlikely that ascertainment could have biased the association with recent vaccine exposure. When combined with prior evidence of excess risk of CVT associated with the AstraZeneca vaccine in other populations, the case-crossover analysis supports a causal interpretation of this association. Although the number of cases exposed to the Pfizer vaccine is too small for any comparison between the two products, an upper bound can still be placed on the absolute risk associated with the Pfizer vaccine.A strength of this study is the complete ascertainment of cases in the population, not reliant on adverse event reporting or diagnostic coding of hospital discharge records. Manual review of scan reports, which typically included a summary of the clinical history allowed some CVT cases to be recoded as secondary to a local lesion such as a tumour or as chronic rather than acute CVT, and allowed the date of onset to be determined from the summary of the clinical history. Accurate assignment of date of onset is required for the case-crossover analysis to be valid. Frequently the onset of symptoms was a few days before presentation to hospital. The time window for onset of vaccine-associated CVT may thus be overestimated in studies that have reported the median time from vaccination to hospitalisation to be more than 10 days , 14.The case-crossover design eliminates confounding by time-invariant factors, which may be strong where vaccine allocation is based on pre-existing risk conditions that were used to allocate priority for vaccination, and thus provides evidence for causality where an association is detected. Note that our analysis assumes an ignorance prior; it ignores any prior evidence of association. Unlike the self-controlled case series design, which has also been used to study the associations of adverse events with COVID-19 vaccine , the casLimitations of our study are that we do not have access to data on platelet counts, D-dimer or platelet factor 4 antibody levels, allowing enumeration of the number of CVT events that are part of the formally defined VITT/TTS syndrome as meeting associated haematological criteria. We note that the Brighton Collaborative however acknowledges that the TTS syndrome definition may be too restrictive by excluding isolated thrombotic events that are causally related to vaccine. Also we did not ascertain venous thromboses at sites other than brain.From this study the background incidence of primary acute CVT in Scotland (excluding the 14-day time window after vaccination) can be estimated as about 12 per million adults per year: this is similar to the estimate of 16 per million per year in Australia in 2016 . For theAdverse event reporting systems can give early warning of unexpected effects but cannot be relied on to estimate incidence rates. By early April 169 CVTs had been reported to the EMA by which time about 34 million doses of Astra Zeneca vaccine had been administered, giving a rate of about 5 per million doses. In the UK as of 9 June 2021 the Yellow Card scheme operated by the MHRA had received 390 reports of cases of “major thromboembolic events with concurrent thrombocytopenia”. Cerebral venous sinus thrombosis was reported in 140 of these. By 2 June 2021 30.1 million doses had been administered, so allowing for a 1-week lag the incidence was 13.0 per million doses for the syndrome and 4.7 per million doses for CVT . A subseThe upper bound of 6.2 per million doses for incidence of CVT within 28 days of vaccination with the AstraZeneca product estimated in this study is rather lower than estimates reported from Scandinavia and Germany. In a study of all 281264 individuals aged 18-65 years in Denmark and Norway who received a first dose of the AstraZeneca vaccine, there were were 7 observed CVT events within 28 days of vaccination compared with 0.3 expected from rates in the general population . A reporThis study based on complete ascertainment of CVT cases makes it possible to set a definitive upper bound on the rate of vaccine-associated CVT in Scotland. The results reinforce the importance of establishing comprehensive surveillance of adverse events occurring after vaccination. By using e-health record systems we were able to obtain all neuroimaging reports for the population and to report preliminary results to public health agencies within a few weeks of initiating this study. This entailed labour-intensive manual coding of scan reports. For longer-term surveillance and scaling to larger populations a natural language processing algorithm could be developed to identify CVTs in imaging reports, but some manual coding would still be required especially to assign the date of onset.Policy on the continued use of the AstraZeneca vaccine has been driven by estimates of the risk/benefit ratio, with risk of TTS estimated from adverse event reporting schemes . Thus onAdditional file 1: Figure S1. Flow diagram for case ascertainment from scan reports.


Vaccine hesitancy remains a public health challenge, especially in Eastern Europe. Our study used a sample of essential workers living in the Republic of North Macedonia to: (1) Describe rates of vaccine hesitancy and risk perception of COVID-19; (2) Explore predictors of vaccine hesitancy; and (3) Describe the informational needs of hesitant and non-hesitant workers. A phone survey was administered in North Macedonia from 4–16 May 2021. Logistic regression explored associations of COVID-19 vaccine hesitancy with sociodemographic characteristics, non-COVID-19 vaccine hesitancy, previous diagnosis of COVID-19, and individual risk perception of contracting COVID-19. Chi-squared analyses compared differences in informational needs by hesitancy status. Of 1003 individuals, 44% were very likely to get the vaccine, and 56% reported some level of hesitancy. Older age, Albanian ethnicity, increased education, previous COVID-19 diagnosis, acceptance of other vaccines, and increased risk perception of COVID-19 infection were negatively associated with vaccine hesitancy. Results indicated significant differences in top informational needs by hesitancy status. The top informational needs of the hesitant were the freedom to choose to be vaccinated without consequences (57% vs. 42%,  COVID-19 is an unprecedented global crisis that has presented unique public health and healthcare challenges to the world. As of 5 December 2021, over 5.2 million people across the globe have died from COVID-19 complications . HoweverAs of January 2022, North Macedonia has reported over 8700 deaths attributed to COVID-19 and over 287,000 cases in a population of approximately 2 million people ,5,6. TheThe causes contributing to these low vaccination numbers are complex, and studies focused on understanding COVID-19 vaccination acceptance in Eastern European and Balkan countries are limited. To our knowledge, Slovenia is the only country in which there are extant scientific publications related to COVID-19 vaccine acceptance. Slovenian nursing students showed an acceptance rate of only 33% and a sample of healthcare workers, not including physicians, showed an acceptance rate lower than the general population (50% versus 57% respectively) . While eThe differences in vaccine acceptance patterns across European and Balkan nations are underscored by the sociopolitical intricacies unique to the region. It is presumed that sentiments against vaccinations can be at least partially attributed to an anti-vaccine and anti-government movement that has been growing in this region. These sentiments may have contributed to the European measles outbreak observed in 2018 . Data deIn this study, we focus on the population of the Republic of North Macedonia. To our knowledge, this is the first study aimed at understanding COVID-19 vaccine hesitancy in this nation with specific reference to essential workers’ informational needs. This study aims to: (1) Describe rates of vaccine hesitancy, (2) Explore predictors of vaccine hesitancy, and (3) Describe the information essential workers would need to make them more likely to accept the vaccine.We conducted a survey of essential workers in North Macedonia to assess their informational needs regarding the COVID-19 vaccine. We defined an essential worker as an individual working in any job category within a list of options as presented in The survey instrument consisted of 35 questions including the following topics/areas: socio-demographics, COVID-19 and other vaccine hesitancy, risk perception about contracting COVID-19, experience with COVID-19, and informational needs about the vaccine. Questions were translated from English into Macedonian and back-translated into English. A copy of the survey instrument is available upon request to the corresponding author.We computed descriptive statistics to describe our sample’s socio-demographics and other variables of interest such as COVID-19 and other vaccine hesitancy, risk perception about contracting COVID-19, experience with COVID-19, and informational needs about the vaccine. Predictors of vaccine hesitancy were explored using three logistic regression models. The dependent variable was derived from the answers to the following question: “If you were offered a COVID-19 vaccine within two months from now at no cost to you—how likely are you to take it?” Answers were coded as 1 (hesitant) if the respondent indicated they were: somewhat likely, not interested now but would consider it later on, somewhat unlikely, or very unlikely; and 0 (non-hesitant) if the respondent indicated they were very likely to take it. In model 1, the independent variables included age, sex, and ethnicity. Model 2 included parameters from model 1 with the addition of education as a proxy for socioeconomic status. Model 3 included the parameters from model 2 with the addition of the previous diagnosis of COVID-19, previous non-COVID-19 vaccine hesitancy, and risk perception of contracting COVID-19.To describe informational needs about the vaccine we analyzed responses to the following question: “What would be important for you to know to make you more likely to take the COVID-19 vaccine?” Respondents were allowed to select the three most important topics to them out of eight possible choices related to vaccine safety and effectiveness, and three choices out of six related to vaccine policies. Chi-Squared tests were used to assess differences in informational needs between hesitant and non-hesitant individuals. The statistical analysis was conducted using SPSS v.28.When asked the following question: “If you were offered a COVID-19 vaccine within two months from now at no cost to you—how likely are you to take it?” Forty-four percent said they were very likely to take it, 14% somewhat likely, 9.5% were not sure, 4% were somewhat unlikely, 13% were very unlikely, 11% reported they would not take it within two months but would consider it later on, and 4.5% refused to answer. Regarding hesitancy towards other vaccines, 12% of respondents reported that in the past they had refused a vaccine that was recommended to them by a healthcare worker.Twenty-five percent of respondents said they had been diagnosed with COVID-19 during the pandemic, 55% of respondents had friends or family members who had tested positive and had no or mild symptoms, 35% did not know of anyone who tested positive, 25% had friends or family members who experienced severe symptoms, and 14% knew a friend or family member who had died from COVID-19. Most respondents were concerned about contracting COVID-19 both at work and outside of work (78%).Model 2 included the parameters from model 1 with the addition of education level, which was used as a proxy for socioeconomic status. When education was included in the regression model, the male sex was no longer a significant predictor of vaccine hesitancy. Respondents with more than secondary school education  had 0.32 times the odds of being hesitant compared to individuals that had less than a secondary school education .Model 3 included parameters from both model 1 and model 2 with the addition of previous non-COVID-19 vaccine hesitancy, previous diagnosis of COVID-19 infection, and degree of concern about contracting COVID-19. In this model, educational attainment remained significantly negatively associated with vaccine hesitancy. Those with prior vaccine hesitancy had lower odds of being hesitant to take the COVID-19 vaccine. Respondents reporting that they had not avoided a vaccine recommended to them in the past had 0.51 times the odds of being hesitant compared to individuals who were hesitant . Individuals who reported they had been diagnosed with COVID-19 in the past had 32% decreased odds of being hesitant compared to individuals who did not receive a diagnosis . Respondents reporting concerns about contracting COVID-19 at either their workplace or home had 0.41 times the odds of being hesitant compared to respondents that were not concerned . Furthermore, respondents reporting concerns about contracting COVID-19 at both their workplace and their homes had 76% decreased odds of being hesitant to vaccination compared to non-concerned individuals .Respondents were asked what would be important for them to know to make them more likely to receive the vaccine. Each respondent was asked to choose the three most important topics that were unordered and mutually exclusive. Among the hesitant, the three most frequently selected topics—related to the safety and effectiveness of the vaccine—for which respondents wanted more information were: (1) The FDA, CDC, and WHO recommend and agree it is safe (35%), (2) the vaccine works in stopping the spread of COVID-19 from one person to another (35%), and (3) The vaccine cannot cause any immediate or long-term harm (33%). Among the non-hesitant respondents, the three most frequently selected topics in this category were (1) My risk of getting sick with COVID-19 is bigger than the risk of side effects from the vaccine (40%), (2) The vaccine works in protecting me from COVID-19 (37%), and 3) The vaccine works in stopping the spread of COVID-19 from one person to another (35%).Respondents were also asked to select topics for which they wanted reassurance related to the vaccine policies. The most frequently selected topics among hesitant respondents were (1) I will be free to choose if I get the vaccine or not with no consequences (57%), (2) Once vaccinated, I will be able to live my life with no restrictions (37%), and (3) Everybody will have equal access to the vaccine regardless of income, race, or insurance status (32%). Interestingly, the non-hesitant shared these same concerns in their top three, however, the order was different: (1) Once vaccinated I will be able to live my life with no restrictions (62%), (2) Everybody will have equal access to the vaccine regardless of income, race, or insurance status (44%), and (3) I will be free to choose if I get the vaccine or not with no consequences (42%).p < 0.01).We also noted which of these top three informational needs had significantly different percentages by hesitancy status. With regard to the vaccine safety-related informational needs of the hesitant, these individuals, compared to the non-hesitant, had a higher percentage of respondents who indicated they wanted to have some reassurance that main international agencies  were recommending the vaccine and that they would be free to choose to get the vaccine or not without consequences  and that the vaccine will protect them from COVID-19 .Regarding the vaccine safety-related informational needs of the non-hesitant, these individuals, compared to the hesitant, had a higher percentage of respondents indicating they needed to be reassured that getting sick with COVID-19 is worse than the risk of side effects from the vaccine . The non-hesitant had a higher percentage of respondents who indicated they wanted to be reassured that they would live with no restrictions once vaccinated (62% vs. 37% p < 0.01) and that everyone would have equal access to the vaccine .While the hesitant and non-hesitant shared the same top three vaccine policy-related informational needs, there were significant differences in their percentages. The hesitant, compared to the non-hesitant, had a higher percentage of respondents who indicated they wanted to be free to choose if they received the vaccine or not with no consequences ” was selected by 29% of hesitant individuals and 25% of non-hesitant individuals. The response, “It is impossible to get COVID-19 or any other disease from the vaccine itself or its components” was selected by 21% of hesitant respondents and 18% of non-hesitant respondents.As of January 2022, approximately 40% of adults in the Republic of North Macedonia have been fully vaccinated against COVID-19. The rate is well short of other Western European countries - most of which have vaccination rates over 60%. The underlying causes contributing to the lack of vaccination coverage in North Macedonia are complex. North Macedonia has struggled with vaccine supply shortages since the early days of its vaccine distribution campaign in mid-February. In addition, limited local capacity in launching timely and tailored awareness campaigns may have affected certain socially disadvantaged groups, such as the Roma population, contributing to a lack of coverage. Overall, general vaccine hesitancy remains one of the largest barriers to vaccine uptake in N. Macedonia ,15.Our survey was conducted in May 2021 when approximately 4% of the country’s 2.1 million residents had been vaccinated, this rate is now close to 40%. As such, the survey was implemented at a time of high demand and low supply, yet our results indicate that vaccine hesitancy may have impacted vaccine uptake at the time of the survey and in subsequent months. Our survey found approximately 56% of respondents reported some degree of hesitancy in getting the vaccine, which is consistent with a similar survey conducted in the United States at the start of the vaccine distribution campaign . When weWe recognize that this study has limitations. First, our sample is not representative of all essential workers in the Republic of North Macedonia. However, a representative sample of essential workers would be very difficult to obtain given the multitude of job categories included in the list of essential workers and the lack of data on how many people work within each category in the nation. Our results should be interpreted taking into consideration the time when the survey was implemented—vaccine hesitancy, as well as its determinants, may have changed since May 2021. The lack of longitudinal data does not allow us to study changes in the willingness to be vaccinated, therefore we do not know if those who were hesitant in May are the same people who are hesitant now, or if the informational needs we identified are reflective of current concerns. While our survey did capture risk perception about COVID-19, we did not include questions regarding awareness of COVID-19 complications, which may impact vaccine hesitancy—future research should aim to address this relationship. Finally, our results may not be comparable to other national polls or surveys because of potential differences in the survey methods, sample populations, and questions related to vaccination intent.Despite its limitations, we believe this study provides useful information to further understand vaccine hesitancy in the Balkans and how to enhance communication strategies that address misinformation. As Balkan governments have scrambled to secure COVID-19 vaccines, this region has become a breeding ground for anti-vaccine movements. The Balkans has long been a hotbed of misinformation, fueled by low levels of trust in government and other institutions . Regardi


Anas platyrhynchos), an important natural vector for avian influenza virus. We show that body temperature, heart rate and leukocyte composition change reliably during an acute phase immune response. Using genome-wide gene expression profiling of whole blood across time points we confirm that immunostimulants activate pathogen-specific gene regulatory networks. By reporting immune response related changes in physiological and behavioural traits that can be studied in free-ranging populations, we provide baseline information with importance to the global monitoring of zoonotic diseases.Monitoring and early detection of emerging infectious diseases in wild animals is of crucial global importance, yet reliable ways to measure immune status and responses are lacking for animals in the wild. Here we assess the usefulness of bio-loggers for detecting disease outbreaks in free-living birds and confirm detailed responses using leukocyte composition and large-scale transcriptomics. We simulated natural infections by viral and bacterial pathogens in captive mallards ( Major modern human diseases such as influenza and salmonellosis are frequently of zoonotic origin2. While zoonoses may have major impact on human health, the natural hosts of these diseases often show little signs of disease upon infection3. This allows for these diseases to persist in reservoir species with a risk of interspecies transmission to more susceptible host-species. Reservoir host physiology and behaviour during infection affect survival and disease duration in the host4 and therefore also affect the spread of infectious diseases5. Still little is known about the immune response of reservoir hosts in their natural environment3. Several hurdles have constrained our understanding of pathogen dynamics and immune responses in wild reservoir hosts. These include, but are not limited to (1) the scarce availability of toolsets available for measuring immune responses in free-ranging non-model species in comparison to those available for well-studied model species7, and (2) lack of detailed experimental data from many reservoir species making it difficult to interpret the results from immunological tests in a field setting7.Wild animals are important reservoirs of a wide range of infectious diseases, of which some have the potential to spill over to humans (zoonoses)8. The APR is triggered by the release of proinflammatory cytokines in immune cells, and is characterised by a suite of molecular, physiological, and behavioural changes10. Animals with an activated APR usually have a febrile response within hours of pathogen exposure11, but the duration and magnitude of the febrile response differ between host species14 and depend on the type of stimuli16. Besides fever, animals usually display sickness behaviours such as lethargy, depression and anorexia during the APR10. The APR has an important role for the recovery and survival of the host during infection17, and may thus affect the risk of further transmission of infectious agents17. Learning about the APR in reservoir species is therefore important for understanding their potential role in the spread of zoonotic diseases, and monitoring their APR in the field could be used as a warning system for disease outbreaks.The innate immune system provides the first line of defence against pathogens. An important part of the innate immune system is the acute-phase response (APR), a rapid and systemic response activated by trauma, inflammation, stress and infection18. Bio-loggers that can detect changes in important characteristics of the APR  are increasingly being used in wildlife research19, and thus provide information that could be used for identifying signs and symptoms of disease in reservoir species. Similarly, rapid advances in and decreased costs of next generation DNA and RNA sequencing technologies now allow researchers to study the underlying mechanisms of the APR in non-model species, and provides disease markers for studying immune status and responses in wild populations22. While these technological advances have been used to study changes in behaviour23, physiology24 and regulation of immune genes in free-living animals25, they have so far not been studied simultaneously during the APR in reservoir species of zoonotic diseases. Studies that define the baseline for these measurements and their deviation during the immune response in reservoir species will provide a valuable resource for disease monitoring in natural populations.Rapid advancement of bio-logging technologies now allows for the study of physiology and behaviour of free-living animals26 and novel candidate genes that are upregulated during different stages of the APR and in response to different pathogens, in tissues that can easily and repeatably be collected in free-living birds. For this purpose, we experimentally induced immune responses in an important natural vector for avian influenza virus (AIV), the mallard (Anas platyrhynchos)28. While mallards show few signs of disease when infected with low pathogenic strains of AIV29, they can show clinical signs and symptoms including fever, anorexia, neurological signs and death when infected with highly pathogenetic AIV31. To study the APR in mallards, we triggered the immune response in captive mallards using non-infectious immunostimulants and monitored the response with state-of-the-art bio-loggers measuring 3D acceleration, heart rate, and body temperature and with high throughput RNA sequencing technologies. Our specific aims were to (1) examine the magnitude and timing of changes in the bio-logged parameters, (2) assess whether a correspondent immune response can be detected in global gene expression profiles from whole blood, and (3) determine the specificity of the immune response to different stimulants, using transcriptomics and gene regulatory network analyses.The aim of this study was to identify reliable and repeatable, integrated multi-scale monitoring methods from several biological fields that will facilitate ecological immunology studies and disease monitoring in wild bird reservoirs. Further we wanted to identify knownStaphylococcus aureus). To determine if the immunostimulants caused physiological and behavioural changes in the mallards, we monitored changes in body temperature, heart rate and activity in three individuals per treatment group using bio-loggers , gram-negative bacteria  and gram-positive bacteria  and was elevated until 18.5 hps—compared to ~ 40–41 °C in the control group.To track the timing of the febrile response, we measured body temperature after administration of immune stimulants. The average body temperature increased in all treatment groups when compared to the control group Fig. a. The maS. aureus group.To assess the stress response, we examined the heart rate in the mallards following stimulation. The average heart rate was elevated in all treatment groups immediately following the stimulation Fig. b. The he32. No clear differences were apparent during the response to treatment, however, ducks showed increased activity upon recovery from the viral mimic  following administration of immune stimulants, we performed full transcriptome sequencing on blood samples from three females and three males for each treatment and timepoint, adding up to a total of 120 samples  analysis to investigate whether certain biological processes and pathways were overrepresented in our list of DEGs. We found a significant overrepresentation of Reactome pathways in the poly I:C treatment group at 3 (n = 25), 6 (n = 29), and 12 h n = 14) ps and in the LPS treatment group 3 (n = 62) and 6 hps (n = 29) (Supplementary Datasets  ps and ihttp://orn-files.iwww.mpg.de/dgeviz) where the gene expression fold changes are illustrated on seven immune related pathways from the Kyoto Encyclopedia of Genes and Genomes (KEGG) database36. The fold changes and corrected p values for all treatments and pathways are available in Supplementary Dataset To further explore the specificity of the immune response to each immunostimulant, we visualised the gene expression profiles for each treatment and time point in the context of gene networks. To facilitate exploration of the results as well as future comparative studies, we built an interactive webpage  and MDA5  were upregulated at early time points, as were the activating protein TRIM25 (Tripartite Motif Containing 25), and downstream signaling molecules TRAF (TNF Receptor Associated Factor) and FADD (Fas Associated Via Death Domain), and interferon regulatory protein IRF7 (Interferon Regulatory Factor 7). Details of the toll-like receptor signaling pathway and the Influenza A pathway are shown and discussed in Supplementary Information  animals39. High definition accelerometers that record acceleration along three axes can be used to detect subtle changes in behaviour, while minimising any potential interference from a human observer’s perspective32. We detected a clear and rapid increase in the core body temperature as well as heart rate in all immune challenged mallards, which correlated well with the timepoints when the white blood cell composition and gene expression profiles were altered in the immune challenged ducks. In contrast, no significant differences in activity levels between the treatment groups were detected during the acute phase response.An acute phase response includes physiological and behavioural changes. Studying such changes during infection in animals is not trivial, and usually includes disturbance of the animals for sampling or observation. One of the main goals of this study was to investigate if the acute phase response (APR) can be monitored using on-animal bio-loggers, technologies that have great applications for remote, long-term disease monitoring in wild populations. For this purpose, we recorded changes in body temperature, heart rate and activity in control and immune challenged mallards using bio-loggers, while simultaneously monitoring the cellular response using blood immune assays. Elevated body temperature can be used to quantify the extent of a fever response in birds40, yet continuous long-term heart rate data are rarely reported in disease ecology studies. It is therefore still unknown, too, whether heart rate and associated energy expenditure increase significantly in birds during common infections, such as AIV28. Here we found that the heart rate of mallards became elevated during the acute phase response in all immunological treatments. As heart rate loggers may become more commonly used in ecological studies in the future19 we expect that we will soon gain additional insights into the magnitude, duration and function of elevated heart rates during infections in wild birds.The cost of an upregulated immune response is debated17. Unexpectedly, we did not find differences in activity patterns between the immune challenged and the control birds. Currently we do not know whether the lack of a difference is a result of the captive conditions of our experimental design, a subclinical course of the APR due to the lower pathogenicity of the stimulant compared to the true pathogen, or whether it also reflects the possibility that the movement patterns of wild mallards are not affected by some infections23. We will follow up on this question in a sequel study that will investigate movement patterns in free-ranging mallards.While a host’s physiological response to an infection is important, a behavioural response such as sickness behaviour is similarly essential and ubiquitous41. Nevertheless, the change in heterophil:lymphocyte (H:L) ratio was more pronounced in all treatment groups than in the control group 42. Likewise, the differential gene expression and the leukocyte count analyses show that the timing of the fever response correlated well with the number of genes that were differentially expressed in the treatment groups  activate pathogen-specific gene regulatory networks in mallards.Our study is a first step to understand the immunocompetence in truly wild animals. While our initial studies examined immune responses of a non-domestic species under controlled conditions, we are aware that subsequent studies will need to be done entirely in the wild. Our strategy here was to reduce the complexity of the environment and to induce an immune response using immunostimulants. These immunostimulants are non-infectious compounds that trigger an immune response, but do not make the animal an infectious carrier. They can therefore be used in field experiments to study the immune response without spreading infectious agentsRSAD2), IFITM1 (Interferon induced transmembrane protein 1), IFIT5 (Interferon induced protein with tricopeptide repeats 5) and OASL (2′–5′ oligoadenylate synthetase-like)43. Poly I:C also induced a rapid and sustained upregulation of the IFI6 (IFN-α-inducible protein 6) in the ducks, an effector that blocks the replication of flaviviruses such as West Nile virus44. This indicates that poly I:C induced a typical antiviral response in the mallards. Several of the most upregulated genes in the LPS treatment group are involved in defence response, including TREM2 (triggering receptor expressed on myeloid cells 2), IL1R2 (Interleukin-1 receptor 2), PTX3 (Pentraxin 3), LYG2 (lysozyme G2) and IL22RA2 (Interleukin 22 Receptor Subunit Alpha 2)  was recently proposed as an important marker to monitor inflammatory conditions in poultry, as it is upregulated in response to bacterial and viral infections in chickens45. While the role of PTX3 is largely unknown in ducks, it was upregulated during early stage of egg drop syndrome virus infection in duck embryo fibroblast cells46. Interestingly, no significant upregulation of PTX3 was detected in the mallards in response to poly I:C or inactivated S. aureus (Supplementary Datasets Escherichia coli) than inactive gram-positive bacteria (S. aureus)47. In line with the results in mice, IL1R2 was differentially expressed in the ducks treated with LPS , was also differentially expressed in the LPS and the poly I:C treatment group  induced an increase in body temperature and heart rate as well as a change in leukocyte composition, only a low number of differentially expressed genes (DEGs) were detected in mallards stimulated with this pyrogen. We think that we are only observing a part of the immune response usually triggered by live S. aureus infection, as live S. aureus activate the NGF 58. Therefore, we suggest that LPS, but not inactivated S. aureus, has a great potential for mimicking bacterial infections in ecological immunology studies in mallards.Results from our gene ontology analyses show that the immunological stimulants induce pathogen-specific changes, which justify their use as surrogates to live pathogens in future manipulative studies. Several antiviral pathways were overrepresented in the poly I:C treatment groups, including the RIG-I/MDA5 signalling pathway Fig. . RIG-I aoup Fig. , as has B4GALNT4  gene which was one of the most upregulated genes in the poly I:C treatment group , a paralog of B4GALNT4, have reduced protection against influenza virus59, the role of B4GALNT4 in the response to viral infections is unknown60. B4GALNT4 is located next to the IFITM3 (Interferon Induced Transmembrane Protein 3) gene, which is known to restrict influenza virus61. IFITM3 is upregulated in mallards during influenza infection61, and was also one of the most upregulated genes in our poly I:C treated mallards  we provide means for future comparisons of the immune response in different species, including species with differences in severity of pathogenesis to AIV. When assessing gene expression in a pathway framework it is important to keep in mind that reference pathways are usually built on knowledge from model species such as human or mouse. The function of certain elements in the pathway used in this study might hence be different in the mallard, or even differ between the mallard and closely related species. One such example with relevance for this study is that birds lack the mammalian TLR6 and TLR962. Other differences that will be of relevance for future comparative immunology studies in birds are that chicken, but not duck, lack the RIG-I and TRIF related adapter molecule  proteins63. TRAM bridges the TLR4 and TRIF in the TLR3- and TLR4-mediated MyD88-independent signalling pathway, and is an important part of the TLR4 pathway64. As more genomic and transcriptomic studies are undertaken, a key next step will be the construction of species-specific immune regulatory networks for species with importance as hosts of EIDs.In many cases, the natural reservoir of EIDs show little to no signs of disease when being infected by the same pathogen that causes serious damage in other species65 combined with appropriate archiving and trans-disciplinary sharing of data (e.g. movebank.org) will facilitate the usage of bio-loggers for disease monitoring in the future.In this study we used attached and implanted bio-loggers as well as blood-based assays to record several characteristics of the immune response simultaneously. While a combination of different measuring techniques is indispensable for obtaining a comprehensive picture of the immune response, each technique has inherent practical limitations. The heart-rate and body temperature bio-loggers used in this study require surgical implantation, which may not be feasible in certain species and field settings. Blood-based assays in turn may be available to more research groups, but baseline information may not be available for many reservoir species hampering the interpretation of results. The technology to use within a particular study thus has to be determined based on the ecology of the species of interest and the research question in mind. We expect that the continuous technological development 66, we can now get closer to understanding the epidemiology of diseases such as AIV in mallards. By analysing the transcriptome after immune stimulation, we did not only gain novel insights into the molecular mechanisms behind this immune reaction but also showed that pathogen-specific immune pathways were upregulated in the blood during the acute phase response.In conclusion, we show that poly I:C and LPS induce a rapid and predictable acute phase response in mallards. We confirm that body temperature and heart rate increase during the acute phase response, and that this can be monitored remotely using on-animal bio-loggers. In combination with GPS data from tracking devices that can record movements of animals on a large scaleAll methods are described in more detail in the Supplementary Information.Anas platyrhynchos) were included in the study. The mallards were housed in groups of three in outdoor aviaries at the Max Planck Institute for Ornithology (MPIO) in Radolfzell, Germany. The aviaries measured 3 m x 4 m × 2.5 m (w × l × h) and contained a water basin and a shelter with nesting material  were used to mimic a Gram-positive bacterial infection. These compounds are all used as common tools for scientific research on the immune response and have all been shown to induce an increase in body temperature in Pekin ducks (Anas platyrhynchos domesticus)16. As body temperature as well as heart rate can be elevated in birds during stress or handling67, the experiment was divided into two parts. The first part of the experiment (Experiment 1) allowed us to monitor changes in body temperature, heart-rate and movement patterns from the individuals without disturbance, while the second part of the experiment (Experiment 2) allowed us to collect blood samples that were used to study differential gene expression and white blood cell composition  in the abdominal cavity of 12 individuals. Four weeks after surgery, we attached acceleration loggers  to the back of the same individuals using a customised backpack68. We divided the individuals into four groups of three individuals each, which then received one of the treatments. The individuals were left in the aviaries with minimal disturbances after stimulation to avoid changes in physiology and behaviour due to handling stress.In Experiment 1 we recorded changes in physiology and behaviour during the acute phase response using bio-loggers. For this purpose, we implanted heart rate and body temperature sensors  were divided into four groups (n = 11) and stimulated with one of the three treatments or the control. Blood samples were collected before stimulation and at a number of time points post stimulation (ps) .In Experiment 2 we repeated the treatments to collect blood samples for leukocyte counts and global gene expression analysis. An additional 32 mallards were included in the second stimulation event to ensure that enough samples were available for further analysis. The treatment was repeated after a minimum of two weeks to avoid potential short-term tolerance effects to the stimulantsFor more detailed description see Supplementary Information Text .70. We fitted generalised additive mixed models (GAMMs) to the data for each physiological measurement, to investigate whether they differed between the treatments. We estimated the mean from the posterior distribution using a Bayesian framework for each measurement.We used bio-loggers to observe changes in physiology and behaviour during the acute phase response. Briefly, we recorded body temperature, electrical activity of the heart, and acceleration data for three individuals per treatment. The data was downloaded remotely using an e-obs base station located outside the aviaries. We calculated the heart rate as beats per minute for every five-minute period from the electrocardiograms. We estimated the activity level of the mallards by calculating the mean of the variance of the acceleration measurements from each axis, followingWe performed a leukocyte differential count and calculated the heterophil:lymphocyte (H:L) ratio to get a better understanding of what changes occur in the leukocyte composition during the acute phase response in mallards. We prepared blood smears for five individuals per treatment and time point and determined the proportion of heterophils, lymphocytes, monocytes, eosinophils, and basophils using light microscopy. Staining and evaluation of blood films was performed by Pendl Lab, Switzerland. We fitted a multinomial model to estimate and compare the proportions of each leukocyte type in the different treatments and time points. The mean of the H:L ratio was estimated from the posterior distribution of the multinomial model.We report the 95% Credible Intervals (CrI) using the 2.5% and 97.5% quantiles from the posterior distribution from each model. The mean of each measurement was considered different from the mean of the control group when the CrI of the treatment group did not include the estimated mean from the control group.For more detailed description see Supplementary Information Text .edgeR71 and limma72 as described in 73. Briefly, we computed empirical Bayes moderated t- and B-statistics, correcting for possible sex and individual differences using fixed and random factors, respectively, to identify genes that were differentially expressed due to treatment. Genes with an FDR adjusted p value < 0.05 were considered differentially expressed. We used Venn diagrams to explore whether the same genes were differentially expressed in the treatment groups and at different time points, and visualised the expression level for the differentially expressed genes (DEGs) using heatmaps. We conducted a gene ontology (GO) analysis in PANTHER74 to retrieve GO IDs75 for the DEGs and to find pathways that were overrepresented in each treatment group.We monitored changes in the whole transcriptome in whole blood before and after the immune-stimulation for six individuals per treatment, using next generation RNA-sequencing to determine whether relevant immune pathways were upregulated in the respective treatments. We sequenced mRNA libraries on the Illumina HiSeq2500, and performed differential gene expression analysis using packages 36 in the VANTED software76. All pathways were compiled into an interactive webpage (http://orn-files.iwww.mpg.de/dgeviz/). The web-based pathway visualizations contain hyperlinks to a description of each gene via the KEGG webpage, including the gene and protein sequence and links to the National Centre for Biotechnology Information (NCBI) and Ensemble.We visualised the gene expression data on seven immune related pathways  from the KEGG databaseFor the poly I:C treatment we validated the RNA-seq results using real-time quantitative PCR (qPCR) (Supplementary Information Text For more detailed description see Supplementary Information Text  and Supphttps://arriveguidelines.org).The experiment was approved by the federal authorities of the German state of Baden-Württemberg . Based on § 42 TierSchVersV  the approval of the authorities has to follow the votum of a commission for animal experiments. This commission is comparable to the ethical committees in other countries, but, according to German legislation, it is not appointed by the research institutes but by the state authorities. The study was carried out in compliance with the ARRIVE guidelines (Supplementary Data 1.Supplementary Data 2.Supplementary Data 3.Supplementary Data 4.Supplementary Data 5.Supplementary Data 6.Supplementary Data 7.Supplementary Information.