Unravelling transmission trees of infectious diseases by combining genetic and epidemiological data.

Knowledge on the transmission tree of an epidemic can provide valuable insights into disease dynamics. The transmission tree can be reconstructed by analysing either detailed epidemiological data (e.g. contact tracing) or, if sufficient genetic diversity accumulates over the course of the epidemic, genetic data of the pathogen. We present a likelihood-based framework to integrate these two data types, estimating probabilities of infection by taking weighted averages over the set of possible transmission trees. We test the approach by applying it to temporal, geographical and genetic data on the 241 poultry farms infected in an epidemic of avian influenza A (H7N7) in The Netherlands in 2003. We show that the combined approach estimates the transmission tree with higher correctness and resolution than analyses based on genetic or epidemiological data alone. Furthermore, the estimated tree reveals the relative infectiousness of farms of different types and sizes.

Risk factors for African swine fever incursion in Romanian domestic farms during 2019.

African swine fever (ASF) entered Georgia in 2007 and the EU in 2014. In the EU, the virus primarily spread in wild boar (Sus scrofa) in the period from 2014-2018. However, from the summer 2018, numerous domestic pig farms in Romania were affected by ASF. In contrast to the existing knowledge on ASF transmission routes, the understanding of risk factors and the importance of different transmission routes is still limited. In the period from May to September 2019, 655 Romanian pig farms were included in a matched case-control study investigating possible risk factors for ASF incursion in commercial and backyard pig farms. The results showed that close proximity to outbreaks in domestic farms was a risk factor in commercial as well as backyard farms. Furthermore, in backyard farms, herd size, wild boar abundance around the farm, number of domestic outbreaks within 2 km around farms, short distance to wild boar cases and visits of professionals working on farms were statistically significant risk factors. Additionally, growing crops around the farm, which could potentially attract wild boar, and feeding forage from ASF affected areas to the pigs were risk factors for ASF incursion in backyard farms.

Intra- and interspecies transmission of H7N7 highly pathogenic avian influenza virus during the avian influenza epidemic in The Netherlands in 2003.

The poultry epidemic of H7N7 highly pathogenic avian influenza (HPAI) virus in the Netherlands in 2003 was probably the result of the introduction of an H7N7 low pathogenic avian influenza (LPAI) virus (by interspecies transmission from wild birds) and the subsequent intraspecies transmission of this virus in poultry. The intraspecies transmission of the ensuing H7N7 HPAI virus was very successful both within and between flocks. Consequently, in the two poultry-dense areas that were affected, the epidemic could only be stopped by eliminating all poultry in the region. According to the spatial models these are the only areas where this was the case in the Netherlands. There was also interspecies transmission to mammals, i.e., to pigs and to humans. For pigs it was shown that possible subsequent intraspecies transmission was negligible (R0 <1). With hindsight the same was probably also true for humans.

The sensitivity and specificity of fecal and cecal culture for the detection of Campylobacter in Dutch broiler flocks quantified by Bayesian analysis.

Dutch broiler flocks are routinely tested for the presence of thermotolerant Campylobacter spp. using a standard cultural procedure for fecal and cecal samples. The objective of this study was to estimate the sensitivity and specificity of fecal and cecal culture for detection of Campylobacter colonization in broiler flocks in absence of a gold standard. Data from 1600 flocks were used from two different populations, whereby only flocks with both fecal and cecal culture results were included in the analysis. Latent class analysis using Bayesian inference was applied to generate the test characteristics of fecal and cecal culture. Two statistical models assuming conditional dependence of both tests on Campylobacter status were used to compare the results. On flock level, the sensitivity of the fecal culture was found to be 21% (95% CI: 12, 31) and 23% (95% CI: 13, 60), and the specificity was 98% (95% CI: 94, 99) and 97% (95% CI: 92, 99) for the two models, respectively. The sensitivity of the cecal culture was 64% (95% CI: 37, 89) and 66% (95% CI: 39, 90), and the specificity was 98% (95 CI: 94, 99) and 95% (95% CI: 72, 99) in respective models. The implications of a low sensitivity as in the case of the fecal culture is important for the design and interpretation of monitoring programmes and may result in excessive false negative test results. Although cecal culture is the more sensitive test, substantial misclassification of infected flocks may still occur.

Urgent request on avian influenza.

Highly pathogenic avian influenza (HPAI) H5N8 is currently causing an epizootic in Europe, infecting many poultry holdings as well as captive and wild bird species in more than 10 countries. Given the clear clinical manifestation, passive surveillance is considered the most effective means of detecting infected wild and domestic birds. Testing samples from new species and non-previously reported areas is key to determine the geographic spread of HPAIV H5N8 2016 in wild birds. Testing limited numbers of dead wild birds in previously reported areas is useful when it is relevant to know whether the virus is still present in the area or not, e.g. before restrictive measures in poultry are to be lifted. To prevent introduction of HPAIV from wild birds into poultry, strict biosecurity implemented and maintained by the poultry farmers is the most important measure. Providing holding-specific biosecurity guidance is strongly recommended as it is expected to have a high impact on the achieved biosecurity level of the holding. This is preferably done during peace time to increase preparedness for future outbreaks. The location and size of control and in particular monitoring areas for poultry associated with positive wild bird findings are best based on knowledge of the wider habitat and flight distance of the affected wild bird species. It is recommended to increase awareness among poultry farmers in these established areas in order to enhance passive surveillance and to implement enhanced biosecurity measures including poultry confinement. There is no scientific evidence suggesting a different effectiveness of the protection measures on the introduction into poultry holdings and subsequent spread of HPAIV when applied to H5N8, H5N1 or other notifiable HPAI viruses.

Development of Disease-specific, Context-specific Surveillance Models: Avian Influenza (H5N1)-Related Risks and Behaviours in African Countries.

Avian influenza virus (H5N1) is a rapidly disseminating infection that affects poultry and, potentially, humans. Because the avian virus has already adapted to several mammalian species, decreasing the rate of avian-mammalian contacts is critical to diminish the chances of a total adaptation of H5N1 to humans. To prevent the pandemic such adaptation could facilitate, a biology-specific disease surveillance model is needed, which should also consider geographical and socio-cultural factors. Here, we conceptualized a surveillance model meant to capture H5N1-related biological and cultural aspects, which included food processing, trade and cooking-related practices, as well as incentives (or disincentives) for desirable behaviours. This proof of concept was tested with data collected from 378 Egyptian and Nigerian sites (local [backyard] producers/live bird markets/village abattoirs/commercial abattoirs and veterinary agencies). Findings revealed numerous opportunities for pathogens to disseminate, as well as lack of incentives to adopt preventive measures, and factors that promoted epidemic dissemination. Supporting such observations, the estimated risk for H5N1-related human mortality was higher than previously reported. The need for multidimensional disease surveillance models, which may detect risks at higher levels than models that only measure one factor or outcome, was supported. To develop efficient surveillance systems, interactions should be captured, which include but exceed biological factors. This low-cost and easily implementable model, if conducted over time, may identify focal instances where tailored policies may diminish both endemicity and the total adaptation of H5N1 to the human species.

Aujeszky's disease (pseudorabies) virus eradication campaign in The Netherlands.

The Dutch Aujeszky's disease virus (ADV) eradication campaign is based on vaccination with glycoprotein E deleted vaccines. In the first stage of the programme, that was started in September 1993, the transmission of ADV must be reduced sharply. Subsequently, the remaining sources of virus need to be traced and eliminated. During the final stage, vaccination should be forbidden. This paper summarizes the observations made during a field study on the eradication of ADV by vaccination and reports the design and preliminary results of the first stage of the Dutch eradication campaign.

The 1997-1998 epidemic of classical swine fever in the Netherlands.

In 1997, the pig husbandry in the Netherlands was struck by a severe epidemic of classical swine fever (CSF). During this epidemic 429 CSF-infected herds were depopulated and approximately 1300 herds were slaughtered pre-emptively. In addition millions of pigs of herds not CSF-infected were killed for welfare reasons (over crowding or overweight). In this paper, we describe the course of the epidemic and the measures that were taken to control it. The first outbreak was detected on 4 February 1997 in the pig dense south-eastern part of the Netherlands. We estimate that CSF virus (CSFV) had already been present in the country by that time for 5-7 weeks and that the virus had been introduced into approximately 39 herds before the eradication campaign started. This campaign consisted of stamping-out infected herds, movement restrictions and efforts to diagnose infected herds as soon as possible. However, despite these measures the rate at which new outbreaks were detected continued to rise. The epidemic faded out only upon the implementation of additional measures such as rapid pre-emptive slaughter of herds in contact with or located near infected herds, increased hygienic measures, biweekly screening of all herds by veterinary practitioners, and reduction of the transportation movements for welfare reasons. The last infected herd was depopulated on 6 March 1998.

Evaluation of tests for detection of antibodies to Aujeszky's disease (pseudorabies) virus glycoprotein E in the target population.

Receiver operating characteristic (ROC) curves assess the quality of tests over the entire range of test signals. We compared the ability of an ELISA to detect antibodies to Aujeszky's disease (pseudorabies) virus gE in colostrum (test A) and in a single droplet of whole blood (test B) with the results obtained in serum (gold standard) in the target population by constructing and analyzing such curves. The area under the ROC curve, which is a quantitative measure of test performance, proved to be significantly (p < 0.01) smaller in test A than in test B or the gold standard. No significant differences in the area under the ROC curve were observed between test B and the gold standard.

Quantification of the transmission of classical swine fever virus between herds during the 1997-1998 epidemic in The Netherlands.

In this study, we describe a method to quantify the transmission of Classical Swine Fever Virus (CSFV) between herds from data collected during the 1997-1998 epidemic in The Netherlands. From the contacts between infected herds and the serological findings shortly before depopulation, we estimated the week of virus introduction and the length of the period over which the herd emitted virus for each CSFV-infected herd. From these data, we estimated the infection-rate parameter beta (the average number of herds infected by one infectious herd during one week) and the herd reproduction ratio, Rh (the average total number of secondary outbreaks caused by one infectious herd, i.e. in its entire infectious period), using a SIR-model for different sets of CSF control measures. When Rh > 1, an epidemic continues to grow. On the other hand, when Rh < 1 an epidemic will fade out. During the phase before the first outbreak was diagnosed and no specific measures had been implemented, beta was estimated at 1.09 and Rh at 6.8. In the subsequent phase infected herds were depopulated, movement restrictions were implemented, infected herds were traced forward and backward and the herds in the protection and surveillance zones were clinically inspected by the veterinary authorities (regional screening). This set of measures significantly reduced beta to 0.38. However, Rh was 1.3 and thus still > 1. Consequently, the number of outbreaks continued to grow. After a number of additional measures were implemented, the value of Rh was reduced to 0.5 and the epidemic came to an end. These measures included pre-emptive slaughter of herds that had been in contact with infected herds or were located near an infected herd, increased hygienic procedures, replacement of transports of pigs for welfare reasons by killing of young piglets and a breeding ban, and regional screening for CSF-infected herds by local veterinary practitioners.

The classical swine fever epidemic 1997-1998 in The Netherlands: descriptive epidemiology.

The objective of this paper is to describe the severe epidemic of classical swine fever (CSF) in The Netherlands in 1997-1998 under a policy of non-vaccination, intensive surveillance, pre-emptive slaughter and stamping out in an area which has one of the highest pig and herd densities in Europe. The primary outbreak was detected on 4 February 1997 on a mixed sow and finishing pig herd. A total of 429 outbreaks was observed during the epidemic, and approximately 700,000 pigs from these herds were slaughtered. Among these outbreaks were two artificial insemination centres, which resulted in a CSF-suspect declaration of 1680 pig herds (mainly located in the southern part of The Netherlands). The time between introduction of CSF virus (CSFV) into the country and diagnosis of CSF in the primary outbreak was estimated to be approximately 6 weeks. It is presumed that CSFV was spread from The Netherlands to Italy and Spain via shipment of infected piglets in the beginning of February 1997, before the establishment of a total stand-still of transportation. In June 1997, CSFV is presumed to be introduced into Belgium from The Netherlands. Pre-emptive slaughter of herds that had been in contact with infected herds or were located in close vicinity of infected herds, was carried out around the first two outbreaks. However, this policy was not further exercised till mid-April 1997, when pre-emptive slaughter became a standard operational procedure for the rest of the epidemic. In total, 1286 pig herds were pre-emptively slaughtered. (approximately 1.1 million pigs). A total of 44 outbreaks (10%) was detected via pre-emptive slaughter. When there were clinical signs, the observed symptoms in infected herds were mainly atypical: fever, apathy, ataxia or a combination of these signs. In 322 out of 429 outbreaks (75%), detection was bases on clinical signs observed: 32% was detected by the farmer, 25% by the veterinary practitioner, 10% of the outbreaks by tracing teams and 8% by screening teams of the veterinary authorities. In 76% of the outbreaks detected by clinical signs, the farmer reported to have seen clinical symptoms for less than 1 week before diagnosis, in 22% for 1-4 weeks before diagnosis, and in 4 herds (1%) the farmer reported to have seen clinical symptoms for more than 4 weeks before diagnosis. Transportation lorries played a major role in the transmission of CSFV before the primary outbreak was diagnosed. It is estimated that approximately 39 herds were already infected before the first measures of the eradication campaign came into force. After the first measures to stop the spread of CSFV had been implemented, the distribution of the most likely routes of transmission markedly changed. In most outbreaks, a neighbourhood infection was indicated. Basically, there were two reasons for this catastrophe. Firstly, there was the extent of the period between introduction of the virus in the region and detection of the first outbreak. As a result, CSFV had opportunities to spread from one herd to another during this period. Secondly, the measures initially taken did not prove sufficient in the swine- and herd-dense region involved.

Transmission of classical swine fever virus within herds during the 1997-1998 epidemic in The Netherlands.

In this paper, we describe the transmission of Classical Swine Fever virus (CSF virus) within herds during the 1997-1998 epidemic in The Netherlands. In seven herds where the infection started among individually housed breeding stock, all breeding pigs had been tested for antibodies to CSF virus shortly before depopulation. Based upon these data, the transmission of CSF virus between pigs was described as exponential growth in time with a parameter r, that was estimated at 0.108 (95% confidence interval (95% CI) 0.060-0.156). The accompanying per-generation transmission (expressed as the basic reproduction ratio, R0) was estimated at 2.9. Based upon this characterisation, a calculation method was derived with which serological findings at depopulation can be used to calculate the period in which the virus was with a certain probability introduced into that breeding stock. This model was used to estimate the period when the virus had been introduced into 34 herds where the infection started in the breeding section. Of these herds, only a single contact with a herd previously infected had been traced. However, in contrast with the seven previously mentioned herds, only a sample of the breeding pigs had been tested before depopulation (as was the common procedure during the epidemic). The observed number of days between the single contact with an infected herd and the day of sampling of these 34 herds fitted well in the model. Thus, we concluded that the model and transmission parameter was in agreement with the transmission between breeding pigs in these herds. Because of the limited sample size and because it was usually unknown in which specific pen the infection started, we were unable to estimate transmission parameters for weaned piglets and finishing pigs from the data collected during the epidemic. However, from the results of controlled experiments in which R0 was estimated as 81 between weaned piglets and 14 between heavy finishing pigs (Laevens et al., 1998a. Vet. Quart. 20, 41-45; Laevens et al., 1999. Ph.D. Thesis), we constructed a simple model to describe the transmission of CSF virus in compartments (rooms) housing finishing pigs and weaned piglets. From the number of pens per compartment, the number of pigs per pen, the numbers of pigs tested for antibodies to CSF virus and the distribution of the seropositive pigs in the compartment, this model gives again a period in which the virus most probably entered the herd. Using the findings in 41 herds where the infection started in the section of the finishers or weaned piglets of the age of 8 weeks or older, and of which only a single contact with a herd previously infected was known, there was no reason to reject the model. Thus, we concluded that the transmission between weaned piglets and finishing pigs during the epidemic was not significantly different from the transmission observed in the experiments.

Spatial and stochastic simulation to evaluate the impact of events and control measures on the 1997-1998 classical swine fever epidemic in The Netherlands. I. Description of simulation model.

The simulation model InterCSF was developed to simulate the Dutch Classical Swine Fever (CSF) epidemic of 1997-98 as closely as possible. InterCSF is a spatial, temporal and stochastic simulation model. The outcomes of the various replications give an estimate of the variation in size and duration of possible CSF-epidemics. InterCSF simulates disease spread from an infected farm to other farms through three contact types (animals, vehicles, persons) and through local spread up to a specified distance. The main disease-control mechanisms that influence the disease spread in InterCSF are diagnosis of the infected farms, depopulation of infected farms, movement-control areas, tracing, and pre-emptive slaughter. InterCSF was developed using InterSpread as the basis. InterSpread was developed for foot-and-mouth disease (FMD). This paper describes the process of modifying InterSpread into InterCSF. This involved changing the assumptions and mechanisms for disease spread from FMD to CSF. In addition, CSF-specific control measures based on the standard European Union (EU) regulations were included, as well as additional control measures that were applied during the Dutch epidemic. To adapt InterCSF as closely as possible to the Dutch 1997/98 epidemic, data from the real epidemic were analysed. Both disease spread and disease-control parameters were thus specifically based on the real epidemic. In general, InterSpread turned out to be a flexible tool that could be adapted to simulate another disease with relative ease. The most difficult were the modifications necessary to mimic the real epidemic as closely as possible. The model was well able to simulate an epidemic with a similar pattern over time for number of detected farms as the real outbreak; but the absolute numbers were (despite many relevant modifications) not exactly the same--but were within an acceptable range. Furthermore, the development of InterCSF provided the researchers with a better insight into the existing knowledge gaps. In part II (see the final paper in this issue), InterCSF was used to compare various control strategies as applied to this epidemic.

Assessment of the effectiveness of vaccination against pseudorabies in finishing pigs.

Whereas the clinical efficacy of vaccination against pseudorabies has been studied extensively, methods to evaluate the influence of vaccination on pseudorabies virus (PRV) transmission have only recently become available. In this study, PRV transmission and growth performance in finishing pigs vaccinated either once or twice were compared. The incidence of PRV infections was significantly (P = 0.039) higher in the group vaccinated once (38%) than in the group vaccinated twice (10%). The reproduction ratio R, which is defined as the average number of new infections caused by 1 infectious individual, was estimated in both groups. This ratio was also significantly (P = 0.025) higher among single vaccinated pigs (R = 3.4) than among pigs that had received double vaccination (R = 1.5). In compartments where serologic evidence of PRV introduction was observed, the mean daily weight gain was significantly (P = 0.029) lower in pigs vaccinated once (698 g/d) than in pigs vaccinated twice (721 g/d). Results of this study document the possibility to objectively evaluate the effect of vaccination on PRV transmission under field conditions. From the results, we concluded that double vaccination is advantageous in populations of finishing pigs at risk for PRV introduction. However, even among pigs vaccinated twice, extensive spread of PRV can occur.

Reduction of the prevalence of pseudorabies virus-infected breeding pigs by use of intensive regional vaccination.

The influence of an intensive vaccination program on prevalence of pseudorabies virus (PRV)-infected breeding pigs was examined in a region where the disease was enzootic. In a 2-year period, significantly (P < 0.005) greater decrease in the prevalence of PRV-infected breeding pigs was observed in herds participating in the regional vaccination program and in herds outside the trial region that intensified PRV vaccination during the study than in herds that applied routine PRV control measures. In the regional vaccination program, introduction of breeding stock from outside the area was significantly (P < 0.05) associated with higher prevalence of PRV-infected pigs at the end of the study. These results indicate that transmission of PRV can be markedly reduced by use of an accurately applied intensive vaccination program.

On-farm euthanasia of broiler chickens: effects of different gas mixtures on behavior and brain activity.

The purpose of this study was to investigate the suitability of gas mixtures for euthanasia of groups of broilers in their housing by increasing the percentage of CO2. The suitability was assessed by the level of discomfort before loss of consciousness, and the killing rate. The gas mixtures injected into the housing were 1) 100% CO2, 2) 50% N2 + 50% CO2, and 3) 30% O2 + 40% CO2 + 30% N2, followed by 100% CO2. At 2 and 6 wk of age, groups of 20 broiler chickens per trial were exposed to increasing CO2 percentages due to the injection of these gas mixtures. Behavior and killing rate were examined. At the same time, 2 broilers per trial equipped with brain electrodes were observed for behavior and brain activity. Ten percent of the 2-wk-old broilers survived the increasing CO2 percentage due to the injection of 30% O2 + 40% CO2 + 30% N2 mixture, therefore this mixture was excluded for further testing at 6 wk of age. At 6 wk of age, 30% of the broilers survived in the 50% N2 + 50% CO2 group. The highest level of CO2 in the breathing air (42%) was reached by the injection of the 100% CO2 mixture, vs. 25% for the other 2 mixtures. In all 3 gas mixtures, head shaking, gasping, and convulsions were observed before loss of posture. Loss of posture and suppression of electrical activity of the brain (n = 7) occurred almost simultaneously. The results of this experiment indicate that euthanasia of groups of 2- and 6-wk-old broilers by gradually increasing the percentage of CO2 in the breathing air up to 40% is possible.

Ambulant 24-hour blood pressure and heart rate of dentists.

PURPOSE: To investigate blood pressure fluctuations in dentists during their daily activities, including dental procedures, in comparison with a non-dentist population. MATERIALS AND METHODS: A pilot study on blood pressure and heart rate fluctuations was performed which involved 26 dentists working at a university clinic. A 24-hour blood pressure registration was obtained using the Oxford Monitoring System. RESULTS: In the dentist group, both blood pressure and heart rate were found to be significantly higher during work than during leisure activities. In the control group, no significant difference in blood pressure was recorded between these periods. This study also showed the feasibility of ambulant 24-hour blood pressure registration on dentists during daily activity. Our results ethically justify a more intensive cardiovascular study involving a larger number of dentists working in private practice in order to establish whether our results are valid for the dental practitioner in general.

Pseudorabies virus eradication by area-wide vaccination is feasible.

This article reviews the rationale for using marker vaccines and companion diagnostic tests in the eradication of pseudorabies virus (PRV). Recent advances in vaccinology and epidemiology indicate that, despite the inability to induce complete immunity, vaccination is a useful tool in the battle against PRV. This review focuses on the effectiveness of vaccination under field conditions and on herd, management and regional factors that are associated with PRV introduction or transmission.

Marked reduction of the prevalence of pseudorabies virus-infected pigs in pig dense regions of The Netherlands during the first year of a nation-wide vaccination campaign.

The breeding and finishing pig populations in four Animal Health Service regions in the Netherlands were monitored with respect to pseudorabies virus (PRV) seroprevalence. Analysis of data of the seroprevalence survey of 1994 indicated that two samples per herd was sufficient to estimate the seroprevalence in both the sow and finishing pig populations. In the northern, eastern, southern, and western regions, 115, 645, 940, and 218 sow herds and 114, 1036, 954, and 323 pig finishing herds were sampled, respectively. In the breeding pig population, the PRV-seroprevalence decreased from 27% to 17% in the eastern region, from 32% to 17% in the southern region, from 18% to 12% in the western region, and from 10% to 6% in the northern region. In the finishing pig population, the PRV-seroprevalence decreased from 15% to 6% in the eastern region, from 19% to 6% in the southern region, and from 12% to 5% in the western region. There was no significant difference in PRV seroprevalence in finishing pigs in the northern region between the survey of 1994 and 1995.

Dynamics of pseudorabies virus infections in vaccinated pig populations: a review.

This paper reviews our studies on the dynamics of pseudorabies virus (PRV) infections in populations of vaccinated pigs. By using mathematical models, experiments, and field observations, we have been able to quantify PRV transmission by the reproduction ratio R, which is defined as the average number of secondary cases caused by one infectious individual. If R is less than 1, PRV infections will fade out in the pig population and eradication is certain. Under experimental conditions, R of double-vaccinated pigs was estimated at 0.3. In the field, R was estimated at 0.7 among multiple-vaccinated breeding pigs, 3.4 among single vaccinated finishing pigs, and 1.5 among double-vaccinated finishing pigs. So far, no risk factors have been identified that explain the difference between the transmission among double-vaccinated pigs in the field and under experimental conditions. The implications of the transmission characteristics of the different types of pigs for the Dutch PRV eradication campaign are discussed. The structure of the PRV research programme described in this paper, in which knowledge of the interaction of the virus with individual pigs is extrapolated to the interaction of the virus with pig populations, can serve as an example for other research programmes that study infectious diseases.