A. Bouma

Avian Influenza A Virus (H7N7) Epidemic in The Netherlands in 2003: Course of the Epidemic and Effectiveness of Control Measures

An epidemic of high-pathogenicity avian influenza (HPAI) A virus subtype H7N7 occurred in The Netherlands in 2003 that affected 255 flocks and led to the culling of 30 million birds. To evaluate the effectiveness of the control measures, we quantified between-flock transmission characteristics of the virus in 2 affected areas, using the reproduction ratio Rh. The control measures markedly reduced the transmission of HPAI virus: Rh before detection of the outbreak in the first infected flock was 6.5 (95% confidence interval [CI], 3.1-9.9) in one area and 3.1 in another area, and it decreased to 1.2 (95% CI, 0.6-1.9) after detection of the first outbreak in both areas. The observation that Rh remained >1 suggests that the containment of the epidemic was probably due to the reduction in the number of susceptible flocks by complete depopulation of the infected areas rather than to the reduction of the transmission by the other control measures.

Risk Maps for the Spread of Highly Pathogenic Avian Influenza in Poultry

Devastating epidemics of highly contagious animal diseases such as avian influenza, classical swine fever, and foot-and-mouth disease underline the need for improved understanding of the factors promoting the spread of these pathogens. Here the authors present a spatial analysis of the between-farm transmission of a highly pathogenic H7N7 avian influenza virus that caused a large epidemic in The Netherlands in 2003. The authors developed a method to estimate key parameters determining the spread of highly transmissible animal diseases between farms based on outbreak data. The method allows for the identification of high-risk areas for propagating spread in an epidemiologically underpinned manner. A central concept is the transmission kernel, which determines the probability of pathogen transmission from infected to uninfected farms as a function of interfarm distance. The authors show how an estimate of the transmission kernel naturally provides estimates of the critical farm density and local reproduction numbers, which allows one to evaluate the effectiveness of control strategies. For avian influenza, the analyses show that there are two poultry-dense areas in The Netherlands where epidemic spread is possible, and in which local control measures are unlikely to be able to halt an unfolding epidemic. In these regions an epidemic can only be brought to an end by the depletion of susceptible farms by infection or massive culling. The analyses provide an estimate of the spatial range over which highly pathogenic avian influenza viruses spread between farms, and emphasize that control measures aimed at controlling such outbreaks need to take into account the local density of farms.

Development of a classical swine fever subunit marker vaccine and companion diagnostic test

The development of a classical swine fever (CSF) subunit marker vaccine, based on viral envelope glycoprotein E2, and a companion diagnostic test, based on a second viral envelope glycoprotein E(RNS), will be described. Important properties of the vaccine, such as onset and duration of immunity, and prevention of horizontal and vertical transmission of virus were evaluated. A single dose of the vaccine protected pigs against clinical signs of CSF, following intranasal challenge with 100LD(50) of virulent classical swine fever virus (CSFV) at 2 weeks after vaccination. However, challenge virus transmission to unvaccinated sentinels was not always completely inhibited at this time point. From 3 weeks up to 6 months after vaccination, pigs were protected against clinical signs of CSF, and no longer transmitted challenge virus to unvaccinated sentinels. In contrast, unvaccinated control pigs died within 2 weeks after challenge. We also evaluated transmission of challenge virus in a setup enabling determination of the reproduction ratio (R value) of the virus. In such an experiment, transmission of challenge virus is determined in a fully vaccinated population at different time points after vaccination. Pigs challenged at 1 week after immunization died of CSF, whereas the vaccinated sentinels became infected, seroconverted for E(RNS) antibodies, but survived. At 2 weeks after vaccination, the challenged pigs seroconverted for E(RNS) antibodies, but none of the vaccinated sentinels did. Thus, at 1 week after vaccination, R1, and at 2 weeks, R=0, implying no control or control of an outbreak, respectively. Vertical transmission of CSFV to the immune-incompetent fetus may lead to the birth of highly viraemic, persistently infected piglets which are one of the major sources of virus spread. Protection against transplacental transmission of CSFV in vaccinated sows was, therefore, tested in once and twice vaccinated sows. Only one out of nine once-vaccinated sows transmitted challenge virus to the fetus, whereas none of the nine twice-vaccinated sows did. Finally, our data show that the E(RNS) test detects CSFV-specific antibodies in vaccinated or unvaccinated pigs as early as 14 days after infection with a virulent CSF strain. This indicates that the E2 vaccine and companion test fully comply with the marker vaccine concept. This concept implies the possibility of detecting infected animals within a vaccinated population.

Efficacy and stability of a subunit vaccine based on glycoprotein E2 of classical swine fever virus.

The purpose of this study was to determine the efficacy and stability of an E2 subunit vaccine against classical swine fever virus (CSFV). The vaccine, which contains E2 produced in insect cells by a baculovirus expression vector is a potential marker vaccine, as it allows discrimination between infected and vaccinated pigs. Several vaccination-challenge experiments were performed to determine the dose that protects 95% of the vaccinated pigs (PD95), and to determine the stability and efficacy of the vaccine several months after production. A single vaccination with a vaccine dose of 32 microg E2 - the estimated PD95 - in a water-oil-water adjuvant prevented clinical signs and mortality due to a CSFV challenge-inoculation three weeks after vaccination. Moreover, virus transmission to susceptible sentinel pigs was prevented in nearly all groups of pigs vaccinated with this dose. The vaccine was stable for at least 18 months, and retained its full potency. These findings indicate that the E2 marker vaccine merits further evaluation for suitability for use in a control program during an outbreak of CSF.

Determination of the onset of the herd-immunity induced by the E2 sub-unit vaccine against classical swine fever virus.

For a recently developed E2 subunit vaccine against classical swine fever (CSF), the reduction in transmission, at different moments after vaccination, was assessed by animal experiments and statistical calculations. Two experiments were performed to estimate the reproduction ratio R. Experiment 1 consisted of three groups and experiment 2 of two groups each of 10 pigs. In four of these groups, all pigs were vaccinated intramuscularly with the vaccine. The pigs in the fifth group remained unvaccinated (control group). After treatment, half of each group was intranasally inoculated with the virulent CSFV strain Brescia. In the vaccine groups, the following vaccination-challenge intervals were applied: 14, 14, 10, and 7 days, respectively. The occurrence of (contact-) infection was determined using the E(rns) ELISA. In the 7-days interval group and in the control group, virus transmission to all contact pigs occurred, indicating R1. Neither in the two 2-week interval groups nor in the 10-day interval group did contact-infections occur. Hence, the estimated R is less than one, which indicates that an epidemic would fade out. Therefore, the E2 subunit vaccine may be an efficacious tool in a control program during an outbreak of CSF as from 10 days after vaccination.

Transmission of pseudorabies virus within pig populations is independent of the size of the population

Abstract Epidemic models are often used to analyse the transmission of infectious diseases. These models, however, differ in whether they assume that transmission of a pathogen increases with population size or not. The purpose of this study was to investigate whether the transmission of pseudorabies virus—expressed as the reproduction ratio R -depends on population size. Experimental groups of either ten or 40 vaccinated pigs per group were housed at equal density. We inoculated half of each group, and we estimated the transmission of the virus from the number of contact-infections, using a stochastic Susceptible-Infectious-Recovered model. We calculated that if the transmission depended on population size, the transmission in the group of 40 pigs would be four times as high as the transmission in the group of ten pigs. However, the transmission of the virus did not differ significantly between the groups, and thus we concluded that the transmission was not influenced by the size of the population. This finding suggests that control measures should not be aimed at reducing the size of the herd.

Estimation of Transmission Parameters of H5N1 Avian Influenza Virus in Chickens

Despite considerable research efforts, little is yet known about key epidemiological parameters of H5N1 highly pathogenic influenza viruses in their avian hosts. Here we show how these parameters can be estimated using a limited number of birds in experimental transmission studies. Our quantitative estimates, based on Bayesian methods of inference, reveal that (i) the period of latency of H5N1 influenza virus in unvaccinated chickens is short (mean: 0.24 days; 95% credible interval: 0.099–0.48 days); (ii) the infectious period of H5N1 virus in unvaccinated chickens is approximately 2 days (mean: 2.1 days; 95%CI: 1.8–2.3 days); (iii) the reproduction number of H5N1 virus in unvaccinated chickens need not be high (mean: 1.6; 95%CI: 0.90–2.5), although the virus is expected to spread rapidly because it has a short generation interval in unvaccinated chickens (mean: 1.3 days; 95%CI: 1.0–1.5 days); and (iv) vaccination with genetically and antigenically distant H5N2 vaccines can effectively halt transmission. Simulations based on the estimated parameters indicate that herd immunity may be obtained if at least 80% of chickens in a flock are vaccinated. We discuss the implications for the control of H5N1 avian influenza virus in areas where it is endemic.

Herd immunity to Newcastle disease virus in poultry by vaccination.

Newcastle disease is an economically important disease of poultry for which vaccination is applied as a preventive measure in many countries. Nevertheless, outbreaks have been reported in vaccinated populations. This suggests that either the vaccination coverage level is too low or that vaccination does not provide perfect immunity, allowing the virus to spread in partially vaccinated populations. Here we study the requirements of an epidemiologically effective vaccination program against Newcastle disease in poultry, based on data from experimental transmission studies. The transmission studies indicate that vaccinated birds with low or undetectable antibody titres may be protected against disease and mortality but that infection and transmission may still occur. In fact, our quantitative analyses show that Newcastle disease virus is highly transmissible in poultry with low antibody titres. As a consequence, herd immunity can only be achieved if a high proportion of birds (>85%) have a high antibody titre (log2 haemagglutination inhibition titre ≥3) after vaccination. We discuss the implications for the control of Newcastle disease in poultry by vaccination.

Experimental non-transmissible marker vaccines for classical swine fever (CSF) by trans-complementation of Erns or E2 of CSFV

Three mutants with deletions in the E2 gene of the infectious DNA copy of the classical swine fever virus (CSFV) strain-C were constructed: one missing the B/C domain of CSFV-E2 between amino acids (aa) 693 and 746, one missing the A domain between aa 800 and 864, and one missing the complete E2 between aa 689 and 1062. All three CSFV-E2 deletion mutants were unable to generate viable virus, indicating that each of the antigenic domains of E2 is essential for viability of CSFV. To rescue the CSFV-E2 deletion mutants SK6 cell lines constitutively expressing glycoprotein E2 of CSFV were generated. The rescued viruses infected and replicated in SK6 cells as demonstrated by expression of viral proteins, but this primary infection did not result in reproduction of infectious virus. Thus, these E2 complemented viruses are considered non-transmissible. In previous experiments, we showed that simultaneous injection of E(rns) complemented virus (Flc23) via intradermal (ID), intramuscular (IM) or intranasal (IN) routes conferred protection to pigs against a lethal challenge with CSFV [J. Virol. 74 (2000) 2973]. Here, we evaluate different routes of application (ID, IM or IN) with E(rns) complemented virus Flc23 in order to find the best route for complemented CSFVs. Intradermal injection with Flc23 protected pigs against a lethal CSFV challenge, whereas intramuscular injection induced partial protection, and intranasal injection did not mediate a protective immune response in pigs at all. We used the intradermal route of vaccination to test the E2 complemented viruses. Vaccination of pigs via the intradermal route with the E2 complemented CSFVs also resulted in the induction of antibodies and in (partial) protection against CSFV challenge. Pigs vaccinated with E2 complemented virus Flc4 (deletion B/C domain) survived a lethal CSFV challenge, whereas partial protection was induced in pigs vaccinated with Flc47 (deletion E2) or Flc48 (deletion A domain) E2 complemented viruses. Serological data demonstrate that these E2 complemented mutant viruses are, in combination with well known diagnostic tests based on E2, potential marker vaccines for CSF.

Reduction of Coxiella burnetii Prevalence by Vaccination of Goats and Sheep, the Netherlands

Recently, the number of human Q fever cases in the Netherlands increased dramatically. In response to this increase, dairy goats and dairy sheep were vaccinated against Coxiella burnetii. All pregnant dairy goats and dairy sheep in herds positive for Q fever were culled. We identified the effect of vaccination on bacterial shedding by small ruminants. On the day of culling, samples of uterine fluid, vaginal mucus, and milk were obtained from 957 pregnant animals in 13 herds. Prevalence and bacterial load were reduced in vaccinated animals compared with unvaccinated animals. These effects were most pronounced in animals during their first pregnancy. Results indicate that vaccination may reduce bacterial load in the environment and human exposure to C. burnetii.