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Conceived and designed the experiments: AB IC KN GK MvB. Performed the experiments: AB KN. Analyzed the data: DK CAD MvB. Contributed reagents/materials/analysis tools: IC KN GK. Wrote the paper: AB DK CAD MvB.

The authors have declared that no competing interests exist.

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.

Outbreaks of highly pathogenic H5N1 avian influenza in poultry first occurred in China in 1996. Since that time, the virus has become endemic in Asia, and has been the cause of outbreaks in Africa and Europe. Although many aspects of H5N1 virus biology have been studied in detail, surprisingly little is known about the key epidemiological parameters of the virus in its avian hosts (the length of time from infection until a bird becomes infectious, the duration of infectiousness, how many birds each infectious bird will infect). In this paper we show, using experimental transmission studies with unvaccinated and vaccinated chickens, that H5N1 avian influenza induces a short duration of infectiousness (∼2 days) and a very short period of time from infection until infectiousness (∼0.25 day) in unvaccinated chickens. Furthermore, while transmission was efficient among unvaccinated birds, no bird-to-bird transmission was observed in vaccinated chickens. Our results indicate that it may be difficult to curb outbreaks by vaccination after an introduction in a flock has been detected. On the other hand, preventive vaccination could be effective in preventing virus introductions and limiting the size of outbreaks.

Highly pathogenic avian influenza virus strains of the H5 or H7 subtypes are noted for being highly contagious among various bird species and inducing high mortality rates in poultry. Although outbreaks of highly pathogenic avian influenza have been reported since the 1950s the current focus is on the H5N1 subtype. The first outbreaks of H5N1 were reported in Hong Kong in 1997

Despite the fact that aspects of H5N1 avian influenza biology have been studied in detail, ranging from molecular studies of host range factors, phylogenetic analyses aimed at unravelling the virus' evolutionary pathways, surveillance of H5N1 in wild birds, studies into the clinical course of H5N1 infections in humans, and vaccine efficacy and safety studies, there is scant information of the basic epidemiological characteristics of H5N1 viruses in their avian hosts. Specifically, little is known about the infectious period of H5N1 in various host species, the duration of the latent period, and the transmissibility of the virus from bird to bird. For a proper understanding of the transmission dynamics of the virus and to be able to assess the potential impact of control measures such as vaccination, however, this information is crucial. For instance, it is well-known that both the invasion prospects of the virus as well as the number of individuals ultimately infected are critically affected by the (distribution of the) infectious period and transmission parameter. The (distribution of the) period of latency is also of importance since it is a key factor affecting the initial growth rate and duration of an epidemic

Here we present and analyze experimental transmission studies with highly pathogenic H5N1 avian influenza virus (A/Chicken/Legok/2003) in chickens to obtain quantitative estimates of key epidemiological parameters. Specifically, we performed experiments in which an artificially infected chicken was placed in a cage with a susceptible contact bird, and in which the transmission chain was monitored by taking daily samples from the trachea and cloaca

The experiments are analyzed by tailored statistical methods based on a SEIR (susceptible-exposed-infectious-removed) epidemiological model. In this way all estimated parameters have a clear-cut biological interpretation (mean and variance of the latent and infectious period, transmission rate, reproduction number). Here we use two different methods of analysis. The first uses final size data i.e. the number of birds that are ultimately infected, and is aimed at estimation of the reproduction number

All inoculated unvaccinated birds (

bird type | days post challenge | |||||||

0 | 1 | 2 | 3 | 4 | 5 | 6 | 7 | |

i | −/− | +/+ | +/+ | |||||

c | −/− | −/− | −/− | +/+ | ||||

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the infection dose is 0.2 ml of 10^{5} EID_{50} intranasally (0.1 ml) and intraocularly (0.1 ml).

i: inoculated bird; c: contact bird; x/y: test result for virus isolation in the trachea/cloaca.

bird died.

the bird was alive at the end of the experiment and tested negative in the serological test.

bird type | days post challenge | |||||||

0 | 1 | 2 | 3 | 4 | 5 | 6 | 7 | |

I | −/− | +/+ | ||||||

C | −/− | −/− | −/− | −/− | ||||

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c | −/− | −/− | −/− | +/+ | ||||

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c | −/− | −/− | −/− | −/− | −/+ | |||

i | −/− | +/+ | ||||||

c | −/− | −/− | −/− | +/+ | ||||

i | −/− | +/+ | −/− | |||||

c | −/− | −/− | +/− | +/+ |

the infection dose is 0.2 ml of 10^{6} EID_{50} intranasally (0.1 ml) and intraocularly (0.1 ml).

i: inoculated bird; c: contact bird; x/y: test result for virus isolation in the trachea/cloaca.

the bird died.

the bird was alive at the end of the experiment and tested negative in the serological test.

In the experiments with vaccinated birds no contact birds were infected and only a few of the inoculated birds shed virus on just a few days. In fact, only 7 out of 66 inoculated birds shed virus for a total of 12 days. Of these, virus was isolated from the trachea only on 11 days and from the trachea and cloaca on a single day. None of the vaccinated birds died in the course of the experiments, and no signs of disease were observed in any of the vaccinated birds. Details of the vaccination experiments are given in

With regard to transmission, the final size analyses indicate that there are significant differences between the experiments with unvaccinated and vaccinated birds.

experiment | final size | |||

low dose (N = 11) | 9 | 9 (1.9–86) | 3.4 (1.3–7.6) | 1.0/1.0 |

high dose (N = 11) | 5 | 1.7 (0.40–6.6) | 1.2 (0.37–2.9) | 0.88/0.77 |

vaccination |
0 | 0 (0–0.80) | 0 (0–0.67) | 0.011/0.0041 |

maximum likelihood estimates of the reproduction number with 95% confidence intervals (between brackets) if the infectious period is exponentially distributed (

p-values of the null hypothesis under the assumption of an exponentially distributed and fixed infectious period.

all experiments with vaccinated birds (

No transmission was observed in all six experiments with vaccinated birds, resulting in a maximum likelihood estimate of the reproduction number of 0. The (two-sided) 95% confidence interval ranges from 0–0.80 or 0–0.67, depending on the assumptions regarding the distribution of the infectious period. Furthermore, the null-hypothesis that the reproduction number is larger than the threshold value 1 can safely be rejected (p = 0.011 in case of an exponentially distributed infectious period, and p = 0.0041 in case of a fixed infectious period). Hence, it is unlikely that an epidemic can occur in vaccinated populations.

The experiments with unvaccinated birds are analyzed using Bayesian methods to obtain estimates of the transmissibility of the virus and the distributions of the latent and infectious periods. ^{−1}^{−1}

Shown are samples from the marginal posterior density of the mean versus variance of the latent period (A), the mean versus variance of the infectious period (B), and the mean infectious period versus transmission rate parameter (C). The contours in (C) correspond to specific values of the reproduction number. See

Shown are samples from the marginal posterior density of the mean versus variance of the latent period (A), the mean versus variance of the infectious period (B), and the mean infectious period versus transmission rate parameter (C). Blue and red dots refer to parameters characterizing the low- and high-dose experiments, respectively. The contours in (C) correspond to specific values of the reproduction number. See

Shown are samples from the marginal posterior density of the mean versus variance of the latent period (A), the mean versus variance of the infectious period (B), and the mean infectious period versus transmission rate parameter (C). Dots refer to parameters characterizing the contact infections. The contours in (C) correspond to specific values of the reproduction number. See

scenario | description | ^{−1} |
||||||

A1 | low-dose experiment | 0.80 | 0.20 | 0.044 | 2.5 | 0.16 | 2.0 | 1.2 |

(0.38–1.5) | (0.049–0.43) | (0.0020–0.45) | (2.2–2.8) | (0.045–0.48) | (0.96–3.6) | (0.94–1.5) | ||

A2 | high-dose experiment | 0.74 | 0.44 | 0.078 | 1.3 | 0.13 | 0.99 | 1.8 |

(0.27–1.6) | (0.14–0.87) | (0.0026–1.1) | (0.92–1.8) | (0.0097–0.66) | (0.38–2.1) | (1.3–2.3) | ||

B | combined analysis | 0.76 | 0.24 | 0.043 | 2.1 | 0.33 | 1.6 | 1.3 |

(0.42–1.2) | (0.099–0.48) | (0.0039–0.36) | (1.8–2.3) | (0.15–0.77) | (0.90–2.5) | (1.0–1.5) | ||

C | differences in the mean infectious period | 0.73 | 0.20 | 0.035 | 2.5 |
0.15 | 1.8 |
1.3 |

(0.43–1.2) | (0.094–0.45) | (0.0030–0.54) | 1.7 |
(0.049–0.58) | 1.2 |
(1.1–1.5) | ||

D | differences between inoculated/contact birds |
0.44 | 0.038 | 1.7 | 0.19 | |||

0.81 | (0.18–0.70) | (0.0011–0.53) | (1.4–2.1) | (0.025–0.58) | 2.0 | 1.2 | ||

(0.44–1.3) | 0.62 | 2.4 | 2.5 | 0.96 | (1.0–3.5) | (1.2–1.3) | ||

(0.21–1.0) | (0.26–4.9) | (1.9–3.3) | (0.31–3.7) |

Cells show the median of the marginal posterior distributions with 95% credible intervals (between brackets).

the upper and lower rows give the parameter estimates for the low and high dose experiments, respectively.

the upper and lower rows give the parameter estimates for the inoculated and contact birds, respectively. The estimate of the reproduction number is based on the infectious period of the contact infected birds.

We then analyzed the data of the low- and high-dose experiments simultaneously to obtain more precise estimates of the parameters of interest. We considered three scenarios (labeled by B, C, and D) that differ with regard to assumptions on the latent and infectious periods (see

Two derived epidemiological measures of interest are the reproduction number _{g}

To explore the implications of the parameter estimates for the dynamics of H5N1 avian influenza in large populations of poultry we have performed stochastic simulations of an SEIR model using the parameter estimates presented in

Parameters values are based on the estimates of ^{−1}^{−1}

Rapid detection of outbreaks of H5N1 highly pathogenic avian influenza virus in poultry is of paramount importance for efficient control within poultry flocks and to be able to minimize the opportunities of virus transmission between flocks

To further investigate the potential for control by vaccination we have carried out simulations using estimates of the epidemiological parameters (

Circles refer to the final size of major outbreaks (error bars: ±2SD) and squares indicate the duration of major outbreaks (error bars: ±2SD). Blue and red lines represent the low and high transmissibility scenarios, respectively (cf.

In this study we have attempted to fill the remarkable void of quantitative information on key epidemiological parameters of H5N1 highly pathogenic avian influenza in chickens. Our results indicate that H5N1 virus induces a short period of latency and a short infectious period. In fact, our estimate of the mean of the latent period varies from 0.20 days (95%CI: 0.049–0.43 days) in scenario A1 to 0.44 days (95%CI: 0.14–0.87 days) in scenario A2 (

Our estimates of the transmission parameter are remarkably similar across the different datasets and model scenarios. The estimate of the transmission parameter is lowest if the data of all experiments are combined (median: 0.73 per day; 95%CI: 0.43–1.2 per day) and highest if the analysis allows for differences between inoculated and contact birds (median: 0.81 per day; 95%CI: 0.44–1.3 per day). In combination with the estimates of the mean infectious period these estimates yield estimates of the reproduction number varying from 0.99 (95%CI: 0.38–2.1) in the high-dose experiment (scenario A2) to 2.0 (95%CI: 0.96–3.6) in the low-dose experiment (scenario A1).

In view of the generally held belief that highly pathogenic avian influenza viruses spread easily and rapidly among chickens

Second, it is not straightforward to extrapolate our results that were obtained in an experimental setting to the situation in the field. This is especially so for estimates of the transmission parameter, which are the result not only of an autonomous process of viral replication and interaction of the pathogen with the immune system within a single host, but also of an interaction between different individuals. Ambient temperature, stocking density, feeding status of the birds, etcetera could all impact on this interaction and critically affect estimates of the transmission parameter. To counter this we have tried to match the conditions in our experiments to those in commercial laying chicken farms. Reassuringly, a recent analysis of transmission of H5N1 in the field

A third point that deserves attention is the fact that housing systems of layer flocks vary from floor systems in which birds can mingle freely to caged systems in which no direct contact between (groups of) birds is possible. In principle, our study is aimed at quantifying transmission in a situation where there is direct contact between birds, corresponding to a floor system. However, the lone study that focused on within-flock transmission (mostly backyard flocks) did not find differences between different housing systems, suggesting that if there are differences in the transmission dynamics they cannot be large

While it is not straightforward to extrapolate from our experimental setting to the field situation, experimental transmission studies also have distinct advantages over field studies. In particular, while field studies often suffer from various sources of bias and confounding, this is not the case in an experimental setting. This allows one to directly ascribe differences between control and treatment groups directly to the treatment (e.g., vaccination) since all other animal and environmental conditions are held constant. Moreover, an experiment has the added advantage over a field study that far fewer birds are needed and that the birds can be sampled more often and efficiently than in a field study. This has allowed us to obtain precise estimates of the key epidemiological parameters of H5N1 highly pathogenic avian influenza in unvaccinated chickens using no more than 50 birds.

Our results show remarkable differences between experiments in which the inoculated bird received a low infection dose (0.2*10^{5} EID_{50}) and experiments in which the inoculated bird received a high dose (0.2*10^{6} EID_{50}). Specifically, while 9 out of 11 birds were infected in case of a low infection dose (^{6} EID_{50} since this yielded comparable infections in inoculated and naturally infected animals

To investigate the implications of our parameter estimates for the dynamics of H5N1 avian influenza virus in large groups of chickens we have carried out stochastic simulations. Since it is not obvious how the transmission parameter as estimated between pairs of chickens can be extrapolated to large populations, we considered a low and high transmissibility scenario (

While H5N1 virus spreads rapidly among unvaccinated chickens, no transmission was observed at all in the experiments with inactivated oil emulsion vaccines (

All experiments were carried out in PT Medion laboratories in Bandung, Indonesia, which have high containment facilities (BSL3). In all experiments, specific pathogen-free (SPF) layer chickens from the animal unit of Medion were used. The birds were hatched and housed in one group until 4 weeks of age. At that age, pairs of birds were housed in cages. Three rooms were available to house the various vaccinated and unvaccinated pairs of birds. Two rows with three levels of cages on top of each other were available in each room. The rows with cages were separated by a corridor of approximately 1 m width. The various rooms as well as the rows with the cages had separate ventilation systems. Each cage had a separate feeding and drinking system. The floor and walls of each cage were covered with plastic to prevent spread of manure or other materials between cages. When sampling the birds, animal caretakers used a new pair of gloves for each cage. Unvaccinated sentinel birds were placed at regular distances between the cages used in the experiments to ensure that no transmission had taken place between cages. All sentinels survived and remained seronegative during the course of the experiments.

The challenge strain used in the experiments was A/Chicken/Legok/2003 H5N1, a highly pathogenic H5N1 strain isolated in Indonesia in 2003 which is genetically very close to strains that circulate in Indonesia in 2008. The strain has been used in experiments carried out at Medion and is able to induce infection, typical signs of disease, and high mortality rates in chickens.

Inactivated oil emulsion vaccines were available from three different manufactures: PT Medion (Bandung, Indonesia), PT Vaksindo (Bogor, Indonesia) and Intervet (Mexico). The vaccines contained either an H5N1 or H5N2 virus strain. The H5N1 vaccines contained A/Chicken/Legok/2003 H5N1, i.e. the vaccine and challenge strains were identical. The H5N2 vaccines contained either A/Turkey/England/N28/73 H5N2 or A/Chicken/Mexico/232/94/CPA H5N2. The protein homologies of the antigenic part of the hemagglutinin (HA1) of the challenge strain to the H5N2 A/Turkey/England/N28/73 and H5N2 A/Chicken/Mexico/232/94/CPA vaccine strains are 92% and 86%, respectively.

All vaccines were re-vialed in coded bottles, and the identity of the vaccines was not known to the staff involved in the experiment. In this manner the experiments were double blinded.

Because the size of a natural infection dose is unknown the inoculum consisted of diluted allantoic fluid containing either 10^{5} EID_{50} per ml (low inoculation dose) or 10^{6} EID_{50} per ml (high inoculation dose). The birds were inoculated both intranasally (0.1 ml) and intratracheally (0.1 ml). Virus titres were confirmed before and after inoculation by titration on embryonated SPF eggs.

Each experiment consisted of a set of 11 trials. In each of the trials an inoculated bird was placed in a cage with an uninfected contact bird, and the transmission chain was monitored daily by virus isolation performed on swabs taken from the trachea and cloaca. In all, a total of eight experiments were carried out. Unvaccinated birds were used in two experiments. In the first of these the inoculated birds received a low infection dose, while in the second the inoculated birds received a high infection dose. The remaining six experiments with vaccinated birds differed with respect to the vaccine used, the manufacturer, and the inoculation dose.

At 4 weeks of age all birds of the vaccination experiments received their first vaccination dose. A second vaccination was carried out at 7 weeks of age. At 10 weeks of age (day 0) one bird was chosen at random per cage, taken from the cage, and infected intratracheally and intranasally. To avoid direct infection of the contact bird by the inoculum the artificially infected birds were placed back in their cages only after a delay of 8 hours.

Tracheal and cloacal swabs were taken daily for 10 days after challenge from all birds. Swabs were incubated for 1 h in one ml of PBS medium containing antibiotics. The medium was subsequently stored at −70°C until testing. Three embryonated SPF chicken eggs were injected with 0.2 ml of the swab medium per egg. After culture for 4 days or when embryos died, the allantoic fluid was harvested and a hemagglutination (HA) assay was performed following standard procedures (

The serological status of the birds was determined just before vaccination, at the start of the experiments just before inoculation (day 0) and, for birds that survived, at the end of the experiments (day 14). Serum blood samples were taken from all birds by puncturing the wing vein. Blood samples were centrifuged and serum was stored at −20°C until tested. The sera were tested in the hemagglutination inhibition (HI) test according procedures described in the Manual of Diagnostic Tests and Vaccines for Terrestrial Animals of the OIE (^{2}log of the serum dilution that caused complete inhibition of agglutination, as specified by OIE guidelines.

Clinical signs of disease were recorded daily for a period of up to 10 days after challenge.

As a first step we estimated the reproduction number

In a second step, we estimated all parameters of interest by Bayesian methods

Further,

With these notational conventions, the contribution of trial

The likelihood contribution in Equation (2) assumes that the latent and infectious periods of inoculated and infectious birds are identically distributed. To investigate the validity of these assumptions we also considered a model which allows for differences between the inoculated and contact birds. In this case, the likelihood contribution becomes

Notice that, since the transmission rate in Equations (1), (2), and (5) is divided by the total size of the population (i.e. 2), the above model assumes frequency dependent transmission (as opposed to density dependent transmission)

As in earlier papers

In our simulations the epidemiological parameters and unobserved transitions were updated by a random-walk Metropolis algorithm. We used Normal proposal distributions with the current value as mean, and a standard deviation of 0.025, 0.05, or 0.1. The transmission parameters and unobserved transitions were updated in blocks, in the order

To choose between models of different complexity we made use of Bayes factors (BF)

A suite of Bayesian analyses were performed for the experiments with unvaccinated birds. First, we analyzed the low- and high-dose experiments of

To explore the implications of the parameters estimated by the above procedures for the pathogen dynamics in large groups of birds, we performed simulations of the stochastic SEIR model using the Sellke construction

Bayesian analysis of the low-dose experiment (

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Bayesian analysis of the high-dose experiment (

(4.80 MB TIF)

Bayesian analysis of the combined experiments (scenario B). Shown are samples of the mean of the latent period (A), variance of the latent period (B), mean of the infectious period (C), variance of the infectious period (D), transmission rate parameter (E), mean of the infectious period versus transmission rate parameter (F), mean versus variance of the latent period (G), and mean versus variance of the infectious period (H).

(4.83 MB TIF)

Bayesian analysis of the combined experiments (scenario C). Shown are samples of the mean of the latent period (A), variance of the latent period (B), mean of the infectious period (C), variance of the infectious period (D), transmission rate parameter (E), mean of the infectious period versus transmission rate parameter (F), mean versus variance of the latent period (G), and mean versus variance of the infectious period (H). Red and blue dots refer to parameters characterizing the low- and high-dose experiments, respectively.

(5.48 MB TIF)

Bayesian analysis of the combined experiments (scenario D). Shown are samples of the mean of the latent period (A), variance of the latent period (B), mean of the infectious period (C), variance of the infectious period (D), transmission rate parameter (E), mean of the infectious period versus transmission rate parameter (F), mean versus variance of the latent period (G), and mean versus variance of the infectious period (H). Blue and red dots refer to parameters characterizing the inoculated and contact birds, respectively.

(6.38 MB TIF)

Overview of the experiments with vaccinated birds.

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Overview of experiment #1 (see

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Overview of experiment #2 (see

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Overview of experiment #3 (see

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Overview of experiment #4 (see

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Overview of experiment #5 (see

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Overview of experiment #6 (see

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Jantien Backer, Bas Engel, Thomas Hagenaars, Jeanet van der Goot (Central Veterinary Institute, Lelystad), Frank van der Meer (University of Calgary), and two anonymous reviewers are gratefully acknowledged for support and constructive comments.