Development and validation of a genotype 3 recombinant protein-based immunoassay for hepatitis E virus serology in swine

Hepatitis E virus (HEV) is classified within the family Hepeviridae, genus Hepevirus. HEV genotype 3 (Gt3) infections are endemic in pigs in Western Europe and in North and South America and cause zoonotic infections in humans. Several serological assays to detect HEV antibodies in pigs have been developed, at first mainly based on HEV genotype 1 (Gt1) antigens. To develop a sensitive HEV Gt3 ELISA, a recombinant baculovirus expression product of HEV Gt3 open reading frame-2 was produced and coated onto polystyrene ELISA plates. After incubation of porcine sera, bound HEV antibodies were detected with anti-porcine anti-IgG and anti-IgM conjugates. For primary estimation of sensitivity and specificity of the assay, sets of sera were used from pigs experimentally infected with HEV Gt3. For further validation of the assay and to set the cutoff value, a batch of 1100 pig sera was used. All pig sera were tested using the developed HEV Gt3 assay and two other serologic assays based on HEV Gt1 antigens. Since there is no gold standard available for HEV antibody testing, further validation and a definite setting of the cutoff of the developed HEV Gt3 assay were performed using a statistical approach based on Bayes' theorem. The developed and validated HEV antibody assay showed effective detection of HEV-specific antibodies. This assay can contribute to an improved detection of HEV antibodies and enable more reliable estimates of the prevalence of HEV Gt3 in swine in different regions.

Development and validation of a genotype 3 recombinant protein-based immunoassay for hepatitis E virus serology in swine.

Hepatitis E virus (HEV) is classified within the family Hepeviridae, genus Hepevirus. HEV genotype 3 (Gt3) infections are endemic in pigs in Western Europe and in North and South America and cause zoonotic infections in humans. Several serological assays to detect HEV antibodies in pigs have been developed, at first mainly based on HEV genotype 1 (Gt1) antigens. To develop a sensitive HEV Gt3 ELISA, a recombinant baculovirus expression product of HEV Gt3 open reading frame-2 was produced and coated onto polystyrene ELISA plates. After incubation of porcine sera, bound HEV antibodies were detected with anti-porcine anti-IgG and anti-IgM conjugates. For primary estimation of sensitivity and specificity of the assay, sets of sera were used from pigs experimentally infected with HEV Gt3. For further validation of the assay and to set the cutoff value, a batch of 1100 pig sera was used. All pig sera were tested using the developed HEV Gt3 assay and two other serologic assays based on HEV Gt1 antigens. Since there is no gold standard available for HEV antibody testing, further validation and a definite setting of the cutoff of the developed HEV Gt3 assay were performed using a statistical approach based on Bayes' theorem. The developed and validated HEV antibody assay showed effective detection of HEV-specific antibodies. This assay can contribute to an improved detection of HEV antibodies and enable more reliable estimates of the prevalence of HEV Gt3 in swine in different regions.

Using egg production data to quantify within-flock transmission of low pathogenic avian influenza virus in commercial layer chickens.

Even though low pathogenic avian influenza viruses (LPAIv) affect the poultry industry of several countries in the world, information about their transmission characteristics in poultry is sparse. Outbreak reports of LPAIv in layer chickens have described drops in egg production that appear to be correlated with the virus transmission dynamics. The objective of this study was to use egg production data from LPAIv infected layer flocks to quantify the within-flock transmission parameters of the virus. Egg production data from two commercial layer chicken flocks which were infected with an H7N3 LPAIv were used for this study. In addition, an isolate of the H7N3 LPAIv causing these outbreaks was used in a transmission experiment. The field and experimental estimates showed that this is a virus with high transmission characteristics. Furthermore, with the field method, the day of introduction of the virus into the flock was estimated. The method here presented uses compartmental models that assume homogeneous mixing. This method is, therefore, best suited to study transmission in commercial flocks with a litter (floor-reared) housing system. It would also perform better, when used to study transmission retrospectively, after the outbreak has finished and there is egg production data from recovered chickens. This method cannot be used when a flock was affected with a LPAIv with low transmission characteristics (R(0)<2), since the drop in egg production would be low and likely to be confounded with the expected decrease in production due to aging of the flock. Because only two flocks were used for this analysis, this study is a preliminary basis for a proof of principle that transmission parameters of LPAIv infections in layer chicken flocks could be quantified using the egg production data from affected flocks.

Transmission between chickens of an H7N1 Low Pathogenic Avian Influenza virus isolated during the epidemic of 1999 in Italy.

The transmissibility of an H7N1 Low Pathogenic Avian Influenza (LPAI) virus isolated from a turkey flock during the large epidemic in Italy in 1999, was experimentally studied in chickens. Four group transmission experiments were performed. Infection and transmission were monitored by means of virus isolation on swab samples and antibody detection in serum samples. From the results of these groups, we estimated the mean infectious period at 7.7 (6.7-8.7) days, the transmission rate parameter at 0.49 (0.30-0.75) infections per infectious chicken per day and the basic reproduction ratio at 3.8 (1.3-6.3). These estimates can be used for the development of surveillance and control programmes of LPAI in poultry.

Transfer of information on palliative home care during the out-of-hours period.

BACKGROUND: Continuity of end-of-life care for patients receiving palliative care is an important challenge for out-of-hours services in general practice. AIM: To investigate how frequent information is transferred on patients receiving palliative care from GPs to the out-of-hours services, to explore the perceptions of GP's on this information transfer and to study the relation between information transfer and the used GP information systems. METHODS: This is a mixed-method design study. The frequency of information transfer to the out-of-hours services was investigated by analyzing a regional out-of-hours database. Barriers and promoting factors for this transfer of information were investigated by using semi-structured interviews among a purposive sample of GPs from the same region. The relation between information transfer and the GP information system was investigated by a postal questionnaire in a national random selection of GPs. RESULTS: When a palliative patient contacted the out-of-hours service, for 20% of these patients, a transfer of information was available and only half of these transfers included an anticipating end-of-life plan. All interviewed GPs considered continuity of care for these patients as important. However, some doubted whether a transfer of information is relevant for the quality of care. There was no relation between the information transfer and the used GP information systems. CONCLUSION: For only a minority of patients receiving palliative care, a transfer of information including an anticipating management plan was present. There is a large variation in the opinions of GPs on how to organize continuity of end-of-life care.

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.

Effect of vaccination on transmission of HPAI H5N1: the effect of a single vaccination dose on transmission of highly pathogenic avian influenza H5N1 in Peking ducks.

The highly pathogenic H5N1 avian influenza virus is widespread among domestic ducks throughout Southeast Asia. Many aspects of the poultry industry and social habits hinder the containment and eradication of AI. Vaccination is often put forward as a tool for the control of AI. However, vaccination will only lead to eradication when it reduces the virus spread to such an extent that herd immunity is obtained. To study the effect of a single vaccination dose on the transmission of H5N1 in domestic ducks we performed experiments in which infected and uninfected ducks were housed together and the infection chain was monitored by means of virus isolation and serology. Specifically, Peking ducks were vaccinated with A/Chicken/Mexico/232/94/ CPA H5N2 and challenged with A/Chicken/GxLA/1204/04 H5N1 one week after vaccination. In both the control and vaccinated groups all inoculated and contact animals were quickly infected. However, the disease signs and mortality differed between the control and treatment groups. This finding may have important implications for the control of H5N1 by means of vaccination.

Quantification of the effect of vaccination on transmission of avian influenza (H7N7) in chickens.

Recent outbreaks of highly pathogenic avian influenza (HPAI) viruses in poultry and their threatening zoonotic consequences emphasize the need for effective control measures. Although vaccination of poultry against avian influenza provides a potentially attractive control measure, little is known about the effect of vaccination on epidemiologically relevant parameters, such as transmissibility and the infectious period. We used transmission experiments to study the effect of vaccination on the transmission characteristics of HPAI A/Chicken/Netherlands/03 H7N7 in chickens. In the experiments, a number of infected and uninfected chickens is housed together and the infection chain is monitored by virus isolation and serology. Analysis is based on a stochastic susceptible, latently infected, infectious, recovered (SEIR) epidemic model. We found that vaccination is able to reduce the transmission level to such an extent that a major outbreak is prevented, important variables being the type of vaccine (H7N1 or H7N3) and the moment of challenge after vaccination. Two weeks after vaccination, both vaccines completely block transmission. One week after vaccination, the H7N1 vaccine is better than the H7N3 vaccine at reducing the spread of the H7N7 virus. We discuss the implications of these findings for the use of vaccination programs in poultry and the value of transmission experiments in the process of choosing vaccine.

Comparison of the transmission characteristics of low and high pathogenicity avian influenza A virus (H5N2).

Low pathogenicity avian influenza A strains (LPAI) of the H5 and H7 type are noted for their ability to transform into highly pathogenic counterparts (HPAI). Here we compare the transmission characteristics in poultry of LPAI H5N2 (A/Chicken/Pennsylvania/83) and corresponding HPAI virus by means of transmission experiments. In the experiments, five inoculated animals are placed in a cage with five contact animals, and the infection chain is monitored by taking blood samples, and samples from the trachea and cloaca. The data are analysed by final size methods and a generalized linear model. The results show that HPAI virus is more infectious and induces a longer infectious period than LPAI. In fact, fully susceptible animals are invariably infected when confronted with HPAI virus and die within six days after infection. Animals previously infected with LPAI virus, on the other hand, survive an infection with HPAI virus or escape infection all together. This implies that a previous infection with LPAI virus effectively reduces susceptibility of the host to infection and decreases transmission of HPAI virus. We discuss the implications of these conclusions for the control and evolution of avian influenza viruses.

Transmission dynamics of low- and high-pathogenicity A/Chicken/Pennsylvania/83 avian influenza viruses.

High-pathogenicity avian influenza (HPAI) viruses emerged from low-pathogenicity avian influenza (LPAI) viruses in Pennsylvania (1983-84), Mexico (1994-95), and Italy (1999-2000). Here we focus on the question of why the HPAI virus supersedes the LPAI virus, once it has appeared during the epidemic. To study this, we used an experimental model in chickens that enabled us to estimate the reproduction ratio (R0). Using this model, we determined the R0 of the A/Chicken/Pennsylvania/21525/83 (LPAI) and of the A/Chicken/Pennsylvania/1370/83 (HPAI). Comparing the R0 of both viruses, we concluded that the R0 of the HPAI virus is significantly higher than the R0 of the LPAI.