Okti Nadia Poetri

A single vaccination of commercial broilers does not reduce transmission of H5N1 highly pathogenic avian influenza

Vaccination of chickens has become routine practice in Asian countries in which H5N1 highly pathogenic avian influenza (HPAI) is endemically present. This mainly applies to layer and breeder flocks, but broilers are usually left unvaccinated. Here we investigate whether vaccination is able to reduce HPAI H5N1 virus transmission among broiler chickens. Four sets of experiments were carried out, each consisting of 22 replicate trials containing a pair of birds. Experiments 1-3 were carried out with four-week-old birds that were unvaccinated, and vaccinated at day 1 or at day 10 of age. Experiment 4 was carried out with unvaccinated day-old broiler chicks. One chicken in each trial was inoculated with H5N1 HPAI virus. One chicken in each trial was inoculated with virus. The course of the infection chain was monitored by serological analysis, and by virus isolation performed on tracheal and cloacal swabs. The analyses were based on a stochastic SEIR model using a Bayesian inferential framework. When inoculation was carried out at the 28th day of life, transmission was efficient in unvaccinated birds, and in birds vaccinated at first or tenth day of life. In these experiments estimates of the latent period (~1.0 day), infectious period (~3.3 days), and transmission rate parameter (~1.4 per day) were similar, as were estimates of the reproduction number (~4) and generation interval (~1.4 day). Transmission was significantly less efficient in unvaccinated chickens when inoculation was carried out on the first day of life. These results show that vaccination of broiler chickens does not reduce transmission, and suggest that this may be due to the interference of maternal immunity.

The large-scale production of an artificial influenza virus-like particle vaccine in silkworm pupae.

We successfully established a mass production system for an influenza virus-like particle (VLP) vaccine using a synthetic H5 hemagglutinin (HA) gene codon-optimized for the silkworm. A recombinant baculovirus containing the synthetic gene was inoculated into silkworm pupae. Four days after inoculation, the hemagglutination titer in homogenates from infected pupae reached a mean value of 0.8 million hemagglutination units (HAU), approximately 2,000 μg HA protein per pupa, more than 50-fold higher than that produced with an embryonated chicken egg. VLPs ranging from 30 nm to 300 nm in diameter and covered with a large number of spikes were detected in the homogenates. The spikes were approximately 14 nm long, similar to an authentic influenza HA spike. Detailed electron micrographs indicated that the VLP spike density was similar to that of authentic influenza virus particles. The results clearly show that the expression of a single HA gene can efficiently produce VLPs in silkworm pupae. When chickens were immunized with the pupae homogenate, the hemagglutination inhibition titer in their sera reached values of 2,048-8,192 after approximately 1 month. This is the first report demonstrating that a large amount of VLP vaccine could be produced by single synthetic HA gene in silkworm pupae. Our system might be useful for future vaccine development against other viral diseases.

Silent spread of highly pathogenic Avian Influenza H5N1 virus amongst vaccinated commercial layers

The aim of this study was to determine whether a single vaccination of commercial layer type chickens with an inactivated vaccine containing highly pathogenic avian influenza virus strain H5N1 A/chicken/Legok/2003, carried out on the farm, was sufficient to protect against infection with the homologous virus strain. A transmission experiment was carried out with pairs of chicken of which one was inoculated with H5N1 virus and the other contact-exposed. Results showed that the majority of the vaccinated birds developed haemagglutination inhibition (HI) titres below 4log2. No clinical signs were observed in the vaccinated birds and virus shedding was limited. However, nearly all vaccinated birds showed a four-fold or higher increase of HI titres after challenge or contact-exposure, which is an indication of infection. This implies that virus transmission most likely has occurred. This study showed that a single vaccination applied under field conditions induced clinical protection, but was insufficient to induce protection against virus transmission, suggesting that silent spread of virus in vaccinated commercial flocks may occur.

Pathotypic characterization of Newcastle disease virus isolated from vaccinated chicken in West Java, Indonesia

Aim: This research was conducted to differentiate and characterize eight Newcastle disease virus (NDV) isolates collected from vaccinated chicken at commercial flocks in West Java, Indonesia, in 2011, 2014 and 2015 by pathotype specific primers. Materials and Methods: A total of eight NDV isolates collected from clinical outbreaks among commercial vaccinated flocks in West Java, Indonesia, in 2011, 2014, and 2015 were used in this study. Reverse transcription-polymerase chain reaction was used to detect and differentiate virulence of NDV strains, using three sets of primers targeting their M and F gene. First primers were universal primers to detect NDV targeting matrix (M) gene. Other two sets of primers were specific for the fusion (F) gene cleavage site sequence of virulent and avirulent NDV strains. Results: Our results showed that three isolates belong to NDV virulent strains, and other five isolates belong to NDV avirulent strains. The nucleotide sequence of the F protein cleavage site showed 112K/R-R-Q/R-K-R/G-F117 on NDV virulent strains and 112G-K/R-Q-G-R-L117 on NDV avirulent strain. Conclusion: Result from the current study suggested that NDV virulent strain were circulating among vaccinated chickens in West Java, Indonesia; this might possess a risk of causing ND outbreaks and causing economic losses within the poultry industry.

Production and characterization of Newcastle disease antibody as a reagent to develop a rapid immunodiagnostic test tool

Aim: This research was conducted to produce and characterize ND antibody as reagent candidate to develop a rapid immunodiagnostic test tool. Materials and Methods: Four New Zealand White rabbits were used in this study and divided into two groups. First group was injected by Sato ND antigen, and second group was injected by genotype VII ND antigen. This study is divided into three steps: (a) ND antibody production, (b) ND antibody purification, and (c) ND antibody characterization. First group was rabbit injected by Sato NDV (5×108.25 egg lethal doses (ELD)50/ml) and second group was injected by genotype VII NDV (5×106.5 ELD50/ml). Antigen induction was performed by subcutaneous administrated for first (day 1) and second (day 14) injection and intravenous administrated for third (day 30) injection. Blood was collected on day 8 after third injection. Results: Antibody production increased on second antigen injection and reached a peak on day 9 after second antigen injection. Sato and genotype VII ND antibody can be produced without adjuvant within 38 days with the highest titer 210. Based on antibody titer data, both antigens induced antibody production in a similar trend. The characterization antibody by SDS-PAGE indicated that molecular weight of immunoglobulin G (IgG) is 154.93 kDa (whole IgG), heavy chain 54.39 kDa, and light chain 27.74 kDa. ND antibodies have specificity to homologous and heterologous NDVs in varying virulence. Conclusion: Sato and genotype VII ND antibodies have been successfully produced within 38 days without adjuvant. Specificity of ND antibodies to NDVs in varying virulence and cross-reaction between Sato ND antibody and genotype VII ND antibody indicates that the characterized ND antibodies can be used as a reagent to develop rapid immunodiagnostic test tools.

Different cross protection scopes of two avian influenza H5N1 vaccines against infection of layer chickens with a heterologous highly pathogenic virus

Avian influenza (AI) virus strains vary in antigenicity, and antigenic differences between circulating field virus and vaccine virus will affect the effectiveness of vaccination of poultry. Antigenic relatedness can be assessed by measuring serological cross-reactivity using haemagglutination inhibition (HI) tests. Our study aims to determine the relation between antigenic relatedness expressed by the Archetti-Horsfall ratio, and reduction of virus transmission of highly pathogenic H5N1 AI strains among vaccinated layers. Two vaccines were examined, derived from H5N1 AI virus strains A/Ck/WJava/Sukabumi/006/2008 and A/Ck/CJava/Karanganyar/051/2009. Transmission experiments were carried out in four vaccine and two control groups, with six sets of 16 specified pathogen free (SPF) layer chickens. Birds were vaccinated at 4weeks of age with one strain and challenge-infected with the homologous or heterologous strain at 8weeks of age. No transmission or virus shedding occurred in groups challenged with the homologous strain. In the group vaccinated with the Karanganyar strain, high cross-HI responses were observed, and no transmission of the Sukabumi strain occurred. However, in the group vaccinated with the Sukabumi strain, cross-HI titres were low, virus shedding was not reduced, and multiple transmissions to contact birds were observed. This study showed large differences in cross-protection of two vaccines based on two different highly pathogenic H5N1 virus strains. This implies that extrapolation of in vitro data to clinical protection and reduction of virus transmission might not be straightforward.