Chicken Enterococcus faecalis-induced immunoglobulin Y as a prophylactic and therapeutic agent against streptococcosis in red tilapia (Oreochromis hybrid).

Background and Aim: Streptococcosis is a common bacterial disease in red tilapia, in which Enterococcus faecalis infection has not been widely reported. This study aimed to evaluate the efficacy of pellets that contain chicken E. faecalis-induced immunoglobulin Y (IgY) to treat and prevent streptococcosis in red tilapia. Materials and Methods: We conducted a 28-day study for immunoprophylaxis and immunotherapy, each using four groups with two replications: Healthy control fish (KS), non-IgY pellets (PA and TA), pellets with 25% egg yolk containing E. faecalis-induced IgY (PB and TB), and pellets with 50% egg yolk containing E. faecalis-induced IgY(PC and TC). Indirect enzyme-linked immunosorbent assay was performed on prototype pellets produced with an IgY suspension at 1.63 mg/mL as the standard optical density curve. For the immunoprophylaxis study, pellets of 3% of the average body weight of the experimental fish (0.50 g per fish per day) were given daily until day 14 before the challenge test with E. faecalis (2.1 × 109 Colony-forming unit/mL peroral) on day 15. The data from the observation period on days 15-28 were analyzed. For the immunotherapy study, pellets of 3% of the average body weight (0.50 g per fish per day) were given daily for 21 days (days 8-28) 7 day spost-infection. The data from the immunotherapy study were collected during the observation period on days 8-28. Statistical analysis was performed on non-specific immune variables: Total leukocytes, monocytes, lymphocytes, neutrophils, phagocytic activity, and macrophage capacity; and the semi-quantitative distribution of melanomacrophage centers (MMCs) in the lymphoid organs, such as spleen and liver. Photomacrographic data were analyzed descriptively and qualitatively by comparing the healing process and clinical signs found between experiments in the immunotherapy study. Results: The pellet with 50% egg yolk with an IgY at 2.43 mg/g pellet, 3% of body weight once daily, was the best formula on experimental fish. The administration of this formulation can also increase non-specific immunity and the distribution of MMCs in the spleen and liver with a survival rate of 55% for 14 days of challenge period in the immunoprophylaxis study and 70% for 21 days of therapy period in the immunotherapy study. Conclusion: Immunoglobulin Y can be a prophylactic and therapeutic agent against streptococcal infections caused E. faecalis in red tilapia with an optimum dosage of 2.43 mg/g pellet.

Molecular Features and Antimicrobial Susceptibilities of Streptococcus equi ssp. equi Isolates from Strangles Cases in Indonesia.

Strangles, caused by Streptococcus equi ssp. equi (S. equi equi), is a highly infectious and frequent disease of equines worldwide. No data are available regarding the molecular epidemiology of strangles in Indonesia. This study aimed to characterize S. equi equi isolates obtained from suspected strangles cases in Indonesia in 2018. Isolates originated from seven diseased horses on four different farms located in three provinces of Indonesia. Whole genome sequences of these isolates were determined and used for seM typing, multilocus sequence typing (MLST), and core genome MLS typing (cgMLST). Genomes were also screened for known antimicrobial resistance genes and genes encoding for the recombinant antigens used in the commercial Strangvac® subunit vaccine. All seven S. equi equi isolates from Indonesia belonged to ST179 and carried seM allele 166. Isolates differed from each other by only 2 to 14 cgSNPs and built an exclusive sub-cluster within the Bayesian Analysis of Population Structure (BAPS) cluster 2 (BAPS-2) of the S. equi equi cgMLST scheme. All isolates revealed predicted amino acid sequence identity to seven and high similarity to one of the eight antigen fragments contained in Strangvac®. Furthermore, all isolates were susceptible to beta-lactam antibiotics penicillin G, ampicillin, and ceftiofur. Our data suggest that the horses from this study were affected by strains of the same novel sublineage within globally distributed BAPS-2 of S. equi equi. Nevertheless, penicillin G can be used as a first-choice antibiotic against these strains and Strangvac® may also be protective against Indonesian strains.

Immunoglobulin yolk targeting spike 1, receptor binding domain of spike glycoprotein and nucleocapsid of SARS-CoV-2 blocking RBD-ACE2 binding interaction.

Coronavirus disease (COVID)-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has become a global pandemic disease that has social and economic chaos. An alternative mitigation strategy may involve the use of specific immunoglobulin (Ig)-Y derived from chicken eggs. Our study aimed to evaluate the neutralizing potential of specific IgY targeting S1, receptor-binding-domain (RBD) of spike glycoprotein and nucleocapsid (N) of SARS-CoV-2 to inhibit RBD and angiotensin-converting-enzyme-2 (ACE2) binding interaction. Hy-Line Brown laying hens were immunized with recombinant S1, RBD spike glycoprotein, and nucleocapsid (N) of SARS-CoV-2. The presence of specific S1,RBD,N-IgY in serum and egg yolk was verified by indirect enzyme-linked immunosorbent assay (ELISA). Specific S1,RBD,N-IgY was purified and characterized from egg yolk using sodium-dodecyl-sulfate-polyacrylamide-gel-electrophoresis (SDS-PAGE), and was subsequently evaluated for inhibition of the RBD-ACE2 binding interaction in vitro. Specific IgY was present in serum at 1 week post-initial immunization (p.i.i), whereas its present in egg yolk was confirmed at 4 weeks p.i.i. Specific S1,RBD,N-IgY in serum was able to inhibit RBD-ACE2 binding interaction between 4 and 15 weeks p.i.i. The results of the SDS-PAGE revealed the presence of bands with molecular weights of 180 kDa, indicating the presence of whole IgY. Our results demonstrated that S1,RBD,N-IgY was able to inhibit RBD-ACE2 binding interaction in vitro, suggesting its potential use in blocking virus entry. Our study also demonstrated proof-of-concept that laying hens were able to produce this specific IgY, which could block the viral binding and large production of this specific IgY is feasible.

Quantity of the antigens of Streptococcus mutans serotype e and Candida albicans and its correlation with the salivary flow rate in early childhood caries.

Background: Streptococcus mutans involved in caries pathogenesis is classified into four serotypes, namely serotypes c, e, f, and k. Candida albicans can be found in the plaque of children with early childhood caries (ECC). Aims: The aim of this study was to analyze the quantity of the antigens of S. mutans serotype e and C. albicans and its correlation with the salivary flow rate in ECC. Materials and Methods: The antigen quantities of caries plaque samples and caries-free were determined using an enzyme-linked immunoassay with 450-nm optical density. Results: There was a significant difference between the quantity of S. mutans serotype e and C. albicans antigens in each salivary flow rate category (P < 0.05). The relationship between the antigen quantity of S. mutans serotype e and C. albicans was r = 0.624 (P > 0.05) for caries plaque samples and r = 0.628 (P > 0.05) for caries-free samples. Conclusion: the antigen quantities of S. mutans serotype e and C. albicans and the salivary flow rate might correlate to the pathogenesis of ECC.

Production of hyperimmune serum against genotype VII Newcastle disease virus in rabbits with several applications.

Objective: This study aimed to produce hyperimmune serum against genotype VII Newcastle disease virus (NDV) with several applications. Materials and Methods: Production of hyperimmune serum against genotype VII NDV was performed on eight New Zealand white rabbits divided into four groups. Rabbits were immunized three times on the 1st day, the 14th day, and the 30th day. Blood sampling was carried out on the 8th day after the third immunization. Results: All groups showed the same pattern of hemagglutination inhibition (HI) titer results. HI titers would peak on the 5th or the 9th day after the second immunization, then decrease until the 3rd day after the third immunization, and increase again on the 5th day after the third immunization. Rabbits immunized intravenously showed higher HI titers than the other groups. These results indicate that the intravenous route for hyperimmune serum production against genotype VII Newcastle disease virus greatly affects the immune response result. Conclusions: The production of hyperimmune serum by intravenous immunization three times was able to produce the highest titer of 210 at 38 days. The agar gel precipitation test and the Western blot assay showed that the hyperimmune serum was specific for the Newcastle disease antigen.

Isolation and molecular characterization of the hemagglutinin gene of H9N2 avian influenza viruses from poultry in Java, Indonesia.

OBJECTIVE: The avian influenza virus (AIV) subtype H9N2 circulating in Indonesia has raised increasing concern about its impact on poultry and its public health risks. In this study, the H9N2 virus from chicken poultry farms in Java was isolated and characterized molecularly. MATERIALS AND METHODS: Thirty-three pooled samples of chicken brain, cloacal swab, trachea, and oviduct were taken from multiple chickens infected with AIV in five regions of Java, Indonesia. The samples were isolated from specific pathogenic-free embryonated eggs that were 9 days old. Reverse transcription polymerase chain reaction and sequencing were used to identify H9N2 viruses. RESULTS: This study was successful in detecting and characterizing 13 H9N2 isolates. The sequencing analysis of hemagglutinin genes revealed a 96.9%-98.8% similarity to the H9N2 AIV isolated from Vietnam in 2014 (A/muscovy duck/Vietnam/LBM719/2014). According to the phylogenetic analysis, all recent H9N2 viruses were members of the lineage Y280 and clade h9.4.2.5. Nine of the H9N2 isolates studied showed PSKSSR↓GLF motifs at the cleavage site, while four had PSKSSR↓GLF. Notably, all contemporary viruses have leucine (L) at position 216 in the receptor-binding region, indicating that the virus can interact with a human-like receptor. CONCLUSION: This study described the features of recent H9N2 viruses spreading in Java's poultry industry. Additionally, H9N2 infection prevention and management must be implemented to avoid the occurrence of virus mutations in the Indonesian poultry industry.

Experimental infection of Enterococcus faecalis in red tilapia (Oreochromis hybrid) revealed low pathogenicity to cause streptococcosis.

Background: Streptococcosis, as a bacterial disease with broad tropism in fish and one of the causes of septicemia. Enterococcus faecalis is one of the causative agents of streptococcosis that can be isolated in tilapia. Aim: This study was undertaken to complete the reporting gap on the pathogenicity profile and clinical symptoms of E. faecalis bacterial infection in red tilapia (Oreochromis hybrid). The study is expected to provide enriching information regarding recognizable clinical signs in the field that can lead to the diagnosis of streptococcosis caused by E. faecalis, especially in the Indonesian aquaculture environment. Methods: The method used in this artificial infection study using red tilapia, which were divided into two types of route groups infection, namely intraperitoneal (IP) and peroral (PO) with bacterial concentrations given for each route of infection to be 2.1 × 108 CFU ml-1; 2.1 × 107 CFU ml-1; and 2.1 × 106 CFU ml-1. One group was given brain heart infusion broth media sterile as a non-infectious control. Clinical symptoms, changes in swimming habits and consuming feed, external and internal organ lesion, and leukocytes profile changes were observed during the observation period along 14 days to evaluate the infectious effect of each treated fish group. The lethal dose 50 (LD50) was estimated with the Spearman-Kärber method. The evaluation of the leukocyte profile was performed to find leukocytosis as the clinical sign of infection. Results: The results showed variations in clinical symptoms inflicted on fish through death or the moribund stage. The highest mortality occurred in the treatment group of 2.1 × 108 CFU ml-1 with the PO route. The bacterial concentration of 2.1 × 107 CFU ml-1 given either as PO or IP can cause mild infection symptoms but did not cause mortality. The LD50 of the PO and IP route was obtained at 1.99 × 108 CFU ml-1 and 0.79 × 108 CFU ml-1, respectively. The total leukocytes in the infected fish group increased significantly (p < 0.05) by twofold when compared with the non-infectious group. The bacteria's discovery on the blood smear examination was taken from fresh dead fish or moribund fish in the treatment group of 2.1 × 108 CFU ml-1, for both PO and IP. Conclusion: Enterococcus faecalis with low pathogenicity can lead to septicemia, characterized by a total increase in leukocytes, bacteria's discovery on the blood smear examination, and various clinical symptoms systemically found in the treated fish.

Phylogenetic studies of Newcastle disease virus isolated from poultry flocks in South Sulawesi Province, Indonesia, in 2019.

OBJECTIVE: Indonesia is one of the Newcastle disease (ND) endemic countries in the world. An outbreak of the ND virus (NDV) was first reported in Indonesia in 1926. This study aimed to detect, isolate, and classify the NDV by molecular approaches from poultry farms in South Sulawesi Province of Indonesia in 2019. MATERIALS AND METHODS: As many as 36 pooling samples from the cloacal swab, trachea swab, proventriculus, and spleen tissues obtained from ND-suspected chickens were isolated in 11-day-old embryonated chicken eggs type-specific antibody-negative. The viruses were confirmed by reverse transcription-polymerase chain reaction (RT-PCR), followed by sequencing. RESULTS: The results showed that 18 out of 36 pooling samples were NDV-positive based on the isolation result and RT-PCR test. The sequencing results showed that 10 NDV isolates had a motif 112R-R-Q-K-R-F117 in the fusion protein cleavage site region, which suggested that the NDV isolates were of virulent pathotype. The phylogenetic studies based on the F gene's partial nucleotide sequence classified the study isolates into NDV virus genotype/subgenotype VII.2. CONCLUSION: These findings are expected to help provide the latest characteristic information of NDV in South Sulawesi Province to determine the seed vaccine for control strategies of ND.

Expression of TNF, IL1B, and iNOS2 in the neural cell after induced by Porphyromonas gingivalis with and without coating antibody anti -Porphyromonas gingivalis.

Porphyromonas gingivalis has virulence factors such as gingipain and lipopolysaccharide, causing bacteremia to reach the brain and activate neuroinflammatory release cytokines. This study analyzed the effect of the co-culture of neuron cells with P. gingivalis coated with anti -P. gingivalis antibodies against cytokines produced by neuron cells. The gene expressions of the TNF, IL1B, NOS2 in neurons was evaluated using RT-qPCR. The results showed that P. gingivalis coated with anti -P. gingivalis antibody before co-culture with neuron cells could decrease the gene expression of TNF, IL1B, and NOS2 of neuron cells.

Production of a polyclonal antibody against acrylamide for immunochromatographic detection of acrylamide using strip tests.

OBJECTIVE: To produce, purify, and characterize a polyclonal antibody against acrylamide (anti-AA) for an application to immunochromatographic strip tests for AA. MATERIALS AND METHODS: Polyclonal anti-AA was prepared by injecting N-acryloxysuccinimideconjugated bovine serum albumin hapten-antigen into New Zealand white rabbits. The antibody was purified using protein A, characterized using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and conjugated with gold nanoparticles (AuNP). The conjugated antibody was then characterized using UV-Vis and FTIR spectroscopy and transmission electron microscopy (TEM). Immunochromatographic strip tests were performed using sample pads, conjugated pads, test zones, control zones, and absorbent pads. Strip tests were finally validated using standard AA solutions followed by the application of various concentrations of coffee samples. RESULTS: Using SDS-PAGE, the purified anti-AA antibody was resolved at 50 and 25 kDa, indicating the presence of heavy and light chains, respectively. The conjugation of anti-AA with AuNP was confirmed using wavelength shifts in UV-Vis and FTIR spectra, and TEM analyses revealed increased diameters of AuNPs after conjugation. The immunochromatographic strip test was sensitive to 1 mgml-1 standard AA. Various concentrations of coffee samples resulted in red color differences in the test zone. High and low coffee concentrations produced thick and thin red lines, respectively. CONCLUSION: Purified anti-AA can be conjugated with AuNP to produce strip tests for detecting AA in coffee samples. The present immunochromatographic strip tests quantitatively showed increasing intensities of red lines with increasing AA concentrations.

Sumateran wild boar (Sus scrofa vittatus) meat antibody production as immunodiagnostic reagent candidate.

AIM: Meat authentication gives significance values in view of religious, food safety, public health, quality assurance, and legal concern. Most of the meat authentication is based on molecular assay; a simpler method to authenticate meat is needed to develop. An immunoassays technique may offer a solution for simpler test. The aim of our current study was to develop a polyclonal antibody of Sus scrofa vittatus (Sumateran wild boar) as an immunodiagnostic reagent candidate. MATERIALS AND METHODS: Three male New Zealand white rabbits were used in this study for antibody production. Antigen used was meat extract of Sumateran wild boar, each rabbit was immunized with meat extract antigen (0.5 mg/ml) emulsified in Freund's complete adjuvant at a 1:1 (v/v) ratio as much as 1 ml at subcutaneous route. Booster was carried out 3 times with interval time of 14 days, using meat extract antigen emulsified in Freund's incomplete adjuvant at a 1:1 (v/v) ratio. Serum samples were taken every week, start from 1 week after the first immunization up to 1 week after the third booster. Antibody purification was performed using ammonium sulfate precipitation and Protein A. The presence of specific antibody was determined using agar gel precipitation test and enzyme-linked immunosorbent assay, while purified specific IgG was characterized using sodium dodecyl sulfate-polyacrylamide gel electrophoresis method. RESULTS: Specific antibody was detected at 14 days after the first immunization and still detected until 2 weeks after the third booster. Highest absorbance of specific antibody was detected 1 week after the third booster. CONCLUSION: The present study demonstrated that specific antibody of Sumateran wild boar is favorable to be produced in rabbit and showed that antibody produced is applicable to detect Sumateran wild boar meat antigen in immunodiffusion assay, indicating that it is promising as a reagent candidate in immunodiagnostic assay/kit.

Genetic characterization of S1 gene of infectious bronchitis virus isolated from commercial poultry flocks in West Java, Indonesia.

BACKGROUND AND AIM: Infectious bronchitis (IB) is still a major problem among poultry industry in Indonesia, IB outbreaks continue to happen even in vaccinated flocks. The emergence of new IB virus (IBV) variants might lead to mismatching between vaccine virus strain and circulating virus strain, this may be a reason of vaccination failure. Information about circulating IBV in a region is important to decide which IB vaccine should be used. However, information about recent IBV strains which circulated in Indonesia and their genetic characters were limited; therefore, the aim of our research was to determine the genetic characterization of S1 gene of IBV isolated from commercial poultry flocks in West Java, Indonesia. MATERIALS AND METHODS: A total of 47 viral isolate samples collected from chickens with clinical sign and reduced in egg production. Six IB live vaccines were used as control, the reference vaccines represent IBV strains including H120, H52, 4/91, CR88, 233A, and 1-96. Primers XCe2+ and XCe2- were used to amplify S1 gene partially. RESULTS: Twenty-six of 47 samples showed positive result to S1 gene of IBV by reverse transcription-polymerase chain reaction. Three IBV isolates, Indonesia/K233A31/18, Indonesia/K4A9/17, and Indonesia/P3/17, were selected for nucleotide sequencing. Phylogenetic analysis of 352 nucleotides of the partial S1 gene shows that isolates Indonesia/K4A9/17 and Indonesia/K233A31/18 have 100% homology with IBV vaccine strain 4/91, while isolate Indonesia/P3/17 has 100% homology with IBV vaccine strain 233A. CONCLUSION: Our result indicates that at least two IBV strains were circulating among poultry in West Java, Indonesia, which is IBV close to vaccine strain 4/91 and 233A. The present study provides updates on the circulating IBV in commercial poultry flocks in West Java, Indonesia, and might use as guidance on selecting a proper IB vaccine strain to improve IB vaccination efficacy in certain region.

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.

Potential risk of viral transmission from flying foxes to domestic animals and humans on the southern coast of West Java, Indonesia.

Flying foxes have been considered to be involved in the transmission of serious infectious diseases to humans. Using questionnaires, we aimed to determine the direct and/or indirect contacts of flying foxes in an Indonesian nature conservation area with domestic animals and humans living in the surrounding area. We surveyed 150 residents of 10 villages in West Java. Villages were classified into 3 groups: inside and/or within 1 km from the outer border of the conservation area and 1-5 km or 5-10 km away from the reserve's outer border. Data were collected by direct interview using a structured questionnaire consisting of the respondent characteristics (age, sex and occupation); histories of contacts between flying foxes and humans, dogs and other domestic animals; and knowledge about infectious diseases, mainly rabies, in flying foxes. We found that flying foxes from the nature conservation area often enter residential areas at night to look for food, especially during the fruit season. In these residential areas, flying foxes had direct contacts with humans and a few contacts with domestic animals, especially dogs. People who encounter flying foxes seldom used personal protective equipment, such as leather gloves, goggles and caps. The residents living around the conservation area mostly had poor knowledge about flying foxes and disease transmission. This situation shows that the population in this region is at a quite high risk for contracting infectious diseases from flying foxes.

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.

Experimental and Field Results Regarding Immunity Induced by a Recombinant Turkey Herpesvirus H5 Vector Vaccine Against H5N1 and Other H5 Highly Pathogenic Avian Influenza Virus Challenges.

Vaccination against H5N1 highly pathogenic avian influenza (AI) virus (HPAIV) is one of the possible complementary means available for affected countries to control AI when the disease has become, or with a high risk of becoming, endemic. Efficacy of the vaccination against AI relies essentially, but not exclusively, on the capacity of the vaccine to induce immunity against the targeted virus (which is prone to undergo antigenic variations), as well as its capacity to overcome interference with maternal immunity transmitted by immunized breeding hens to their progeny. This property of the vaccine is a prerequisite for its administration at the hatchery, which assures higher and more reliable vaccine coverage of the populations than vaccination at the farm. A recombinant vector vaccine (Vectormune® AI), based on turkey herpesvirus expressing the hemagglutinin gene of an H5N1 HPAIV as an insert, has been used in several experiments conducted in different research laboratories, as well as in controlled field trials. The results have demonstrated a high degree of homologous and cross protection against different genetic clades of the H5N1 HPAIV. Furthermore, vaccine-induced immunity was not impaired by the presence of passive immunity, but on the contrary, cumulated with it for improved early protection. The demonstrated levels of protection against the different challenge viruses exhibited variations in terms of postchallenge mortality, as well as challenge virus shedding. The data presented here highlight the advantages of this vaccine as a useful and reliable tool to complement biosecurity and sanitary policies for better controlling the disease due to HPAIV of H5 subtypes, when the vaccination is applied as a control measure.

Effect of topical anti-Streptococcus mutans IgY gel on quantity of S. mutans on rats' tooth surface.

This study aims to evaluate the effect of anti-Streptococcus mutans IgY gel on quantity of S. mutans on rats' tooth surface. Sprague Dawley rats were exposed intra-orally with S. mutans Xc and were fed a caries-inducing diet 2000. The 24 rats were divided into four groups: group A had their teeth coated with IgY gel; group B received sterilized water as a control; group C had their teeth coated with IgY gel starting on the 29(th) day; and group D had their teeth coated with a gel without IgY. Plaque samples were swabbed from the anterior teeth for S. mutans colony quantification, and saliva was collected to measure immunoreactivity by enzyme-linked immunosorbent assay. The results indicated that the quantity of S. mutans in rats treated with IgY gel showed significant difference compared with the controls. After coating with IgY anti-S. mutans gel, the mean immunoreactivity in rat saliva was higher than that of the no treatment group. In conclusion, topical application with anti-S. mutans IgY gel reduced the quantity of S. mutans on the tooth surface.

Effects of soybean milk, chitosan, and anti-Streptococcus mutans IgY in malnourished rats' dental biofilm and the IgY persistency in saliva.

OBJECTIVE: This study aims to evaluate the eff ect of soybean milk containing a combination of anti-Streptococcus mutans IgY and chitosan to the colonization of S. mutans in the saliva and to the IgY persistency in the saliva. MATERIALS AND METHODS: Experimental malnourished Sprague-Dawley rats were fed with soybean milk that is enriched with anti-S. mutans IgY and chitosan. After 15 days of feeding, we evaluated the S. mutans in dental biofilm, in addition to the persistency level of anti-S. mutans IgY. RESULTS: The rats that received soybean milk supplemented with anti-S. mutans IgY had the lowest number of S. mutans colonies (p < 0.05). Anti-S. mutans IgY was detected in saliva after 15 days of feeding. CONCLUSIONS: Soybean milk supplemented with anti-S. mutans IgY and chitosan could signifi cantly reduce S. mutans biofilm, and the supplemented anti-S. mutans IgY persisted in these rats' saliva following the feeding period.

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.

Efficacy of a recombinant HVT-H5 vaccine against challenge with two genetically divergent Indonesian HPAI H5N1 strains.

The swift evolution rate of avian influenza (AI) H5N1 virus demands constant efforts to update inactivated vaccines to match antigenically with the emerging new field virus strains. Recently, a recombinant turkey herpesvirus (rHVT)-AI vaccine, rHVT-H5, expressing the HA gene of a highly pathogenic avian influenza (HPAI) H5N1 clade 2.2 A/Swan/Hungary/499/ 2006 strain inserted into FC-126 strain of HVT vector, has been developed to combat current threats in poultry industry. Here, we present the results of two trials where rHVT-H5 was tested alone or in combination with inactivated H5N1 vaccines (the latter vaccines contained antigens produced by using a clade 2.1.3 HPAI H5N1 virus [A/Ck/WestJava-Nagrak/2007] in the first trial or mixture of antigen produced by strain A/Ck/WestJava-Nagrak/2007 and A/Ck/Banten-Tangerang/2010 [bivalent vaccine] for second trial) in broiler chickens (Gallus gallus domesticus) carrying maternally derived antibodies to H5N1 and then challenged with Indonesian HPAI H5N1 field isolates. The effectiveness of vaccination was evaluated on the basis of clinical protection (morbidity and mortality) and measurement of virus shedding after challenge. Immune response to vaccination was followed by serology. In the first experiment, chickens were vaccinated at the day of hatch with rHVT-H5 alone (Group 1) or combined with inactivated vaccine at day old (Group 2) or at 10 days of age (Group 3). The chickens along with nonvaccinated hatch-mates were challenged at 28 days of age with the HPAI H5N1 field isolate dade 2.1.3 A/Chicken/WestJava-Subang/29/2007. Eighty, 100%, and 80% clinical protection was recorded in Group 1, 2, and 3, respectively. A similar experiment was performed a second time, but the chicks in Group 3 received the inactivated vaccine earlier, at 7 days of age. Challenge was performed at 28 days of age using a different H5N1 isolate, clade 2.1.3 A/Ck/Purwakarta-Cilingga/142/10. Clinical protection achieved in the second trial was 95%, 75%, and 90% in Group 1, 2, and 3, respectively. Shedding of challenge virus was significantly lower in the vaccinated groups compared with controls in both experiments. Vaccinated birds developed hemagglutination inhibition antibody response to H5N1 by the time of challenge. These experiments confirmed that the rHVT-H5 vaccine applied alone or in combination with inactivated H5N1 vaccines could provide high level (> 80%) clinical protection against divergent HPAI H5N1 field isolates after single immunization by 4 wk of age and a significant reduction in the excretion of challenge virus.