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.

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.

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.

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.

Protection from avian influenza H5N1 virus infection with antibody-impregnated filters.

There is worldwide concern over the possibility of a new influenza pandemic originating from the highly pathogenic avian H5N1 influenza viruses. We herein demonstrate that functional air filters impregnated with ostrich antibodies against the hemagglutinin of the H5N1 virus protect chickens from death by H5N1 transmission. These results suggest that the use of ostrich antibody-impregnated filters might be a powerful way to prevent the transmission of H5N1.