Enzyme characterization of lactic acid bacteria isolated from duck excreta.

Background and Aim: The production of lignocellulosic biomass waste in the agricultural sector of Indonesia is quite high annually. Utilization of lignocellulosic biomass waste through fermentation technology can be used as feed and biofuel. Fermentation technology requires the involvement of micro-organisms such as bacteria (lactic acid bacteria or LAB). LABs can be isolated from various sources, such as duck excreta. However, there have not been many reports of LAB from duck excreta. The present study aimed to characterize LAB enzymes isolated from duck excreta and obtain LAB enzymes with superior fermentation properties. Materials and Methods: A total of 11 LAB cultures obtained from duck excreta in Yogyakarta, Indonesia, were tested. Enzyme characterization of each LAB was performed using the API ZYM kit (BioMérieux, Marcy-I'Etoile, France). The bacterial cell suspension was dropped onto the API ZYM™ cupule using a pipette and incubated for 4 h at 37°C. After incubation, ZYM A and ZYM B were dripped onto the API ZYM cupule, and color changes were observed for approximately 10 s under a strong light source. Results: Esterase activity was moderate for all LABs. The activity of α-chymotrypsin, ß-glucuronidase, α-fucosidase, and α-mannosidase was not observed in a total of 10 LAB. The phosphohydrolase and amino peptidase enzyme activity of seven LABs was strong. Only six LAB samples showed protease activity. The glycosyl hydrolase (GH) activity was observed in a total of 8 LAB, while the activity of 2 LAB was strong (Lactococcus lactis subsp. lactis K5 and Lactobacillus brevis M4A). Conclusion: A total of 2 LABs have superior properties. L. lactis subsp. lactis K5 and L. brevis M4A have a high potential to be used in fermentation. They have the potential for further research, such as their effectiveness in fermentation, lignocellulose hydrolysis, feed additives, molecular characterization to detect specific enzymes, and their specific activities.

Isolation, genetic characterization, and virulence profiling of different Aeromonas species recovered from moribund hybrid catfish (Clarias spp.).

Background and Aim: The high diversity of Aeromonas spp. results in various pathogenicity levels. This group of bacteria causes a serious disease named motile Aeromonas septicemia (MAS) in catfish (Clarias spp.). This study aimed to characterize the species and virulence gene diversity of Aeromonas spp. isolated from diseased catfish. Materials and Methods: Nine Aeromonas spp. were isolated from infected catfish cultivated in Java, Indonesia, and they were identified at the phenotypic and molecular levels (16S rDNA). The virulence genes assessed included aer/haem, alt, ast, flaA, lafA, and fstA. Results: Phylogenetic analysis identified nine isolates of Aeromonas spp.: Aeromonas hydrophila (11.11%), Aeromonas caviae (11.11%), Aeromonas veronii bv. veronii (44.44%), and Aeromonas dhakensis (33.33%). Virulence genes, such as aer/haem, alt, ast, flaA, lafA, and fstA, were detected in all isolates at frequencies of approximately 100%, 66.67%, 88.89%, 100%, 55.56%, and 66.67%, respectively. This study is the first report on A. dhakensis recovered from an Indonesian catfish culture. Furthermore, our study revealed the presence of A. veronii bv veronii, a biovar that has not been reported before in Indonesia. Conclusion: This finding confirms that MAS was caused by multiple species of Aeromonas, notably A. dhakensis and A. veronii bv veronii, within Indonesian fish culture. The presence of these Aeromonas species with multiple virulence genes poses a significant threat to the freshwater aquaculture industry.

Genetic characterization and distribution of the virus in chicken embryo tissues infected with Newcastle disease virus isolated from commercial and native chickens in Indonesia.

Background and Aim: Newcastle disease (ND) is a viral infectious disease that affects commercial and native chickens, resulting in economic losses to the poultry industry. This study aimed to examine the viral strains circulating in commercial and native chickens by genetic characterization and observe the distribution of Newcastle disease virus (NDV) in chicken embryonic tissue. Materials and Methods: ND was detected using a quantitative reverse transcription-polymerase chain reaction. Genetic characterization of the fusion (F) and hemagglutinin-neuraminidase (HN) genes from the eight NDVs was performed using specific primers. The sequence was compared with that of other NDVs from GenBank and analyzed using the MEGA-X software. The distribution of NDV in chicken embryos was analyzed based on lesions and the immunopositivity in immunohistochemistry staining. Results: Based on F gene characterization, velogenic NDV strains circulating in commercial and native chickens that showed varying clinical symptoms belonged to genotype VII.2. Lentogenic strains found in chickens without clinical symptoms were grouped into genotype II (unvaccinated native chickens) and genotype I (vaccinated commercial chickens). Amino acid variations in the HN gene, namely, the neutralization epitope and antigenic sites at positions 263 and 494, respectively, occurred in lentogenic strains. The NDV reaches the digestive and respiratory organs, but in lentogenic NDV does not cause significant damage, and hence embryo death does not occur. Conclusion: This study showed that velogenic and lentogenic NDV strains circulated in both commercial and native chickens with varying genotypes. The virus was distributed in almost all organs, especially digestive and respiratory. Organ damage in lentogenic infection is not as severe as in velogenic NDV. Further research is needed to observe the distribution of NDV with varying pathogenicity in chickens.

Human Papillomavirus Type 16 L2 Gene Sequence Variation Analysis in Indonesian Cervical Cancer Specimens.

BACKGROUND: Human papillomavirus type 16 (HPV16) is the most prevalent etiology of cervical cancer in Indonesian women. The L2 minor capsid protein has considerable potential as a broad-protective antigen target of the cervical cancer vaccine strategies, yet the data on L2 gene variation is still minimal. In this research, we determined the variations of the HPV16 L2 gene sequences in Indonesian cervical cancer specimens. METHODS: We cross-sectionally observed 23 DNA isolates of HPV16 positive cervical cancer specimens stored in the laboratory of the Center for Diagnostic and Research on Infectious Diseases (PDRPI Lab), Faculty of Medicine, Universitas Andalas, Padang, Indonesia. We detected and amplified the HPV16 L2 gene sequences in the samples, followed by sequencing, DNA alignment, single nucleotide polymorphisms (SNPs) analysis, and phylogenetic tree reconstruction. RESULTS: As many as 35 SNPs were found, consist of 18 synonymous SNPs (sSNPs) and 17 non-synonymous SNPs (nsSNPs). Amino acid variations were mostly detected at S269P (100%) and L330F (43.48%) with no variation in the immuno-protective region near L2 N-terminus. A total of 5 HPV16 phylogenetic sub-lineages were found closely related to A1 (n=5), A2 (n=12), A3 (n=2), A4 (n=3), and C (n=1). CONCLUSION: The variations of HPV16 L2 gene sequences are mainly located in the central region of the L2 sequences, and the cross-protective region near the L2 N-terminus is remarkably conserved. This study should enhance the information about HPV16 L2 gene variation in Indonesia.

The Distribution of M2 Macrophage and Treg in Nasopharyngeal Carcinoma Tumor Tissue and the Correlation with TNM Status and Clinical Stage.

OBJECTIVE: This study aimed to identify the distribution of M2 macrophage and Treg in Nasopharyngeal Carcinoma (NPC) tumor tissue samples. The presence of these two groups of cells was further correlated to clinical stage, tumor size, the lymphatic node involvement, and metastasis. METHODS: The total of 50 formalin-fixed paraffin-embedded (FFPE) NPC tissue samples was collected retrospectively (27 samples) and prospectively (23 samples). Samples were FFPE tissue slices. Immunohistochemistry was done on the FFPE tissue slides using anti-CD-163 and anti-FoxP-3 antibodies for M2 macrophage and Treg detection, respectively. The M2 macrophage interpretation was performed by eye-balling method and the score was divided into 0 (negative), 1 (scant), 2 (focal), and 3 (abundant). The average number of Treg FOXP3+ cells in 5 high power fields (HPF) was calculated. The relationship of M2 macrophage and Treg was tested with Spearman's correlation. The relationship between M2 macrophage and Treg with clinical stage, tumor size, node involvement and metastasis was tested by chi square, with p<0.1. RESULTS: M2 macrophage and Treg were positive correlated (r=0.469, p<0.001). The presence of M2 macrophage and regulatory T cell (Treg) was significantly correlated to tumor size (p= 0.091 for M2 macrophage and p=0.022 for Treg) and clinical stage (p= 0.030 for M2 macrophage and p= 0.002 for Treg), but did not correlate with lymphatic node involvement and metastasis. CONCLUSIONS: In Epstein-Barr virus related NPC tumor microenvironment, the presence of M2 macrophage was correlated with Treg, and both types of the cells were correlated with tumor size and clinical stages.

The intrasulcular application effect of bisphosphonate hydrogel toward osteoclast activity and relapse movement.

INTRODUCTION: Orthodontic relapse occurs after orthodontic treatment and shifting of teeth to unfavorable positions. Bisphosphonates' effects on bone resorption and relapse prevention have been extensively investigated. However, topical administration, which results in local effect, is still a problem. OBJECTIVE: This study aimed to investigate the effect of risedronate with gelatin hydrogel as a carrier to prevent relapse movement by inhibiting osteoclast activity. METHODS: Lower incisors of 75 guinea pigs were moved distally using an orthodontic appliance until ±3 mm length. Gelatin hydrogel was fabricated to obtain a semisolid controlled release of 250 (Bis-CR250) and 500 mmol/L risedronate (Bis-CR500) and then applied intrasulcularly into the mesial subperiosteal area of 50 guinea pigs (25 in each group) every 3 days; the rest were the control (Bis-CR000). After 14 days of stabilization, the apparatus was removed. The distance decrease between incisors and the osteoclast number with TRAP staining at 0, 3, 7, 14, and 21 days were measured. ANOVA was used to determine the differences among the different time and experimental groups. RESULTS: Both treatments showed significantly less relapse movement compared to the control (p < 0.05) at 14 and 21 days. Bis-CR500 more effectively inhibited the relapse movement than Bis-CR250 on day 21, indicating a dose dependency in the inhibition. Both treatments showed less osteoclast numbers than control (p < 0.05). CONCLUSION: Controlled release of bisphosphonate risedronate with a topically administered gelatin hydrogel has shown to be effective in decreasing the tooth relapse movement and osteoclast activity.

Antimicrobial sensitivity of Avibacterium paragallinarum isolates from layers in the special region of Yogyakarta, Indonesia.

BACKGROUND AND AIM: Infectious coryza (IC) is an upper respiratory disease of chicken caused by Avibacterium paragallinarum. Its clinical symptoms are swollen face and malodorous sinus exudate. This study was conducted to determine the antimicrobial sensitivity of A. paragallinarum isolates from layers in the Special Region of Yogyakarta, Indonesia. MATERIALS AND METHODS: The samples used in this study were 30 layers that showed IC symptoms. The colony and cell morphology were observed with Gram staining; then, biochemical tests (catalase, oxidase, urease, indole, and motility tests, and carbohydrate fermentation tests using lactose, maltose, mannitol, and sorbitol) were performed to the suspected colony to identify A. paragallinarum. An antibiotic sensitivity test was performed using several antibiotic disks against A. paragallinarum isolates that were cultured on Mueller-Hinton Agar. RESULTS: Out of 30 samples, 24 samples (80%) were found positive for A. paragallinarum. All isolates were sensitive to ampicillin (AMP) and amoxicillin (AML) (100%), and chloramphenicol (C) (91.6%). The antibiotics with intermediate sensitivity were enrofloxacin (79.2%), fosfomycin (75%), and ciprofloxacin (54.2%). The isolates were most resistant to erythromycin (100%), followed by tetracycline (87.5%), streptomycin (83.3%), doxycycline and kanamycin (70.8%), and trimethoprim (62.5%). CONCLUSION: Out of the total samples, 24 samples (80%) from layers with IC symptoms were identified biochemically as A. paragallinarum. It was sensitive to AMP, AML, and C.

Proliferation of Fibroblast Cells in Periradicular Tissue Following Intentional Replantation of Vertical Root Fractures Using Two Materials.

AIM: This study aimed to investigate the fibroblast cells proliferation in periradicular tissue using mineral trioxide aggregate (MTA) and self-adhesive resin cement as the adhesive material for vertical root fracture fragments following intentional replantation. MATERIALS AND METHODS: This study used 27 male New Zealand rabbits aged 8-12 weeks. The mandibular incisor of each rabbit was extracted, and to simulate vertical fracture, the incisor tooth was sectioned vertically from the cervical to the 2/3 apical. The samples were randomly divided into three groups of nine each. Group 1 (control group), no application of any material. Group 2, the fracture line was sealed using MTA. Group 3, with self-adhesive resin cement. All teeth in all groups were then inserted back (replanted) into the socket of the rabbits. Each group was further divided into three subgroups according to the duration of replantation, namely, group A: week 1, group B: week 2, and group C: week 3. Rabbits were sacrificed according to each duration of replantation for histological preparations. The number of fibroblast cells was evaluated by counting at the three viewpoints under the light-microscope (400× magnification) and Optilab camera; finally, the calculation results were averaged. Data were analyzed using a two-way ANOVA and post hoc LSD test, with a significance level of 95%. RESULTS: Following MTA application in the third week produced the highest number of fibroblast cells (104 + 29.5) compared to other groups. Conversely, the lowest number of fibroblast cells occurred in the control group in the first week of observation (4.33 + 3.5). CONCLUSION: MTA produced the greatest fibroblast cell proliferation than self-adhesive resin cement, particularly in week 3 of vertical root fractures replantation. CLINICAL SIGNIFICANCE: As the adhesive material for vertical root fracture fragments, MTA generated greater fibroblast proliferation than self-adhesive resin cement. Therefore, it is recommended to use MTA to attach vertical root fracture fragments.

Complete Genome Sequences of 11 Newcastle Disease Virus Isolates of Subgenotype VII.2 from Indonesia.

We report the complete genome sequences of 11 virulent Newcastle disease viruses. The isolates were obtained from vaccinated broiler and layer chickens in three different provinces of Indonesia in 2013 and 2014. Phylogenetic analysis revealed that all isolates belong to subgenotype VII.2 in the class II cluster.

Mortality risk of bloodstream infection caused by either Escherichia coli or Klebsiella pneumoniae producing extended-spectrum ß-lactamase: a prospective cohort study.

OBJECTIVE: Several studies reported that infection of extended-spectrum ß lactamase (ESBL)-producing Escherichia coli (E. coli) or Klebsiella pneumoniae (K. pneumoniae) contributed to higher mortality rates but others found it was not associated with mortality. A prospective cohort study which involved 72 patients was conducted to assess the risk of mortality of bloodstream infection due to ESBL-producing K. pneumoniae or E. coli as compared to those infected by either K. pneumoniae or E. coli which not produce ESBL. RESULT: Mortality in the group of patients infected with ESBL-producing bacteria was 30.6%, whereas in another group which was infected with non ESBL-producing bacteria was 22.2% (p = 0.59). Kaplan-Meier's analysis showed that the survival rate during 14-days follow-up among these two group was not significantly different (p = 0.45) with hazard ratio 1.41 (95% CI 0.568-3.51). Stratification analysis found that adult and elderly patients, patients with sign of leukocytosis, and patients treated with carbapenem were modifier effect variables.

Combinations of herbs and probiotics as an alternative growth promoter: An in vitro study.

BACKGROUND AND AIM: Antibiotic growth promoters (AGPs) are added to animal feed to stimulate growth and increase livestock productivity. However, the regular use of antibiotics in animal diets has a considerable contribution to the occurrence of antibiotic resistance in livestock and humans. This study aimed to investigate the feasibility of red ginger (Zingiber officinale var. Rubrum), turmeric (Curcuma domestica), and wild ginger (Curcuma xanthorrhiza), Lactobacillus acidophilus, and Lactobacillus brevis as an alternative to AGPs. MATERIALS AND METHODS: The antibacterial activities and probiotic stimulatory effects of herbs were screened through the disk diffusion method and optical densitometry. The inhibitory ability of probiotics against pathogens was also tested through the disk diffusion method. The adhesion ability of probiotics was tested by mixing the optimal herbal combinations with broiler intestinal epithelial cells (105 cells/ml). The cells were then subjected to Gram staining, and the number of adherent bacteria was calculated. RESULTS: The test results showed that 3.13% ethanolic wild ginger extract had the highest inhibitory activity against Salmonella Enteritidis, followed by ethanolic red ginger extract and aqueous wild ginger extract at the same concentration. The three extracts also supported the growth of L. acidophilus and L. brevis. Further tests showed that the combination of 3.13% ethanolic red ginger extract had the highest inhibitory activity against S. Enteritidis, followed by ethanolic and aqueous wild ginger extract at the same concentration. The three extracts also supported the growth of L. acidophilus and L. brevis. Further tests showed that the combination of 3.13% ethanolic red ginger extract and 3.13% aqueous wild ginger extract had the best inhibitory effect on the growth of S. Enteritidis. The stimulatory effect of the combinations of herbal extract on the growth of L. acidophilus (0.18±0.00) and L. brevis (0.21±0.01) was better than those of individual extract, positive controls, and the glucose control. L. acidophilus and L. brevis had a weak inhibitory effect on the growth of S. Enteritidis (<6 mm). The adhesion ability of L. acidophilus (420.00±28.21) and L. brevis (259.33±24.03) was stronger than that of S. Enteritidis (202.00±14.00) under treatment with combined extracts. CONCLUSION: The tested combinations of herbs and probiotics can adhere to the intestinal tract. Given this characteristic, herb and probiotic combinations may be developed as an alternative to conventional AGPs.

Sero-diagnostic potential of Plasmodium falciparum recombinant merozoite surface protein (MSP)-3 expressed in silkworm.

Plasmodium falciparum is a blood protozoan parasite, transmitted by Anopheles mosquitoes vectors, that can cause morbidity and even leads to mortality in tropical countries. Strategies are directed to combat malaria including development of diagnostic tools, serological markers and vaccinations. A target under intensive studies is Merozoite Surface Protein (MSP)-3. The aim of this study is to express and purify recombinant MSP3 of P. falciparum (rPfMSP3) using silkworm expression system as a host for its large-scale production and to investigate its potential effectiveness for sero-diagnosis. The rPfMSP3 formed oligomers in a blue-native PAGE and its N-glycosylation was confirmed by periodic acid-Schiff staining and PNGase F treatment. The amyloid-like morphology of the rPfMSP3 oligomers was observed. Enzyme-linked immunosorbent assay showed that 60-70% of human samples from subjects living in malaria endemic areas in Indonesia detected the rPfMSP3. Western blot results showed that the rPfMSP3 was recognized by a malaria infected human serum but not by an uninfected human serum. The rPfMSP3 was successfully expressed in silkworm as a soluble protein and has the potential to be used in serological measurement for detecting PfMSP3-specific antibodies in sera from individuals living in endemic areas.

Molecular characterization of pathogenic 4/91-like and QX-like infectious bronchitis virus infecting commercial poultry farms in Indonesia.

BACKGROUND AND AIM: Existing data on the characteristics of infectious bronchitis virus (IBV) gathered throughout Indonesia have been recognized to indicate variants similar to globally distributed vaccine strains. Despite past and current intensive vaccination programs, IBV infections in the country's poultry industry have not been effectively controlled. Therefore, this study aimed to investigate the genotype of several isolates based on partial S1 gene sequences. In particular, the investigation is directed to focus on layer chickens in actively vaccinated farms indicating IBV symptoms. MATERIALS AND METHODS: Samples were isolated from ten different layer chicken flocks experiencing respiratory problem, drops in egg production, and a "penguin-like" stance, which were collected from commercial poultry farms in Central Java and Yogyakarta regions, Indonesia, within the periods of 2012-2018. Fragment of the S1 gene of IBV sampled from actively vaccinated commercial poultry farms was amplified using primer 5'-aca tgg taa ttt ttc aga tgg-3' (forward) and 5'-cag att gct tac aac cac c-3' (reverse) with the length of polymerase chain reaction (PCR) product at 383 bp. The sequence of samples was then compared with the sequence of reference S1 gene nucleotides of IBV from NCBI GenBank database. The amino acid analysis and multiple alignment sequence were conducted using Mega X. RESULTS: During necropsy, enlargement of the oviduct and swollen kidney were observed. Reverse transcription-PCR diagnosis of their 383 bp S1 gene showed that all samples were IBV positive. Phylogenetic analysis of the S1 gene discovered seven samples to be clustered as 4/91-like strains. Meanwhile, the remaining three samples were grouped in QX-like strain cluster. CONCLUSION: This study is a pioneering report providing molecular evidence of pathogenic QX-like and 4/91-like strains circulating in Indonesia. Findings discovered, in this study, strongly suggested the importance of improving protections by available IBV vaccines through updated circulating strain clusters. It is critical to ensure the delivery of an effective control measurement of and vaccination protocols against IBV infections in the country's commercial poultry industry in particular and worldwide in general.

Efficacy of Artemisinin Base Combination Therapy and Genetic Diversity of Plasmodium falciparum from Uncomplicated Malaria Falciparum Patient in District of Pesawaran, Province of Lampung, Indonesia.

BACKGROUND: Malaria is an infectious disease caused by Plasmodium sp., that still prevalence in some part of Indonesia. District of Pesawaran is one of malaria endemic area in the Province of Lampung. The purpose of this study was to evaluate the efficacy of the ACT treatment in the District of Pesawaran Province of Lampung, Indonesia from Dec 2012 to Jul 2013 and the genetic variation of the Plasmodium falciparum also studied. METHODS: This study was observational analytic study of falciparum malaria patients treated with ACT and primaquine (DHP-PQ and AAQ-PQ) at Hanura Primary Health Centre (Puskesmas). DNA isolation was done with QIAmp DNA Mini Kit. Amplification of PfMDR1, MSP1, and MSP2 genes was done with appropriate forward and reverse primer and procedures optimized first. PCR Product of PfMDR1 gene was prepared for sequencing. Data analysis was done with MEGA 6 software. RESULTS: The results of this research are DHP-PQ effectiveness was still wellness among falciparum malaria patients in District of Pesawaran, Province of Lampung, Indonesia. There is Single-nucleotide mutation of N86Y of PfMDR1 gene. The dominant alleles found are MAD20 and 3D7 alleles with Multiplicity of Infection (MOI) are low. CONCLUSION: Therapy of DHP-PQ as an antimalarial falciparum in Pesawaran District, Lampung, Indonesia is still good. The genetic variation found was the SNP on the N86Y PfMDR1 gene, with dominant allele MAD20 and 3D7.

Cloning and sequencing gB, gD, and gM genes to perform the genetic variability of bovine herpesvirus-1 from Indonesia.

AIM: Previous research has shown that bovine herpesvirus-1 (BHV-1) in Indonesia was closely related to subtype-1 based on glycoprotein D genes. This study aimed to analyze the genetic variability of the BHV-1 isolated from the recent case in Indonesia not only based on gD but also other genes such as gB and gM and to study the homology and similarity of the sample to other BHV-1 isolated in other countries or regions. MATERIALS AND METHODS: Samples were drawn from the tracheal organ in recent field case and prepared for DNA extraction. The gB, gD, and gM were amplified using nested polymerase chain reaction (nPCR) with our specifically designed primer pair and based on the specified bands of 350 bp gB, 325 bp gD, and 734 bp gM confirmed as BHV-1. The PCR product was ligated into pGEM-T and transformed into competent Escherichia coli. The purified plasmid was subsequently sequenced. RESULTS: The virus sample isolated from the recent field case of infectious bovine rhinotracheitis (IBR) from Indonesia showed variability based on the gB, gD, and gM sequences. However, all of the genes had high similarity (98-100%) to BHV-1.2. CONCLUSION: The recent field case of IBR in Indonesia was similar to BHV-1.2.

Field effectiveness of highly pathogenic avian influenza H5N1 vaccination in commercial layers in Indonesia.

Although vaccination of poultry for control of highly pathogenic avian influenza virus (HPAIV) H5N1 has been practiced during the last decade in several countries, its effectiveness under field conditions remains largely unquantified. Effective HPAI vaccination is however essential in preventing incursions, silent infections and generation of new H5N1 antigenic variants. The objective of this study was to asses the level and duration of vaccine induced immunity in commercial layers in Indonesia. Titres of H5N1 haemagglutination inhibition (HI) antibodies were followed in individual birds from sixteen flocks, age 18-68 week old (wo). The study revealed that H5N1 vaccination had highly variable outcome, including vaccination failures, and was largely ineffective in providing long lasting protective immunity. Flocks were vaccinated with seven different vaccines, administer at various times that could be grouped into three regimes: In regime A, flocks (n = 8) were vaccinated two or three times before 19 wo; in regime B (n = 2), two times before and once after 19 wo; and in regime C (n = 6) three to four times before and two to three times after 19 wo. HI titres in regime C birds were significantly higher during the entire observation period in comparison to titres of regime A or B birds, which also differed significantly from each other. The HI titres of individual birds in each flock differed significantly from birds in other flocks, indicating that the effectiveness of field vaccination was highly variable and farm related. Protective HI titres of >4log2, were present in the majority of flocks at 18 wo, declined thereafter at variable rate and only two regime C flocks had protective HI titres at 68 wo. Laboratory challenge with HPAIV H5N1 of birds from regime A and C flocks confirmed that protective immunity differed significantly between flocks vaccinated by these two regimes. The study revealed that effectiveness of the currently applied H5N1 vaccination could be improved and measures to achieve this are discussed.

Regulatory elements of stx2 gene and the expression level of Shiga-like toxin 2 in Escherichia coli O157:H7.

BACKGROUND/PURPOSE: Shiga-like toxin (Stx) is an important factor in the pathogenesis of Escherichia coli O157:H7 infection and is responsible for some severe complications. Stx2 is usually associated with hemolytic uremic syndrome in humans. Its expression is regulated by elements located upstream of the stx2 gene, including stx2-promoter sequence, ribosome binding site, and the antiterminator q gene. The present study aimed to find the correlation between regulatory elements and the expression level of Stx2 in two local isolates of E. coli O157:H7. METHODS: Two local E. coli O157:H7 strains SM-25(1) and KL-48(2), originating from human and cattle feces, respectively, and an E. coli reference strain, ATCC 43894, were investigated. The complete stx2 gene covering the sequences of promoter, ribosome binding site, and open reading frame and q gene of each strain was analyzed. The magnitude of Stx2 production was detected with a reverse passive latex agglutination method and Stx mediated cellular damage was determined with the Vero cell assay. RESULTS: A comparison of the complete stx2 gene contained stx2-promoter, ribosome binding site, and q genes of two local strains KL-48(2) and SM25(1), and the E. coli ATCC 43894 showed that the amino acid sequences were identical. Both local isolates were Stx negative in the reverse passive latex agglutination test and nontoxic in the Vero cell assay. CONCLUSION: The expression level of Shiga-like toxin of the two local isolates of E. coli O157:H7 did not only depend on the regulatory elements of the stx2 gene.

Phenotype and genotype of lactic acid bacteria (LAB) isolated from the tiger grouper Epinephelus fuscoguttatus alimentary tract.

Lactic acid bacteria (LAB) have been isolated successfully from the tiger grouper Epinephelusfuscoguttatus intestine. However, their genus or species have not been identified. Therefore, the objective of the present study was to characterize the three isolated LAB (KSBU-12C, KSBU-5Da, and KSBU-9) based on their phenotype and genotype. The LAB phenotype was examined by observing morphological features including cell morphology, spore production and motility. The physiological tests were performed in 6.5% NaCl at the  temperatures of 10 oC and 45 oC, and the biochemical tests were evaluated based on the production of enzymes catalase, oxidase and arginine dehydrolase, following  the Standard Analytical Profile Index, API 50 CH kit.  The genotype was examined based on 16S rDNA gene sequence analysis , and the products were analyzed using the BLAST (Basic Local Alignment Search Tool) NCBI database. The three isolates (KSBU-5Da, KSBU-12C, and KSBU-9) were categorized into the genus Enterococcus. 16S rDNA sequence analysis indicated that the isolates had 99% similarity to E. hirae ATCC 9790, registered in GenBank with accession number NR_075022.1. It was concluded that the three LAB isolates taken from the tiger grouper Epinephelus fuscoguttatus are E. hirae.

Use of M2e ELISAs for longitudinal surveillance of commercial poultry in Indonesia vaccinated against highly pathogenic avian influenza.

In countries where highly pathogenic avian influenza virus (HPAIV) H5N1 is endemic and controlled by vaccination, post-vaccination serological monitoring is essential to differentiate vaccinated poultry from those that are infected. The objectives of this study were to validate two experimental ELISAs that detect antibodies raised against the M2e protein of avian influenza virus that can be used for DIVA purposes. Results from the sM2e and tM2e ELISAs were compared with other conventional tests for the detection of H5N1influenza virus (virus isolation and RT-PCR) using samples collected from 16 commercial flocks in Indonesia. These comprised vaccinated layers aged between 18 and 68 weeks old that were sampled at ten-weekly intervals. A small number of sera were positive in sM2e and tM2e ELISA, 14 (0.6%) and 17 (0.7%) respectively, with low OD420 (0.1-0.3), but only 4 sera were positive in both tests. At the flock level, the incidence of M2e positive sera was low (4%), well below previously established minimum of 40% for an HPAIV H5N1-infected flock. Conventional M and H5 gene RT-PCRs indicated that none of 16 flocks were infected at any time during the study. No virus was isolated from any of the 480 pooled swab samples, except from one, for which the combined data analysis suggest to be the result of a laboratory cross-contamination. Clinical disease, mortalities or reduction in production performance, indicative of field H5N1 challenge, were not observed either in any of the flocks. Birds from two surveyed flocks, challenged in the laboratory with an Indonesian HPAIV H5N1 developed M2e antibodies in 50% and 55% of surviving birds with OD420 in the range of 0.35-1.47 in tM2e ELISA, confirming the validity of the criteria established for use of M2e ELISA for DIVA purposes. Overall these results showed that the tM2e ELISA could be a useful monitoring tool to ascertain freedom from H5N1 infections in vaccinated commercial poultry.

Hepatitis E virus infection in two different regions of Indonesia with identification of swine HEV genotype 3.

Hepatitis E is an emerging disease with a high incidence globally. Few data are available on hepatitis E virus (HEV) infection in Indonesia. To obtain molecular information on HEV infection in two regions of Indonesia with different customs and swine breeding conditions, serum samples from 137 swine farm workers, 100 blood donors and 100 swine (27 fecal samples also obtained) in Yogyakarta (Central Java) and from 12 and 64 swine farm workers, 42 and 135 local residents and 89 and 119 swine in Tulungagung (East Java) and Mengwi (Bali), respectively, from our previous study, were compared.Serological tests for anti­HEV antibodies by ELISA, HEV­RNA detection by RT­PCR and phylogenetic analysis were performed. The total prevalence of anti­HEV antibodies in humans was higher in Bali(11.6%) than in Java (5.1%; P=0.015). No significant differences in anti­HEV prevalence among swine farm workers and local residents in Java were found. The finding of swine HEV genotype 3 in specimens from Yogyakarta and genotype 4 from Tulungagung and Bali is somewhat different from other reports.We suggest other factors in addition to close contact with swine might play an important role in HEV transmission of non­endemic/related custom groups. To the best of our knowledge, this is the first report on swine HEV genotype 3 in Indonesia.