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.

Abstract. Widodo A, Lamid M, Effendi MH, Khairullah AR, Riwu KHP, Yustinasari LR, Kurniawan SC, Ansori ANM, Silaen OSM, Dameanti FNAEP. 2022. Antibiotic sensitivity profile of multidrug-resistant (MDR) Escherichia coli isolated from dairy cow's milk in Probolinggo, Indonesia. Biodiversitas 23: 4971-4976. The presence of resistant bacteria in animal products such as milk can be a new threat because it is directly related to the human food chain. Resistant Escherichia coli has been widely studied and detected in farms in developing countries. The aim of present study was to determine the antibiotic resistance profile of E. coli bacteria from dairy cows taken during the milking process from several dairy farms in Probolinggo district, Indonesia. A total of 150 milk samples were obtained from farms and E. coli was isolated and identified on Eosin methylene blue (EMB) media and biochemical test, such as Triple Sugar Iron Agar (TSIA) and indole test, methyl red test, Voges-Proskauer test, and citrate test (IMViC) were also performed. The antibiotic sensitivity profile was screened using the Kirby-Bauer test and results were interpreted according to the CLSI standard. The results showed that 124/150 (82.67%) E. coli bacteria exhibited highest percentage of antibiotic resistance to tetracycline (13.71%), streptomycin (9.68%), trimethoprim (8.87%), chloramphenicol (0.87%), and aztreonam (1.61%). A total of 9/124 (7.26%) E. coli isolates were detected as multidrug-resistant (MDR) and 1/9 (0.81%) E. coli isolate was suspected as extended spectrum ?-lactamase (ESBL) bacteria which was resistant to aztreonam antibiotic. Thus, the threat of multidrug-resistant (MDR) E. coli can come from milk which can affect public health.

Study on sialidases as antiviral agents has been widely performed, but many types of sialidase have not been tested for their antiviral activity. Pasteurella multocida NanB sialidase is one such sialidase that has never been isolated for further research. In this study, the activity of NanB sialidase was investigated in silico by docking the NanB sialidase of Pasteurella multocida to the Neu5Acα(2–6)Gal and Neu5Acα(2–3)Gal ligands. Additionally, some local isolates of Pasteurella multocida, which had the NanB gene were screened, and the proteins were isolated for further testing regarding their activity in hydrolyzing Neu5Acα(2–6)Gal and Neu5Acα(2–3)Gal. Silico studies showed that the NanB sialidase possesses an exceptional affinity towards forming a protein–ligand complex with Neu5Acα(2–6)Gal and Neu5Acα(2–3)Gal. NanB sialidase of Pasteurella multocida B018 at 0.129 U/mL and 0.258 U/mL doses can hydrolyze Neu5Acα(2–6)Gal and Neu5Acα(2–3)Gal better than other doses. In addition, those doses can inhibit effectively H9N2 viral binding to red blood cells. This study suggested that the NanB sialidase of Pasteurella multocida B018 has a potent antiviral activity because can hydrolyze sialic acid on red blood cells surface and inhibit the H9N2 viral binding to the cells.

Background and Aim: Fowl avidenoviruses (FAdVs) are generally considered ubiquitous, but certain serotypes and strains are known to be associated with primary diseases, such as inclusion body hepatitis (IBH). Since 2018, the outbreak of IBH has been reported in part provinces of Indonesia. This study aimed to isolate and molecularly characterize the FAdV from Banten and West Java Provinces of Indonesia and described the phylogenetic relationship with the FAdV that has been characterized in other countries. Materials and Methods: A total of 25 FAdV archive samples have been collected from January to August 2019 from clinical cases of FAdV infection in Banten and West Java Provinces, Indonesia. Collected samples were inoculated in 10-day-old specific-pathogenic-free chicken embryonated eggs. Hexon gene of FAdV was detected using polymerase chain reaction (PCR) with a primer set from previous study. To gain a better understanding of the FAdV genetic properties and construct the phylogeny tree, the PCR products were sequenced and subjected to a BLAST search and inferred using the neighbor-joining method by bootstrap test 1000×. Results: FAdV-D and FAdV-E are present in Banten, Indonesia. The phylogenetic analysis of 850 nucleotides that encode 289 amino acid of the partial hexon gene shows that the isolates Broiler/MSL/Ciputat-149/18, Broiler/MSL/Lebak-151/18, and Broiler/MSL/Ciputat-29/19 have 100% homology with FAdV-E TR/BVKE/R/D-1 from Turkey, whereas the isolates Layer/MSL/Ciputat-20/19 and Broiler/MSL/Ciputat-30/19 have 100% homology with FAdV-D strain 685 from Canada. Conclusion: The present study provides updates of the circulating FAdV in commercial poultry flocks in Banten and West Java Provinces, Indonesia. Since the FAdV vaccine was unavailable in Indonesia, this result might be used as guidance to select a proper FAdV vaccine strain. Our result indicates that at least two FAdV species were circulating among poultry in Banten and West Java Provinces, Indonesia; they are FAdV-D and FAdV-E.

Abstract. Widodo A, Lamid M, Effendi MH, Khailrullah AR, Kurniawan SC, Silaen OSM, Riwu KHP, Yustinasari LR, Afnani DA, Dameanti FNAEP, Ramandinianto SC. 2022. Antimicrobial resistance characteristics of multidrug resistance and extended spectrum beta-lactamase producing Escherichia coli from several dairy farms in Probolinggo, Indonesia. Biodiversitas 23: 215-221. Escherichia coli bacteria initially reside in the digestive tract of humans and animals but are able to adapt to new environments that are different from their initial habitat. The pathogenicity of E. coli can occur when these bacteria grow more than normal limits, produce toxins, and are resistant to certain types of antibiotics. The purpose of this study was to investigate the antimicrobial resistance characteristics of MDR and ESBL-producing E. coli from several dairy farms in the Probolinggo district of East Java province, Indonesia. A total of 150 samples consisting of 109 milk and 41 environmental samples from 41 dairy farms were used for isolation. TSIA and IMViC biochemical tests were used to identify E. coli bacteria. Escherichia coli resistance profile was obtained through disc diffusion test on several antibiotics, namely tetracycline, streptomycin, trimethoprim, chloramphenicol and aztreonam. Escherichia coli that was resistant to 3 or more antibiotics was defined as MDR. The results of isolation and identification obtained 124 (82.6%) isolates characterizing E. coli bacteria. The antimicrobial susceptibility test of E. coli showed 9 (7.26%) MDR isolates and 2 (22.22%) ESBL isolates by double-disc synergy test (DDST). MDR E. coli was dominated by the pattern of antimicrobial drug resistance TE-S-W (tetracycline, streptomycin, trimethoprim) with a total of 8 (38.10%) isolates, followed by antimicrobial drug resistance pattern TE-S-W-ATM (tetracycline, streptomycin, trimethoprim, aztreonam) with one (4.76%) E. coli isolates. The pattern of antimicrobial drugs of ESBL E. coli showed in one (11.11%) sample of ESBL E. coli from a milk sample with the pattern of TE-S-W-ATM (tetracycline, streptomycin, trimethoprim, aztreonam) and one (11.11%) sample ESBL E. coli (AL 30) from the environmental sample with a pattern of TE-S-W pattern (tetracycline, streptomycin, trimethoprim). The discovery of MDR E. coli isolates and ESBL E. coli from milk and environmental samples at several dairy farms in Probolinggo district, East Java, Indonesia is a matter of concern and requires real action to reduce antibiotic resistance.

Objective: The Newcastle disease virus (NDV) is an infectious disease that causes very high eco¬nomic losses due to decreased livestock production and poultry deaths. The vaccine’s ineffec¬tiveness due to mutation of the genetic structure of the virus impacts obstacles in controlling the disease, especially in some endemic areas. This study aimed to provide an alternative treatment for NDV infection by observing the viral replication inhibitor activity of Clostridium perfringens sialidase in primary chicken embryo fibroblast (CEF) cells. Materials and Methods: The virus was adapted in CEF monolayer cells, then collected thrice using the freeze–thaw method and stored at −20°C for the next step in the challenge procedure. C. perfringens crude sialidase was obtained, but it was further purified via stepwise elution in ion exchange using Q Sepharose® Fast Flow and affinity chromatography with oxamic acid agarose. The purified sialidase was tested for its toxicity, ability to breakdown sialic acid, stopping viral replication, and how treated cells expressed their genes. Results: According to this study, purified C. perfringens sialidase at dosages of 187.5, 93.75, and 46.87 mU effectively hydrolyzes CEF cells’ sialic acid and significantly inhibits viral replication on the treated cells. However, sialidase dosages of 375 and 750 mU affected the viability of mono¬layer CEF cells. Interestingly, downregulation of toll-like receptor (TLR)3 and TLR7 (p < 0.05) in the sialidase-treated group indicates viral endocytosis failure. Conclusions: By stopping endocytosis and viral replication in host cells, sialidase from C. perfrin¬gens can be used as an alternative preventive treatment for NDV infection.

Methicillin-resistant Staphylococcus aureus (MRSA) is a S. aureus strain resistant to β-lactam antibiotics and is often associated with livestock, known as livestock-associated (LA)-MRSA. Using molecular typing with multi-locus sequence typing, MRSA clones have been classified in pigs, including clonal complex 398. Livestock-associated-methicillin-resistant S. aureus was first discovered in pigs in the Netherlands in 2005. Since then, it has been widely detected in pigs in other countries. Livestock-associated-methicillin-resistant S. aureus can be transmitted from pigs to pigs, pigs to humans (zoonosis), and humans to humans. This transmission is enabled by several risk factors involved in the pig trade, including the use of antibiotics and zinc, the size and type of the herd, and the pig pen management system. Although LA-MRSA has little impact on the pigs' health, it can be transmitted from pig to pig or from pig to human. This is a serious concern as people in direct contact with pigs are highly predisposed to acquiring LA-MRSA infection. The measures to control LA-MRSA spread in pig farms include conducting periodic LA-MRSA screening tests on pigs and avoiding certain antibiotics in pigs. This study aimed to review the emerging LA-MRSA strains in pig farms.

Study on sialidases as antiviral agents has been widely performed, but many types of sialidase had not been tested for their antiviral activity. One of such sialidase is the NanB sialidase of Pasteurella multocida, which has never been isolated for further study. In this study, the activity of NanB sialidase was investigated in silico by docking the NanB sialidase of Pasteurella multocida to the Neu5Acα(2-6)Gal ligand. Additionally, some local isolates of Pasteurella multocida, which had the NanB gene were screened, and the proteins were isolated for further testing regarding their activity in hydrolyzing Neu5Acα(2-6)Gal. In silico studies showed that the NanB sialidase possesses an exceptional affinity towards forming a protein-ligand complex with Neu5Acα(2-6)Gal. This was further confirmed by showing that a dose of 0.258 U/ml (100%) NanB sialidase of Pasteurella multocida B018 can hydrolyze up to 44.28% of Neu5Acα(2-6)Gal in chicken red blood cells and 81.95% in rabbit red blood cells. This study suggested that the NanB sialidase of Pasteurella multocida B018 has a potent antiviral activity that can inhibit avian influenza virus infection.