Identify genes encoding resistance to tetracycline (TE) and plasmid-mediated resistance to quinolones in Escherichia coli isolates from clinical cases of avian colibacillosis in Sukabumi, Indonesia

Background and Aim: Clostridium toxins are widely used as medicinal agents. Many active metabolic enzymes, including sialidase (neuraminidase), hyaluronidase, and collagenase, contribute to the mechanism of action of these toxins. Sialidase from Clostridium perfringens recognizes and degrades sialic acid receptors in the host cell glycoprotein, glycolipid, and polysaccharide complexes. Sialic acid promotes the adhesion of various pathogens, including viruses, under pathological conditions. This study aimed to investigate the potential of C. perfringens sialidase protein to inhibit Newcastle disease virus (NDV) infection in ovo model. Materials and Methods: C. perfringens was characterized by molecular identification through polymerase chain reaction (PCR) and is cultured in a broth medium to produce sialidase. In addition, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis was conducted to characterize the sialidase protein. In contrast, enzymatic activity and protein concentration were carried out using a neuraminidase assay kit and Bradford to obtain suitable active substances. Furthermore, embryonated chicken egg models were used to observe the toxicity of several sialidase doses. Then, the hemagglutination (HA) titer was obtained, and absolute quantitative reverse transcription–PCR assay was performed to measure the viral replication inhibitory activity of sialidase against NDV. Results: Each isolate had a specific sialidase gene and its product. The sialidase derived from C. perfringens could hydrolyze the sialic acid receptor Neu5Ac (2,6)-Gal higher than Neu5Ac (2,3)Gal in chicken erythrocytes, as observed by enzyme-linked lectin assay. A significant difference (p = 0.05) in the HA titer in the pre-challenge administration group at dosages of 375 mU, 187.5 mU, and 93.75 mU in the competitive inhibition experiment suggests that sialidase inhibits NDV reproduction. Quantification of infective viral copy confirmed the interference of viral replication in the pre-challenge administration group, with a significant difference (p = 0.05) at the treatment doses of 750 mU, 375 mU, and 46.87 mU. Conclusion: The potency of sialidase obtained from C. perfringens was shown in this study, given its ability to reduce the viral titer and copy number in allantoic fluids without adversely impacting the toxicity of the chicken embryo at different concentrations.

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.

The Avian influenza virus is an infectious agent that may cause global health problem issues in poultry and potentially zoonotic. In recent decades, bacterial-derived sialidases have been extensively studied for their ability to inhibit avian influenza virus infections. In this study the antiviral activity of NanB sialidase from Pasteurella multocida was investigated through in vitro analysis using MDCK cells. NanB sialidase was purified from P. multocida for testing its toxicity and its ability to hydrolyze its sialic acid receptors on MDCK cells. H9N2 challenge virus was propagated in MDCK cells until cytopathic effects (CPE) appeared. Antiviral activity of NanB sialidase was conducted using MDCK cells, and then observed based on cell morphology, viral copy number, and expression of apoptosis-mediating genes. NanB sialidase effectively hydrolyzes Neu5Acα(2-6)Gal sialic acid at the dose of 129 mU/ml, while at 258 mU/ml it cause toxicity on MDCK cells. Antiviral activity of sialidase is evident based on the significantly decrease in viral copy number at all doses administrated. The increase of p53 and caspase-3 expression was observed in infected cells without sialidase. Our study demonstrates the ability of NanB sialidase to inhibit H9N2 virus replication based on observations of sialic acid hydrolysis, reduction in viral copy number, and expression of apoptosis-related genes. The future application of sialidase may be considered as an antiviral strategy against avian influenza H9N2 virus infections.

Necrotic enteritis causes significant losses in the global poultry industry, necessitating accurate diagnosis for effective intervention. This study aimed to develop a diagnostic tool for detecting necrotic enteritis in poultry based on the presence of Clostridium perfringens (C. perfringens) Alpha-toxin in poultry feces. The reagent of the detection kit was developed by conjugation of IgG against C. perfringens toxin and Staphylococcus cells containing protein A. The IgG antibody was derived from an 8-month-old thin-tailed male sheep immunized with purified 2 ml of C. perfringens Alpha-toxin. Sensitivity assays were carried out to determine the detection limit, while Escherichia coli (E. coli) and Salmonella enteritidis (S. enteritidis) were used to identify specificity. A purified Alpha-toxin with a protein concentration of 2.8 mg/ml and a specific molecular weight of 43 kDa was successfully obtained. A strong reaction of the hyperimmune antibody (IgG) was also detected in the thin-tailed male sheep serum. The developed rapid detection kit in this study indicated C. perfringens Alpha-toxin with a lower concentration (12 ng/ml). Agglutination reactions could differentiate positive control from negative without significant cross-reactivity towards other bacteria (S. enteritidis and E. coli). Keywords: Agglutination, Clostridium perfringens, Detection, Necrotic Enteritis, Toxin

Newcastle disease (ND) is an acute poultry disease caused by Paramyxovirus group. It has characteristic neurological symptoms, called torticollis. The molecular assay to find out the presence of viral genes in the brain can be an option in detecting ND virus infections since it penetrates brain barrier system. The purpose of this study was to identify ND viruses in the brain of chickens with torticollis symptoms, to analyze its phylogenetic and to characterize its virulence genetic code. Samples used were 12 dead chickens with historically had torticollis symptoms, obtained from poultry farms at several areas in West Java and Banten. Chicken brains were prepared for reverse transcriptasepolymerase chain reaction (RT-PCR) test. All positive samples then sequenced to obtain its nucleotide sequences from some of Fusion (F) genes analyzed its phylogenetic by comparing with Indonesian ND isolate virus from GenBank using Mega X software. The results of RT-PCR test showed that only one sample (Virus MSL.03) contained genes of ND virus. Based on homology tests and phylogenetic analysis, the virus belonged to subgenotype VIIh with an identical level of 95.34-95.86% when compared to several isolates from Indonesia. The MSL.03 ND virus has 112RRRKRF117 pattern in F0 indicatest its virulent category.