Rapid testing of antibiotic residues to increase food safety awareness of animal origin.

Background and Aim: Antibiotics are used to improve growth, reduce disease, and decrease mortality in animals grown for food. The government regulates and prohibits the use of antibiotics, in particular, the use of antibiotic growth promoter (AGP) in livestock; however, it is not yet known whether the use of antibiotics is in accordance with regulations so that there are no antibiotic residues in food of animal origin. To ensure food safety of animal origin and to raise awareness of food safety, it is necessary to detect antibiotic residues in fish, eggs, and chicken meat from Yogyakarta Special Province through monitoring and monitoring. To ensure food safety and regulatory compliance in food samples, antibiotic residue screening techniques are essential. A number of methods, such as time-consuming and costly chromatographic and spectroscopic methods, have been developed for the detection of antibiotic residues in food samples; however, not all laboratories have these facilities. Therefore, a rapid diagnosis of food of animal origin is required. The purpose of this study was to rapidly test antibiotic residues by using Premi®test kits (R-Biopharm AG, Germany) to increase awareness of food safety of animal origin. Materials and Methods: We tested 345 animal-based food samples from traditional markets, supermarkets, and central markets in five districts of Yogyakarta Special Province for antibiotic residues using rapid test kits and observation questionnaires to identify risk factors. Results: The presence of antibiotic residues in food-animal origin samples from the Yogyakarta region had an antibiotic residue level of 9.28% (32/345), consisting of fish samples 11.3% (18/97), eggs 15.65% (1/114), and chicken meat samples 0.87% (13/102). The highest percentage of samples positive for residual antibiotics was 21.9% (7/32) from supermarket meat samples. The highest amounts of antibiotic residues were found in fish samples collected from Sleman Regency, up to 25% (8/32), whereas in supermarket fish samples, there were as high as 18.8% (6/32). Conclusion: Antibiotic residues in animal-based food can be attributed to various factors, including product source, transportation conditions, and environmental conditions. The widespread distribution of antibiotic residues in fish comes from environmental conditions during maintenance, distribution, and retailing. Monitoring antibiotic residue prevalence in food-animal origins, particularly chicken meat, eggs, and fish, is crucial for improving animal food quality and safety.

Examination of macroscopic and microscopic lesions in IBDV-infected organs and molecular characterization of IBDV VP1 gene fragments obtained from commercial broiler farms in Indonesia.

Background and Aim: Infectious bursal disease (IBD) is an infectious immunosuppressive disease that affects young chickens. Instead of strict biosecurity practices, vaccination is used to control IBD. However, the disease has not been effectively managed. Variations in the observed clinical symptoms lead to confounding diagnoses. The study aimed to obtain pathological lesion data from chickens suspected of IBD virus (IBDV) infection by gross pathology, confirm IBDV infection through molecular diagnostics, and genotype the VP1 gene fragments of circulating IBDV in the field. Materials and Methods: The bursa of Fabricius, thymus, spleen, proventricular-ventricular junction, thigh muscles, and kidneys samples were collected from chickens suspected of IBDV infection from four commercial broiler farms in Central Java and The Yogyakarta Special Region Province between 2021 and 2022. The collected samples were examined histopathologically. Infectious bursal disease virus RNA was extracted from the bursa of Fabricius and VP1 gene was identified by reverse-transcriptase polimerase chain reaction (RT-PCR). The RT-PCR positive sample were sequenced and analyzed in Mega X for homology search and phylogenetic tree analysis. Results: Macroscopic pathological lesions in the bursa of Fabricius were demonstrated by enlarged edema and thickened plica, presence of gelatinous exudate, hemorrhage, atrophy, and caseous exudate in the lumen. Moreover, the thymus had atrophy and small gray foci were observed in the spleen. Petechiae or hemorrhage was detected on the thigh muscle, and the kidney was dull and pale. Hemorrhage in the proventricular-ventricular junction was distinct. The histopathological examination of the bursa of Fabricius showed follicular vacuolization, edema, heterophilic infiltration, follicular atrophy, congestion, and hemorrhage. The thymus and spleen showed the presence of multifocal necrosis. Hemorrhage was observed in thigh muscle and mucosal part of proventricular-ventricular junction. Vacuolization was seen in renal tubules (nephrosis). Reverse transcriptase-PCR of 26 bursa of Fabricius samples from chickens suspected of IBDV infection showed four negative and 22 positive samples. Phylogenetic analysis of the VP1 gene fragment has indicated very virulent IBD (vvIBD) and belonged to B2 genotype. Conclusion: Infectious bursal diseases virus infection in broiler chicken generated macroscopic and microscopic primary lesions in the bursa of Fabricius and thigh muscle. Other organs such as the spleen, thymus, proventricular-ventricular junction, and kidney, were also involved. Molecular analysis of the VP1 gene confirmed the causative agent and grouped the virus into vvIBD and B2 genotype. All samples were collected from vaccinated birds therefore, the efficacy of available vaccine is required for urgent evaluation. Since most studies only focused on VP1, further exploration on VP2 gene is suggested notably for new-generation vaccines. Monitoring clinical signs' transformation over time could assist field diagnostics.

Analysis of antibody response to an epitope in the haemagglutinin subunit 2 of avian influenza virus H5N1 for differentiation of infected and vaccinated chickens.

The H5N1 subtype of highly pathogenic avian influenza virus has been circulating in poultry in Indonesia since 2003 and vaccination has been used as a strategy to eradicate the disease. However, monitoring of vaccinated poultry flocks for H5N1 infection by serological means has been difficult, as vaccine antibodies are not readily distinguishable from those induced by field viruses. Therefore, a test that differentiates infected and vaccinated animals (DIVA) would be essential. Currently, no simple and specific DIVA test is available for screening of a large number of vaccinated chickens. Several epitopes on E29 domain of the haemagglutinin H5N1 subunit 2 (HA2) have recently been examined for their antigenicity and potential as possible markers for DIVA in chicken. In this study, the potential of E29 as an antigen for DIVA was evaluated in detail. Three different forms of full-length E29 peptide, a truncated E29 peptide (E15), and a recombinant E29 were compared for their ability to detect anti-E29 antibodies. Preliminary ELISA experiments using mono-specific chicken and rabbit E29 sera, and a mouse monoclonal antibody revealed that the linear E29 peptide was the most antigenic. Further examination of the E29 antigenicity in ELISA, using several sera from experimentally infected or vaccinated chickens, revealed that the full-length E29 peptide had the greatest discrimination power between infected and vaccinated chicken sera while providing the least non-specific reaction. This study demonstrates the usefulness of the HPAI H5N1 HA2 E29 epitope as a DIVA antigen in HPAI H5N1-vaccinated and -infected chickens.RESEARCH HIGHLIGHTS E29 (HA2 positions 488-516) epitope is antigenic in chickens.Antibodies to E29 are elicited following live H5N1 virus infection in chickens.E29 epitope is a potential DIVA antigen for use in ELISA.

Characterisation of the antigenic epitopes in the subunit 2 haemagglutinin of avian influenza virus H5N1.

Monitoring avian influenza (AI) infection and detecting silent infection in vaccinated chickens has been challenging due to the lack of effective serological diagnostic assays to differentiate between vaccinated and infected animals. Very few studies have identified suitable proteins in AI virus that can be used in successfully differentiating infected from vaccinated animals (DIVA). An HA2 peptide: HA2 position 197-201 (HA position 488-516) described by Khurana et al. (J Virol 85(23):12455-12463, 2011), was shown to have DIVA ability by differentiating H5N1-infected human sera in ELISA. In order to explore the capacity of the HA2 protein, as a DIVA reagent in chickens, four overlapping recombinant HA2 proteins, were expressed in E. coli and tested for reaction with H5N1 sera obtained from infected and vaccinated chickens. Recombinant protein HA2_B2 (380-461) was able to generate a detectable reaction with both H5N1 infected and vaccinated chicken sera but recombinant protein HA2_B4 (483-565) reacted strongly only with sera obtained from chickens infected with live virus, confirming its suitability as a DIVA antigen. Further analysis of the HA2 using several overlapping peptides suggested that positions 380-461 and 483-565 were antigenic in mouse and chicken. This study, for the first time, identified novel antigenic epitopes on the H5N1 HA2 subunit. Two epitopes, found in the HA2 ectodomain, have never been described for AIV infection in any animal species. Also one HA2 epitope was found to have high potential as a DIVA antigen.