Determination of oxolinic acid residues in three species of Perciformes

Hsun-Lung Chan

Abstract

The purpose of this study was to assess oxolinic acid residues in grouper (Epinephelus coioides), silver perch (Lates calcarifer) and black seabream (Acanthopagrus schlegelii) and generate a basis for determining withdrawal periods, which will be used as references during the review of newly approved aquatic animal drugs and their proper administration. Based on the standardized tests established by the Ministry of Health and Welfare, oxolinic acid residues in fish tissue were analyzed by ultra-performance liquid chromatography combined with electrospray ionization triple quadrupole tandem mass spectrometry (UPLC–MS/MS) and quantification was achieved by using multiple reaction monitoring (MRM). Under standard analytical conditions, calibration curves of oxolinic acid showed good linearity within the range of 0.005-1 μg/mL, with square correlation coefficients of 0.99996, and limits of detection (LOD) and limits of quantification (LOQ) of 0.003 μg/g and 0.005 μg/g, respectively. At spiked levels of 10-100 μg/kg oxolinic acid, muscle tissue (including skin) displayed average recovery rates of 72.19-93.60% while liver recovery rates ranged from 82.67-99.30%, in the three species of Perciformes. The intra-assay relative standard deviations (RSDs) of the muscle and liver assays were 1.31-9.20% and 1.49-6.83% respectively, while the inter-assay RSDs were 4.34-13.31% and 2.32-8.80%, respectively. We measured oxolinic acid residues in these three fish species which were orally administered low and high oxolinic acid doses of 30 and 60 mg/kg respectively, for five days. Muscle and liver samples from fish were both collected at definitive time points on the 1st, 3rd, 5th, 7th, 10th, 14th and 21st days. The results showed that oxolinic ace residue levels in muscle and liver tissues in fish administered with low and high doses were both under the LOD (0.003 μg/g) by the 5^th day in black seabream, the 7^th day in silver perch, and by the 10^th day in grouper. The withdrawal time is calculated as the time it takes for oxolinic acid residue to be below the LOD from the time of oral administration, plus half of this time. These results thus suggest recommended withdrawal periods of 8 days for black seabream, 11 days for silver perch, and 15 days for grouper.

Evaluation of a challenge strain for Newcastle disease vaccine inspection

Bing-Yi Chen

Abstract

At present, the Newcastle disease virus (NDV) genotype VII strain is one of the most prevalent strains of NDV circulating worldwide, but currently the standard strain of Newcastle disease (ND) vaccine inspections via the efficacy test is the Sato strain of NDV genotype III. The purpose of this study was to evaluate whether the NDV genotype VII strain can be used as a challenge strain for the routine testing of ND vaccines. The NDV genotype VII strain was used to infect SPF chickens to determine the median lethal dose（LD₅₀）, minimal lethal dose（MLD）, transmission ability, and the survival rate of SPF chickens after immunization with live vaccines. The test was carried out in the vaccine inspection animal house (inspection amimal house) and the veterinary genetic modification product house (GMO animal house). The LD₅₀ and MLD of the virus was found to be 10^10.5 LD₅₀/mL and 10¹⁰ MLD/mL, respectively. Transmissivity of the virus was found to proceed rapidly through the air and to successfully infect SPF chickens in an adjacent animal breeding room as well as in semi-closed cages, causing symptoms such as diarrhea, depression, neurological symptoms, and death. However, these symptoms were not present in SPF chickens challenged with the NDV Sato strain. The live vaccine strains of ND administered in drinking water to SPF chickens were LaSota, B1, Clone30, VH, VG/GA, C2, Ulster 2C and 6/10. The protective results after NDV genotype VII challenge showed that the Ulster 2C strain and 6/10 vaccine strains did not to meet the 75% survival rate specified by the inspection standard. However, spray-immunization may be a more effective route for administration. Moreover, we considered roughly 30 to 50 batches of ND vaccines for inspections per month, and the number of test chickens in each batch of 10 to 12 will require more than 10 million NT dollars for the purchase of animal feeding equipment and for the improvement of animal biosafety facilities. Nevertheless, we recommend that the Sato strain remains as a challenge strain for ND vaccine inspections.

Diagnosis and  monitoring of highly pathogenic avian influenza in wild birds in Japan

Li-Hsuan Chen, Wan-Chen Li

Abstract

There are strict regulations on the prevention of avian influenza in Japan. In addition to the regulation that high and low pathogenic avian influenza virus strains must be completely absent in poultry, , the presence of avian influenza virus antibodies are also not allowed. Although highly pathogenic avian influenza viruses are still sporadically detected in wild birds and poultry in Japan during the bird migratory season, all of the outbreaks are effectively eliminated. This visit focused on the monitoring scheme for highly pathogenic avian influenza in populations of wild birds, the proper protocols and procedures for diagnosis during outbreaks, and the current methodological tools used in avian influenza research in Japan. The National Institute of Animal Health (NIAH) was the main host institute. At the NIAH, we learned about the application of high-throughput sequencers in avian influenza research, the pretreatment method (unclear what kind of pretreatment method?!), and other related analysis tools. The second visited unit was the Livestock Hygiene Service Center (LHSC) in Ibaraki Prefecture. There, we learned about their monitoring and diagnostic operations for avian influenza as part of many of Japan's first-line of defense disease control units. The third visited site, was the National Institute of Environmental Studies (NIES), which oversees the National Wild Bird Avian Influenza Monitoring Program. It is noteworthy that avian influenza monitoring and diagnostics are carried out by the local LHSC, which determine whether the subtype avian influenza virus is H5 or H7, isolate the viruses, and determine the necessary actions for epidemic prevention. This centralized work-flow procedure cuts down sample transfer times and avoids communication errors. In terms of wild bird monitoring, highly pathogenic avian influenza has never been detected  in feces samples from wild birds but primarily within dead birds. The wild birds monitored are not just limited to migratory waterfowl but also other commonly occurring wild bird species in Japan. This report can thus be used as a reference for the effective design of an avian influenza control program in Taiwan.