Cytotoxicity associated with prolonged room temperature storage of serum and proposed methods for reduction of cytotoxicity.

Canine serum preserved at room temperature (25°C) for longer than 24h is known to exhibit significant cytotoxicity. This phenomenon is one of the major reasons for the failure of virus neutralization tests. In this study, a method for reducing this cytotoxicity was investigated by applying several treatments to dog, cat and human serum prior to room temperature storage. Additionally, the identity of the cytotoxic factor generated during room temperature storage was investigated. Heat-inactivation at 56°C or 65°C and the addition of protease inhibitor prior to storage were found to be effective for reducing cytotoxicity in the serum. Furthermore, heat-inactivation at 65°C reduced the cytotoxicity that was induced under room temperature storage. Several protein factors in serum were suspected to play a role in the observed cytotoxicity. According to this study, the membrane-attack-complex in serum was not involved in the cytotoxicity. This study provides useful information for development and improvement of cell culture and virus neutralization tests.

Neutralizing antibody response in dogs and cats inoculated with commercial inactivated rabies vaccines.

In Japan, the import quarantine regulation against rabies has required from 2005 that dogs and cats should be inoculated with the rabies vaccine and that the neutralizing antibody titer should be confirmed to be at least 0.5 international units (IU)/ml. The fluorescent antibody virus neutralization (FAVN) test is used as an international standard method for serological testing for rabies. To achieve proper immunization of dogs and cats at the time of import and export, changes in the neutralizing antibody titer after inoculation of the rabies vaccine should be understood in detail. However, few reports have provided this information. In this study, we aimed to determine evaluated, such changes by using sera from experimental dogs and cats inoculated with the rabies vaccine, and we tested samples using the routine FAVN test. In both dogs and cats, proper, regular vaccination enabled the necessary titer of neutralizing antibodies to be maintained in the long term. However, inappropriate timing of blood sampling after vaccination could result in insufficient detected levels of neutralizing antibodies.

Determination of critical factors causing cytotoxicity in the virus neutralization test.

The fluorescent antibody virus neutralization (FAVN) test, an international standard method for serological testing for rabies, has been adopted by many countries. However, some dog serum samples inhibit the formation of cell monolayers by BHK-21 cells used in the test, resulting in failures to determine antibody titers. This inhibition of cell monolayer formation was defined as cytotoxicity. In this study, critical factors that induce cytotoxicity of the dog serum in BHK-21 cells were determined, and the effective ways to prevent cytotoxicity were also established. Specifically, some anticoagulants, anti-BHK-21 cell IgG antibodies, and serum storage at temperatures of >25°C were found to induce cytotoxicity. On the other hand, several treatments of the dog serum, including the absorption by BHK-21 cells or kaolin, incubation with trypsin-EDTA, and the use of collagen- or gelatin-coated plates, were shown to reduce cytotoxicity. Based on these results, the FAVN test may be modified to enhance its performance.

Prevalence and molecular epidemiological characterization of antimicrobial-resistant Escherichia coli isolates from Japanese black beef cattle.

We investigated the prevalence of antimicrobial-resistant Escherichia coli in Japanese black beef cattle from the three major production regions of Japan. We collected and examined 291 fecal samples from Japanese black beef cattle in Hokkaido, Chubu, and Kyushu. Of the 3,147 E. coli isolates, 1,397 (44.4%) were resistant to one or more antibiotics; these included 553 (39.8%) of 1,388 isolates from Hokkaido, 352 (54.4%) of 647 isolates from Chubu, and 492 (44.2%) of 1,112 isolates from Kyushu. The difference in resistance rates between the three regions was significant. The antibiotics with the highest rates of resistance were oxytetracycline and dihydrostreptomycin (35.8% each), followed by ampicillin (21.4%). Further, E. coli isolates from calves had higher resistance rates than those from growing cattle and mature cattle, and the calf isolates showed high rates of resistance to gentamicin (20.2%), enrofloxacin (9.4%), and ceftiofur (4.2%). In addition, the high degrees of similarity in the genotypes of the isolates and in the resistance patterns on each farm suggest that resistance bacteria and resistance genes were horizontally transferred. Most isolates, in each of the three regions, harbored resistance genes such as blaTEM, strA, strB, aphA1, aphAI-IAB, and catI. In contrast to the isolates from Kyushu, most of which harbored aacC2, tetB, and dfrA12, the isolates from Hokkaido and Chubu harbored a variety of resistance genes. Furthermore, the prevalence of genes for resistance to dihydrostreptomycin, gentamicin, chloramphenicol, and trimethoprim differed significantly between the regions. This is the first large-scale study describing and comparing antimicrobial-resistant bacteria from different regions in Japan. The results will contribute to improving food safety and promoting careful usage of antimicrobial agents.

Antimicrobial resistance in fecal Escherichia coli isolated from growing chickens on commercial broiler farms.

To investigate the effects of rearing practices of commercial broiler chickens on the incidence of antimicrobial resistance in commensal Escherichia coli isolates, fecal E. coli isolates obtained in 4 farms were screened for anitimicrobial resistance. Ten E. coli isolates were recovered from each of the fecal samples collected from 10 birds in the farms at the ages of 2 days, 14-17 days, and 47-50 days. In 2 out of the 4 farms, no antimicrobials were used during the rearing period. In the other two farms, following collection of the fecal samples at 14 and 15 days of age, oxytetracycline (OTC), sulfadimethoxine (SDMX), and tylosin were given to birds on one farm and SDMX was used in the other. Isolates resistant to ampicillin and OTC that were obtained from an untreated flock at different sampling times were closely related to each other by pulsed-field gel electrophoresis patterns (PFGE) of XbaI-digested chromosomal DNA. PFGE analysis together with in vitro conjugation experiments suggested that diversity of resistance phenotypes within a clone may be resulted from the acquisition and loss of R-plasmids in an untreated and a treated flock. The numbers of resistance phenotypes observed among fecal isolates increased during the growth of the chickens in all the farms. The results in the present study suggest that persistence of commensal E. coli strains resistant to antimicrobials even in the absence of antimicrobial administration. It is also hypothesized that horizontal transmission of resistance determinants resulted in the emergence of different resistance phenotypes in those farms.

Determination of ethoxyquin by high-performance liquid chromatography with fluorescence detection and its application to the survey of residues in food products of animal origin.

An analytical method using HPLC with fluorescence detection (HPLC-FL) has been developed and applied for the survey of residue levels of ethoxyquin in a variety of food products of animal origin. HPLC was performed using a silica octadecylsilane column, butylhydroxytoluene-acetonitrile-water (0.05 + 800 + 200, v/v/v) mobile phase, and detection at excitation and emission wavelengths of 370 and 415 nm, respectively. HPLC/MS was used to confirm whether a chromatographic peak was ethoxyquin. The LOQ of the foods was 0.01 microg/g, except for pig fat and cow's milk, and the RSD (n=6) at 0.1 microg/mL of the standard solution was 1.12%. The accuracy of the calculated data of the standard solution was within the range of 94.0 to 101.2%. Recoveries of ethoxyquin from the food products of cattle, pigs, chickens, and salmon were more than 71.0% with an RSD of < 9.3%, except for chicken liver at different concentration levels, including the lower LOQ, the maximum residue limit (MRL), and in some tissues, twice the MRL. Residue levels of ethoxyquin in 33 commercially available food products of animal origin that were purchased on the west side of the Tokyo metropolitan area were surveyed. Contents of ethoxyquin residues in three chicken fat samples by the HPLC-FL method were 0.08, 0.03, and 0.04 microg/g, all less than the MRL (5 microg/g).

Simultaneous determination of azaperone and azaperol in animal tissues by HPLC with confirmation by electrospray ionization mass spectrometry.

A simple method is described for the determination of azaperone and its metabolite, azaperol, in animal tissues by high-performance liquid chromatography with ultraviolet detection (HPLC/UV). Chromatography was performed using an ODS column, an acetonitrile-0.025% aqueous diethylamine mixture (2:3, v/v) as a mobile phase and UV detection at 250 nm. Peak heights were found linearly related to the concentrations injected from 0.05 to 2 microg/mL (r>0.999). Azaperone and azaperol spiked into several animal tissues were solubilized in 1 mol/L NaOH, extracted with hexane, transferred to 0.1 mol/L H(2)SO(4) and re-extracted with hexane in a mild basic condition. Recoveries of both compounds from 12 types of samples (swine muscle, swine adipose tissue, swine liver, bovine muscle, bovine adipose tissue, bovine liver, poultry muscle, poultry adipose tissue, poultry liver, bovine milk, poultry egg, and salmon muscle) were more than 72%. The lower limit of quantification of was 0.025 microg/g. Azaperone and azaperol at 0.1 microg/g were confirmed by LC/MS. In conclusion, we found this method is both simple and useful for the determination of azaperone and azaperol in a variety of animal tissues for food safety and veterinary applications.

Hepatic lesions caused by migrating larvae of Ascaris suum in chickens.

Group A consisted of chickens infected with a single dose of Ascaris suum and group B of chickens infected with two successive doses. At days 1, 3, 7, 14 and 21 after the first or second infection dose, six chickens from each group were sacrificed. In both groups, larvae were recovered from the livers on days 1, 3, and 7 and lungs on days 3 and 7. No larvae were detected in chickens on day 14. Clear white lesions were noticed only on the livers from chickens of group B at day 7 but had disappeared at day 14. A comparison with group B showed mild histological changes that developed relative to the livers from group A.

Liquid chromatography-tandem mass spectrometric method for determination of mosapride citrate in equine tissues.

A simple method for determination of mosapride citrate and its metabolite, des-p-fluorobenzyl mosapride (M-1), in equine muscle, liver, kidney, adipose tissue and intestine by liquid chromatography-tandem mass spectrometry has been developed. (+/-)-4-Amino-5-chloro-2-ethoxy-N-[[4-(2-chlorobenzyl)morpholinyl]methyl]benzamide was used as an internal standard. The analytes and internal standard were spiked and extracted from tissues by acetonitrile. The chromatographic separation was performed on a reversed-phase TSK-GEL SUPER ODS column with a mobile phase of acetonitrile-0.05% (v/v) formic acid containing 5 mmol/L nonafluoropentanoic acid (2:3, v/v). The method exhibited a large linear range from 0.0005 to 0.2 microg/mL for both mosapride citrate and M-1 (r>0.9976). In the intra-day assay (n=5), the relative standard deviations (RSDs) ranged from 1.1 to 7.8% for mosapride citrate and 1.6 to 7.2% for M-1. In the inter-day assay (n=3), the RSDs ranged from 1.0 to 13% for mosapride citrate and 0.8 to 11% for M-1. The extraction recovery at 1.28 microg/g of mosapride citrate from equine tissues ranged from 97 to 107%. The lower limit of quantification for mosapride citrate was found to be 0.004 microg/g. Stability studies were carried out at different storage conditions. The method reported is reliable, precise, and accurate and it has the capacity to be used for determination of mosapride citrate and its metabolite in tissue samples.

Establishment of a potency test by ELISA for a rabies vaccine for animal use in Japan.

The ELISA we developed was able to determine the antigen content and was suitable for a potency test, and we described a relative potency assay method which determines the potency of test vaccines by comparing the ELISA value of a test vaccine to that of a reference vaccine. In the present study, we standardized the reference vaccine used for determining the potencies of test vaccines, and established a potency test by ELISA. We evaluated the proposed reference vaccine by the neutralizing antibody responses in dogs after vaccination, by the challenge protection test in guinea pigs (GP potency test), which is the earlier official potency test used in Japan, and by the NIH potency test, which is widely used throughout the world. The results showed that a 4-fold dilution of the proposed reference vaccine induced sufficient immunity in dogs. A 3-fold dilution of the proposed reference vaccine passed the GP potency test. The international units (IU) calibrated by the NIH potency test were 3.7 IU/dose. From the results and the WHO recommendation that veterinary rabies vaccines should have a potency of at least 1.0 IU/dose, we determined to dilute the proposed reference vaccine by 3 fold and regarded it as the reference vaccine. Finally, we confirmed that there is a good agreement between the results of the potency test by ELISA and the results of the GP potency test. The establishment of the potency test by ELISA has made it possible to monitor the potency in the production process and has contributed to the stable production of the vaccine.