著者

相馬 武久

出版者

麻布大学

巻号頁・発行日

2001

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Along with a recent trend to characterize pet dog as companion animal, further improvement has been required in veterinary medicine based essentially on an accurate diagnosis. Regarding the diagnosis of infectious diseases, neutralizing test (NT) is the most reliable antibody test widely used; however, due to disadvantages in lacking rapidity, simplicity and economic efficiency, the test is not suitable for routine laboratory practice. Hemagglutination inhibition (HI) test provides simplicity along with the specificity equivalent to that of the NT, yet it is only applicable to detect viruses demonstrating hemagglutination activity. Enzyme-linked immunosorbent assay (ELISA) has recently been demonstrated as a simple and accurate detecting method and is currently utilized most frequently in the field of veterinary medicine. However, because of the non-specific reactions, ELISA sometimes yields an ambiguous result to determine. Immunoperoxidase plaque staining (IP) test was demonstrated in 1982 to compensate for the defect associated with ELISA. In this enzyme antibody technique, plaque, a cluster of virus-infected cells, is used as an antigen and raised in a testing well concomitantly with an uninfected cell layer, providing readily distinguishable appearance of non-specific reactions. The test procedures are similar to those of ELISA. In this study, to examine the diagnostic application of the IP test to canine virus infections, we established antibody test systems for canine distemper virus (CDV), canine coronavirus (CCoV) and canine herpesvirus (CaHV), in which the HI test is less applicable, and examined the possibility to apply the system to the field of canine practice. The epidemiological surveys on these viruses in Japan have not yet been completed; therefore, we conducted the field studies to examine the prevalence of antibodies to these viruses using the IP test.1. Establishment of antibody test system by IP test First, conditions of antigen plates were examined to establish a diagnostic system with the IP test. Vero, CRFK and A-72 cells, which provided clearly observable plaque, were used for cell culture in the CDV-, CCoV- and CaHV-IP tests, respectively. These were infected with each virus at titers of 30 plaque forming units (PFU)/well, providing an appropriate viral density per well for easy observation. The infected cells were incubated for 40 hours on a methylcellulose underlay to support plaque formation and to simplify subsequent operations, followed by fixation with ethanol and these plates were used for the antibody test. For determination, the wells were stained with o-dianisidine to reveal plaque. A test sample was determined positive when brown-colored plaques were observed. The highest serum dilution showing this color development was regarded as IP titer. The color reaction given by the plaques was clear, even when some color developed on uninfected cells in the background owing to non-specific reactions, because the coloration of the plaques was much stronger. We also examined the influences of long-term storage and lot-to-lot variation on the quality of the IP antigen plates. The antigen titers for CDV, CCoV and CaHV were well preserved in the IP plates stored in -20℃ for six months after the production, and were not affected by lot-to-lot variation. These results demonstrate that the IP test constantly provides stable antibody determination.2. Specificity of IP test The specificity of the IP tests was examined by exposing the CDV-, CCoV- and CaHV-IP antigen plates to antisera containing several antibodies against canine viruses. There were no cross-reactions with the antisera in each IP plate, indicating that the IP test specifically detects the target antibody.3. Application of IP test to CDV3-1. Antibody responses of CDV-vaccinated SPF dogs When the antibody response was examined in 84 specific pathogen-free (SPF) dogs that had received CDV vaccine and 30 unimmunized SPF dogs, all the sera from unimmunized SPF dogs that had negative NT titers showed negative IP titers. All the sera from the vaccinated SPF dogs that had positive NT titers showed positive IP titers. There was a strong correlation between the IP and NT titers (r=0.953). The regression line was as follows: y= 0.884x-0.252. Then, when 12 SPF dogs were inoculated with CDV vaccines, both titers followed a similar course after the inoculation in all the dogs. These findings indicate that the IP test has specificity similar to that of the NT. Based on the above regression line, an NT titer of 1:32, which was estimated as a criterion for defensive ability against canine distemper (CD), was then converted into an IP titer of 1:160 to establish the evaluation criterion for the IP test.3-2. Prevalence of antibody in companion dogs The IP test was used to investigate the prevalence of the antibody in sera from 584 healthy dogs. The percentages of sera that had positive IP titers (≧1:10) and that reached the criterion (≧1:160) were 76.5% and 62.5% of all sera tested, respectively. These percentages tended to decline in dogs aged over 9 year. In particular, a marked decrease was observed in the percentage of those reaching the criterion for defensive ability. Aging has recently been progressing so rapidly in the pet community that an increased concern has been raised about the age-related impaired immune system. Our results confirmed this age-related problem and emphasized the need for an aged dog to undergo regular antibody testing and appropriate vaccination based on the result. The results also demonstrated that the IP test was extremely suitable for examining a large number of samples, for instance, in the case of medical checkup.3-3. Comparison of IP test and NT in companion dogs The antibody titers were investigated in 50 healthy dogs and 186 suspected cases of CD (107 dogs showed neurological signs and 83 dogs did not). AII the sera that had positive NT titers showed positive IP titers, and all the sera that had negative NT titers showed negative IP titers in the healthy dogs. In contrast, 71.7% and 65.5% of the sera that had negative NT titers showed positive IP titers in the suspected cases of CD with and without neurological signs, respectively. Hence, the IP test was clearly demonstrated as an effective antibody test for the diagnosis of CD.3-4. Estimation of IP-NT score Furthermore, we examined a possible diagnostic index in terms of the IP titer. The difference between IP and NT titers (IP-NT score) was estimated in the healthy dogs and the suspected cases of CD. The score was indicated as the mean±SD; that is, 0.616±0.385 in the healthy dogs, 1.631±0.834 in the suspected cases of CD with neurological signs and 1.348±0.837 in the suspected cases of CD without neurological signs, resulting in a significantly increased score in the suspected cases of CD (p<0.01). Based on this result, we analyzed the mean±3SD, which 99% of the samples tested are within, and determined the upper limit (mean+3SD) for the healthy dogs as 1.8 of IP-NT score. This upper limit was exceeded in 41.4% of the suspected cases of CD with neurological signs and 31.5% of the suspected cases of CD without neurological signs, indicating that an IP-NT score of > 1.8 is available as one of the diagnostic indices. In a follow-up study conducted on 15 dogs displaying an IP-NT score of > 1.8 at the first examination, 12 of 15 dogs developed neurological signs within one month. These results suggest that the IP-NT score is useful for predicting the onset of neurological signs as well as for diagnosing CD.4. Application of IP test to CCoV4-1. Changes in antibody titers in CCoV-experimentally infected SPF dogs When 3 SPF dogs that were inoculated with CCoV, IP and NT titers followed a similar course after the inoculation.4-2. Comparison of IP test and NT in companion dogs A comparison between the IP test and the NT was performed on 240 healthy dogs and 187 diarrheic dogs (115 dogs aged under one year and 72 dogs aged over one year). All the sera that had negative NT titers showed negative IP titers and all the sera that had positive NT titers showed positive IP titers in the healthy dogs. In contrast, 20.3% and 2.1% of the sera that had negative NT titers showed positive IP titers in the diarrheic dogs aged under one year and aged over one year, respectively. Hence, the IP test should be useful for the diagnosis of CCoV-infection.4-3. Estimation of IP-NT score The score was indicated as the mean±SD; that is, 0.982±0.447 in the healthy dogs, 2.350±0.931 in the diarrheic dogs aged under one year and 1.404±0.896 in the diarrheic dogs aged over one year, resulting in significantly increased scores in the diarrheic dogs (under one year; p<0.01, over one year; p<0.05). The upper limit (mean+3SD) for the healthy dogs was 2.3 of IP-NT score. This upper limit was exceeded in 57.1% of the diarrheic dogs aged under one year and 24.0% of the diarrheic dogs aged over one year, indicating that an IP-NT score of > 2.3 is available as one of the diagnostic indices for CCoV-infection.4-4. Prevalence of antibody in companion dogs The percentages of the sera which had positive IP titers were 25.0%, 48.7% and 34.7% in 240 healthy dogs, 115 diarrheic dogs aged under one year and 72 diarrheic dogs aged over one year, respectively. The percentage of the diarrheic dogs under one year of age was significantly higher than that of the healthy dogs (p<0.01), indicating that CCoV contributes to diarrhea in many juvenile dogs. Further analysis was performed to determine the prevalence of CCoV antibody in the diarrheic dogs with or without anti-canine parvovirus (CPV)-IgM antibody. CCoV-antibodies were identified in 38.1% of sera positive for CPV-IgM antibody and in 62.2% of those negative for the IgM, the latter being significantly higher than the former (p<0.05) as well as that in the healthy dogs (p<0.01). This suggests that the antibody test for CCoV should be performed concurrently with that for CPV when diagnosing diarrhea in dogs.5. Application of IP test to CaHV5-1. Comparison of IP test and NT in laboratory and companion dogs A comparison between the IP test and the NT was performed on 53 dogs from a CaHV-contaminated laboratory, 590 healthy dogs and 35 dogs with chronically respiratory signs. In these animals, all the sera that had positive NT titers showed positive IP titers; however, 23.8%, 0.5% and 3.5% of each group, which had negative NT titers, showed positive IP titers, implying that the IP test detects the antibody more accurately than the NT. The results indicate that the IP test is highly applicable to the serodiagnosis of CaHV-infection.5-2. Estimation of IP-NT score The IP-NT score was indicated as the mean±SD; that is, 1.280±0.520 in the laboratory dogs, 1.220±0.441 in the healthy dogs and 1.106±0.334 in the dogs with respiratory signs. There were no significant differences among the laboratory dogs, the healthy dogs and the dogs with respiratory signs.5-3. Prevalence of antibody in companion dogs Investigation of CaHV-antibody in sera from 590 healthy dogs demonstrated an extremely low prevalence, that is, 3.1%. However, the prevalence in 35 dogs with chronically respiratory signs was estimated to be 20.0%, and was significantly higher than that of the healthy dogs (p<0.01). The result was consistent with a general tendency concerning the relationships between CaHV and respiratory diseases.6. Prevalence of antibody in multi-dog households Additionally, we analyzed the prevalence of CCoV- and CaHV-antibody in mass-breeding environments. CCoV-antibodies were identified in 100%, 100%, 90.0%, 0% and 0% of the respective samples from 5 breeding facilities; the percentages in the 3 facilities exceeded that of the healthy dogs (p<0.01). CaHV-antibodies were identified in 100%, 69.8%, 0% and 0% of the respective samples from 4 breeding facilities; the percentages in the 2 facilities exceeded that of the healthy dogs (p<0.01). These results imply that once CCoV and CaHV contaminate a mass-breeding facility, they spread extensively and the antibodies are detected in a considerable percentage of animals. Therefore, prior to the transportation and intermixing of dog populations, CCoV- and CaHV-infections should be examined using the IP test. In this study, the IP test was used for the first time to diagnose canine virus infections, indicating its high specificity as well as simplicity and rapidity compared to those of the conventional NT and ELISA. We established a useful diagnostic method for CDV and CCoV infections using a combination of IP test and NT. No such information has previously been reported. Furthermore, we demonstrated the prevalence of the CDV-, CCoV- and CaHV-antibodies in the pet population, which has not previously been thoroughly investigated in Japan. Taken together, these results suggest that the IP test is highly applicable to the diagnosis of canine virus infections.