Pathological analysis of batch safety testing of veterinary vaccines using small laboratory animals

Batch safety tests (BSTs) of veterinary vaccines are conducted using small laboratory animals to assure the safety of vaccines according to several criteria, including clinical signs and change in body weight. Although the latter is used as an evaluation index in BSTs, there have been no reports on the internal changes that affect body weight during the test period. Therefore, we analyzed BST via pathological examination of the tested animals. Here, BSTs were performed for 176 batches using mice and 126 batches using of guinea pigs. Most of the gross findings could be classified into four lesion types (nodules, adhesions, ascites, no apparent signs), with only one vaccine inducing lesions that could not be classified into any of these four types. Histopathological examination revealed that the reactions caused by BST were pyogenic and/or granulomatous inflammation. Nodular or adhesive lesions comprised more severe pyogenic granulomatous inflammation than ascites or cases with no apparent gross lesions. These nodular or adhesive lesions were more frequently induced by vaccines that contained an adjuvant than by vaccines that did not contain an adjuvant. The cases with “exceptional” gross findings histologically presented severe necrosis of the hematopoietic system. Additional testing showed that these “exceptional” lesions were induced when a specific type of light liquid paraffin was injected along with other vaccine additives. Our results show that body weight loss and/or lesions during BST were induced by proinflammatory properties of the tested vaccines and that BST is a sensitive method for detecting unexpected effects of vaccine components.     

黄金鲈与伊犁鲈杂交后代在气提泵循环水养殖系统中生长分析

研究通过黄金鲈(Perca flavescens)雌鱼与伊犁鲈(Perca schrenki)雄鱼进行杂交,以期加快鲈鱼的生长速度,推迟其性成熟和提高上市规格.为减少饲养环境条件和人为管理差异造成生长试验误差,研究采用试验鱼同池混养的方法进行生长比较.在气提泵循环水养殖系统中,经690 d的生长比较试验.结果表明:杂交鲈体重增长比黄金鲈快33.91%;杂交鲈的性腺发育被推迟或性腺发育不良;黄金鲈和杂交鲈的成活率分别为87.50%和82.08%,二者在成活率上无显著差异(P=0.29).杂交鲈适宜在高密度循环水养殖条件下生长,杂交优势较为显著,可以取代黄金鲈进行集约化养殖生产.     

Potassium behavior and clay mineral composition in the soil with low effectiveness of potassium application

ABSTRACT

Increasing exchangeable potassium (ExK) content in soil to an appropriate level is important to mitigate the transfer of radioactive cesium to crops. We focused on a buckwheat (Fagopyrum esculentum Moench) field with a low ExK content, despite the application of K, in Fukushima Prefecture, Japan (Field A), following the Tokyo Electric Power Company Fukushima Dai-ichi (No. 1) Nuclear Power Plant accident in March 2011. We examined the relationship between K concentration and clay mineral composition in the soil of Field A and compared the findings with another field in Fukushima Prefecture (Field B) to clarify whether K applied to the soil was leached or remaining fixed. Pot experiments showed that K concentration in water seepage from pots following irrigation was significantly lower in pots from Field A than in those from Field B. Soil ExK content after soybean cultivation was lower in soils of Field A than those of Field B. These results indicate that K applied to Field A was fixed in the soil. Analysis of clay mineral composition confirmed the distinctive vermiculitic nature of Field A soils. This clay mineralogy would be associated with the higher K fixation ability of Field A than Field B soils. This study demonstrated that K fixation in vermiculite was a factor preventing the increase in ExK content from K application to Field A.     

Community structure of bacteria on different types of mineral particles in a sandy soil

Various types of mineral particles in a soil probably provide different microenvironments for microorganisms. The purpose of this study is to investigate whether different types of mineral in a soil harbor different bacterial populations. DNA was extracted from five types (quartz, feldspar, pyroxene, magnetite, iron-coated reddish brown particles) of sand-size mineral particles separated from a sandy soil, and was amplified for partial 16 S rRNA gene by polymerase chain reaction (PCR). Twenty-nine to 69 amplicons per each type of mineral were cloned and sequenced, followed by phylogenetic affiliation of the sequences. As a result, some types of bacteria were detected on all of the types of mineral including the orders Rhizobiales, Bacillales, and Acidobacteriales. In the case of Acidobacteriales, higher percentages were found on magnetite and quartz. Some taxa were restricted to specific types of mineral; the class Actinobacteria was found on pyroxene but not on quartz, and rarely on magnetite and feldspar. Bacterial diversity at the order level estimated by Chao1 value was higher in feldspar and pyroxene than the other three types of mineral. The UniFrac Significance test indicated that the differences in bacterial communitiy structures among the particles were suggestive except that between feldspar and pyroxene. These results support the idea that different communities of bacteria were associated with each of the mineral types.     

Influences of indigenous phototrophs on methane emissions from a straw-amended paddy soil

A pot experiment was conducted to investigate the influences of indigenous phototrophs on methane (CH₄) emissions from a paddy soil where rice straw was incorporated or was surface-applied. During the cultivation, half of the pots were covered with aluminum foil, except for the minimum space for rice plants, to prevent ambient light reaching the floodwater or the soil surface. Growth of oxygen-producing phototrophs was hardly observed in the unilluminated plots, whereas intensive growth of algae, duckweed and hydrophytes was found in the illuminated ones. Plant growth was not affected by the different treatments. Seasonal changes in CH₄ emission determined by a closed chamber method indicated that illumination had no or only minor effects on CH₄ emissions when rice straw was incorporated or was not applied, but significantly reduced CH₄ emissions when rice straw was surface-applied. Methanogenesis occurring in the soil-floodwater interface was further investigated in two lab-scale model experiments measuring methanogenic activity. As a result, more activated methanogenesis was found in the surface-applied rice straw and the soil around the straw compared with the soil incubated without rice straw. The magnitude of the methanogenic activity in the rice straw incubated under illuminated conditions was significantly lower than that incubated in the dark. Consequently, this study demonstrates that methanogenesis in paddy soil occurs even in the soil-floodwater interface if plant residues like rice straw exist, and such methanogenesis is likely to be suppressed by growth of indigenous phototrophs under illumination.     

Temporal shifts in diversity and quantity of nirS and nirK in a rice paddy field soil

Denitrification is an important part of the nitrogen cycle in the environment, and diverse bacteria, archaea, and fungi are known to have denitrifying ability. Rice paddy field soils have been known to have strong denitrifying activity, but the microbes responsible for denitrification in rice paddy field soils are not well known. Present study analyzed the diversity and quantity of the nitrite reductase genes (nirS and nirK) in a rice paddy field soil, sampled four times in one rice-growing season. Clone library analyses suggested that the denitrifier community composition varied over sampling time. Although many clones were distantly related to the known NirS or NirK, some clones were related to the NirS from Burkholderiales and Rhodocyclales bacteria, and some were related to the NirK from Rhizobiales bacteria. These denitrifiers may play an important role in denitrification in the rice paddy field soil. The quantitative PCR results showed that nirK was more abundant than nirS in all soil samples, but the nirK/nirS ratio decreased after water logging. These results suggest that both diversity and quantity changed over time in the rice paddy field soil, in response to the soil condition.     

Cono- and seminiphagous insects of Abies alba Mill. in the Gorce National Park in Poland during 1996?–?1998

A study was conducted on the insects infesting seeds and cones of Abies alba Mill. in the Gorce National Park, in southern Poland during 1996–1998. A total of 856 cones were collected from 66 trees growing at 11 sites located in partially or strictly protected forest reserves. Of this sample, 217 cones with 54,958 seeds were dissected. This study yielded 5,816 specimens of insects, namely: seminiphagous Resseliella piceae Seitn., Megastigmus suspectus Borr. as well as conophagous Earomyia impossibile Morge, Barbara herrichiana Obr. and Dioryctria abietella (Den. et Schiff.). The most abundant species was R. piceae with 4,186 individuals. The index of cone infestation was calculated. It showed great variability between forest reserves and study-years. The numbers of insect individuals, numbers of viable and infertile seeds, and seeds damaged by M. suspectus and R. piceae, in both strict and partial reserves in the consecutive years were compared statistically. The test of significant differences between two frequencies at 0.05 level was used in statistical analysis. In both strict and partial reserves, statistically significant differences were found between studied variables.     

PCR detection of Porcine circovirus type 2 DNA in whole blood,serum, oropharyngeal swab,nasal swab,and feces from experimentally infected pigs and field cases

Porcine circovirus type 2 (PCV2) shedding patterns were investigated by polymerase chain reaction (PCR) for the detection of PCV2 DNA, and the diagnostic suitability of a sample for the PCR was examined by using different types of samples. In the experimental infection, sixteen pigs were inoculated intranasally with PCV2. The samples, including oropharyngeal and nasal swabs, feces, whole blood and serum became positive for PCV2 DNA by PCR immediately after the inoculation, and almost all samples remained positive during the observation period, post-inoculation-day 70. Field samples were collected from 313 pigs in five different age groups. The overall percentages of positive samples in the whole blood, nasal swabs, and feces detected by PCR were 30.4%, 19.2%, and 20.4%, respectively. The frequency of positive samples increased after the nursery stages and reached a peak in the 3 to 4-month-old pigs. These results indicate that PCV2 infection may occur after weaning, that PCV2 DNA may be present in whole blood for a long period after infection, and that whole blood and serum are the most suitable sample types for the PCR analysis of PCV2.     

Balenine,imidazole dipeptide,induces activation of superoxide dismutase in myotubes

Balenine is one of the endogenous imidazole dipeptides. It is composed of beta-alanine and 3-methyl-l-histidine, which exist mainly in the muscles and the brain. The exact biological properties of balenine are still not well known, although the antioxidant activity of carnosine, another imidazole dipeptide, is known. In this study we investigated whether balenine exhibits antioxidant activity. It was found to decrease the superoxide anion (O₂⁻) and increased hydrogen peroxide (H₂O₂) generation. We found that SOD activity increased in balenine-treated C2C12 myotubes, although balenine did not increase expression of SOD mRNA. On the other hand, there were no changes in other antioxidant enzymes, CAT and GPX activity, and mRNA levels. In an in vitro assay, the direct activation of SOD treated by balenine was significantly higher than with carnosine treatment. Moreover, balenine constituent amino acids did not have the ability to activate SOD. Our results suggest that balenine contributes to antioxidant effects through activation of SOD.

     

Effects of Inoculation of Phototrophic Purple Bacteria on Grain Yield of Rice and Nitrogenase Activity of Paddy Soil in a Pot Experiment

Phototrophic purple bacteria (PPB) are one group of dinitrogen (N₂)-fixing bacteria often found in the anoxic and photic zone of paddy soil. In the present study, the effects of inoculation of PPB on grain yield of rice and changes in their populations and nitrogenase activity were investigated with and without surface application of rice straw in a pot experiment. As an inoculant, Rhodopseudomonas palustris strain KN122 isolated from a paddy soil was selected, and the cell suspension was inoculated into the floodwater once or three times during the cultivation. As a result, the inoculation of PPB increased the grain yield of rice. Compared to the control, the grain yield was 9% higher in the inoculated plot without rice straw application. The treatment was more effective in combination with rice straw application. In the plots where PPB were inoculated with rice straw, grain yields were 21% (single inoculation) and 29% (triple inoculation) higher. Populations of PPB in the plots without rice straw application significantly increased by the inoculation, unlike those in the plots with rice straw application, except for the bulk soil. Phototrophic and heterotrophic nitrogenase activities (acetylene-reducing activity) associated with the soils or residues of the rice straw were not affected by PPB inoculation throughout the experiment. This indicates that the inoculation of PPB into floodwater may not be effective for enhancing N₂ fixation in paddy soils and that the beneficial effect of PPB inoculation on the grain yield of rice may be due to unidentified functions of PPB other than biological N₂ fixation.