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.     

Mitigation of pyrexia by a Th-1-biased IgG subclass response after infection with equine herpesvirus type 1 in horses pre-immunized with inactivated vaccine

The immunoglobulin G (IgG) subclass response was investigated in horses with or without pyrexia after natural infection with equine herpesvirus type 1 (EHV-1) in the field. All horses were kept at the training centers of the Japan Racing Association and were immunized with an inactivated EHV-1 vaccine before EHV-1 infection. An IgG subclass response dominated by IgGa and IgGb was induced in horses without pyrexia after EHV-1 infection. In contrast, horses that developed pyrexia showed increased IgGc and IgG (T) subclass production in addition to IgGa and IgGb. Although inactivated EHV-1 vaccines are considered to induce a mainly Th-2-biased response, these results indicated that the responses in horses inoculated with inactivated EHV-1 vaccine were not uniform, and that horses with a Th-1-biased response were likely to be protected from pyrexia.     

Thermal tolerance of a thermally selected strain of rainbow trout <Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis> and the pedigrees of its F1 and F2 generations indicated by their critical thermal maxima

A thermally selected strain of rainbow trout has been established by selective breeding since 1966 in Miyazaki, Japan. In the present study, we compared the critical thermal maxima (CTMs), the temperatures at which organisms reach a predefined sublethal endpoint and lose their equilibrium, between a thermally selected and two normal (Donaldson) strains of rainbow trout. The CTM of one normal strain from Nikko (Nikko strain) acclimated to 20 °C (29.7 °C) was significantly lower than those of the thermally selected strain (30.0 °C) and the other Donaldson strain from Aomori (29.9 °C) (P?<?0.05). The F1 generations, F1T and F1N, were produced by crossing thermally selected strain females with Nikko strain males and Nikko strain females with thermally selected strain males, respectively. No significant difference was observed in the CTM between F1T [30.1?±?0.15 °C (n?=?30)] and F1N [30.1?±?0.16 °C (n?=?30)] (P?>?0.05) for fish acclimated to 20 °C, suggesting that the F1 offspring inherited the thermal tolerance trait from one thermally selected strain parent irrespective of whether it was the male or female. F2 offspring of F1T or F1N also showed the thermal tolerance trait. The coefficients of variation for CTM were also compared among all the datasets obtained in the present study and their values for F1 hybrids were lower than those of the parental generation of the Nikko strain (P?<?0.05). In contrast, the coefficients of variation of F2s were the same as those of their parental generation. Furthermore, the thermally selected strain and Nikko strain as a reference family provide a F2 generation for segregating phenotypes, which is required for in-depth genetic analysis of the thermally selected rainbow trout strain.     

Tracking the Northern Pacific sea star <Emphasis Type="Italic">Asterias amurensis</Emphasis> with acoustic transmitters in the scallop mariculture field of Hokkaido,Japan

The Northern Pacific sea star Asterias amurensis has a major negative impact on scallop mariculture. In northern Japan, fishermen clean up sea stars before releasing young scallops in the mariculture field; however, new sea stars constantly invade the field from outside areas to feed on scallops. Thus, it is important to determine the migration speed and seasonal behavioral patterns of the Northern Pacific sea star to implement effective density control measures. Here, we set out to quantify these parameters using acoustic telemetry. In a rearing experiment, acoustic transmitters were retained on sea stars for up to 71 days using nylon fishing line. In the field experiment, we showed that the moving distance of the Northern Pacific sea star over a 1-week period was significantly further in spring (90.9 ± 49.9 m) than in summer (25.1 ± 18.9 m), and that the moving speed was significantly faster in spring (18.1 ± 15.2 m/day) than in summer (4.3 ± 9.1 m/day). Our results are the first to present the two-dimensional movement of Northern Pacific sea star individuals in spring and summer. We suggest that sea star extermination practices should be extended beyond the immediate culture area.     

Use of water-treatment residue containing polysilicate-iron to stabilize arsenic in flooded soils and attenuate arsenic uptake by rice (Oryza sativa L.) plants

A pot experiment was conducted to examine how soil amendment with water-treatment residue (WTR) containing polysilicate-iron affected dissolved arsenic (As) in flooded soils and As uptake by rice plants (Oryza sativa L.). The WTR was applied at a rate of 0 (control), 5, 10 or 20 t ha^?1. Simple linear regression analyses showed significant negative relationships between the concentrations of dissolved As in soil solution and WTR application rates at all sampling times, probably due to adsorption of As onto ferrihydrite in the WTR. Compared to As concentrations in rice plants grown on control soil, the concentrations in plants grown on WTR-treated soils decreased by 20.1–41.6% in straw (stems and leaves), 19.8–31.7% in husk and 18.6–21.0% in grain. The regression analyses demonstrated that the concentration and content of As in rice are negatively correlated with WTR application rate. Total As content was 16.5–32.0% lower in rice shoots grown on WTR-treated soils than on control soil. The percentage of each As species in grain decreased in the following order: As(III) » dimethylarsinic acid » As(V). The application of WTR did not change the As speciation in grain. Silicon contents in shoot significantly increased with application of WTR, indicating the potency of WTR as a silicate fertilizer. Taken together, our results indicate that WTR containing polysilicate-iron promises to be a practical amendment for stabilizing As and attenuating As uptake by rice plants.     

Two new species,Phytophthora nagaii sp. nov. and P. fragariaefolia sp. nov., causing serious diseases on rose and strawberry plants,respectively, in Japan

A new disease of rose was noticed in Chiba Prefecture of Japan in 1968, and the pathogen was initially identified as Phytophthora megasperma based on morphological characteristics. Similar Phytophthora isolates have since been collected from rose plants in Chiba, Kanagawa, and Shizuoka Prefectures. In 2005, several Phytophthora isolates were recovered from crowns of strawberry plants in Hokkaido Prefecture. These were considered to be members of a new species. In this study, we re-examined all these isolates using morphological and physiological studies and a multilocus phylogenetic analysis. The rose and strawberry isolates were mostly similar morphologically and physiologically, with some exceptions. The rose isolates differed significantly from P. megasperma sensu stricto and other related Phytophthora species. The rose and strawberry isolates had external proliferation of sporangia, characteristic funnel-shaped oogonia, predominantly paragynous antheridia, and fast growth rates of 10.5 mm/24 h at an optimum temperature of 28 °C. In the multilocus phylogenetic tree constructed using sequences from the rDNA ITS regions, rDNA LSU, and the translation elongation factor 1-α, β-tubulin and coxI genes, they formed a distinct monophyletic group in clade 7 with strong bootstrap support. The rose and strawberry isolates separated into two distinct groups. The results indicate that the rose and strawberry isolates constitute two separate species, designated here as Phytophthora nagaii and P. fragariaefolia.     

Partial characterization and ontogenetic development of pancreatic digestive enzymes in Japanese eel Anguilla japonica larvae

The pancreatic digestive enzymes, trypsin, chymotrypsin, lipase and amylase were partially characterized, and changes in their activities were examined during the initial ontogeny of Japanese eel Anguilla japonica larvae from 5 to 34 days post-hatching (dph). The pH optima of the eel larval enzymes were narrower than those other fish species; trypsin activity was highest at pH 9, chymotrypsin and amylase activities were highest at pH 7 and 8, and lipase activity was highest at pH 8 and 9. In an analysis of thermal profiles, the larval pancreatic enzymes had a high optimal temperature and high thermal stability, which are typical of fish from the tropics. At 12 and 13 dph, lipase activity and gene expression levels of trypsin (-a and -b), lipase and amylase decreased markedly, suggesting a marked change in larval metabolism at that time. These data could be useful in the development of artificial larval diets in Japanese eel.     

Habitat differentiation between sei (Balaenoptera borealis) and Bryde's whales (B. brydei) in the western North Pacific

Two closely related baleen whale species, sei and Bryde's whales, in the western North Pacific were studied to identify differences in habitat use. Data were obtained from May to August 2004 and 2005. This study examined the relationship between oceanographic features derived from satellite data and the distribution of sei and Bryde's whales using basic statistics. We investigated oceanographic features including sea surface temperature (SST), sea surface chlorophyll a (Chl‐a), sea surface height anomalies (SSHAs), and depth of the habitat. These two whale species used habitats with different SST, Chl‐a, and SSHA ranges. The 0.25 mg m^?3 Chl‐a contour (similar to the definition of the Transition Zone Chlorophyll Front) was a good indicator that separated the habitats of sei and Bryde's whales. Then generalized linear models were used to model the probabilities that the whale species would be present in a habitat and to estimate their habitat distribution throughout the study area as a function of environmental variables. The potential habitats of the two species were clearly divided, and the boundary moved north with seasonal progression. The habitat partitioning results indicated that SST contributed to the patterns of habitat‐use and might reflect differences in prey species between the two whales. This study showed that the habitats of the sei and Bryde's whales were clearly divided and their potential habitat‐use changed seasonally.     

Resistance in leaf blades assessed by counting conidia correlates with whole-plant-specific resistance in leaf sheaths in a compatible rice–Magnaporthe oryzae interaction

Resistance in the leaf blades of rice plants against a virulent race of the rice blast fungus Magnaporthe oryzae was quantitatively examined using a modified spot inoculation method. Numbers of conidia produced in the lesions were affected by plant age and paralleled the frequency of resistance infection types, which is indicative of whole-plant-specific resistance (WPSR), in the inoculated leaf sheaths of the corresponding plants. Exogenous abscisic acid treatment, which suppresses WPSR, also increased the susceptibility of the leaf blades. These results indicate a correlation between the resistance of the leaf blades and the WPSR in the leaf sheaths.     

Comparison of the amount of thiotrophic symbionts in the deep-sea mussel <Emphasis Type="Italic">Bathymodiolus septemdierum</Emphasis> under different sulfide levels using fluorescent in situ hybridization

Various invertebrates inhabiting hydrothermal vents harbor thiotrophic endosymbionts that provide the host with nutrients and are possibly involved in the detoxification of harmful sulfides. In this study, we first determined the partial 16S rRNA gene sequence of the thiotrophic symbiont of the deep-sea mussel Bathymodiolus septemdierum, a dominant species at hydrothermal vents in the Izu–Ogasawara (Bonin) area. We then designed a new probe, Bsob692, for fluorescent in situ hybridization (FISH) using regions completely conserved among thiotrophic symbionts of all bathymodiolin mussels and established the protocol for FISH to compare the distribution and amount of the symbiont using an image analysis program that is commercially available. We compared fluorescent intensity in the gill of the mussels collected at different sites and found a higher intensity in specimens collected from a site with higher sulfide concentration. We also compared mussels reared in the presence and absence of sulfide and found that the former had a higher fluorescent intensity.