Viral nervous necrosis in hatchery-reared larvae and juveniles of Japanese parrotfish, Oplegnathus fasciatus (Temminck & Schlegel)

Abstract. Mass mortalities of hatchery-reared Japanese parrotfish larvae and juveniles, Oplegnathus fasciatus (Temminck & Schlegel), have occurred in Nagasaki Prefecture. Light and electron microscopic examinations showed that the only consistent histopathological feature was extensive nervous necrosis in the spinal cord, spinal ganglia and brain. Numerous non-enveloped virus particles, icosahedral in morphology and measuring about 34 nm in diameter, were found in the cytoplasm of affected neurones and glial cells. Such nervous necrosis is believed to be the major cause of the mass mortalities of hatchery-reared Japanese parrotfish larvae and juveniles.     

Inactivation of Escherichia coli in soil by solarization

Contamination of agricultural soil by fecal pathogenic bacteria poses a potential risk of infection to humans. For the biosafety control of field soil, soil solarization in an upland field was examined to determine the efficiency of solarization on the inactivation of Escherichia coli inoculated into soil as a model microorganism for human pathogenic bacteria. Soil solarization, carried out by sprinkling water and covering the soil surface with thin plastic sheets, greatly increased the soil temperature. The daily average temperature of the solarized soil was 4–10°C higher than that of the non-solarized soil and fluctuated between 31 and 38°C. The daily highest temperature reached more than 40°C for 8 days in total in the solarized soil during the second and third weeks of the experiment. Escherichia coli in the solarized soil became undetectable (< 0.08 c.f.u. g⁻¹ dry soil) within 4 weeks as a result, whereas E. coli survived for more than 6 weeks in the non-solarized soil. Soil solarization, however, had little influence on the total direct count and total viable count of bacteria in the soil. These results indicate that soil solarization would be useful for the biosafety control of soil contaminated by human pathogens via immature compost or animal feces.     

Digestive enzyme activities of pancreas and intestinal digesta in streptozotocin-induced diabetic piglets

Six (Landrace × Large White) × Duroc crossbred, castrated weanling piglets were used to evaluate the effect of a streptozotocin (STZ) injection to induce diabetes on the activities of digestive enzymes derived from the pancreas (lipase, amylase, trypsin and chymotrypsin) in the pancreas and intestinal digesta. Fasting plasma glucose after the administration of STZ was maintained at the level of 302–491 mg/dL during this experiment, compared with the level of 89–125 mg/dL in the control group, and they were defined as the STZ‐induced diabetic piglets for about 7 weeks. Although their bodyweight increased in proportion to a quadratic curve (P < 0.0001) during 49 days after the administration of STZ, the growth of the STZ‐induced diabetic piglets was slower compared with the control. The STZ‐injection did not affect the percentage of the pancreas in bodyweight. The administration of STZ in the piglets tended to accelerate the activity of lipase (P = 0.06) and depress the activity of amylase (P = 0.15) or chymotrypsin (P = 0.18), as units/kg bodyweight, in the pancreas. In the case of measurement as units/kg bodyweight, the activities of intestinal digesta in the STZ group showed a tendency to be higher than those in control group, irrespective of the sort of enzymes. In conclusion, STZ‐induced diabetic piglets have a moderate digestive ability and the administration of exogenous digestive enzymes is not necessary when they are used as a diabetic animal model.     

Urinary free metanephrines measurement in dogs with adrenal gland diseases using a new simple liquid chromatography tandem mass spectrometry method

Measurement of urinary metanephrines in spot samples is used for the diagnosis of canine pheochromocytoma (PC). We describe a simple analytical method based on liquid chromatography tandem mass spectrometry (LC-MS/MS) for measuring free metanephrine (MN) and normetanephrine (NMN) in spot urine samples. Using the developed method, we evaluated the stability of urinary free-MN and free-NMN at various storing conditions. In addition, we assessed the feasibility of urinary free-MN and -NMN measurement for diagnosing PC. Urine samples were mixed with stable isotope internal standards and thereafter purified by ultrafiltration. The purified samples were analyzed by LC-MS/MS in the multiple reaction monitoring mode after separation on a multimode octa decyl silyl column. The coefficient of variation of free-MN and -NMN measurement was 7.6% and 5.5%, respectively. The linearity range was 0.5–10 µg/l for both analytes. Degradation was less than 10% for both analytes under any of the storage conditions. The median free-NMN ratio to creatinine of 9 PC dogs (595, range 144–47,961) was significantly higher (P<0.05) than that of 13 dogs with hypercortisolism (125, range 52–224) or 15 healthy dogs (85, range 50–117). The developed method is simple and may not require acidification of spot urine. The results of this preliminary retrospective study suggest that the measurement of urinary free metanephrines is a promising tool for diagnosing canine PC.     

Significance of mitochondria for porphyrin and heme biosynthesis

It is concluded that mitochondria are involved in three steps of porphyrin and heme biosynthesis-first, in the formation of delta-aminolevulinic acid from glycine and active succinate; second, in the synthesis of protoporphyrin; third, in the incorporation of iron into the porphyrin ring-that is, in heme formation.     

Pathogenicity of Bordetella bronchiseptica isolated from apparently healthy rabbits in guinea pig,rat, and mouse

Bordetella bronchiseptica (B. bronchiseptica) is associated with respiratory tract infections in laboratory animals. In our laboratory animal facility, B. bronchiseptica was isolated from 21 of 27 apparently healthy rabbits obtained from a breeding farm contaminated with B. bronchiseptica. Restriction fragment length polymorphism (RFLP) analysis showed that the flagellin genotype of isolates from the laboratory animal facility and breeding farm was type A, which is seen relatively frequently in rabbits in Europe. To examine its pathogenicity, guinea pigs, rats, and mice were inoculated intranasally with a representative strain isolated in the laboratory animal facility. Following inoculation of 10⁷ colony forming unit (cfu), severe inflammation was observed in the lungs of guinea pig and mice, although the inflammation was less severe in rats. The strain was recovered from the trachea and lungs of these species after inoculation with lower dose such as 10³ or 10⁴ cfu. These results suggest that the isolated strain causes respiratory tract infection in guinea pigs, rats, and mice, and that its pathogenicity higher in mice than in rats. This study extends our knowledge of interpreting the microbiologic status of laboratory animals, which will contribute to the development of reliable and reproducible animal experiments.     

Characterization of ISPsy2 and ISPsy3, Newly Identified Insertion Sequences in Pseudomonas syringae pv. eriobotryae

Two new active insertion sequences, ISPsy2 and ISPsy3, were isolated from Pseudomonas syringae pv. eriobotryae, the causal agent of stem cankers of loquat trees. ISPsy2 is 1194-bp long, has 16-bp imperfect terminal inverted repeats, and generates a 4-bp target site duplication upon insertion into the selective cartridge of the entrap vector pSHI1063. The nucleotide sequence of ISPsy2 is completely identical with that of the previously identified IS-like element located adjacent to the virulence gene psvA of Pseudomonas syringae pv. eriobotryae NAE6. The single open reading frame of ISPsy2 encodes a 323-amino-acid protein that has similarity to the transposase of the IS5 subgroup of the IS5 family. The ISPsy3 belonging to the IS91 family is 1507 bp in length, does not duplicate its target sequence, GAAC, and presents an 81% sequence homology with IS801 in P. s. pv. phaseolicola. The transposase of ISPsy3 possesses the conserved amino acid motifs found in the rolling-circle replication protein. Southern blot analysis indicated that multiple copies of ISPsy2 and ISPsy3 are present in the genomes of P. s. pv. eriobotryae and some of the other P. s. pathovars tested. Received 16 August 2001/ Accepted in revised form 19 October 2001     

Immunohistochemical detection of virus antigen in the nasal planum of rabid dogs

The rabies virus is one of the most neurotropic of all viruses infecting mammals. During the terminal phases of infection, the virus spreads to peripheral tissues, including the skin. The external skin of the nose, called the nasal planum, is a sensory organ where numerous nerve bundles and terminal nerves are distributed. Therefore, the nasal planum is expected to serve as a postmortem diagnostic material. However, the distribution of rabies virus antigens in the nasal planum in rabid animals has not yet been studied. In this study, the nasal planum was obtained from 45 rabid dogs. In all rabid dogs, the viral antigen was detected in the peripheral nerve tissues, Merkel cells, and squamous cells. The viral antigen in the epidermis exhibited three patterns: first, a diffuse positive pattern from the basal layer to the squamous layer; second, a reticular positive pattern along the cell membrane in the squamous layer; and third, a basal layer pattern of the epidermis. In the dermis, viral antigens were detected more often in lamellated corpuscles just beneath the rete pegs. These results suggest that the nasal planum could serve as a useful alternative source for postmortem diagnosis in rabies endemic countries.     

Mixed germ cell-sex cord-stromal tumor with a concurrent interstitial cell tumor in a ferret

A 5-year-old male ferret presented with an enlarged canalicular testis in the left inguinal region. Microscopically, the enlarged testis consisted of a diffuse intimately admixed proliferation of c-kit-positive germ cell-like and Wilms tumor-1 protein-positive Sertoli cell-like components, but no Call-Exner body was detected. In addition, the compact proliferation of steroidogenic acute regulatory protein-intense positive interstitial cells was identified in a separate peripheral area of the mass. Based on histopathological and immunohistochemical findings, the tumor was diagnosed as a mixed germ cell-sex cord-stromal tumor with a concurrent interstitial cell tumor.