Hyaluronidase is not essential for the lethality of Erysipelothrix rhusiopathiae infection in mice

To investigate the role of hyaluronidase in the pathogenicity of Erysipelothrix rhusiopathiae, transposon Tn916 was transferred from Enterococcus faecalis CG110 to a virulent strain of E. rhusiopathiae, and hyaluronidase-deficient mutants were isolated. A virulence assay in the mice showed that of the seven hyaluronidase-deficient mutants tested, six mutants were avirulent, but that one mutant, designated AST121, was as virulent as its parental strain. Western immunoblotting with a monoclonal antibody specific to the capsule, a major virulence factor of the organism, revealed that all of the avirulent mutants had lost the capsular antigen, whereas the mutant AST121 did not. These results suggest that the lack of virulence of the six hyaluronidase-negative mutants could be due to a loss of the capsule and that hyaluronidase does not contribute to the lethality of E. rhusiopathiae infection in mice.     

Evaluation of isotonic sodium bicarbonate solution for alkalizing effects in conscious calves

After intravenous (i.v.) infusion of various volumes of 1.35%-isotonic sodium bicarbonate solution (ISB), acid-base equilibrium, blood pressure, plasma volume and biochemical parameters in healthy Holstein calves were studied. Four calves each were randomly assigned to the low-dose (LD; i.v. infusion of 5 ml/kg ISB), middle-dose (MD; i.v. infusion of 10 ml/kg ISB) and the high-dose groups (HD; i.v. infusion of 15 ml/kg ISB). Administration volumes of ISB in the LD, MD and HD groups were decided based on the first half volumes of 5, 10 and 15 mEq of base requirement by the acceptable equation. Systemic, pulmonary artery and central venous pressures, cardiac output and plasma osmotic pressure were not changed by ISB infusion and remained constant throughout the experiment for all groups. There was good correlation (r(2) = 0.950) between relative changes in base excess and infused volume of bicarbonate (y=2.491x). The coefficient of distribution for bicarbonate ions was calculated to be 0.401 (=1/2.491). Therefore, it is suggested that a value of 0.4 would be most appropriate when calculating the base requirements in calves. Therefore, the first half volumes of ISB correcting base deficits of 5, 10 and 15 mEq in calves were estimated to be 6, 12 and 18 ml/kg, respectively. On the basis of the findings in this study, ISB may be used to correct metabolic acidosis without altering the plasma osmotic pressure, hemodynamic status and respiratory function in the calves.     

Isolation and characterisation of fetal bovine brain cells in primary culture

Primary cultures and cryopreservation procedure of bovine brain cells were established as in vitro experimental systems to study the responses of bovine brain cells to neuropathogenic agents. Brain cells were dissociated by papain from the cerebellum of a bovine fetus at 90 to 120 days old, and were cultured in different media. In a medium containing 1 per cent fetal bovine serum (FBS), neuronal cells were maintained and they formed clusters on glial and fibroblastic cell sheets. In a medium containing 10 per cent FBS, the proportion of neurones decreased, and fibroblastic and microglial cells dominated. In a serum-free medium containing epidermal growth factor, the highest neuronal proportion was obtained. Optimal cryopreservation condition for the brain tissues was investigated by changing the concentrations of DMSO and FBS. Brain cells could be cultured from cryopreserved tissue with only slightly reduced growth profiles and varying cell proportions in comparison to those prepared from fresh tissue.     

Preferential infection of neuronal and astroglia cells by Akabane virus in primary cultures of fetal bovine brain

Akabane virus is a member of the genus Bunyavirus; it is pathogenic for ruminants and transmitted by arthropod vectors. Infection of adult cattle and sheep causes a transient viremia without obvious clinical signs, while infection of pregnant animals often causes fetal abnormalities including hydranencephaly, poliomyelitis and arthrogryposis. Infectious virus or viral antigens is present in the brain, spinal cord and skeletal muscle of infected fetuses. To understand the interaction between Akabane virus and bovine brain cells, we investigated the viral tropism using primary cultures of fetal bovine brain. The cultured neuronal cells, astroglia cells and microglia cells were distinguished by cell type specific antisera. Akabane virus was found to infect neuronal cells and astroglia cells, which led to degenerative death. No microglia cells were found infected. In some brain cultures, we observed different sensitivities of the cells to two Akabane virus strains: an attenuated strain infected and spread more readily than wild type virus. This difference was not observed in a hamster fibroblast cell line. Both viral and host determinants might be involved in the different susceptibility of brain cells to Akabane virus infection.     

Immunogenic properties of a bovine herpesvirus-1 (BHV-1) recombinant expressing major pseudorabies virus (PrV) glycoproteins in combination

A recombinant bovine herpesvirus type 1 (BHV-1), designated BHV-1/TF17-1, which expresses pseudorabies virus (PrV) glycoproteins gB, gC, gD, gE and gI in combination was constructed. To test the protective immunity, 10 mice were inoculated with BHV-1/TF17-1 and three weeks later 10 mice were intraperitoneally (i.p.) challenged with 20 LD50 virulent PrV (YS-81). BHV-1/TF17-1 protected all the mice from the PrV lethal challenge while all the control mice died in around 3 days. Mice vaccinated with BHV-1/TF17-1 acquired high PrV-neutralizing antibody titers and demonstrated strong delayed type hypersensitivity responses and moderate in vitro lymphocyte proliferative responses to PrV antigen. Since the major PrV glycoproteins were integrated into virions (probably into viral envelope), BHV-1/17-1 was neutralized with anti-PrV antiserum. However, the susceptibility of BHV-1/TF17-1 to anti-PrV antiserum is 2- to 4-fold lower than that of PrV vaccine lines. Our results demonstrated the possibility of BHV-1/17-1 as a vaccine to protect piglets from Audjesky's disease where maternal antibodies against PrV interfere attenuated live PrV vaccines.     

Sequential observation of 2,6-dimethylaniline-induced nasal lesions in a rat two-stage nasal carcinogenesis model after initiation with N-bis(2-hydroxypropyl) nitrosamine

Male F344 rats received diet containing 3,000 ppm 2,6-dimethylaniline (DMA) after initiation with a single subcutaneous injection of 2,400 mg/kg of N-bis(2-hydroxypropyl)nitrosamine (DHPN), and histological and electron microscopic examinations of the nasal cavity were performed at 4, 13, 26 and 52 weeks to examine sequential changes induced by DMA. Severe atrophy of Bowman's glands and epithelial disarrangement were apparent from week 4, followed by dilatation and/or proliferation of Bowman's glands, degeneration of epithelial cells, and proliferation of undifferentiated epithelial cells from week 13. Focal glandular hyperplasias, dysplastic foci, and adenomas were observed from week 26, and carcinomas at 52 week. These nasal lesions were mostly evident in the olfactory mucosa in the nasal cavity, and their severity and/or incidences, other than atrophy of Bowman's glands, increased with the treatment period. Electron microscopically, carcinoma cells demonstrated desmosomes, dense secretory granules identical to those in normal Bowman's glands, a basement membrane, and microvilli. These results suggest that Bowman's glands are the target of DMA, giving rise to nasal carcinomas after DHPN-initiation.     

Apoptosis-independent Poor Morphology of Bovine Embryos Produced by Multiple Ovulation

In multiple ovulation and embryo transfer (MOET) programmes in cattle, a considerable number of morphologically poor-quality embryos continue to be produced; this is one of the limiting factors of the technique. Apoptosis has often been implicated in developmental arrest and fragmentation; these are regarded as poor traits of embryonic quality in mammalian pre-implantation embryos. In the present study, apoptosis was assessed in morphologically poor-quality embryos in comparison with good-quality embryos that were recovered from a MOET programme. Retarded embryos (two to 16 cell stage), morulae with severe fragmentation and morphologically good-quality morulae recovered from superstimulated cows at day 7 post-insemination were subjected to TdT-mediated dUTP nick-end labelling (TUNEL) and Hoechst staining. Cell nuclei that showed both TUNEL staining and apoptotic morphology were considered to be apoptotic. Apoptotic index (AI) was calculated as the percentage of apoptotic cells per embryo. Fifteen of 17 retarded embryos and 10 of 15 morphologically poor-quality morulae did not show signs of apoptosis. The mean AIs in the morphologically poor-quality embryos (two to 16 cell stage, 2.2%; poor morulae, 1.3%) were as low as that in the good-quality embryos (2.9%). These results suggest that another mode of developmental arrest and/or fragmentation that is independent of apoptosis occurs in morphologically poor-quality embryos recovered from MOET programmes.     

PCR-based differentiation of Fusarium oxysporum ff. sp. lycopersici and radicis-lycopersici and races of F. oxysporum f. sp. lycopersici

The pathogenic type (form and race) of Fusarium oxysporum, which generates wilt symptoms on tomato, was rapidly identified with a polymerase chain reaction (PCR)-based technique. We compared the partial nucleotide sequences of endo polygalacturonase (pg1) and exo polygalacturonase (pgx4) genes from isolates of F. oxysporum ff. sp. lycopersici (FOL) and radicis-lycopersici (FORL) from Japan and designed specific primer sets (uni, sp13, sp23, and sprl) based on the nucleotide differences that appeared among the pathogenic types. PCR with the uni primer set amplified a 670∼672-bp fragment from all isolates of FOL and FORL. With the sp13 primer set, an amplicon of 445 bp was obtained only from isolates of FOL race 1 and 3. With the sp23 primer set, a 518-bp fragment was obtained from isolates of FOL race 2 and 3. The sprl primer set yielded a 947-bp fragment from isolates of FORL, but not from FOL. A combination of amplifications with these primer sets effectively differentiated the pathogenic types of F. oxysporum in tomato.     

The effect of exogenous purine supply on the endogenous excretion of purine derivatives in the urine of growing lambs

An experiment was made to determine the absorption of purine metabolites in dietary nucleic acids through the digestive tract, and also to determine the utilization of nucleic acids absorbed in the body, using growing lambs. Two pairs of 120‐ and 180‐day‐old twin female lambs with a bodyweight of 18.2–19.0 kg were kept in metabolism crates and fed on purine‐free milk replacement (MR) with supplements of exogenous purine (purine base or purine nucleoside) at a level of 0.2 mmol/BW^0.75/d for 5 consecutive days, and thereafter they were maintained in the crates for 4 days. The daily amount of exogenous purine supply was calculated based on the urinary excreted purine derivatives (PD) in lambs fed on milk replacement alone. A urine sample was collected daily for 9 consecutive days, and the urinary excretion of PD was determined daily. Urinary PD excretion opened to increase within 24 h after the dose of purine bases, and the level was recovered on 3 days after ceasing the exogenous purine supply. The recovery of PD in the urine was about 70% of the purine supplement. When purine nucleosides were added to the feed, urinary PD excretion was initiated within 24 h after dosing, and the values were recovered after ending the purine nucleoside supply. The recovery rate of PD in the urine was only 30% of the supplemented purine. The plasma allantoin levels were almost similar after feeding purine bases and purine nucleosides, and the values were mostly in the range (40–60 µmol/L). These findings indicate that an exogenous purine can be directly incorporated into the body, and the purine as nucleoside is more effectively utilized for the synthesis of nucleic acids than as a purine base in the body of growing lambs.