Investigation for presence of Neospora caninum, Toxoplasma gondii and Brucella-species infection in killer whales (Orcinus orca) mass-stranded on the coast of Shiretoko, Hokkaido, Japan

Twelve killer whale (Orcinus orca) were hemmed in by ice floes, and nine died on the Aidomari coast in the Nemuro Strait in Rausu, Shiretoko, Hokkaido, Japan on 8 February 2005. Tissue samples collected from 8 whales were tested for Neospora caninum, Toxoplasma gondii, and Brucella species DNA by polymerase chain reaction (PCR) assay. Gamma-globulin isolated from blood samples by ammonium sulfate precipitation was tested for antibodies to these pathogens by means of agglutination tests and immunoblotting. None of the 8 tissue samples had antibodies to the pathogens, when subjected to agglutination tests. In immunoblotting, one sample (sample No.5) showed antibody binding to N. caninum antigens. In the PCR assay, none of the samples was positive. Further study is necessary to examine the prevalence of the pathogens in marine mammals inhabiting this area.     

Muscle type‐specific effect of myostatin deficiency on myogenic regulatory factor expression in adult double‐muscled Japanese Shorthorn cattle

To clarify muscle type‐specific effect of myostatin on myogenic regulatory factors (MRFs), we examined mRNA expression of MRFs in five skeletal muscles of normal (NM) and myostatin‐deficient double‐muscled (DM) adult Japanese Shorthorn cattle by quantitative reverse‐transcribed PCR. Among the four MRFs, namely, Myf5, MyoD, myogenin, and MRF4, MyoD expression was different among the muscles of the DM cattle (P < 0.01) but not of the NM cattle. Meanwhile, MyoD expression was significantly elevated only in masseter (MS) muscle in the DM cattle due to the myostatin deficiency (P < 0.05). Myf5 and MRF4 expression in semitendinosus (ST) was higher in the DM than in the NM cattle (P < 0.05). According to analysis of myosin heavy chain (MyHC) isoform expression, more MyHC‐2x and ‐2a and less ‐slow isoforms were expressed in the longissimus and ST muscles compared to the MS muscle in both cattle (P < 0.05), but no significant difference in MyHC expression was observed between the NM and DM cattle. Taken together, myostatin has influences on Myf5 and MRF4 expression in faster‐type muscles and on MyoD expression in slower‐type muscles, suggesting a possible muscle type‐specific effect of myostatin in skeletal muscle growth and maintenance.     

Regional specialization of the Ganglion cell density in the retina of the Ostrich (Struthio camelus)

In this study, retinal whole-mount specimens were prepared and stained with 0.1% cresyl violet for the ganglion cell study in the Ostrich (Struthio camelus) . The total number, distribution, and size of these cells were determined in different retinal regions. The mean total number of ganglion cells (three retinas) was 1 435 052 with an average density of 652 cells/mm². The temporo – nasal area of the retina with high cell density were identified with the peak of 7525 cells/mm² in the central area. The size of most ganglion cells ranged from 113–403 µm², with smaller cells predominating along the temporo-nasal streak above the optic disc and larger cells comprising more of the peripheral regions. The average thickness of the retina was 196 µm. The central area was the thickest area (268.6 µm), whereas the peripheral area was the thinnest area. Thus, the specialization of ganglion cell densities, their sizes and the thickness of the retina support the notion that the conduction of visual information towards the brain from all regions of the retina is not uniform, and suggests that the temporo – nasal streak is the fine quality area for vision in ostriches.     

The cellular differentiation of M cells from crypt undifferentiated epithelial cells into microvillous epithelial cells in follicle-associated epithelia of chicken cecal tonsils

To clarify the cellular origin and the fate of M cells, detailed distributions of the epithelial cells were investigated scanning electron microscopically on the follicle-associated epithelia (FAE) of chicken cecal tonsils. The distribution of M cells was closely related with the situation of the crypt orifices in chicken cecal tonsils. In undeveloped cecal tonsils, the intestinal crypts were localized at the periphery of the FAE. In these tonsils, M cells without microvilli (M(0)) were predominantly populated in the basal region of the FAE, whereas goblet cells and microvillous epithelial cells (MV) were more distributed in the middle to the apical region of the FAE. A few M cells with short microvilli were dispersed throughout the FAE. Significantly shrunk MV (MVs) clustered together in transitional portions from the lateral face to the roof of the FAE. In well-developed cecal tonsils, the crypts also opened at the lateral surface in addition to the periphery of the FAE. In these tonsils, the M(0) accumulated densely in the small areas around the crypt orifices exclusively. No sign of exfoliation of apoptotic epithelial cells was found in the M(0)-accumulated areas and at their peripheral boundaries. The MVs were often clustered in the central regions among the crypt orifices in addition to the roof of the FAE. These findings suggest that M cells are directly derived from the undifferentiated crypt epithelial cells, not fall into apoptotic cell death and further differentiate into MV in the FAE of chicken cecal tonsils.     

Morphological reproductive characteristics of testes and fertilization capacity of cryopreserved sperm after the Fukushima accident in raccoon (Procyon lotor)

Since the Fukushima Daiichi Nuclear Power Plant (FDNPP) accident, we have established an archive system of livestock and wild animals from the surrounding ex-evacuation zone. Wildlife within the alert zone have been exposed to low-dose-rate (LDR) radiation for a long continuous time. In this study, we analysed the morphological characteristics of the testes and in vitro fertilization (IVF) capacity of cryopreserved sperm of racoons from the ex-evacuation zone of the FDNPP accident. The radioactivity of caesium-137 (¹³⁷Cs) was measured by gamma-ray spectrometry, and the measured radioactivity concentration was 300–6,630 Bq/kg in the Fukushima raccoons. Notably, normal spermatogenesis was observed in the seminiferous tubules of the testes, with the germinal epithelium composed of a spermatogenic cell lineage with no evident ultrastructural alterations; freeze-thawing sperm penetration ability was confirmed using the interspecific zona pellucida-free mouse oocytes IVF assays. This study revealed that the chronic and LDR radiation exposure associated with the FDNPP accident had no adverse effect on the reproductive characteristics and functions of male raccoons.     

Effective combinations of functional groups in chemically modified kraft lignins for reduction of aluminum toxicity

Plant growth tests were performed with radish (Raphanus sativa var. radicula Pers.) in culture solutions containing low molecular weight compounds in the presence of aluminum to determine the types of functional groups in kraft lignin (KL) modified with ozone and alkali that contributed to reducing aluminum toxicity. The low molecular weight compounds used in this study contained carboxyl, formyl, methoxyl, alcohol hydroxyl, and phenolic hydroxyl groups. The compounds that had adjacent two carboxyl groups (oxalic acid), carboxyl/alcohol hydroxyl groups (glycolic acid), or carboxyl/formyl groups (glyoxylic acid) were effective in reducing aluminum toxicity. Malonic acid, having two carboxyl groups, also reduced aluminum toxicity. The ability of ozone-treated KLs to reduce aluminum toxicity was considered to be partly due to these chemical structures. Protocatechuic acid, having two adjacent phenolic hydroxyl groups, was also effective in reducing aluminum toxicity. This indicated that the effectiveness of the alkaline-treated KL was partly due to its catechol structure. This report was presented in part at the 58th Annual Meeting of the Japan Wood Research Society, Tsukuba, Japan, March 2008     

Effect of dietary high amount of calcium and phosphorous on reducing the prevalence of morphological deformities in GH-transgenic Nile tilapia

To satisfy the world’s growing need for animal protein, it is important to develop or select fish species which are suitable for optimal cultivation in closed recirculating aquaculture systems (CRASs) as an environment-friendly aquaculture technology for providing safe and secure aquaculture food products. We have developed an ‘all fish’ growth-hormone (GH)-transgenic Nile tilapia Oreochromis niloticus (GHTi) which appears to be a predominant candidate for CRAS since it has been found to exhibit two important characteristics, namely, a shorter rearing duration to a market size and lowered N and P discharge load. However, these fish have undergone morphological changes, resulting in a short body which is less acceptable to consumers as an edible fish. One possible solution to reducing the prevalence of such morphological deformities is to determine the association between changes in the nutritional conditions of these fish. To this end, we first calculated the ideal total calcium (Ca) and phosphorous (P) intake (g/kg), ideal daily Ca and P intake and retention (g/kg/day) and ideal daily feeding rate (%) for the fourth filial (F₄) offspring of GHTi. Our hypothesis was that GHTi would achieve the same total retention of Ca (14.6 g/kg) or P (7.69 g/kg) during a 69-day shorter rearing period as their production-matched pedigree common domestic strain (CTi). We estimated that when GHTi were fed conventional commercial diets to satiety, those with a body weight (BW) of more than approximately 230 g could not retain sufficient Ca, while those with a BW of more than approximately 429 g could not retain sufficient P. We then attempted to reduce the prevalence of morphological deformities in GHTi via dietary compound regulation by supplying a high amount of dietary Ca [6.82 % in the diet with Ca supplementation (Ca-S)] or P [4.75 % in the diet with P supplementation (P-S)]. Both the Ca-S and P-S diet improved mineral retention, while those fed on a Ca-S diet showed a lower prevalence of deformities, close to the size-matched CTi. These results suggest that the requirements of dietary Ca and P were significantly higher in GHTi than in CTi and, consequently, that the conventional levels of Ca and P in commercial diets for CTi cannot meet the mineral requirement of GHTi. We therefore propose to develop an optimal feed to meet the altered nutritional requirements of GH-transgenic Nile tilapia for growth-promoting optimization. This may become prerequisite for rearing this GHTi line in CRASs.     

Persorption mechanisms of luminal antigenic particulates via apoptotic epithelial cells of intestinal villi into systemic blood circulation in orally immunized rats

The possibility of persorption of prefixed bovine serum albumin-coated sheep erythrocytes (BSA-SEs) from mucous epithelial cells and its mechanisms were investigated in rats orally immunized by BSA for 14 consecutive days. On the day after the final oral immunization, the rats were duodenally perfused by BSA-SEs or non-coated SEs. BSA-SEs were also duodenally perfused in non-immunized rats. Thirty min after perfusion, BSA-SEs were significantly more engulfed by late-apoptotic-stage villous columnar epithelial cells in the orally immunized rats than those in other experiments. The specific antibody (SpAb) was detected on the surfaces of BSA-SEs in rats with oral immunization. In Peyer's patches of all animals, no SEs reached the follicle-associated epithelium, because of the close attachment of follicle-associated intestinal villi and the thick mucous layer. BSA-SEs were more frequently persorbed into portal blood in the orally immunized rats than in other rats. Small numbers of BSA-SEs or SEs were detected in the systemic blood of all animals. BSA-SEs were also histologically found in the blood vessels of the liver, but not in mesenteric lymph nodes. These findings suggest that sensitized antigenic particulates are taken up by late-apoptotic-stage villous columnar epithelial cells in the small intestine and are finally persorbed into the systemic blood circulation. The uptake of antigenic particulates might be mediated by its luminal SpAb.     

Comparisons of ribosomal DNA-internal transcribed spacer sequences and biological features among <Emphasis Type="Italic">Olpidium bornovanus</Emphasis> isolates from Cucurbitaceae-cultivating soil in Japan

The chytrid fungus Olpidium bornovanus is an obligate plant parasite that acts as a vector to transmit Melon necrotic spot virus in cultivated soil. Here, we conducted a molecular phylogenetic analysis of 16 isolates of O. bornovanus taken from soil in which Cucurbitaceae plants had been grown in various locations throughout Japan. The ribosomal DNA and internal transcribed spacer region sequences of the 16 O. bornovanus isolates were divided into four molecular phylogenetic groups, designated O.bor-A to O.bor-D. Biological features of O. bornovanus isolates such as host specificity varied consistently with their molecular phylogenetic groups.