Inclusion of novel bacteria in rumen microbiology: Need for basic and applied science

Rumen microbiology has made a significant contribution to the understanding of ruminant nutrition. However, further progress in research has been hindered by the incomplete analysis of the rumen microbiota comprised of bacteria, protozoa and fungi, most of which remain uncharacterized due to the difficulties in their isolation and cultivation. In order to maximize rumen fiber digestion, it is necessary to understand the community structure of rumen microbes, especially bacteria, and the factors that influence their composition. Recent advances in molecular biology techniques allow the analysis of such bacteria without cultivation, thereby identifying many functional, but uncultured, bacteria as new targets for basic and applied research. Specific uncultured bacterial groups are being considered as important members of a fibrolytic consortium in the rumen, judging by their ecologic distribution. The inclusion of such uncharacterized bacteria in analyses is crucial for understanding the rumen microbial community and its manipulation. In addition, these bacteria could potentially be candidates as probiotics and sources of enzymes for animal feed and other industrial uses.     

Ecological characterization of three different phylogenetic groups belonging to the cellulolytic bacterial species Fibrobacter succinogenes in the rumen

To study the group‐dependent ecology of Fibrobacter succinogenes in the rumen, real‐time polymerase chain reaction assays for two phylogenetic groups (groups 2 and 3) of F. succinogenes were newly established and applied to rumen samples. Both the assays targeting the bacterial 16S rDNA were sensitive and accurate, showing wide quantifiable ranges (10⁴?10⁹ and 10²?10⁹ copies of 16S rDNA) and high recoveries of known amounts of added DNA (96.9 and 98.0%). The quantity of group 1 was confirmed to be numerable by subtracting assay values of groups 2 and 3 from that of F. succinogenes species (groups 1–3). By using the developed assays and the above subtractive calculation, the quantities of all three groups were evaluated in solid and liquid fractions of the rumen content and also on hay stems. In the solid fraction, groups 1 and 2 were abundantly present, compared with group 3 (P < 0.05). On untreated hay stems, group 1 was dominant throughout 48 h. In addition, group 1 showed growth even on the cellulase‐treated hay stems, unlike the other two groups. These results suggest that F. succinogenes group 1 greatly contributes to rumen fiber digestion, even for less degradable materials.     

Production of transgenic cloned pigs expressing the far-red fluorescent protein monomeric Plum

Monomeric Plum (Plum), a far-red fluorescent protein with photostability and photopermeability, is potentially suitable for in vivo imaging and detection of fluorescence in body tissues. The aim of this study was to generate transgenic cloned pigs exhibiting systemic expression of Plum using somatic cell nuclear transfer (SCNT) technology. Nuclear donor cells for SCNT were obtained by introducing a Plum-expression vector driven by a combination of the cytomegalovirus early enhancer and chicken beta-actin promoter into porcine fetal fibroblasts (PFFs). The cleavage and blastocyst formation rates of reconstructed SCNT embryos were 81.0% (34/42) and 78.6% (33/42), respectively. At 36–37 days of gestation, three fetuses systemically expressing Plum were obtained from one recipient to which 103 SCNT embryos were transferred (3/103, 2.9%). For generation of offspring expressing Plum, rejuvenated PFFs were established from one cloned fetus and used as nuclear donor cells. Four cloned offspring and one stillborn cloned offspring were produced from one recipient to which 117 SCNT embryos were transferred (5/117, 4.3%). All offspring exhibited high levels of Plum fluorescence in blood cells, such as lymphocytes, monocytes and granulocytes. In addition, the skin, heart, kidney, pancreas, liver and spleen also exhibited Plum expression. These observations demonstrated that transfer of the Plum gene did not interfere with the development of porcine SCNT embryos and resulted in the successful generation of transgenic cloned pigs that systemically expressed Plum. This is the first report of the generation and characterization of transgenic cloned pigs expressing the far-red fluorescent protein Plum.     

Distribution of the sex chromosome during mouse spermatogenesis in testis tissue sections

During mammalian spermatogenesis, spermatogenic cells undergo mitotic division and are subsequently divided into haploid spermatids by meiotic division, but the dynamics of sex chromosomes during spermatogenesis are unclear in vivo. To gain insight into the distribution of sex chromosomes in the testis, we examined the localization of sex chromosomes before and after meiosis in mouse testis sections. Here, we developed a method of fluorescence in situ hybridization (FISH) using specific probes for the X and Y chromosomes to obtain their positional information in histological testis sections. FISH analysis revealed the sex chromosomal position during spermatogenesis in each stage of seminiferous epithelia and in each spermatogenic cell. In the spermatogonia and leptotene spermatocytes, sex chromosomes were distantly positioned in the cell. In the zygotene and pachytene spermatocytes at prophase I, X and Y chromosomes had a random distribution. After meiosis, the X and Y spermatids were random in every seminiferous epithelium. We also detected aneuploidy of sex chromosomes in spermatogenic cells using our developed FISH analysis. Our results provide further insight into the distribution of sex chromosomes during spermatogenesis, which could help to elucidate a specific difference between X and Y spermatids and sex chromosome-specific behavior.     

Therapeutic effects of vitamin E supplementation in 4 dogs with poor semen quality and low superoxide dismutase activity in seminal plasma

Four dogs with poor semen quality, low seminal plasma superoxide dismutase (SOD) activity and low blood plasma testosterone (T) levels were orally administered one vitamin E tablet containing 50 mg α-tocopheryl acetate per dog daily for 4 weeks. The mean values of semen quality were temporarily improved after the start of vitamin E treatment and the values of 4, and 5 weeks after that were significantly different from those before the treatment (P<0.05–0.001). The mean blood plasma T and seminal plasma SOD activity values slightly increased in the 4 dogs after the treatment. The results of the present study indicate that poor semen quality in dogs with low seminal plasma SOD can be improved by vitamin E treatment.     

Swine abortion caused by Candida parapsilosis

In July 2020, a sow in a breeding herd in the Chiba Prefecture, Japan, suffered abortion. A necropsy revealed pale pulmonary foci scattered in the two fetuses. Histologically, multifocal pulmonary necrosis was detected with numerous yeasts. The yeast was positively stained using the periodic acid-Schiff reaction and Grocott’s silver stain. Molecular identification indicated that the yeast was Candida parapsilosis. In conclusion, our results suggested that C. parapsilosis caused multifocal necrotizing pneumonia in the two fetuses. This study is the first report of a swine abortion with C. parapsilosis infection.     

Allelopathy of the moss Hypnum plumaeforme by the production of momilactone A and B

Extract of soil under colonies of Hypnum plumaeforme inhibited the growth of roots and shoots of cress, lettuce, lucerne, ryegrass, timothy, Digitaria sanguinalis and Echinochloa crus-galli . Increasing the extract concentration increased the inhibition, which suggest that the soil may contain growth inhibitory substances and possess allelopathic potential. The extract of the soil under H. plumaeforme was purified and two main inhibitory substances were isolated and determined by MS and ¹H- and ¹³C-NMR spectral data as momilactone A and B. Momilactone A and B inhibited hypocotyls and roots of cress seedlings at concentrations >10 and 1 μ m respectively. The endogenous concentration of momilactone A and B in H. plumaeforme was 58.7 and 23.4 μg g⁻¹ dry weight respectively and the concentration of momilactone A and B in MS growth medium of H. plumaeforme was 4.3 and 6.4 μg g⁻¹ dry weight of H. plumaeforme , respectively. These results suggest that momilactone A and B were probably secreted into the medium during the incubation and momilactone A and B found in the soil under H. plumaeforme may have been released by the moss. Therefore, growth inhibitory activity of the soil under H. plumaeforme may be caused by momilactone A and B, which may act as allelopathic agents of H. plumaeforme .     

Molecular Phylogenetic Analysis of Ribosomal DNA Internal Transcribed Spacer Regions and Comparison of Fertility in <Emphasis Type="Italic">Phomopsis </Emphasis>Isolates from Fruit Trees

Sequences of the ribosomal DNA (rDNA) internal transcribed spacer (ITS) regions were examined to infer a molecular phylogeny of small-spored Phomopsis isolates, designated W-type (mainly white colony, weakly virulent, bearing both alpha and beta conidia at 25°C on PDA) and G-type (mainly gray colony, highly virulent, bearing only alpha conidia at 25°C on PDA), and P. amygdali from fruit trees. Phomopsis G-type and P. amygdali were a monophyletic group distinct from the W-type. The W-type isolates were divided into two monophyletic groups. Diaporthe citri, D. tanakae, P. asparagi, P. viticola, P. vitimegaspora and D. nomurai, which are morphologically distinguishable from W- and G-types, differed from the W- and G-types in molecular phylogenetic analyses. PCR-RFLP analysis of rDNA ITS regions was useful to distinguish each of the Phomopsis species and groups using three restriction enzymes. In mating tests, W-type isolates from fruit trees were heterothallic and inter-fertile even between isolates belonging to the different monophyletic groups. Isolates of the G-type and P. amygdali collected in Japan were cross-fertile. Some isolates from Lunaria annua, Ulmus glabra and Juglans regia belonged to one of the two monophyletic groups of the W-type and were cross-fertile with W-type isolates from Rosaceous fruit trees. Received 27 September 1999/ Accepted in revised form 27 January 2000     

Web Blight of Fan Columbine, <Emphasis Type="Italic">Aquilegia flabellata, </Emphasis>Caused by <Emphasis Type="Italic">Rhizoctonia solani </Emphasis>AG1-IB : A New Disease Record

A new disease, found on fan columbine in August of 1997, first appeared as necrotic spots on leaves and within a week caused wilting of all the leaves. Fungal mycelia bound aerial parts of the plants together, formed mats of mycelia and eventually killed the plants. The pathogen, isolated from the infected leaves and stalks, was identified as Rhizoctonia solani AG1-IB in respect to hyphal anastomosis and culture types. The common name of web blight is proposed for this new occurrence on fan columbine (Kumonosu-byo in Japanese). Received 24 November 1999/ Accepted in revised form 4 February 2000     

Characteristic computed tomographic myelography findings in 23 Thoroughbred horses

The purpose of this observational study was to determine the characteristic computed tomographic (CT) myelography findings of cervical vertebral stenotic myelopathy (CVSM) lesions in Thoroughbred horses. A total of 23 Thoroughbred horses (age range, 155–717 days on CT examination; mean, 410.9 days) were analyzed. All 23 Thoroughbred horses underwent unenhanced radiography, radiographic myelography, and CT myelography. Unenhanced radiographs were observed the presence of cervical vertebral malalignment and osseous lesions. Radiographic myelograms were observed for signs of cervical spinal cord compression; additionally, CT myelograms were used to detect cervical vertebral osseous lesions. Ventral compressions were frequently observed in the cranial cervical vertebrae (C2–C4), whereas dorsal compressions were frequently observed in the caudal cervical vertebrae (C5–C7). Furthermore, osseous lesions of the caudal articular process developed more frequently than those of the cranial articular process. CT myelography in Thoroughbred horses is a useful method for detecting CVSM changes.